Evolution of Darwin’s Peloric Gloxinia (Sinningia speciosa) Is Caused by a Null Mutation in a Pleiotropic TCP Gene

Evolution of Darwin’s Peloric Gloxinia (Sinningia speciosa) Is Caused by a Null Mutation in a... Abstract Unlike most crops, which were domesticated through long periods of selection by ancient humans, horticultural plants were primarily domesticated through intentional selection over short time periods. The molecular mechanisms underlying the origin and spread of novel traits in the domestication process have remained largely unexplored in horticultural plants. Gloxinia (Sinningia speciosa), whose attractive peloric flowers influenced the thoughts of Darwin, have been cultivated since the early 19th century, but its origin and genetic basis are currently unknown. By employing multiple experimental approaches including genetic analysis, genotype–phenotype associations, gene expression analysis, and functional interrogations, we showed that a single gene encoding a TCP protein, SsCYC, controls both floral orientation and zygomorphy in gloxinia. We revealed that a causal mutation responsible for the development of peloric gloxinia lies in a 10-bp deletion in the coding sequence of SsCYC. By combining genetic inference and literature searches, we have traced the putative ancestor and reconstructed the domestication path of the peloric gloxinia, in which a 10-bp deletion in SsCYC under selection triggered its evolution from the wild progenitor. The results presented here suggest that a simple genetic change in a pleiotropic gene can promote the elaboration of floral organs under intensive selection pressure. peloric gloxinia, Sinningia speciosa, domestication, molecular mechanism, floral horizontal orientation, pleiotropic gene Introduction The interaction between humans and plants is best characterized by horticultural domestication, which is intimately associated with the development of modern civilization over the last 300 years (Janick 2005; Kingsbury 2009). Horticultural plants were domesticated by humans through intentional selection, usually by crossing different varieties to alter morphological characters for ornamental, medicinal or religious purposes, over a relatively short time period (Doebley et al. 2006; Kingsbury 2009). Some horticultural plants, like Petunia and Primula, were wildly used as model system to develop genetic toolkits for experimental studies in evolution of floral traits and speciation under natural selection regime (Hoballah et al. 2007; Amrad et al. 2016; Li et al. 2016; Sheehan et al. 2016). However, the origin of horticultural characters arising from domestication have remained practically unexplored in most horticultural plants at the molecular level. Gloxinia is a tropical gesneriacious plant native to Brazil and was domesticated in Great Britain in the early 19th century for its large attractive peloric flowers. The peloric gloxinia was originally documented in 1845, and famously became known as Darwin’s peloric gloxinia. Distinct from the peloric variety, the wild progenitors produce nodding zygomorphic flowers that is adapted to insect pollination (fig. 1) (Loddiges 1817; Harrison 1847; Darwin 1868). The genetic control of floral zygomorphy was best characterized in the model species Antirrhinum majus with isolation and function investigation of two paralogous TCP (TEOSINTE BRANCHED1, CYCLOIDEA, and PCF) genes CYCLOIDEA (CYC) and DICHOTOMA (DICH) (Luo et al. 1996, 1999). CYC and DICH establish zygomorphic floral patterning by specifying the dorsal organ identities in the second and third whorls (Luo et al. 1996, 1999). In the Gesneriaceae, the CYC-like genes were demonstrated to be involved in the development of zygomorphic flowers, with diverse morphological variation (Song et al. 2009; Yang et al. 2012, 2015). Previously, Citerne et al. (2000) isolated a partial coding sequence of GCYC from a peloric gloxinia cultivar and observed a single adenine base deletion immediately downstream of the TCP domain. It was therefore proposed that the deletion and resulting truncated protein promotes the development of the peloric phenotype (Citerne et al. 2000). However, subsequent resequencing of GCYC from other peloric gloxinia accessions showed that the adenine deletion is probably a PCR artefact (Zaitlin 2011; also see Smith et al. 2004), implying that the molecular mechanism underlying the peloric gloxinia is more complicated than previously hypothesized. Fig. 1. View largeDownload slide Phenotypic analysis of flower character development in the wild-type and cultivated peloric gloxinia. (A) Plant architecture of wild-type gloxinia (WT-PF), showing the horizontally oriented zygomorphic flower. (B) Front view of WT-PF flower. (C) Stamen of WT-PF, the red arrow denotes the dorsal staminode. (D) Side view of WT-PF floral tube (sepals are removed), showing the gibbous structure (arrow). (E) Plant architecture of cultivated peloric gloxinia (MU-WB), showing the upright actinomorphic flower. (F) Front view of MU-WB flower. (G) Stamen of MU-WB. (H) Side view of MU-WB floral tube (sepals are removed), showing the loss of the gibbous structure. (I–N) Floral developmental series of wild-type gloxinia (WT-PF) revealed by SEM. (I) The sepals initiated and developed equally. (J) The petal primordia arise subsequently inside the five sepals (removed). (K) The stamen primordia emerge inside the five petals in an alternating pattern. (L–N) As the floral organs advance in development, the floral zygomorphy manifests with the two dorsal petals slightly smaller than the lateral and ventral ones (L and M), and the dorsal stamen is finally suppressed into a staminode in development (N). (O–T) Floral developmental series of peloric gloxinia (MU-WB) revealed by SEM. (O) The sepals initiated and developed equally. (P) Petal primordia emerge subsequently inside the five sepals (removed). (Q) The stamen primordia arise inside the five petals in an alternative pattern. (R, S) As the development program proceeds, all the floral organs in each whorl develop equally. (T) The five stamens are equally differentiated into fertile stamens. Scale bar in (A, E), 10cm; (B–D and F–H), 1cm; (I–T), 100μm. Fig. 1. View largeDownload slide Phenotypic analysis of flower character development in the wild-type and cultivated peloric gloxinia. (A) Plant architecture of wild-type gloxinia (WT-PF), showing the horizontally oriented zygomorphic flower. (B) Front view of WT-PF flower. (C) Stamen of WT-PF, the red arrow denotes the dorsal staminode. (D) Side view of WT-PF floral tube (sepals are removed), showing the gibbous structure (arrow). (E) Plant architecture of cultivated peloric gloxinia (MU-WB), showing the upright actinomorphic flower. (F) Front view of MU-WB flower. (G) Stamen of MU-WB. (H) Side view of MU-WB floral tube (sepals are removed), showing the loss of the gibbous structure. (I–N) Floral developmental series of wild-type gloxinia (WT-PF) revealed by SEM. (I) The sepals initiated and developed equally. (J) The petal primordia arise subsequently inside the five sepals (removed). (K) The stamen primordia emerge inside the five petals in an alternating pattern. (L–N) As the floral organs advance in development, the floral zygomorphy manifests with the two dorsal petals slightly smaller than the lateral and ventral ones (L and M), and the dorsal stamen is finally suppressed into a staminode in development (N). (O–T) Floral developmental series of peloric gloxinia (MU-WB) revealed by SEM. (O) The sepals initiated and developed equally. (P) Petal primordia emerge subsequently inside the five sepals (removed). (Q) The stamen primordia arise inside the five petals in an alternative pattern. (R, S) As the development program proceeds, all the floral organs in each whorl develop equally. (T) The five stamens are equally differentiated into fertile stamens. Scale bar in (A, E), 10cm; (B–D and F–H), 1cm; (I–T), 100μm. It has long been recognized that floral orientation is intimately associated with floral symmetry (Sprengel 1793; Robertson 1888; Darwin 1865). However, the floral orientation has received little attention to date, despite the fact that floral symmetry and the evolution of zygomorphy is an important trend in angiosperm radiation (Dilcher 2000; Preston and Hileman 2009; Hileman 2014a). The floral horizontal orientation has evolved as an adaption to enable efficient pollination, likely representing the first step towards floral specialization (Fenster et al. 2009; Wang et al. 2014). In fact, the floral horizontal orientation and zygomorphy usually covary in angiosperms, particularly in sympetalous groups (supplementary table S1, Supplementary Material online). Deciphering whether these highly linked traits are underpinned by multiple factors or simple genetics would promote an understanding of the complex mechanisms behind floral evolution in angiosperms. In this study, we explore the molecular mechanisms underlying the evolution of the peloric characteristics of cultivated gloxinias, and further determine its origin by employing a highly combinatorial experimental approach. We have characterized a 10-bp deletion in the coding sequence of TCP-coding gene Sscyc that likely results in the production of a nonfunctional truncated protein with 45 amino acid (aa) in length. We demonstrate that this mutation is causally responsible for the development of peloric flowers in gloxinia. The putative ancestor of modern peloric gloxinias was further traced back to a particular wild collection from Rio de Janeiro State in 1820 (G. caulescens, now a synonym of S. speciosa) by literature review, and genetic inference combined with phylogenetic analysis. The results show that one mutation in a single gene causes a complex morphological shift from a horizontally orientated, and zygomorphic flower, to an upright, actinomorphic form in gloxinia. Our findings suggest that pleiotropy may play a crucial role in the coordinated evolution of complex floral organs in angiosperms. Results Phenotypic and Genetic Analysis of the Peloric Character in Gloxinia Wild-type gloxinia flowers are zygomorphic (bilaterally symmetric) and horizontally orientated (fig. 1A). The floral zygomorphy is weak in the second whorl, where the dorsal petals are only slightly smaller than the lateral and ventral ones, and strong in the third whorl, where the dorsal stamen is present as a staminode (fig. 1A–C and supplementary fig. S1, Supplementary Material online). The horizontal orientation of the flower is caused by a ventral–dorsal asymmetric growth at the base of dorsal corolla which produces a convex, gibbous structure (fig. 1D). Meanwhile, the peloric flowers are actinomorphic (radially symmetric) and upright without the development of a gibbous structure (fig. 1E–H and supplementary fig. S1, Supplementary Material online). In order to determine whether these two floral traits are tightly correlated in a broad range of taxonomic groups, we conducted a morphological survey covering all families of sympetalous angiosperms, and found that the horizontal placement of the floral tube is largely associated with the zygomorphic state of flowers (supplementary table S1, Supplementary Material online). This result suggests that the combination of these morphological traits conferred a selective advantage during floral evolution in angiosperms. To determine the genetics of the peloric phenotype, we crossed the wild-type zygomorphic gloxinia “Pink Flower” (WT-PF) with peloric gloxinia “White Bell” (MU-WB) (Materials and Methods). All 13 F1 plants exhibited a WT phenotype with a typical horizontally orientated zygomorphic flower (fig. 2A), implying that the peloric phenotype is recessive. Representatives of F1 plants were selfed to produce an F2 segregating population with a 3:1 ratio of WT to peloric phenotypes (141: 41; χ2 = 0.593, f = 1, P = 0.441) (fig. 2A), suggesting that peloria in gloxinia is a Mendelian character. We observed a perfect association (100%) between the floral zygomorphy and a horizontally orientated floral tube in the F2 segregating population (fig. 2A), indicating that the peloric phenotype, that is, floral actinomorphy and standing upright, is controlled by recessive mutation(s) in a single gene, or closely linked genes. Fig. 2. View largeDownload slide Genetic and association analysis of peloric flower in gloxinia. (A) Representatives of F1 and F2 plants of WT-PF × MU-WB. (B) Genotyping of F2 plants by CAPS. 16 representatives of F2 progenies are shown in the gel. Control experiment (Control) was conducted with SsCYC homozygous PCR product with no Nde I treatment. (C) Genotype–phenotype association analysis with variation sites in the 3,111-bp sequence of the SsCYC locus. In the upper panel, haplotypes of the significantly associated variation sites with n denote the number of sequences that are included in each haplotype; the variation sites that have consistent association between genotype and phenotype are colored in blue and red, respectively. The middle panel illustrates the gene structure of SsCYC. The TCP domain and R domain are shaded in orange and pink, respectively. The significant associated SNPs and Indel are indicated by red bars and triangles, respectively. In the bottom panel, grey circles represent variation sites that are not significantly associated with the peloric flower character, whilst the red circles represent SNPs or Indel significantly associated with the peloric flower character. PC, Peloric Cultivars; WA, Wild Accessions; ZC, Zygomorphic-flowered Cultivars; p, peloric flower; Z, zygomorphic flower. Scale bar in (A), 1cm. Fig. 2. View largeDownload slide Genetic and association analysis of peloric flower in gloxinia. (A) Representatives of F1 and F2 plants of WT-PF × MU-WB. (B) Genotyping of F2 plants by CAPS. 16 representatives of F2 progenies are shown in the gel. Control experiment (Control) was conducted with SsCYC homozygous PCR product with no Nde I treatment. (C) Genotype–phenotype association analysis with variation sites in the 3,111-bp sequence of the SsCYC locus. In the upper panel, haplotypes of the significantly associated variation sites with n denote the number of sequences that are included in each haplotype; the variation sites that have consistent association between genotype and phenotype are colored in blue and red, respectively. The middle panel illustrates the gene structure of SsCYC. The TCP domain and R domain are shaded in orange and pink, respectively. The significant associated SNPs and Indel are indicated by red bars and triangles, respectively. In the bottom panel, grey circles represent variation sites that are not significantly associated with the peloric flower character, whilst the red circles represent SNPs or Indel significantly associated with the peloric flower character. PC, Peloric Cultivars; WA, Wild Accessions; ZC, Zygomorphic-flowered Cultivars; p, peloric flower; Z, zygomorphic flower. Scale bar in (A), 1cm. Expression of SsCYC Is Correlated with the Development of Horizontally Orientated Zygomorphic Flowers CYCLOIDEA (CYC)-like TCP genes are known to be involved in the genetic control of floral zygomorphy in a broad array of species (Luo et al. 1996; see review in Preston and Hileman 2009). In Gesneriaceae, an ancient gene duplication event occurred prior to the split of the Gesnerioideae and the Cyrtandroideae subfamilies generating GCYC1 and GCYC2 paralogous lineages (Citerne et al. 2000; Smith et al. 2004). We cloned the full length Sinningia speciosa CYC (SsCYC) gene, and performed a phylogenetic analysis. The results indicate that the Gesnerioideae subfamily, which includes the Sinningia genus, has lost the GCYC1 lineage in evolution and harbours only one copy of the CYC-like genes, belonging to the GCYC2 clade, which is homologous to AmCYC/AmDICH (supplementary fig. S2, Supplementary Material online). Ontogenetic analysis shows that the floral organ development of WT-PF and MU-WB exhibits a similar pattern before the stamen initiation with all primordium developed equally (fig. 1I–K and 1O–Q). Later on, the developmental differences between them become evident in the dorsal floral organs with their growth suppressed in WT-PF (fig. 1L–N and 1R–T). Correlatively, SsCYC shows a weak dorsal specific expression pattern in the petal whorl, and strong asymmetric expression in the stamen whorl, with an extremely high level of expression in the staminode in WT-PF (fig. 3B, C, and G). In contrast, all floral organs develop equally in MU-WB, and no Sscyc expression can be detected during petal and stamen development (figs. 1O–T and 3E and H). In the floral tube of WT-PF, the expression of SsCYC seems to be exceptionally high in the gibbous structure compared with other sampled tissues, whereas its expression in the Dorsal Floral Tube (DFT) is only slightly higher relative to those in the Ventral Counterparts of the gibbous structure (VC) and the Ventral Floral Tube (VFT) (fig. 4I and J). In MU-WB, only a residual Sscyc expression signal can be detected in the sampled tissues of the floral tube (fig. 4J). The RNA in situ hybridization shows that the SsCYC expression exhibits an asymmetric gradient pattern in the developing gibbous structure, in which strong signals are observed in the inner cell layers whilst almost no expression can be detected in the outer cell layers (fig. 4A, B, D, and F). In contrast, no expression signal of SsCYC can be detected in all ventral counterparts of the gibbous structure (fig. 4C, E, and G). Concomitantly, the cell growth in the inner part of the gibbous structure is significantly suppressed while the cells in the outer layers and the ventral counterparts are expanded almost equally (fig. 4K–R and supplementary fig. S3, Supplementary Material online). As outlined above, the specific expression of SsCYC is correlated with a suppression of growth in the dorsal floral organs, and a suppression of cell expansion in the inner part of the gibbous structure. Fig. 3. View largeDownload slide Comparative expression analysis of SsCYC/Sscyc in WT-PF and MU-WB. (A) The SsCYC mRNA can be detected across the apex of the young floral meristems of WT-PF (triangle). (B) In WT-PF, SsCYC is evenly distributed in the petal and stamen primordia with an extremely high level detected in dorsal staminode. (C) As the developmental program proceeds, the SsCYC mRNA is specifically accumulated in the dorsal stamens and is undetectable in the other floral organs of WT-PF. (D) Sscyc mRNA is evenly distributed in the young floral meristems of MU-WB. (E) Its transcripts can no longer be detected in the subsequent development stages of MU-WB. (F) Control experiment with sense SsCYC probe generated no signal in WT-PF flower of stage similar to (C). (G) Expression analysis of SsCYC in WT-PF floral organs. (H) Expression analysis of Sscyc in MU-WB floral organs. DS/LS/VS, Dorsal/Lateral/Ventral Sepals; DP/LP/VP, Dorsal/Lateral/Ventral Petals; DSt/LSt/VSt, Dorsal/Lateral/Ventral Stamen; DFT/VFT, Dorsal/Ventra Floral Tube; GS, Gibbous Structure; VC, Ventral Counterpart of the GS. FD, Floral Discs; Error bars in (G and H) indicate S.D. of three biological replicates. Scale bars in (A–F), 100 μm. Fig. 3. View largeDownload slide Comparative expression analysis of SsCYC/Sscyc in WT-PF and MU-WB. (A) The SsCYC mRNA can be detected across the apex of the young floral meristems of WT-PF (triangle). (B) In WT-PF, SsCYC is evenly distributed in the petal and stamen primordia with an extremely high level detected in dorsal staminode. (C) As the developmental program proceeds, the SsCYC mRNA is specifically accumulated in the dorsal stamens and is undetectable in the other floral organs of WT-PF. (D) Sscyc mRNA is evenly distributed in the young floral meristems of MU-WB. (E) Its transcripts can no longer be detected in the subsequent development stages of MU-WB. (F) Control experiment with sense SsCYC probe generated no signal in WT-PF flower of stage similar to (C). (G) Expression analysis of SsCYC in WT-PF floral organs. (H) Expression analysis of Sscyc in MU-WB floral organs. DS/LS/VS, Dorsal/Lateral/Ventral Sepals; DP/LP/VP, Dorsal/Lateral/Ventral Petals; DSt/LSt/VSt, Dorsal/Lateral/Ventral Stamen; DFT/VFT, Dorsal/Ventra Floral Tube; GS, Gibbous Structure; VC, Ventral Counterpart of the GS. FD, Floral Discs; Error bars in (G and H) indicate S.D. of three biological replicates. Scale bars in (A–F), 100 μm. Fig. 4. View largeDownload slide Gene expression and cell morphology analysis of the gibbous structure in WT-PF. (A–H) SsCYC expression analysis in the developing GS and floral tube of WT-PF. (A) SsCYC signals are specifically localized in the inner cell layer (red arrows) of GS but not in the VC (enlarged in B and C, respectively). (D and E) In late floral developmental stages when the anthers were differentiated, SsCYC expression can still be detected in the inner cell layers (red arrow) of the GS (D, enlarged in F), but not in the VC (E, enlarged in G). (H) Control experiment using sense SsCYC probe generates no signals. (I) Graphic view of the floral tissues sampled for expression analysis in (J). (K) Cross sections of the GS. A single cell layer is marked in red to show the cell size variation tendency from the inner epidermis to the outer epidermis (red arrow). (L and M) SEM analysis of the epidermal cells of WT-PF GS showing that cell expansion is extremely suppressed in the inner epidermis (L) compared with the outer epidermis (M). (O) Cross sections of the VC. A single cell layer is marked in blue to show no obvious variation of cell size from the inner epidermis to the outer epidermis. (P and Q) SEM analysis of the epidermal cells of ventral counterparts of gibbous structure showing that no cell expansion difference can be detected. (R) Scatterplot of cell size (μm2) and relative position of the cell from the inner epidermis (0%) to the outer epidermis (100%). Gaussian Fitting was calculated to show the variation tendency of cell size along the dorso-ventral axis of the tissue. GS, gibbous structure; VC, Ventral Counterpart of the GS; FD, Floral Disc. Scale bar in (A–H), 150 μm; (K and O), 100 μm; (L, M, P, and Q), 50 μm; (I), 0.5 cm. Fig. 4. View largeDownload slide Gene expression and cell morphology analysis of the gibbous structure in WT-PF. (A–H) SsCYC expression analysis in the developing GS and floral tube of WT-PF. (A) SsCYC signals are specifically localized in the inner cell layer (red arrows) of GS but not in the VC (enlarged in B and C, respectively). (D and E) In late floral developmental stages when the anthers were differentiated, SsCYC expression can still be detected in the inner cell layers (red arrow) of the GS (D, enlarged in F), but not in the VC (E, enlarged in G). (H) Control experiment using sense SsCYC probe generates no signals. (I) Graphic view of the floral tissues sampled for expression analysis in (J). (K) Cross sections of the GS. A single cell layer is marked in red to show the cell size variation tendency from the inner epidermis to the outer epidermis (red arrow). (L and M) SEM analysis of the epidermal cells of WT-PF GS showing that cell expansion is extremely suppressed in the inner epidermis (L) compared with the outer epidermis (M). (O) Cross sections of the VC. A single cell layer is marked in blue to show no obvious variation of cell size from the inner epidermis to the outer epidermis. (P and Q) SEM analysis of the epidermal cells of ventral counterparts of gibbous structure showing that no cell expansion difference can be detected. (R) Scatterplot of cell size (μm2) and relative position of the cell from the inner epidermis (0%) to the outer epidermis (100%). Gaussian Fitting was calculated to show the variation tendency of cell size along the dorso-ventral axis of the tissue. GS, gibbous structure; VC, Ventral Counterpart of the GS; FD, Floral Disc. Scale bar in (A–H), 150 μm; (K and O), 100 μm; (L, M, P, and Q), 50 μm; (I), 0.5 cm. The Peloric Phenotype Is Associated with a 10-bp Deletion in the Coding Sequence of Sscyc To determine whether the peloric mutation maps to the SsCYC locus, we genotyped 182 F2 plants of WT-PF × MU-WB using Cleaved Amplified Polymorphic Sequences (CAPS). The SsCYC allele (wild-type) perfectly cosegregated with the phenotype of horizontally orientated zygomorphic flowers, and all upright peloric flowers were homozygous for the mutant Sscyc allele (fig. 2B). In addition, we also genotyped 36 modern accessions of gloxinia with either zygomorphic or peloric phenotype from various geographic locations around the globe. This result confirmed the genotype–phenotype correlation revealed from the analysis of the F2 population (supplementary table S2 and fig. S4, Supplementary Material online). A careful comparison of the 3,111-bp genomic DNA sequences (including 1992-bp 5′ promoter sequence) between WT-PF and MU-WB revealed 56 SNPs and one Indel (data not shown), but failed to resolve the causal mutation for peloria. In order to find the causal mutation, we took a SNP-phenotype association analysis using a panel of 75 gloxinia accessions (52 cultivated accessions and 23 wild collections) collected from different geographic regions (supplementary table S2, Supplementary Material online). We identified a total of 29 distinct haplotypes in that seven SNPs and one Indel were significantly associated with the peloric phenotype (P < 0.01) (fig. 2C and supplementary table S3, Supplementary Material online). Further analysis of these sites within six haplotypes showed that four SNPs in the regulatory region (SNP96, SNP98, SNP190, and SNP192) and two in the coding region (SNP251 and SNP253) are completely or partially shared between peloric cultivars and three or more zygomorphic accessions, thus excluding them from having a functional relevance to the Sscyc allele (fig. 2C). Of the remaining two sites, SNP203 (A/C, +46 bp) is unlikely to be responsible for peloria, as the amino acid substitution (Serine/Tyrosine) lies outside of the TCP domain, and therefore is unlikely to alter the activity of SsCYC. The Indel (-CTTCAATCTC, +48 bp) causes a frameshift in the coding sequence, which results in a truncated protein with 45-amino acids in length. This truncated protein is likely nonfunctional as it lacks the entire TCP domain and the downstream C terminus (fig. 2C). Functional Analyses of SsCYC/Sscyc Confirm Its Role in Peloric Gloxinia Development To validate the biological function of SsCYC and Sscyc, we fused the coding sequences of the alleles with the CaMV 35S promoter and transformed Arabidopsis thaliana. T1 transgenic plants were selfed to produce T2 populations (see methods). The heritability of the transgene was confirmed by RT-PCR (supplementary fig. S5, Supplementary Material online). Compared with WT plants, the SsCYC overexpression lines showed retarded plant development (fig. 5A) with smaller leaves (supplementary figs. S6 and S8, Supplementary Material online) and smaller floral organs (supplementary figs. S7 and S8, Supplementary Material online). Meanwhile, Sscyc overexpression generated no obvious phenotypic changes (fig. 5A and supplementary figs. S6–S8, Supplementary Material online). Further SEM analysis of the epidermis of leaves and petals showed that cell expansion was repressed in SsCYC overexpression lines (fig. 5B and C and supplementary fig. S6, Supplementary Material online), which is consistent with our hypothesis that dorsal-specific expression of SsCYC suppresses the growth of the dorsal floral organs (figs. 1A–C and 4K–M). In contrast, no evident changes were observed regarding the cell morphologies in leaves or petals in Sscyc overexpressing lines, indicating that Sscyc protein is nonfunctional (fig. 5B and D and supplementary figs. S6 and S8, Supplementary Material online). Therefore, the development of the peloric gloxinia may be attributed to the 10-bp deletion in Sscyc and the resultant truncated non-functional protein. Fig. 5. View largeDownload slide Functional analysis of SsCYC/Sscyc in Arabidopsis and CRES-T analysis of SsCYC in WT-PF. (A) Overexpression of SsCYC in Arabidopsis retards plant development while overexpression of Sscyc in Arabidopsis generates no phenotypic alteration. The inserts show the flowers of each genotype. SEM analysis of the petal epidermal cells of WT plants (B), SsCYC overexpressor (C) and Sscyc overexpressor (D). In (B–D), a total of 20 epidermal cells are shaded in yellow to indicate the cell size difference. (E) Plant architecture of the transgenic control plants. (F) Side view of the horizontally oriented flower character of the transgenic control plants. (G) Front view of the wild-type zygomorphic flower of the transgenic control plants. (H) Plant architecture of the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1), showing the upright peloric flowers. (I) Side view of the upright peloric flower from the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1). (J) Front view of the peloric flower from the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1). Scale bar in (A), 5 cm; inserted graphs in (A), 1 mm; (B–D), 150 μm; (E, H), 7 cm; (F, G, I, J), 1 cm. Fig. 5. View largeDownload slide Functional analysis of SsCYC/Sscyc in Arabidopsis and CRES-T analysis of SsCYC in WT-PF. (A) Overexpression of SsCYC in Arabidopsis retards plant development while overexpression of Sscyc in Arabidopsis generates no phenotypic alteration. The inserts show the flowers of each genotype. SEM analysis of the petal epidermal cells of WT plants (B), SsCYC overexpressor (C) and Sscyc overexpressor (D). In (B–D), a total of 20 epidermal cells are shaded in yellow to indicate the cell size difference. (E) Plant architecture of the transgenic control plants. (F) Side view of the horizontally oriented flower character of the transgenic control plants. (G) Front view of the wild-type zygomorphic flower of the transgenic control plants. (H) Plant architecture of the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1), showing the upright peloric flowers. (I) Side view of the upright peloric flower from the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1). (J) Front view of the peloric flower from the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1). Scale bar in (A), 5 cm; inserted graphs in (A), 1 mm; (B–D), 150 μm; (E, H), 7 cm; (F, G, I, J), 1 cm. Chimeric Repressor Silencing Technology (CRES-T) was recently developed as a powerful tool to knock-down endogenous genes by converting transcriptional activators into strong repressors when the target gene is fused to the ERF-associated Amphiphilic Repression (EAR) motif (SRDX, Hiratsu et al. 2003). Considering that SsCYC is the only copy of the CYC-like gene in the gloxinia genome (supplementary fig. S2, Supplementary Material online), we next tried to repress SsCYC function by producing transgenic plants with 35S: SsCYC: SRDX in WT-PF gloxinias (Materials and Methods) (supplementary fig. S9, Supplementary Material online). The plants regenerated from untransformed leaf cultures produce flowers with the typical WT zygomorphic appearance with nodding floral tubes (fig. 5E–G). Among ten independent transgenic plants expressing the 35S: SsCYC: SRDX fusion protein, three developed upright actinomorphic flowers with fully ventralized petals and stamens, perfectly phenocopying the flowers of peloric gloxinias (fig. 5H–J). In addition, we did not observe any phenotypic alterations in these transgenic plants other than floral symmetry changes, suggesting the SsCYC: SRDX fusion protein specifically targets the SsCYC pathway in the flowers (Compare fig. 5E and H). The results of functional knock-down of SsCYC by CRES-T also indicate that the GCYC2 member of Gesnerioideae exerts similar function as the GCYC1 members of Cyrtandroideae in repressing cell expansion in the dorsal floral organs (Yang et al. 2012; Liu et al. 2014). Taken together, these findings indicate that a single pleiotropic gene, SsCYC, controls both the floral orientation and floral symmetry through its activity in the basal floral tube, petals and stamens. A 10-bp deletion in the coding sequence of Sscyc results in the production of a nonfunctional truncated protein, and is causally responsible for the development of peloric flowers in the gloxinia. Development of Peloric Gloxinia Involves a Two-Fold Molecular Mechanism Our previous study shows that the persistent dorsal specific expression of CYC-like gene depends on the activity of an auto-regulatory loop of CYC-like genes (Yang et al. 2012). Therefore, both the functional CYC protein and CYC binding sites (CBS) in the promoter are indispensable for the establishment of floral zygomorphy (Yang et al. 2012). For instance, in Antirrhinum majus, the loss-of-function change in amino acid coding region in the cyc-608 mutant produces partially ventralized peloric flowers (Luo et al. 1996). In addition, the lack of a CBS in the promoter sequence in Arabidopsis thaliana leads to transient expression of AtTCP1 in the dorsal floral primordium in the early stages, thereby producing actinomorphic flowers (Cubas et al. 2001). Curiously, in the F1 plants of WT-PF × MU-WB, we observed the restoration of dorsal specific expression of Sscyc by allele-specific expression analysis (fig. 6A). Given that initiation of Sscyc expression is normal in the peloric floral meristems as in wild-type (fig. 3A and D) and the dorsal specific expression is subsequently lost during floral organ development in the peloric flowers (fig. 3B and E), we hypothesized that the loss of dorsal specific expression of Sscyc in the peloric flowers resulted from the disruption of the auto-regulatory loop by the nonfunctional Sscyc protein (Yang et al. 2012). To test this idea, we compared the ∼2.0 kb promoter sequence between SsCYC and Sscyc, and found that the putative SsCYC interacting element (CYC-binding Site, CBS) is conserved between WT-PF and MU-WB, that is, they share the same CBS sequence (GGGGCCC) (fig. 6E). Furthermore, an Electrophoresis Mobility Shift Assay (EMSA) analysis showed that SsCYC could interact with a probe containing the CBS, this binding could be effectively competed by adding excessive amounts of unlabeled CBS-containing DNA probes (fig. 6B and C). In sharp contrast, the mutated Sscyc protein failed to bind to this CBS-containing DNA sequence (fig. 6D). Fig. 6. View largeDownload slide Allele specific expression of Sscyc in F1 hybrids and in vitro DNA–Protein interaction by EMSA. (A) Expression analysis of the peloric Sscyc allele in floral organs of the F1 hybrid of WT-PF×MU-WB by allele-specific real-time qPCR, showing that the dorsal specific expression pattern of Sscyc was restored. (B) Oligonucleotide probe sequence used for EMSA analysis, the putative CYC-Binding Site (CBS) sequence is shown in bold capital letters. (C) EMSA analysis of the CBS and SsCYC recombinant protein shows that the interaction of SsCYC with CBS results in a retarded band in the gel (arrow, lane 3); the interaction of the SsCYC with CBS is abolished when excessive amounts of unlabeled probe is added (lane 4). (D) EMSA analysis of the CBS and Sscyc recombinant protein shows that the Sscyc protein has lost its ability to interact with the CBS sequence. (E) The promoter sequences of SsCYC and Sscyc. Both contain the same sequence matching the consensus CYC-Binding Sites (CBS) (boxed) Fig. 6. View largeDownload slide Allele specific expression of Sscyc in F1 hybrids and in vitro DNA–Protein interaction by EMSA. (A) Expression analysis of the peloric Sscyc allele in floral organs of the F1 hybrid of WT-PF×MU-WB by allele-specific real-time qPCR, showing that the dorsal specific expression pattern of Sscyc was restored. (B) Oligonucleotide probe sequence used for EMSA analysis, the putative CYC-Binding Site (CBS) sequence is shown in bold capital letters. (C) EMSA analysis of the CBS and SsCYC recombinant protein shows that the interaction of SsCYC with CBS results in a retarded band in the gel (arrow, lane 3); the interaction of the SsCYC with CBS is abolished when excessive amounts of unlabeled probe is added (lane 4). (D) EMSA analysis of the CBS and Sscyc recombinant protein shows that the Sscyc protein has lost its ability to interact with the CBS sequence. (E) The promoter sequences of SsCYC and Sscyc. Both contain the same sequence matching the consensus CYC-Binding Sites (CBS) (boxed) Under both artificial and natural selection regimes, adaptive morphological traits can be produced by either change in protein function (Li et al. 2006; Hoballah et al. 2007; Pourkheirandish et al. 2015) or regulatory modifications (Chan et al. 2010; Studer et al. 2011; Dong et al. 2014). The observations presented here strongly suggest that the evolution of the peloric gloxinia involves a 2-fold mechanism. That is, the 10-bp deletion brings about the loss-of-function of Sscyc protein, which in turn, further disrupts the auto-regulatory loop of Sscyc leading to a complete loss of dorsal specific expression. SsCYC Was Targeted by Artificial Selection during the Domestication Process of Peloric Gloxinia For crops, domestication occurs when a single favoured haplotype associated with favorable morphological evolution is targeted by selection and fixed over time (Gross and Olsen 2010). To evaluate the impact of artificial selection on the SsCYC locus, we analyzed the DNA polymorphisms in a ∼3.1 kb genomic region of SsCYC in a panel of 40 peloric accessions from diverse geographic locations, and 23 wild collections with zygomorphic flowers from Brazil (supplementary table S2, Supplementary Material online). In the wild gloxinia population, the average nucleotide diversity of SsCYC DNA sequence is π = 0.0164, indicative of considerable sequence polymorphism at the SsCYC locus (supplementary fig. S10, Supplementary Material online). However, all 40 peloric accessions share the same haplotype (π = 0) including the fixed 10-bp deletion in the ∼3.1 kb SsCYC genomic sequence (supplementary fig. S10, Supplementary Material online), indicating that intensive artificial selection has targeted this mutation by removing rare sequence variants in this locus. To trace the origin of peloric gloxinias, we constructed the SsCYC phylogeny based on the ∼1,250-bp genomic sequence. All 40 peloric gloxinias are grouped together with two wild gloxinias “Cardoso Moreira” in a single well supported clade (fig. 7). In contrast, the tree of the putative neutral marker, nuclear chloroplast-expressed Glutamine Synthetase (ncpGS), showed that the peloric accessions are dispersed into multiple branches nested in wild gloxinias (supplementary fig. S11, Supplementary Material online). These results suggest that the SsCYC locus in peloric gloxinias was targeted by artificial selection, and the peloric Sscyc allele was derived from the sequence of “Cardoso Moreira” that is distinct from all other wild gloxinia accessions by a single domestication event. Fig. 7. View largeDownload slide Phylogenetic analysis of the SsCYC locus in Sinningia speciosa. Majority rule consensus tree of 235 most parsimonious trees generated from SsCYC sequences of S. speciosa. Bootstrap (BS) values and Bayesian Posterior Probabilities (PP) are indicated above and below the branches, respectively. The cultivated peloric gloxinia accessions (Cultivated, CV) and wild gloxinia accessions (Wild Accession, WA) are indicated by blue and red dots, respectively. The red star indicates the close relationship of the peloric gloxinia accessions with two wild gloxinia collections (S. speciosa “Cardoso Moreira pink mutant” and S. speciosa “Cardoso Moreira”). Fig. 7. View largeDownload slide Phylogenetic analysis of the SsCYC locus in Sinningia speciosa. Majority rule consensus tree of 235 most parsimonious trees generated from SsCYC sequences of S. speciosa. Bootstrap (BS) values and Bayesian Posterior Probabilities (PP) are indicated above and below the branches, respectively. The cultivated peloric gloxinia accessions (Cultivated, CV) and wild gloxinia accessions (Wild Accession, WA) are indicated by blue and red dots, respectively. The red star indicates the close relationship of the peloric gloxinia accessions with two wild gloxinia collections (S. speciosa “Cardoso Moreira pink mutant” and S. speciosa “Cardoso Moreira”). Reconstructing the Domestication History of the Peloric Gloxinia In the literature, the first peloric gloxinia with upright flowers was documented in 1845 under the name Gloxinia fyfiana (synonym of S. speciosa) produced by an Englishman called Fyfe by crossing G. maxima and G. caulescens in 1844 (both synonyms of S. speciosa) (fig. 8A) (Harrison 1847; Louis 1848; Fyfe 1879). Gloxinia caulescens was introduced from Brazil into England in 1820, published in 1827. It was widely cultivated and frequently crossed to G. speciosa due to its extravagant ornamental flowers (fig. 8E) (Edwards 1827; Paxton 1838; Maund and Henslow 1839; Harrison 1847; Johnson and Landreth 1847). Gloxinia maxima was recorded as a hybrid between G. candida and G. speciosa (both synonyms of S. speciosa) and published in 1838 (fig. 8B) (Paxton 1838). Gloxinia speciosa has horizontally orientated, zygomorphic flowers, and was introduced from Brazil into cultivation in England in 1815, published in 1817 (fig. 8C) (Loddiges 1817). Gloxinia candida first appeared in an exhibition in 1832 as a garden origin hybrid with no detailed character description except for white slipper flowers. The first “gloxinia” with white flowers, that is, G. speciosa var. albiflora, was published in 1833 (Hooker 1833). In 1839, another hybrid with white slipper flowers, similar to that of G. candida, was described in detail with a clear record that this hybrid was produced from a cross between G. speciosa var. albiflora and G. caulescens (fig. 8D) (Maund and Henslow 1839). Since hybridization and breeding among “gloxinia” species was fashionable from 1820 to 1840 in Great Britain (Paxton 1838; Maund and Henslow 1839; Johnson 1847), it is possible that a hybrid with similar white slipper flowers was repeatedly produced from the crosses between G. speciosa var. albiflora and G. caulescens by gardeners at the time. Therefore, G. candida is probably a hybrid of G. speciosa var. albiflora × G. caulescens. Fig. 8. View largeDownload slide Historical paintings of Gloxinia species and the genealogy of the first peloric gloxinia flower. (A) The original depiction of G. fyfiana in 1848 by Louis. (B) The original depiction of G. maxima in 1838 by Paxton. (C) The original depiction of G. speciosa in 1817 by Loddiges. (D) The original depiction of G. speciosa-caulescens hybrid in 1839 by Maund and Henslow. (E) The original depiction of G. caulescens in 1827 by Edwards. (F) Plant architecture of a flowering S. speciosa “Cardoso Moreira”, showing the great morphological similarities to G. fyfiana (A) and G. caulescens (E) in terms of plant architecture. The photo is courtesy of Mr. Lin Ruei Chau. (G) The genealogy of the first peloric Gloxinia. The genotype and first appearance year of each “species” is indicated below, respectively. The peloric allele Sscyc and the mutated cultivar of G. caulescens are labeled in red. Note: the original imported G. caulenscens plants (1820) were supposed to be homozygous for SsCYC, as we failed to detect the mutated Sscyc allele in the wild populations. The Sscyc allele might have originated from the de novo mutation of 10-bp deletion in the cultivation process of G. caulenscens from 1820 to 1832 and kept a heterozygous (SsCYC/Sscyc) status in G. candida and G. maxima in the following 12 years before meeting with the mutated cultivar of G. caulenscens again in 1844. All the latin names presented in (G) are synonyms of S. speciosa. Fig. 8. View largeDownload slide Historical paintings of Gloxinia species and the genealogy of the first peloric gloxinia flower. (A) The original depiction of G. fyfiana in 1848 by Louis. (B) The original depiction of G. maxima in 1838 by Paxton. (C) The original depiction of G. speciosa in 1817 by Loddiges. (D) The original depiction of G. speciosa-caulescens hybrid in 1839 by Maund and Henslow. (E) The original depiction of G. caulescens in 1827 by Edwards. (F) Plant architecture of a flowering S. speciosa “Cardoso Moreira”, showing the great morphological similarities to G. fyfiana (A) and G. caulescens (E) in terms of plant architecture. The photo is courtesy of Mr. Lin Ruei Chau. (G) The genealogy of the first peloric Gloxinia. The genotype and first appearance year of each “species” is indicated below, respectively. The peloric allele Sscyc and the mutated cultivar of G. caulescens are labeled in red. Note: the original imported G. caulenscens plants (1820) were supposed to be homozygous for SsCYC, as we failed to detect the mutated Sscyc allele in the wild populations. The Sscyc allele might have originated from the de novo mutation of 10-bp deletion in the cultivation process of G. caulenscens from 1820 to 1832 and kept a heterozygous (SsCYC/Sscyc) status in G. candida and G. maxima in the following 12 years before meeting with the mutated cultivar of G. caulenscens again in 1844. All the latin names presented in (G) are synonyms of S. speciosa. According to our results, the peloric allele is recessive and hence the peloric flower is homozygous for the Sscyc allele. In order to produce the peloric flower of G. fyfiana, both G. maxima and G. caulescens must possess a heterozygous SsCYC/Sscyc genotype. As for G. maxima, theoretically, it could have inherited the peloric allele from either G. candida or G. speciosa. As G. speciosa is a wild gloxinia introduced from Brazil, and because the probability of the same peloric mutation (10-bp deletion) happening independently in G. caulescens and G. speciosa is extremely low, we hypothesize that G. speciosa is unlikely to be the donor of the recessive Sscyc allele. It is more likely that G. maxima inherited the peloric allele from G. candida if we consider that the mutation occurred only once in G. caulescens specifically. Taken together, the genetic inference above suggests that the causal mutation (10-bp deletion) leading to peloric gloxinia initially occurred in a cultivar of G. caulescens during the cultivation process between its importation in 1820 and the first appearance of the white-flowered hybrid (G. candida) in 1832 (fig. 8G). This mutation then passed from G. candida to G. maxima in 1838 and was kept in a heterozygous state for the following 6 years (1844) until G. maxima merging with the mutated heterozygous cultivar of G. caulescens in a cross generated by Mr. Fyfe (fig. 8G). Discussion On the Origin of the Peloric Gloxinia The floral characteristics of ornamental plants have undergone an unprecedented explosion in color and morphology by deliberate hybridization-assisted selection over the past 200 years (Darwin 1868; Crane and Lawrence 1934; Kingsbury 2009). However, even for species whose domestication is clearly documented, showing major morphological transitions (e.g., Chinese primrose) (Crane and Lawrence 1934), the origin and spread of novel traits cannot be well understood until the genetic factors underlying these changes have been identified. Based on literature review and genetic inferences, we propose that the causal mutation (10-bp deletion in Sscyc) leading to peloria, occurred only once, in a cultivar of G. caulescens. However, in the phylogenetic tree based on the ∼1,250-bp of the SsCYC genomic sequence, the modern peloric gloxinias grouped together with two wild gloxinia collections from “Cardoso Moreira”, suggesting that modern peloric gloxinia are derived from these two wild collections, or related populations. In fact, the wild “Cardoso Moreira” collections are closely related to the initial peloric gloxinia morphologically in plant architecture of extremely robust stems and one or two (or only several) flowers produced in the top leaf axils (fig. 8A, E, and F), suggesting that they are morphologically related. In addition, G. caulescens was originally recorded in wet rocks at the base of Corcovado Montain in Rio de Janeiro State in 1838 (Brackenridge 1886; Zaitlin 2011), adjacent to the location where the “Cardoso Moreira” samples were collected. Taken together, the genetic inferences and textual examination of the literature presented above strongly suggest that the “Cardoso Moreira” plants, or a related population of the same race, may be the gloxinia plants (designated as G. caulescens) exported from Brazil to Great Britain in 1820, which are the direct ancestors of the modern peloric gloxinia. SsCYC Is a Pleiotropic Gene Controlling Multiple Floral Characters Flowers are considered to be an essential model for studying the genetics of speciation, as the floral traits are intimately associated with prezygotic isolation (Smith 2016). Floral traits are often integrated as functional modules, which show correlated variation as the results of adaptation to specific pollination strategies (Fenster et al. 2009; Smith 2016). The genetic mechanism proposed to account for this complex morphological integration is that different characteristic elements are controlled by the same developmental gene with pleiotropic effects (Wagner and Zhang 2011; Smith 2016). In plants, genetic studies have identified a number of genomic regions responsible for multiple floral traits (Bradshaw et al. 1998; Wessinger et al. 2014). One caveat of these studies is that colocalization of multiple traits to the same genetic locus does not necessarily mean that the underlying genes or mutations are exactly the same, as a single locus may contain a large number of genes (Smith 2016). In addition, genes with pleiotropic effects identified in the functional analysis of model species are proposed to act as hot spots that facilitate phenotypic evolution (Wagner and Zhang 2011). However, direct molecular evidence for the evolution of functionally integrated phenotypic modules with the contribution of pleiotropic genes remain rare in plants, and the only examples are from animals (Linnen et al. 2013; Chung et al. 2014; Smith 2016). In Sinningia speciosa, our analysis provides three pieces of concrete evidence for the involvement of a pleiotropic gene in the development of an integrated floral character complex, that is, the nodding zygomorphic flower. Firstly, the anatomic examination shows that the nodding flower phenotype is caused by the development of a gibbous structure at the base of dorsal corolla. There is a simultaneous loss of the gibbous structure and zygomorphy in the peloric gloxinia, which lacks morphological recombination in the F2 segregating population. This suggests that these characters are controlled by the same genetic regulator. Secondly, SsCYC exhibits dorsal specific expression in the floral organs (i.e., the dorsal petals and staminode) and inner parts of the basal dorsal floral tubes, which are correlated with the retardation of growth in the dorsal floral organs, and restricted cell expansion in the inner parts of the gibbous structure. Thirdly, the knock-down of SsCYC by dominant repression produces transgenic plants with perfectly ventralized actinomorphic flowers with loss of the gibbous structure, implying that the SsCYC is a pleiotropic gene responsible for the development of both morphological traits. The discovery of a CYC-like gene involved in the development of both floral symmetry and orientation provides empirical evidence that a simple genetic change in a pleiotropic gene with selective advantage would promote coordinated evolution of the highly integrated floral organs. In angiosperms, the evolution of zygomorphic flowers is considered to be a major morphological innovation that led to the diversification of species (Dilcher 2000). It has been recently demonstrated that horizontal-orientated and zygomorphic flowers, with the two traits acting as a functional unit, confer a selective adaptive advantage, as they direct the pollinator movement within the flowers, and therefore, enable effective and precise pollen transfer to the stigma (Ushimaru and Hyodo 2005; Fenster et al. 2009; Wang et al. 2014). In fact, the association between the floral horizontal orientation and floral zygomorphy was recognized very early by Robertson in 1888. However, the floral orientation has long been neglected since then (Fenster et al. 2009). To our knowledge, this is the first time to report the role of CYC-like genes in controlling the floral orientation, expanding the function of CYC-like genes from controlling the floral symmetry to regulating both floral orientation and symmetry. This finding provides critical insights into how the high frequency of speciation with subsequent rapid diversification has occurred in zygomorphic lineages. In general, the floral horizontal orientation can be produced either by the asymmetrical growth of the floral tube or the bending of the pedicels relative to the stem. The floral zygomorphy coupled with floral horizontal orientation may have been independently evolved through different pathways. We also notice some cases of floral zygomorphy disassociated from horizontal orientation, such as some species of Mimulus (Phrymaceae) and Agalinis (Orobanchaceae) with zygomorphic but upright flowers. Further exploring the genetic basis relating to alternative pathways of the coupling between floral zygomorphy and horizontal orientation and the adaptive scenario for the decoupling between them in given taxa would shed new light on the mechanisms that underlie the vast morphological diversity of floral zygomorphy in angiosperms. Materials and Methods Plant Samples, Growth Condition and Artificial Hybridization We chose Pink Flower (WT-PF) and White Bell (MU-WB) as plant materials for expression and functional analysis. WT-PF is a wild-type gloxinia that is native to Brazil and produces large, horizontally oriented pink zygomorphic flowers with a white band decorated with dark purple spots in the ventral corolla throat. MU-WB is a popular cultivated variety widely cultivated in China, which bears upright white actinomorphic bell-shaped flowers. Various domesticated gloxinia accessions (supplementary table S2, Supplementary Material online) were either obtained from the Gesneriad Society (www.gesneriadsociety.org) or from commercial sources. The wild gloxinia (S. speciosa) collections and two Sinningia species (S. tubiflora and S. rupicola) were obtained as seed from Mauro Peixoto’s Brazil Plants Organization (Mogi das Cruzes, SP, Brazil). The seeds were germinated on 1/2 Murashige and Skoog (MS) medium at 26 °C. The 1-month seedlings were then transplanted to 7-cm pots containing a mixture of moss substrate, vermiculite and perlite (1:1:1) in the glasshouse of Institute of Botany, the Chinese Academy of Sciences. Growth conditions were long-day photo period (16-h light/8-h dark) at 28 °C with a relative humidity of 70% and 60% shading. Arabidopsis thaliana (ecotype Columbia-0) used in this study was germinated on 1/2 MS medium at 23 °C. The seedlings were transplanted in the soil under 16-h-light (200 μmol m−2 s−1, 23 °C) and 8-h-dark (20 °C) conditions. To generate the hybrid gloxinias, we pollinated the emasculated maternal plants (MU-WB) with pollen from the paternal plants (WT-PF) at anthesis. The resultant hybrids were germinated and transplanted into the glasshouse as described above. Morphological Analysis and Scanning Electron Microscopy (SEM) The floral organs of WT-PF and MU-WB at anthesis were dissected and the morphology of floral organs was recorded with Nikon D7100 camera. For SEM, young inflorescences of WT-PF and MU-WB were fixed in FAA and infiltrated under vacuum. The respective floral meristems from distinct developmental stages were dissected with a needle in 70% ethanol under a light microscope. The materials were dried with critical point of CO2 and the floral organs were examined using a Hitachi S-4800 scanning electron microscope (SEM) as previously described (Zhou et al. 2008). To quantify cell size in the gibbous structure, the basal floral tube (including the gibbous structure) was fixed in FAA and embedded into paraffin (Sigma, USA). 8-μm sections were prepared using a rotary microtome. Section images were captured and processed by Image J (1.50b) software. For the gibbous structure, a total of 600 cells (12 cell layers, each layer five cells, ten sections) were examined for cell size variation, and a total of 500 cells (ten cell layers, each layer five cells, ten sections) were recorded in the ventral counterpart of the gibbous structure. Cell size was calculated according to the scale bars to generate the real size. Scatterplot analysis of cell size versus cell layer was conducted by Minitab17 (Minitab, Inc.), Gaussian Fitting (Curve Fitting Tool) was stimulated to show the variation tendency of cell size along the dorso-ventral axis of the gibbous structures and the ventral counterparts, respectively. Genotyping and Association Analysis The differences between SsCYC and Sscyc in the coding sequence allowed genotype specific CAPS primers to be designed (supplementary table S4, Supplementary Material online). A 624-bp fragment of SsCYC coding sequence was amplified by PCR and the purified products were subjected to NdeI digestion. For Sscyc, the 624-bp fragment is digested into 422 and 202-bp, whereas the SsCYC fragment cannot be digested and remains 624-bp after digestion. The segregation ratio and statistical analysis of the F2 plants were performed using SPSS 14.0 software. For SNP-phenotype association analysis, we isolated the 1922-bp 5′ promoter sequence by TAIL-PCR and sequenced 3,111-bp gDNA from a panel of 75 gloxinia accessions (supplementary table S2, Supplementary Material online). The initial sequences were aligned using Clustal X software (Thompson et al. 1997). The matrix was adjusted manually by using BioEdit Software (Hall 1999). Then, the matrix was imported into DnaSP 5.10 software (Librado and Rozas 2009) to generate the haplotype matrix by considering Indels as informative sites. The floral characters of 75 gloxinia accessions were recorded as peloric (1) and zygomorphic (0). The association of SNPs/Indels with phenotype was conducted by Tassel 5.0 software (Bradbury et al. 2007) under General Linear Model (GLM). For annotation of the regulatory SNP in the promoters, we used online software TSSP (www.softberry.com) and PLACE (sogo.dna.affrc.go.jp) to predict the putative regulatory motifs in the 20-bp sequence including the significant associated SNPs. RNA Extraction and Expression Analysis Each floral organ was dissected and immediately frozen in liquid nitrogen. Total RNA was extracted using SV Total RNA Isolation System, and DNase I was added to digest the genomic DNA (Promega, USA) following the manufacturer’s instructions. Complimentary DNA (cDNA) was synthesized using a RevertAid H Minus First-Strand cDNA Synthesis Kit (Thermo, USA) according to the manufacturer’s instructions. For real-time qPCR and allele specific gene expression of SsCYCs/Sscyc, we designed primers that specifically anchored to the 10-bp deletion difference between SsCYC and Sscyc (supplementary table S4, Supplementary Material online). Before conducting expression analysis, the specificity of the primers was verified by PCR and sequencing. The efficiency of the primers (95–105%) was determined by creating standard curve. The SYBR Premix ExTaq (TaKaRa, China) was used to perform real-time qPCR with ROX as a reference dye on a StepOne Plus Real-Time PCR System (Life Technology, USA). The CT value of each gene was determined by normalizing the fluorescence threshold. The relative expression level of the target gene was determined using the ratio = 2−ΔCTmethod, and SsACT was used as an internal control (Pfaffl 2001). For RNA in situ hybridization, a 423-bp sequence targeted the 3′ coding sequence and 3′UTR of SsCYC was amplified using primers (supplementary table S4, Supplementary Material online) designed to enhance the specificity and avoid cross-hybridization with other TCP genes. Digoxygenin-labeled probes were generated using an in vitro transcription system (Roche, Switzerland). RNA in situ hybridization experiments were conducted as previously described with minor modifications (Bradley et al. 1993). Briefly, young inflorescence and flowers of WT-PF and MU-WB were fixed in FAA and embedded in paraffin (Sigma, USA). 10-μm sections were prepared using a rotary microtome. After removing the paraffin, samples were hybridized with the antisense/sense probe of SsCYC at 42 °C for 16 h. Stringent formamide washings for nonspecific probes were performed after hybridization. The AP-conjugated anti-DIG antibodies (Roche, Switzerland) were then mounted on the samples for 2 h. After removing the nonspecific antibody, the samples were then incubated with the NBT/NCIP solution (Roche, Switzerland) for staining at room temperature for 10 h. The slides were dried and mounted with CC/mount medium (Sigma, USA). Samples were analyzed using the Zeiss Axio Imager A microscopy (Carl Zeiss, Germany). Recombinant Protein Production and EMSA A DNA fragment of 665-bp from the start codon of SsCYC or Sscyc was amplified from the WT-PF and MU-WB gDNA, respectively. The PCR product was digested with BamH I and Hind III and inserted into the pET30α vector (Merck, Germany). Constructed plasmids were verified by sequencing and then introduced into BL21 Escherichia coli cells. The His-tagged recombinant proteins were purified from the soluble fraction of the cell lysate using Ni sepharose (GE Healthcare, USA). For EMSA, the 20 bp biotin-labeled probes were generated by Sangon Company (Sangon, Shanghai). EMSA was performed using nonradioactive NF-κB EMSA Kit (Thermo, USA) following the manufacturer’s instructions. After the reaction, electrophoresis was conducted on a 6.5% nondenaturing polyacrylamide gel at 175 V in 0.25× TBE (22.25 mM Tris–HCl, 22.25 mM boric acid, and 5 mM EDTA, pH8.0) buffer at 4 °C for 1 h. The reaction products were transferred to the binding membrane at 394 mA in 0.5× TBE) at room temperature for 40 min. The probes were detected according to the manufacturer’s instructions using the Imager Apparatus (Alpha, Canada). Two independent experiments were carried out to ensure that probe–protein interactions were specific. Transgenic Analysis For dominant repression of the SsCYC protein, the full length 1,035-bp SsCYC-CDS was fused in-frame with the EAR repression domain from the SUPERMAN gene and inserted downstream of the CaMV 35S promoter of the pCAMBIA 1301 vector to construct the SsCYC-SRDX vector. The vector was verified by sequencing and introduced into the Agrobacterium tumefaciens strain LBA4404 by electroporation. The transformation of Sinningia speciosa followed the methods described in Li et al. (2013) and Liu et al. (2014) with minor modifications (Li et al. 2013; Liu et al. 2014). Leaf discs from 8-weeks-old plantlets were precultured on MS medium containing 2 mg l−1 2,4-dichlorophenoxy acetic acid (2,4-D) for 2 days. Discs were then subjected to infiltration with Agrobacterium tumefaciens (O.D. 600, 0.3) at room temperature for 15 min, transferred to the coculture MS medium containing 100 μM Acetosyringone (AS), and kept in the dark for 3 days. Resultant explants were transferred to selection MS medium containing 2 mg l−1 6-Benzylaminopurine, 0.2 mg l−1 Naphthylacetic acid, 5 mg l−1 Hygromycin (Hyg) and 200 mg l−1 Cefotaxime (Cef). After six 2-week rounds of selection, regenerated Hyg-resistant adventitious shoots were obtained. The shoots were then transferred to MS medium for root induction. Discs of untransformed cultures were carried through the regeneration process as the wild type control. Transgenic and control plants were transplanted into the glasshouse as described above. For SsCYC-SRDX, a total of seven individual transgenic lines were obtained. The flower morphology of the transgenic plants was recorded with a Nikon D7100 camera. For overexpression of SsCYC/Sscyc in Arabidopsis, the 1,267-bp of SsCYC/Sscyc g-DNA sequence encompassing the entire ORF was isolated from WT-PF and MU-WB, and inserted downstream of the CaMV 35S promoter of pCAMBIA 1301. Vectors were verified by sequencing and then introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation. The resultant Agrobacterium was infiltrated into Arabidopsis using the floral dipping method (Clough and Bent 1998). Positive primary transformants were selected on 1/2 MS medium containing 40 mg/ml Hyg and 250 mg/ml Cef. Among the 45 SsCYC-OE T1 transformants, 39 showed a retarded plant development phenotype, with smaller rosette leaves and reproductive organs when compared with WT plants, whilst all 36 Sscyc-OE T1 transformants exhibited wild-type characteristics without any phenotypic changes. For each transgene, we randomly chose at least ten independent T1 transgenic plants, and selfed them to generated T2 plants. The phenotypic analysis was performed on the T2 population. To record leaf and petal parameters, the seventh leaf and sixth flower were collected for measurement by using a vernier. The SEM of leaf and petal epidermal cells was conducted as mentioned above. Phylogenetic Analysis We isolated the ∼1,250-bp SsCYC from 23 wild gloxinia collections with WT phenotypes, 40 peloric gloxinias and two members of Sinningia (S. tubiflora and S. rupicola) which were used as an outgroup (supplementary table S2, Supplementary Material online). The sequences were aligned using Clustal X software and adjusted manually with the software Geneious version 7.1.4 (Kearse et al. 2012). Parsimony analysis was implemented in PAUP*4.0B10 (Swofford 2003). Bayesian inference analyses were carried out in MrBayes version 3.2.2 (Ronquist and Huelsenbeck 2003). Mrmodeltest version 2.3 (Nylander 2004) was used to select an appropriate model of sequence evolution for each DNA data set in Bayesian inference analyses. Bootstrap values of parsimony analysis and posterior probabilities (PP) obtained from the analysis were used to test the credibility of various branches. For the neutral marker ncpGS, we isolated a ∼710-bp sequence from 20 wild gloxinia collections, 19 peloric gloxinias, and two members of Sinningia (S. tubiflora and S. rupicola) which were used as outgroups (supplementary table S2, Supplementary Material online). The Phylogenetic analysis were conducted as aforementioned methods. For the phylogenetic analysis of CYC-like genes from Gesneriaceae species, the full-length protein sequences were downloaded from GenBank database and aligned with Clustal X software. The protein matrix was determined autoomatically in RAxML and 1000 bootstrap replicates were conducted in ML analysis. The Maximum likelihood (ML) tree with bootstrap support value was generated based on Protein sequence matrix by RAxML on the CIPRES Science Gateway Portal (Miller et al. 2010). Population Genetic Analysis We isolated the 3,111-bp gDNA sequence of SsCYC from 40 peloric gloxinia accessions from diverse locations around the globe and 23 wild gloxinia collections from Brazil (supplementary table S2, Supplementary Material online). Sequences were aligned by Clustal X software to generate the matrix (Thompson et al. 1997). Then the matrix was adjusted manually using BioEdit software and inputted into DnaSP 5.10 software (Hall 1999; Librado and Rozas 2009). Values of genetic diversity per base pair (π) were estimated for the domesticated peloric gloxinia and wild gloxinia groups. Sliding window analysis of genetic diversity was calculated using 100-bp window with a 25-bp step with average pairwise difference per base pair between sequences. Acknowledgments We thank André Kuhn, Feng-Xian Guo, Heather Bland, James F. Smith, Lukasz Langowski, Lars Østergaard, Pauline Stephenson, Rebecca Mosher, and three anonymous reviewers for their constructive comments. This study is funded by the National Natural Science Foundation of China (Grants 31470333, 31530003 to Yin-Zheng Wang and Grands 31400205 to Yang Dong) and the Financial Grant from the China Postdoctoral Science Foundation (Grants 2014M550878 and Grants 2015T80151 to Yang Dong). Author’s Contributions Y.Z.W. initiated, conceived, designed, supervised the research and wrote the article. Y.D. conceived, designed and performed all the research, analyzed the data, and wrote the article. 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Evolution of Darwin’s Peloric Gloxinia (Sinningia speciosa) Is Caused by a Null Mutation in a Pleiotropic TCP Gene

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Abstract

Abstract Unlike most crops, which were domesticated through long periods of selection by ancient humans, horticultural plants were primarily domesticated through intentional selection over short time periods. The molecular mechanisms underlying the origin and spread of novel traits in the domestication process have remained largely unexplored in horticultural plants. Gloxinia (Sinningia speciosa), whose attractive peloric flowers influenced the thoughts of Darwin, have been cultivated since the early 19th century, but its origin and genetic basis are currently unknown. By employing multiple experimental approaches including genetic analysis, genotype–phenotype associations, gene expression analysis, and functional interrogations, we showed that a single gene encoding a TCP protein, SsCYC, controls both floral orientation and zygomorphy in gloxinia. We revealed that a causal mutation responsible for the development of peloric gloxinia lies in a 10-bp deletion in the coding sequence of SsCYC. By combining genetic inference and literature searches, we have traced the putative ancestor and reconstructed the domestication path of the peloric gloxinia, in which a 10-bp deletion in SsCYC under selection triggered its evolution from the wild progenitor. The results presented here suggest that a simple genetic change in a pleiotropic gene can promote the elaboration of floral organs under intensive selection pressure. peloric gloxinia, Sinningia speciosa, domestication, molecular mechanism, floral horizontal orientation, pleiotropic gene Introduction The interaction between humans and plants is best characterized by horticultural domestication, which is intimately associated with the development of modern civilization over the last 300 years (Janick 2005; Kingsbury 2009). Horticultural plants were domesticated by humans through intentional selection, usually by crossing different varieties to alter morphological characters for ornamental, medicinal or religious purposes, over a relatively short time period (Doebley et al. 2006; Kingsbury 2009). Some horticultural plants, like Petunia and Primula, were wildly used as model system to develop genetic toolkits for experimental studies in evolution of floral traits and speciation under natural selection regime (Hoballah et al. 2007; Amrad et al. 2016; Li et al. 2016; Sheehan et al. 2016). However, the origin of horticultural characters arising from domestication have remained practically unexplored in most horticultural plants at the molecular level. Gloxinia is a tropical gesneriacious plant native to Brazil and was domesticated in Great Britain in the early 19th century for its large attractive peloric flowers. The peloric gloxinia was originally documented in 1845, and famously became known as Darwin’s peloric gloxinia. Distinct from the peloric variety, the wild progenitors produce nodding zygomorphic flowers that is adapted to insect pollination (fig. 1) (Loddiges 1817; Harrison 1847; Darwin 1868). The genetic control of floral zygomorphy was best characterized in the model species Antirrhinum majus with isolation and function investigation of two paralogous TCP (TEOSINTE BRANCHED1, CYCLOIDEA, and PCF) genes CYCLOIDEA (CYC) and DICHOTOMA (DICH) (Luo et al. 1996, 1999). CYC and DICH establish zygomorphic floral patterning by specifying the dorsal organ identities in the second and third whorls (Luo et al. 1996, 1999). In the Gesneriaceae, the CYC-like genes were demonstrated to be involved in the development of zygomorphic flowers, with diverse morphological variation (Song et al. 2009; Yang et al. 2012, 2015). Previously, Citerne et al. (2000) isolated a partial coding sequence of GCYC from a peloric gloxinia cultivar and observed a single adenine base deletion immediately downstream of the TCP domain. It was therefore proposed that the deletion and resulting truncated protein promotes the development of the peloric phenotype (Citerne et al. 2000). However, subsequent resequencing of GCYC from other peloric gloxinia accessions showed that the adenine deletion is probably a PCR artefact (Zaitlin 2011; also see Smith et al. 2004), implying that the molecular mechanism underlying the peloric gloxinia is more complicated than previously hypothesized. Fig. 1. View largeDownload slide Phenotypic analysis of flower character development in the wild-type and cultivated peloric gloxinia. (A) Plant architecture of wild-type gloxinia (WT-PF), showing the horizontally oriented zygomorphic flower. (B) Front view of WT-PF flower. (C) Stamen of WT-PF, the red arrow denotes the dorsal staminode. (D) Side view of WT-PF floral tube (sepals are removed), showing the gibbous structure (arrow). (E) Plant architecture of cultivated peloric gloxinia (MU-WB), showing the upright actinomorphic flower. (F) Front view of MU-WB flower. (G) Stamen of MU-WB. (H) Side view of MU-WB floral tube (sepals are removed), showing the loss of the gibbous structure. (I–N) Floral developmental series of wild-type gloxinia (WT-PF) revealed by SEM. (I) The sepals initiated and developed equally. (J) The petal primordia arise subsequently inside the five sepals (removed). (K) The stamen primordia emerge inside the five petals in an alternating pattern. (L–N) As the floral organs advance in development, the floral zygomorphy manifests with the two dorsal petals slightly smaller than the lateral and ventral ones (L and M), and the dorsal stamen is finally suppressed into a staminode in development (N). (O–T) Floral developmental series of peloric gloxinia (MU-WB) revealed by SEM. (O) The sepals initiated and developed equally. (P) Petal primordia emerge subsequently inside the five sepals (removed). (Q) The stamen primordia arise inside the five petals in an alternative pattern. (R, S) As the development program proceeds, all the floral organs in each whorl develop equally. (T) The five stamens are equally differentiated into fertile stamens. Scale bar in (A, E), 10cm; (B–D and F–H), 1cm; (I–T), 100μm. Fig. 1. View largeDownload slide Phenotypic analysis of flower character development in the wild-type and cultivated peloric gloxinia. (A) Plant architecture of wild-type gloxinia (WT-PF), showing the horizontally oriented zygomorphic flower. (B) Front view of WT-PF flower. (C) Stamen of WT-PF, the red arrow denotes the dorsal staminode. (D) Side view of WT-PF floral tube (sepals are removed), showing the gibbous structure (arrow). (E) Plant architecture of cultivated peloric gloxinia (MU-WB), showing the upright actinomorphic flower. (F) Front view of MU-WB flower. (G) Stamen of MU-WB. (H) Side view of MU-WB floral tube (sepals are removed), showing the loss of the gibbous structure. (I–N) Floral developmental series of wild-type gloxinia (WT-PF) revealed by SEM. (I) The sepals initiated and developed equally. (J) The petal primordia arise subsequently inside the five sepals (removed). (K) The stamen primordia emerge inside the five petals in an alternating pattern. (L–N) As the floral organs advance in development, the floral zygomorphy manifests with the two dorsal petals slightly smaller than the lateral and ventral ones (L and M), and the dorsal stamen is finally suppressed into a staminode in development (N). (O–T) Floral developmental series of peloric gloxinia (MU-WB) revealed by SEM. (O) The sepals initiated and developed equally. (P) Petal primordia emerge subsequently inside the five sepals (removed). (Q) The stamen primordia arise inside the five petals in an alternative pattern. (R, S) As the development program proceeds, all the floral organs in each whorl develop equally. (T) The five stamens are equally differentiated into fertile stamens. Scale bar in (A, E), 10cm; (B–D and F–H), 1cm; (I–T), 100μm. It has long been recognized that floral orientation is intimately associated with floral symmetry (Sprengel 1793; Robertson 1888; Darwin 1865). However, the floral orientation has received little attention to date, despite the fact that floral symmetry and the evolution of zygomorphy is an important trend in angiosperm radiation (Dilcher 2000; Preston and Hileman 2009; Hileman 2014a). The floral horizontal orientation has evolved as an adaption to enable efficient pollination, likely representing the first step towards floral specialization (Fenster et al. 2009; Wang et al. 2014). In fact, the floral horizontal orientation and zygomorphy usually covary in angiosperms, particularly in sympetalous groups (supplementary table S1, Supplementary Material online). Deciphering whether these highly linked traits are underpinned by multiple factors or simple genetics would promote an understanding of the complex mechanisms behind floral evolution in angiosperms. In this study, we explore the molecular mechanisms underlying the evolution of the peloric characteristics of cultivated gloxinias, and further determine its origin by employing a highly combinatorial experimental approach. We have characterized a 10-bp deletion in the coding sequence of TCP-coding gene Sscyc that likely results in the production of a nonfunctional truncated protein with 45 amino acid (aa) in length. We demonstrate that this mutation is causally responsible for the development of peloric flowers in gloxinia. The putative ancestor of modern peloric gloxinias was further traced back to a particular wild collection from Rio de Janeiro State in 1820 (G. caulescens, now a synonym of S. speciosa) by literature review, and genetic inference combined with phylogenetic analysis. The results show that one mutation in a single gene causes a complex morphological shift from a horizontally orientated, and zygomorphic flower, to an upright, actinomorphic form in gloxinia. Our findings suggest that pleiotropy may play a crucial role in the coordinated evolution of complex floral organs in angiosperms. Results Phenotypic and Genetic Analysis of the Peloric Character in Gloxinia Wild-type gloxinia flowers are zygomorphic (bilaterally symmetric) and horizontally orientated (fig. 1A). The floral zygomorphy is weak in the second whorl, where the dorsal petals are only slightly smaller than the lateral and ventral ones, and strong in the third whorl, where the dorsal stamen is present as a staminode (fig. 1A–C and supplementary fig. S1, Supplementary Material online). The horizontal orientation of the flower is caused by a ventral–dorsal asymmetric growth at the base of dorsal corolla which produces a convex, gibbous structure (fig. 1D). Meanwhile, the peloric flowers are actinomorphic (radially symmetric) and upright without the development of a gibbous structure (fig. 1E–H and supplementary fig. S1, Supplementary Material online). In order to determine whether these two floral traits are tightly correlated in a broad range of taxonomic groups, we conducted a morphological survey covering all families of sympetalous angiosperms, and found that the horizontal placement of the floral tube is largely associated with the zygomorphic state of flowers (supplementary table S1, Supplementary Material online). This result suggests that the combination of these morphological traits conferred a selective advantage during floral evolution in angiosperms. To determine the genetics of the peloric phenotype, we crossed the wild-type zygomorphic gloxinia “Pink Flower” (WT-PF) with peloric gloxinia “White Bell” (MU-WB) (Materials and Methods). All 13 F1 plants exhibited a WT phenotype with a typical horizontally orientated zygomorphic flower (fig. 2A), implying that the peloric phenotype is recessive. Representatives of F1 plants were selfed to produce an F2 segregating population with a 3:1 ratio of WT to peloric phenotypes (141: 41; χ2 = 0.593, f = 1, P = 0.441) (fig. 2A), suggesting that peloria in gloxinia is a Mendelian character. We observed a perfect association (100%) between the floral zygomorphy and a horizontally orientated floral tube in the F2 segregating population (fig. 2A), indicating that the peloric phenotype, that is, floral actinomorphy and standing upright, is controlled by recessive mutation(s) in a single gene, or closely linked genes. Fig. 2. View largeDownload slide Genetic and association analysis of peloric flower in gloxinia. (A) Representatives of F1 and F2 plants of WT-PF × MU-WB. (B) Genotyping of F2 plants by CAPS. 16 representatives of F2 progenies are shown in the gel. Control experiment (Control) was conducted with SsCYC homozygous PCR product with no Nde I treatment. (C) Genotype–phenotype association analysis with variation sites in the 3,111-bp sequence of the SsCYC locus. In the upper panel, haplotypes of the significantly associated variation sites with n denote the number of sequences that are included in each haplotype; the variation sites that have consistent association between genotype and phenotype are colored in blue and red, respectively. The middle panel illustrates the gene structure of SsCYC. The TCP domain and R domain are shaded in orange and pink, respectively. The significant associated SNPs and Indel are indicated by red bars and triangles, respectively. In the bottom panel, grey circles represent variation sites that are not significantly associated with the peloric flower character, whilst the red circles represent SNPs or Indel significantly associated with the peloric flower character. PC, Peloric Cultivars; WA, Wild Accessions; ZC, Zygomorphic-flowered Cultivars; p, peloric flower; Z, zygomorphic flower. Scale bar in (A), 1cm. Fig. 2. View largeDownload slide Genetic and association analysis of peloric flower in gloxinia. (A) Representatives of F1 and F2 plants of WT-PF × MU-WB. (B) Genotyping of F2 plants by CAPS. 16 representatives of F2 progenies are shown in the gel. Control experiment (Control) was conducted with SsCYC homozygous PCR product with no Nde I treatment. (C) Genotype–phenotype association analysis with variation sites in the 3,111-bp sequence of the SsCYC locus. In the upper panel, haplotypes of the significantly associated variation sites with n denote the number of sequences that are included in each haplotype; the variation sites that have consistent association between genotype and phenotype are colored in blue and red, respectively. The middle panel illustrates the gene structure of SsCYC. The TCP domain and R domain are shaded in orange and pink, respectively. The significant associated SNPs and Indel are indicated by red bars and triangles, respectively. In the bottom panel, grey circles represent variation sites that are not significantly associated with the peloric flower character, whilst the red circles represent SNPs or Indel significantly associated with the peloric flower character. PC, Peloric Cultivars; WA, Wild Accessions; ZC, Zygomorphic-flowered Cultivars; p, peloric flower; Z, zygomorphic flower. Scale bar in (A), 1cm. Expression of SsCYC Is Correlated with the Development of Horizontally Orientated Zygomorphic Flowers CYCLOIDEA (CYC)-like TCP genes are known to be involved in the genetic control of floral zygomorphy in a broad array of species (Luo et al. 1996; see review in Preston and Hileman 2009). In Gesneriaceae, an ancient gene duplication event occurred prior to the split of the Gesnerioideae and the Cyrtandroideae subfamilies generating GCYC1 and GCYC2 paralogous lineages (Citerne et al. 2000; Smith et al. 2004). We cloned the full length Sinningia speciosa CYC (SsCYC) gene, and performed a phylogenetic analysis. The results indicate that the Gesnerioideae subfamily, which includes the Sinningia genus, has lost the GCYC1 lineage in evolution and harbours only one copy of the CYC-like genes, belonging to the GCYC2 clade, which is homologous to AmCYC/AmDICH (supplementary fig. S2, Supplementary Material online). Ontogenetic analysis shows that the floral organ development of WT-PF and MU-WB exhibits a similar pattern before the stamen initiation with all primordium developed equally (fig. 1I–K and 1O–Q). Later on, the developmental differences between them become evident in the dorsal floral organs with their growth suppressed in WT-PF (fig. 1L–N and 1R–T). Correlatively, SsCYC shows a weak dorsal specific expression pattern in the petal whorl, and strong asymmetric expression in the stamen whorl, with an extremely high level of expression in the staminode in WT-PF (fig. 3B, C, and G). In contrast, all floral organs develop equally in MU-WB, and no Sscyc expression can be detected during petal and stamen development (figs. 1O–T and 3E and H). In the floral tube of WT-PF, the expression of SsCYC seems to be exceptionally high in the gibbous structure compared with other sampled tissues, whereas its expression in the Dorsal Floral Tube (DFT) is only slightly higher relative to those in the Ventral Counterparts of the gibbous structure (VC) and the Ventral Floral Tube (VFT) (fig. 4I and J). In MU-WB, only a residual Sscyc expression signal can be detected in the sampled tissues of the floral tube (fig. 4J). The RNA in situ hybridization shows that the SsCYC expression exhibits an asymmetric gradient pattern in the developing gibbous structure, in which strong signals are observed in the inner cell layers whilst almost no expression can be detected in the outer cell layers (fig. 4A, B, D, and F). In contrast, no expression signal of SsCYC can be detected in all ventral counterparts of the gibbous structure (fig. 4C, E, and G). Concomitantly, the cell growth in the inner part of the gibbous structure is significantly suppressed while the cells in the outer layers and the ventral counterparts are expanded almost equally (fig. 4K–R and supplementary fig. S3, Supplementary Material online). As outlined above, the specific expression of SsCYC is correlated with a suppression of growth in the dorsal floral organs, and a suppression of cell expansion in the inner part of the gibbous structure. Fig. 3. View largeDownload slide Comparative expression analysis of SsCYC/Sscyc in WT-PF and MU-WB. (A) The SsCYC mRNA can be detected across the apex of the young floral meristems of WT-PF (triangle). (B) In WT-PF, SsCYC is evenly distributed in the petal and stamen primordia with an extremely high level detected in dorsal staminode. (C) As the developmental program proceeds, the SsCYC mRNA is specifically accumulated in the dorsal stamens and is undetectable in the other floral organs of WT-PF. (D) Sscyc mRNA is evenly distributed in the young floral meristems of MU-WB. (E) Its transcripts can no longer be detected in the subsequent development stages of MU-WB. (F) Control experiment with sense SsCYC probe generated no signal in WT-PF flower of stage similar to (C). (G) Expression analysis of SsCYC in WT-PF floral organs. (H) Expression analysis of Sscyc in MU-WB floral organs. DS/LS/VS, Dorsal/Lateral/Ventral Sepals; DP/LP/VP, Dorsal/Lateral/Ventral Petals; DSt/LSt/VSt, Dorsal/Lateral/Ventral Stamen; DFT/VFT, Dorsal/Ventra Floral Tube; GS, Gibbous Structure; VC, Ventral Counterpart of the GS. FD, Floral Discs; Error bars in (G and H) indicate S.D. of three biological replicates. Scale bars in (A–F), 100 μm. Fig. 3. View largeDownload slide Comparative expression analysis of SsCYC/Sscyc in WT-PF and MU-WB. (A) The SsCYC mRNA can be detected across the apex of the young floral meristems of WT-PF (triangle). (B) In WT-PF, SsCYC is evenly distributed in the petal and stamen primordia with an extremely high level detected in dorsal staminode. (C) As the developmental program proceeds, the SsCYC mRNA is specifically accumulated in the dorsal stamens and is undetectable in the other floral organs of WT-PF. (D) Sscyc mRNA is evenly distributed in the young floral meristems of MU-WB. (E) Its transcripts can no longer be detected in the subsequent development stages of MU-WB. (F) Control experiment with sense SsCYC probe generated no signal in WT-PF flower of stage similar to (C). (G) Expression analysis of SsCYC in WT-PF floral organs. (H) Expression analysis of Sscyc in MU-WB floral organs. DS/LS/VS, Dorsal/Lateral/Ventral Sepals; DP/LP/VP, Dorsal/Lateral/Ventral Petals; DSt/LSt/VSt, Dorsal/Lateral/Ventral Stamen; DFT/VFT, Dorsal/Ventra Floral Tube; GS, Gibbous Structure; VC, Ventral Counterpart of the GS. FD, Floral Discs; Error bars in (G and H) indicate S.D. of three biological replicates. Scale bars in (A–F), 100 μm. Fig. 4. View largeDownload slide Gene expression and cell morphology analysis of the gibbous structure in WT-PF. (A–H) SsCYC expression analysis in the developing GS and floral tube of WT-PF. (A) SsCYC signals are specifically localized in the inner cell layer (red arrows) of GS but not in the VC (enlarged in B and C, respectively). (D and E) In late floral developmental stages when the anthers were differentiated, SsCYC expression can still be detected in the inner cell layers (red arrow) of the GS (D, enlarged in F), but not in the VC (E, enlarged in G). (H) Control experiment using sense SsCYC probe generates no signals. (I) Graphic view of the floral tissues sampled for expression analysis in (J). (K) Cross sections of the GS. A single cell layer is marked in red to show the cell size variation tendency from the inner epidermis to the outer epidermis (red arrow). (L and M) SEM analysis of the epidermal cells of WT-PF GS showing that cell expansion is extremely suppressed in the inner epidermis (L) compared with the outer epidermis (M). (O) Cross sections of the VC. A single cell layer is marked in blue to show no obvious variation of cell size from the inner epidermis to the outer epidermis. (P and Q) SEM analysis of the epidermal cells of ventral counterparts of gibbous structure showing that no cell expansion difference can be detected. (R) Scatterplot of cell size (μm2) and relative position of the cell from the inner epidermis (0%) to the outer epidermis (100%). Gaussian Fitting was calculated to show the variation tendency of cell size along the dorso-ventral axis of the tissue. GS, gibbous structure; VC, Ventral Counterpart of the GS; FD, Floral Disc. Scale bar in (A–H), 150 μm; (K and O), 100 μm; (L, M, P, and Q), 50 μm; (I), 0.5 cm. Fig. 4. View largeDownload slide Gene expression and cell morphology analysis of the gibbous structure in WT-PF. (A–H) SsCYC expression analysis in the developing GS and floral tube of WT-PF. (A) SsCYC signals are specifically localized in the inner cell layer (red arrows) of GS but not in the VC (enlarged in B and C, respectively). (D and E) In late floral developmental stages when the anthers were differentiated, SsCYC expression can still be detected in the inner cell layers (red arrow) of the GS (D, enlarged in F), but not in the VC (E, enlarged in G). (H) Control experiment using sense SsCYC probe generates no signals. (I) Graphic view of the floral tissues sampled for expression analysis in (J). (K) Cross sections of the GS. A single cell layer is marked in red to show the cell size variation tendency from the inner epidermis to the outer epidermis (red arrow). (L and M) SEM analysis of the epidermal cells of WT-PF GS showing that cell expansion is extremely suppressed in the inner epidermis (L) compared with the outer epidermis (M). (O) Cross sections of the VC. A single cell layer is marked in blue to show no obvious variation of cell size from the inner epidermis to the outer epidermis. (P and Q) SEM analysis of the epidermal cells of ventral counterparts of gibbous structure showing that no cell expansion difference can be detected. (R) Scatterplot of cell size (μm2) and relative position of the cell from the inner epidermis (0%) to the outer epidermis (100%). Gaussian Fitting was calculated to show the variation tendency of cell size along the dorso-ventral axis of the tissue. GS, gibbous structure; VC, Ventral Counterpart of the GS; FD, Floral Disc. Scale bar in (A–H), 150 μm; (K and O), 100 μm; (L, M, P, and Q), 50 μm; (I), 0.5 cm. The Peloric Phenotype Is Associated with a 10-bp Deletion in the Coding Sequence of Sscyc To determine whether the peloric mutation maps to the SsCYC locus, we genotyped 182 F2 plants of WT-PF × MU-WB using Cleaved Amplified Polymorphic Sequences (CAPS). The SsCYC allele (wild-type) perfectly cosegregated with the phenotype of horizontally orientated zygomorphic flowers, and all upright peloric flowers were homozygous for the mutant Sscyc allele (fig. 2B). In addition, we also genotyped 36 modern accessions of gloxinia with either zygomorphic or peloric phenotype from various geographic locations around the globe. This result confirmed the genotype–phenotype correlation revealed from the analysis of the F2 population (supplementary table S2 and fig. S4, Supplementary Material online). A careful comparison of the 3,111-bp genomic DNA sequences (including 1992-bp 5′ promoter sequence) between WT-PF and MU-WB revealed 56 SNPs and one Indel (data not shown), but failed to resolve the causal mutation for peloria. In order to find the causal mutation, we took a SNP-phenotype association analysis using a panel of 75 gloxinia accessions (52 cultivated accessions and 23 wild collections) collected from different geographic regions (supplementary table S2, Supplementary Material online). We identified a total of 29 distinct haplotypes in that seven SNPs and one Indel were significantly associated with the peloric phenotype (P < 0.01) (fig. 2C and supplementary table S3, Supplementary Material online). Further analysis of these sites within six haplotypes showed that four SNPs in the regulatory region (SNP96, SNP98, SNP190, and SNP192) and two in the coding region (SNP251 and SNP253) are completely or partially shared between peloric cultivars and three or more zygomorphic accessions, thus excluding them from having a functional relevance to the Sscyc allele (fig. 2C). Of the remaining two sites, SNP203 (A/C, +46 bp) is unlikely to be responsible for peloria, as the amino acid substitution (Serine/Tyrosine) lies outside of the TCP domain, and therefore is unlikely to alter the activity of SsCYC. The Indel (-CTTCAATCTC, +48 bp) causes a frameshift in the coding sequence, which results in a truncated protein with 45-amino acids in length. This truncated protein is likely nonfunctional as it lacks the entire TCP domain and the downstream C terminus (fig. 2C). Functional Analyses of SsCYC/Sscyc Confirm Its Role in Peloric Gloxinia Development To validate the biological function of SsCYC and Sscyc, we fused the coding sequences of the alleles with the CaMV 35S promoter and transformed Arabidopsis thaliana. T1 transgenic plants were selfed to produce T2 populations (see methods). The heritability of the transgene was confirmed by RT-PCR (supplementary fig. S5, Supplementary Material online). Compared with WT plants, the SsCYC overexpression lines showed retarded plant development (fig. 5A) with smaller leaves (supplementary figs. S6 and S8, Supplementary Material online) and smaller floral organs (supplementary figs. S7 and S8, Supplementary Material online). Meanwhile, Sscyc overexpression generated no obvious phenotypic changes (fig. 5A and supplementary figs. S6–S8, Supplementary Material online). Further SEM analysis of the epidermis of leaves and petals showed that cell expansion was repressed in SsCYC overexpression lines (fig. 5B and C and supplementary fig. S6, Supplementary Material online), which is consistent with our hypothesis that dorsal-specific expression of SsCYC suppresses the growth of the dorsal floral organs (figs. 1A–C and 4K–M). In contrast, no evident changes were observed regarding the cell morphologies in leaves or petals in Sscyc overexpressing lines, indicating that Sscyc protein is nonfunctional (fig. 5B and D and supplementary figs. S6 and S8, Supplementary Material online). Therefore, the development of the peloric gloxinia may be attributed to the 10-bp deletion in Sscyc and the resultant truncated non-functional protein. Fig. 5. View largeDownload slide Functional analysis of SsCYC/Sscyc in Arabidopsis and CRES-T analysis of SsCYC in WT-PF. (A) Overexpression of SsCYC in Arabidopsis retards plant development while overexpression of Sscyc in Arabidopsis generates no phenotypic alteration. The inserts show the flowers of each genotype. SEM analysis of the petal epidermal cells of WT plants (B), SsCYC overexpressor (C) and Sscyc overexpressor (D). In (B–D), a total of 20 epidermal cells are shaded in yellow to indicate the cell size difference. (E) Plant architecture of the transgenic control plants. (F) Side view of the horizontally oriented flower character of the transgenic control plants. (G) Front view of the wild-type zygomorphic flower of the transgenic control plants. (H) Plant architecture of the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1), showing the upright peloric flowers. (I) Side view of the upright peloric flower from the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1). (J) Front view of the peloric flower from the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1). Scale bar in (A), 5 cm; inserted graphs in (A), 1 mm; (B–D), 150 μm; (E, H), 7 cm; (F, G, I, J), 1 cm. Fig. 5. View largeDownload slide Functional analysis of SsCYC/Sscyc in Arabidopsis and CRES-T analysis of SsCYC in WT-PF. (A) Overexpression of SsCYC in Arabidopsis retards plant development while overexpression of Sscyc in Arabidopsis generates no phenotypic alteration. The inserts show the flowers of each genotype. SEM analysis of the petal epidermal cells of WT plants (B), SsCYC overexpressor (C) and Sscyc overexpressor (D). In (B–D), a total of 20 epidermal cells are shaded in yellow to indicate the cell size difference. (E) Plant architecture of the transgenic control plants. (F) Side view of the horizontally oriented flower character of the transgenic control plants. (G) Front view of the wild-type zygomorphic flower of the transgenic control plants. (H) Plant architecture of the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1), showing the upright peloric flowers. (I) Side view of the upright peloric flower from the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1). (J) Front view of the peloric flower from the SsCYC-SRDX transgenic plants (SsCYC-SRDX-L1). Scale bar in (A), 5 cm; inserted graphs in (A), 1 mm; (B–D), 150 μm; (E, H), 7 cm; (F, G, I, J), 1 cm. Chimeric Repressor Silencing Technology (CRES-T) was recently developed as a powerful tool to knock-down endogenous genes by converting transcriptional activators into strong repressors when the target gene is fused to the ERF-associated Amphiphilic Repression (EAR) motif (SRDX, Hiratsu et al. 2003). Considering that SsCYC is the only copy of the CYC-like gene in the gloxinia genome (supplementary fig. S2, Supplementary Material online), we next tried to repress SsCYC function by producing transgenic plants with 35S: SsCYC: SRDX in WT-PF gloxinias (Materials and Methods) (supplementary fig. S9, Supplementary Material online). The plants regenerated from untransformed leaf cultures produce flowers with the typical WT zygomorphic appearance with nodding floral tubes (fig. 5E–G). Among ten independent transgenic plants expressing the 35S: SsCYC: SRDX fusion protein, three developed upright actinomorphic flowers with fully ventralized petals and stamens, perfectly phenocopying the flowers of peloric gloxinias (fig. 5H–J). In addition, we did not observe any phenotypic alterations in these transgenic plants other than floral symmetry changes, suggesting the SsCYC: SRDX fusion protein specifically targets the SsCYC pathway in the flowers (Compare fig. 5E and H). The results of functional knock-down of SsCYC by CRES-T also indicate that the GCYC2 member of Gesnerioideae exerts similar function as the GCYC1 members of Cyrtandroideae in repressing cell expansion in the dorsal floral organs (Yang et al. 2012; Liu et al. 2014). Taken together, these findings indicate that a single pleiotropic gene, SsCYC, controls both the floral orientation and floral symmetry through its activity in the basal floral tube, petals and stamens. A 10-bp deletion in the coding sequence of Sscyc results in the production of a nonfunctional truncated protein, and is causally responsible for the development of peloric flowers in the gloxinia. Development of Peloric Gloxinia Involves a Two-Fold Molecular Mechanism Our previous study shows that the persistent dorsal specific expression of CYC-like gene depends on the activity of an auto-regulatory loop of CYC-like genes (Yang et al. 2012). Therefore, both the functional CYC protein and CYC binding sites (CBS) in the promoter are indispensable for the establishment of floral zygomorphy (Yang et al. 2012). For instance, in Antirrhinum majus, the loss-of-function change in amino acid coding region in the cyc-608 mutant produces partially ventralized peloric flowers (Luo et al. 1996). In addition, the lack of a CBS in the promoter sequence in Arabidopsis thaliana leads to transient expression of AtTCP1 in the dorsal floral primordium in the early stages, thereby producing actinomorphic flowers (Cubas et al. 2001). Curiously, in the F1 plants of WT-PF × MU-WB, we observed the restoration of dorsal specific expression of Sscyc by allele-specific expression analysis (fig. 6A). Given that initiation of Sscyc expression is normal in the peloric floral meristems as in wild-type (fig. 3A and D) and the dorsal specific expression is subsequently lost during floral organ development in the peloric flowers (fig. 3B and E), we hypothesized that the loss of dorsal specific expression of Sscyc in the peloric flowers resulted from the disruption of the auto-regulatory loop by the nonfunctional Sscyc protein (Yang et al. 2012). To test this idea, we compared the ∼2.0 kb promoter sequence between SsCYC and Sscyc, and found that the putative SsCYC interacting element (CYC-binding Site, CBS) is conserved between WT-PF and MU-WB, that is, they share the same CBS sequence (GGGGCCC) (fig. 6E). Furthermore, an Electrophoresis Mobility Shift Assay (EMSA) analysis showed that SsCYC could interact with a probe containing the CBS, this binding could be effectively competed by adding excessive amounts of unlabeled CBS-containing DNA probes (fig. 6B and C). In sharp contrast, the mutated Sscyc protein failed to bind to this CBS-containing DNA sequence (fig. 6D). Fig. 6. View largeDownload slide Allele specific expression of Sscyc in F1 hybrids and in vitro DNA–Protein interaction by EMSA. (A) Expression analysis of the peloric Sscyc allele in floral organs of the F1 hybrid of WT-PF×MU-WB by allele-specific real-time qPCR, showing that the dorsal specific expression pattern of Sscyc was restored. (B) Oligonucleotide probe sequence used for EMSA analysis, the putative CYC-Binding Site (CBS) sequence is shown in bold capital letters. (C) EMSA analysis of the CBS and SsCYC recombinant protein shows that the interaction of SsCYC with CBS results in a retarded band in the gel (arrow, lane 3); the interaction of the SsCYC with CBS is abolished when excessive amounts of unlabeled probe is added (lane 4). (D) EMSA analysis of the CBS and Sscyc recombinant protein shows that the Sscyc protein has lost its ability to interact with the CBS sequence. (E) The promoter sequences of SsCYC and Sscyc. Both contain the same sequence matching the consensus CYC-Binding Sites (CBS) (boxed) Fig. 6. View largeDownload slide Allele specific expression of Sscyc in F1 hybrids and in vitro DNA–Protein interaction by EMSA. (A) Expression analysis of the peloric Sscyc allele in floral organs of the F1 hybrid of WT-PF×MU-WB by allele-specific real-time qPCR, showing that the dorsal specific expression pattern of Sscyc was restored. (B) Oligonucleotide probe sequence used for EMSA analysis, the putative CYC-Binding Site (CBS) sequence is shown in bold capital letters. (C) EMSA analysis of the CBS and SsCYC recombinant protein shows that the interaction of SsCYC with CBS results in a retarded band in the gel (arrow, lane 3); the interaction of the SsCYC with CBS is abolished when excessive amounts of unlabeled probe is added (lane 4). (D) EMSA analysis of the CBS and Sscyc recombinant protein shows that the Sscyc protein has lost its ability to interact with the CBS sequence. (E) The promoter sequences of SsCYC and Sscyc. Both contain the same sequence matching the consensus CYC-Binding Sites (CBS) (boxed) Under both artificial and natural selection regimes, adaptive morphological traits can be produced by either change in protein function (Li et al. 2006; Hoballah et al. 2007; Pourkheirandish et al. 2015) or regulatory modifications (Chan et al. 2010; Studer et al. 2011; Dong et al. 2014). The observations presented here strongly suggest that the evolution of the peloric gloxinia involves a 2-fold mechanism. That is, the 10-bp deletion brings about the loss-of-function of Sscyc protein, which in turn, further disrupts the auto-regulatory loop of Sscyc leading to a complete loss of dorsal specific expression. SsCYC Was Targeted by Artificial Selection during the Domestication Process of Peloric Gloxinia For crops, domestication occurs when a single favoured haplotype associated with favorable morphological evolution is targeted by selection and fixed over time (Gross and Olsen 2010). To evaluate the impact of artificial selection on the SsCYC locus, we analyzed the DNA polymorphisms in a ∼3.1 kb genomic region of SsCYC in a panel of 40 peloric accessions from diverse geographic locations, and 23 wild collections with zygomorphic flowers from Brazil (supplementary table S2, Supplementary Material online). In the wild gloxinia population, the average nucleotide diversity of SsCYC DNA sequence is π = 0.0164, indicative of considerable sequence polymorphism at the SsCYC locus (supplementary fig. S10, Supplementary Material online). However, all 40 peloric accessions share the same haplotype (π = 0) including the fixed 10-bp deletion in the ∼3.1 kb SsCYC genomic sequence (supplementary fig. S10, Supplementary Material online), indicating that intensive artificial selection has targeted this mutation by removing rare sequence variants in this locus. To trace the origin of peloric gloxinias, we constructed the SsCYC phylogeny based on the ∼1,250-bp genomic sequence. All 40 peloric gloxinias are grouped together with two wild gloxinias “Cardoso Moreira” in a single well supported clade (fig. 7). In contrast, the tree of the putative neutral marker, nuclear chloroplast-expressed Glutamine Synthetase (ncpGS), showed that the peloric accessions are dispersed into multiple branches nested in wild gloxinias (supplementary fig. S11, Supplementary Material online). These results suggest that the SsCYC locus in peloric gloxinias was targeted by artificial selection, and the peloric Sscyc allele was derived from the sequence of “Cardoso Moreira” that is distinct from all other wild gloxinia accessions by a single domestication event. Fig. 7. View largeDownload slide Phylogenetic analysis of the SsCYC locus in Sinningia speciosa. Majority rule consensus tree of 235 most parsimonious trees generated from SsCYC sequences of S. speciosa. Bootstrap (BS) values and Bayesian Posterior Probabilities (PP) are indicated above and below the branches, respectively. The cultivated peloric gloxinia accessions (Cultivated, CV) and wild gloxinia accessions (Wild Accession, WA) are indicated by blue and red dots, respectively. The red star indicates the close relationship of the peloric gloxinia accessions with two wild gloxinia collections (S. speciosa “Cardoso Moreira pink mutant” and S. speciosa “Cardoso Moreira”). Fig. 7. View largeDownload slide Phylogenetic analysis of the SsCYC locus in Sinningia speciosa. Majority rule consensus tree of 235 most parsimonious trees generated from SsCYC sequences of S. speciosa. Bootstrap (BS) values and Bayesian Posterior Probabilities (PP) are indicated above and below the branches, respectively. The cultivated peloric gloxinia accessions (Cultivated, CV) and wild gloxinia accessions (Wild Accession, WA) are indicated by blue and red dots, respectively. The red star indicates the close relationship of the peloric gloxinia accessions with two wild gloxinia collections (S. speciosa “Cardoso Moreira pink mutant” and S. speciosa “Cardoso Moreira”). Reconstructing the Domestication History of the Peloric Gloxinia In the literature, the first peloric gloxinia with upright flowers was documented in 1845 under the name Gloxinia fyfiana (synonym of S. speciosa) produced by an Englishman called Fyfe by crossing G. maxima and G. caulescens in 1844 (both synonyms of S. speciosa) (fig. 8A) (Harrison 1847; Louis 1848; Fyfe 1879). Gloxinia caulescens was introduced from Brazil into England in 1820, published in 1827. It was widely cultivated and frequently crossed to G. speciosa due to its extravagant ornamental flowers (fig. 8E) (Edwards 1827; Paxton 1838; Maund and Henslow 1839; Harrison 1847; Johnson and Landreth 1847). Gloxinia maxima was recorded as a hybrid between G. candida and G. speciosa (both synonyms of S. speciosa) and published in 1838 (fig. 8B) (Paxton 1838). Gloxinia speciosa has horizontally orientated, zygomorphic flowers, and was introduced from Brazil into cultivation in England in 1815, published in 1817 (fig. 8C) (Loddiges 1817). Gloxinia candida first appeared in an exhibition in 1832 as a garden origin hybrid with no detailed character description except for white slipper flowers. The first “gloxinia” with white flowers, that is, G. speciosa var. albiflora, was published in 1833 (Hooker 1833). In 1839, another hybrid with white slipper flowers, similar to that of G. candida, was described in detail with a clear record that this hybrid was produced from a cross between G. speciosa var. albiflora and G. caulescens (fig. 8D) (Maund and Henslow 1839). Since hybridization and breeding among “gloxinia” species was fashionable from 1820 to 1840 in Great Britain (Paxton 1838; Maund and Henslow 1839; Johnson 1847), it is possible that a hybrid with similar white slipper flowers was repeatedly produced from the crosses between G. speciosa var. albiflora and G. caulescens by gardeners at the time. Therefore, G. candida is probably a hybrid of G. speciosa var. albiflora × G. caulescens. Fig. 8. View largeDownload slide Historical paintings of Gloxinia species and the genealogy of the first peloric gloxinia flower. (A) The original depiction of G. fyfiana in 1848 by Louis. (B) The original depiction of G. maxima in 1838 by Paxton. (C) The original depiction of G. speciosa in 1817 by Loddiges. (D) The original depiction of G. speciosa-caulescens hybrid in 1839 by Maund and Henslow. (E) The original depiction of G. caulescens in 1827 by Edwards. (F) Plant architecture of a flowering S. speciosa “Cardoso Moreira”, showing the great morphological similarities to G. fyfiana (A) and G. caulescens (E) in terms of plant architecture. The photo is courtesy of Mr. Lin Ruei Chau. (G) The genealogy of the first peloric Gloxinia. The genotype and first appearance year of each “species” is indicated below, respectively. The peloric allele Sscyc and the mutated cultivar of G. caulescens are labeled in red. Note: the original imported G. caulenscens plants (1820) were supposed to be homozygous for SsCYC, as we failed to detect the mutated Sscyc allele in the wild populations. The Sscyc allele might have originated from the de novo mutation of 10-bp deletion in the cultivation process of G. caulenscens from 1820 to 1832 and kept a heterozygous (SsCYC/Sscyc) status in G. candida and G. maxima in the following 12 years before meeting with the mutated cultivar of G. caulenscens again in 1844. All the latin names presented in (G) are synonyms of S. speciosa. Fig. 8. View largeDownload slide Historical paintings of Gloxinia species and the genealogy of the first peloric gloxinia flower. (A) The original depiction of G. fyfiana in 1848 by Louis. (B) The original depiction of G. maxima in 1838 by Paxton. (C) The original depiction of G. speciosa in 1817 by Loddiges. (D) The original depiction of G. speciosa-caulescens hybrid in 1839 by Maund and Henslow. (E) The original depiction of G. caulescens in 1827 by Edwards. (F) Plant architecture of a flowering S. speciosa “Cardoso Moreira”, showing the great morphological similarities to G. fyfiana (A) and G. caulescens (E) in terms of plant architecture. The photo is courtesy of Mr. Lin Ruei Chau. (G) The genealogy of the first peloric Gloxinia. The genotype and first appearance year of each “species” is indicated below, respectively. The peloric allele Sscyc and the mutated cultivar of G. caulescens are labeled in red. Note: the original imported G. caulenscens plants (1820) were supposed to be homozygous for SsCYC, as we failed to detect the mutated Sscyc allele in the wild populations. The Sscyc allele might have originated from the de novo mutation of 10-bp deletion in the cultivation process of G. caulenscens from 1820 to 1832 and kept a heterozygous (SsCYC/Sscyc) status in G. candida and G. maxima in the following 12 years before meeting with the mutated cultivar of G. caulenscens again in 1844. All the latin names presented in (G) are synonyms of S. speciosa. According to our results, the peloric allele is recessive and hence the peloric flower is homozygous for the Sscyc allele. In order to produce the peloric flower of G. fyfiana, both G. maxima and G. caulescens must possess a heterozygous SsCYC/Sscyc genotype. As for G. maxima, theoretically, it could have inherited the peloric allele from either G. candida or G. speciosa. As G. speciosa is a wild gloxinia introduced from Brazil, and because the probability of the same peloric mutation (10-bp deletion) happening independently in G. caulescens and G. speciosa is extremely low, we hypothesize that G. speciosa is unlikely to be the donor of the recessive Sscyc allele. It is more likely that G. maxima inherited the peloric allele from G. candida if we consider that the mutation occurred only once in G. caulescens specifically. Taken together, the genetic inference above suggests that the causal mutation (10-bp deletion) leading to peloric gloxinia initially occurred in a cultivar of G. caulescens during the cultivation process between its importation in 1820 and the first appearance of the white-flowered hybrid (G. candida) in 1832 (fig. 8G). This mutation then passed from G. candida to G. maxima in 1838 and was kept in a heterozygous state for the following 6 years (1844) until G. maxima merging with the mutated heterozygous cultivar of G. caulescens in a cross generated by Mr. Fyfe (fig. 8G). Discussion On the Origin of the Peloric Gloxinia The floral characteristics of ornamental plants have undergone an unprecedented explosion in color and morphology by deliberate hybridization-assisted selection over the past 200 years (Darwin 1868; Crane and Lawrence 1934; Kingsbury 2009). However, even for species whose domestication is clearly documented, showing major morphological transitions (e.g., Chinese primrose) (Crane and Lawrence 1934), the origin and spread of novel traits cannot be well understood until the genetic factors underlying these changes have been identified. Based on literature review and genetic inferences, we propose that the causal mutation (10-bp deletion in Sscyc) leading to peloria, occurred only once, in a cultivar of G. caulescens. However, in the phylogenetic tree based on the ∼1,250-bp of the SsCYC genomic sequence, the modern peloric gloxinias grouped together with two wild gloxinia collections from “Cardoso Moreira”, suggesting that modern peloric gloxinia are derived from these two wild collections, or related populations. In fact, the wild “Cardoso Moreira” collections are closely related to the initial peloric gloxinia morphologically in plant architecture of extremely robust stems and one or two (or only several) flowers produced in the top leaf axils (fig. 8A, E, and F), suggesting that they are morphologically related. In addition, G. caulescens was originally recorded in wet rocks at the base of Corcovado Montain in Rio de Janeiro State in 1838 (Brackenridge 1886; Zaitlin 2011), adjacent to the location where the “Cardoso Moreira” samples were collected. Taken together, the genetic inferences and textual examination of the literature presented above strongly suggest that the “Cardoso Moreira” plants, or a related population of the same race, may be the gloxinia plants (designated as G. caulescens) exported from Brazil to Great Britain in 1820, which are the direct ancestors of the modern peloric gloxinia. SsCYC Is a Pleiotropic Gene Controlling Multiple Floral Characters Flowers are considered to be an essential model for studying the genetics of speciation, as the floral traits are intimately associated with prezygotic isolation (Smith 2016). Floral traits are often integrated as functional modules, which show correlated variation as the results of adaptation to specific pollination strategies (Fenster et al. 2009; Smith 2016). The genetic mechanism proposed to account for this complex morphological integration is that different characteristic elements are controlled by the same developmental gene with pleiotropic effects (Wagner and Zhang 2011; Smith 2016). In plants, genetic studies have identified a number of genomic regions responsible for multiple floral traits (Bradshaw et al. 1998; Wessinger et al. 2014). One caveat of these studies is that colocalization of multiple traits to the same genetic locus does not necessarily mean that the underlying genes or mutations are exactly the same, as a single locus may contain a large number of genes (Smith 2016). In addition, genes with pleiotropic effects identified in the functional analysis of model species are proposed to act as hot spots that facilitate phenotypic evolution (Wagner and Zhang 2011). However, direct molecular evidence for the evolution of functionally integrated phenotypic modules with the contribution of pleiotropic genes remain rare in plants, and the only examples are from animals (Linnen et al. 2013; Chung et al. 2014; Smith 2016). In Sinningia speciosa, our analysis provides three pieces of concrete evidence for the involvement of a pleiotropic gene in the development of an integrated floral character complex, that is, the nodding zygomorphic flower. Firstly, the anatomic examination shows that the nodding flower phenotype is caused by the development of a gibbous structure at the base of dorsal corolla. There is a simultaneous loss of the gibbous structure and zygomorphy in the peloric gloxinia, which lacks morphological recombination in the F2 segregating population. This suggests that these characters are controlled by the same genetic regulator. Secondly, SsCYC exhibits dorsal specific expression in the floral organs (i.e., the dorsal petals and staminode) and inner parts of the basal dorsal floral tubes, which are correlated with the retardation of growth in the dorsal floral organs, and restricted cell expansion in the inner parts of the gibbous structure. Thirdly, the knock-down of SsCYC by dominant repression produces transgenic plants with perfectly ventralized actinomorphic flowers with loss of the gibbous structure, implying that the SsCYC is a pleiotropic gene responsible for the development of both morphological traits. The discovery of a CYC-like gene involved in the development of both floral symmetry and orientation provides empirical evidence that a simple genetic change in a pleiotropic gene with selective advantage would promote coordinated evolution of the highly integrated floral organs. In angiosperms, the evolution of zygomorphic flowers is considered to be a major morphological innovation that led to the diversification of species (Dilcher 2000). It has been recently demonstrated that horizontal-orientated and zygomorphic flowers, with the two traits acting as a functional unit, confer a selective adaptive advantage, as they direct the pollinator movement within the flowers, and therefore, enable effective and precise pollen transfer to the stigma (Ushimaru and Hyodo 2005; Fenster et al. 2009; Wang et al. 2014). In fact, the association between the floral horizontal orientation and floral zygomorphy was recognized very early by Robertson in 1888. However, the floral orientation has long been neglected since then (Fenster et al. 2009). To our knowledge, this is the first time to report the role of CYC-like genes in controlling the floral orientation, expanding the function of CYC-like genes from controlling the floral symmetry to regulating both floral orientation and symmetry. This finding provides critical insights into how the high frequency of speciation with subsequent rapid diversification has occurred in zygomorphic lineages. In general, the floral horizontal orientation can be produced either by the asymmetrical growth of the floral tube or the bending of the pedicels relative to the stem. The floral zygomorphy coupled with floral horizontal orientation may have been independently evolved through different pathways. We also notice some cases of floral zygomorphy disassociated from horizontal orientation, such as some species of Mimulus (Phrymaceae) and Agalinis (Orobanchaceae) with zygomorphic but upright flowers. Further exploring the genetic basis relating to alternative pathways of the coupling between floral zygomorphy and horizontal orientation and the adaptive scenario for the decoupling between them in given taxa would shed new light on the mechanisms that underlie the vast morphological diversity of floral zygomorphy in angiosperms. Materials and Methods Plant Samples, Growth Condition and Artificial Hybridization We chose Pink Flower (WT-PF) and White Bell (MU-WB) as plant materials for expression and functional analysis. WT-PF is a wild-type gloxinia that is native to Brazil and produces large, horizontally oriented pink zygomorphic flowers with a white band decorated with dark purple spots in the ventral corolla throat. MU-WB is a popular cultivated variety widely cultivated in China, which bears upright white actinomorphic bell-shaped flowers. Various domesticated gloxinia accessions (supplementary table S2, Supplementary Material online) were either obtained from the Gesneriad Society (www.gesneriadsociety.org) or from commercial sources. The wild gloxinia (S. speciosa) collections and two Sinningia species (S. tubiflora and S. rupicola) were obtained as seed from Mauro Peixoto’s Brazil Plants Organization (Mogi das Cruzes, SP, Brazil). The seeds were germinated on 1/2 Murashige and Skoog (MS) medium at 26 °C. The 1-month seedlings were then transplanted to 7-cm pots containing a mixture of moss substrate, vermiculite and perlite (1:1:1) in the glasshouse of Institute of Botany, the Chinese Academy of Sciences. Growth conditions were long-day photo period (16-h light/8-h dark) at 28 °C with a relative humidity of 70% and 60% shading. Arabidopsis thaliana (ecotype Columbia-0) used in this study was germinated on 1/2 MS medium at 23 °C. The seedlings were transplanted in the soil under 16-h-light (200 μmol m−2 s−1, 23 °C) and 8-h-dark (20 °C) conditions. To generate the hybrid gloxinias, we pollinated the emasculated maternal plants (MU-WB) with pollen from the paternal plants (WT-PF) at anthesis. The resultant hybrids were germinated and transplanted into the glasshouse as described above. Morphological Analysis and Scanning Electron Microscopy (SEM) The floral organs of WT-PF and MU-WB at anthesis were dissected and the morphology of floral organs was recorded with Nikon D7100 camera. For SEM, young inflorescences of WT-PF and MU-WB were fixed in FAA and infiltrated under vacuum. The respective floral meristems from distinct developmental stages were dissected with a needle in 70% ethanol under a light microscope. The materials were dried with critical point of CO2 and the floral organs were examined using a Hitachi S-4800 scanning electron microscope (SEM) as previously described (Zhou et al. 2008). To quantify cell size in the gibbous structure, the basal floral tube (including the gibbous structure) was fixed in FAA and embedded into paraffin (Sigma, USA). 8-μm sections were prepared using a rotary microtome. Section images were captured and processed by Image J (1.50b) software. For the gibbous structure, a total of 600 cells (12 cell layers, each layer five cells, ten sections) were examined for cell size variation, and a total of 500 cells (ten cell layers, each layer five cells, ten sections) were recorded in the ventral counterpart of the gibbous structure. Cell size was calculated according to the scale bars to generate the real size. Scatterplot analysis of cell size versus cell layer was conducted by Minitab17 (Minitab, Inc.), Gaussian Fitting (Curve Fitting Tool) was stimulated to show the variation tendency of cell size along the dorso-ventral axis of the gibbous structures and the ventral counterparts, respectively. Genotyping and Association Analysis The differences between SsCYC and Sscyc in the coding sequence allowed genotype specific CAPS primers to be designed (supplementary table S4, Supplementary Material online). A 624-bp fragment of SsCYC coding sequence was amplified by PCR and the purified products were subjected to NdeI digestion. For Sscyc, the 624-bp fragment is digested into 422 and 202-bp, whereas the SsCYC fragment cannot be digested and remains 624-bp after digestion. The segregation ratio and statistical analysis of the F2 plants were performed using SPSS 14.0 software. For SNP-phenotype association analysis, we isolated the 1922-bp 5′ promoter sequence by TAIL-PCR and sequenced 3,111-bp gDNA from a panel of 75 gloxinia accessions (supplementary table S2, Supplementary Material online). The initial sequences were aligned using Clustal X software (Thompson et al. 1997). The matrix was adjusted manually by using BioEdit Software (Hall 1999). Then, the matrix was imported into DnaSP 5.10 software (Librado and Rozas 2009) to generate the haplotype matrix by considering Indels as informative sites. The floral characters of 75 gloxinia accessions were recorded as peloric (1) and zygomorphic (0). The association of SNPs/Indels with phenotype was conducted by Tassel 5.0 software (Bradbury et al. 2007) under General Linear Model (GLM). For annotation of the regulatory SNP in the promoters, we used online software TSSP (www.softberry.com) and PLACE (sogo.dna.affrc.go.jp) to predict the putative regulatory motifs in the 20-bp sequence including the significant associated SNPs. RNA Extraction and Expression Analysis Each floral organ was dissected and immediately frozen in liquid nitrogen. Total RNA was extracted using SV Total RNA Isolation System, and DNase I was added to digest the genomic DNA (Promega, USA) following the manufacturer’s instructions. Complimentary DNA (cDNA) was synthesized using a RevertAid H Minus First-Strand cDNA Synthesis Kit (Thermo, USA) according to the manufacturer’s instructions. For real-time qPCR and allele specific gene expression of SsCYCs/Sscyc, we designed primers that specifically anchored to the 10-bp deletion difference between SsCYC and Sscyc (supplementary table S4, Supplementary Material online). Before conducting expression analysis, the specificity of the primers was verified by PCR and sequencing. The efficiency of the primers (95–105%) was determined by creating standard curve. The SYBR Premix ExTaq (TaKaRa, China) was used to perform real-time qPCR with ROX as a reference dye on a StepOne Plus Real-Time PCR System (Life Technology, USA). The CT value of each gene was determined by normalizing the fluorescence threshold. The relative expression level of the target gene was determined using the ratio = 2−ΔCTmethod, and SsACT was used as an internal control (Pfaffl 2001). For RNA in situ hybridization, a 423-bp sequence targeted the 3′ coding sequence and 3′UTR of SsCYC was amplified using primers (supplementary table S4, Supplementary Material online) designed to enhance the specificity and avoid cross-hybridization with other TCP genes. Digoxygenin-labeled probes were generated using an in vitro transcription system (Roche, Switzerland). RNA in situ hybridization experiments were conducted as previously described with minor modifications (Bradley et al. 1993). Briefly, young inflorescence and flowers of WT-PF and MU-WB were fixed in FAA and embedded in paraffin (Sigma, USA). 10-μm sections were prepared using a rotary microtome. After removing the paraffin, samples were hybridized with the antisense/sense probe of SsCYC at 42 °C for 16 h. Stringent formamide washings for nonspecific probes were performed after hybridization. The AP-conjugated anti-DIG antibodies (Roche, Switzerland) were then mounted on the samples for 2 h. After removing the nonspecific antibody, the samples were then incubated with the NBT/NCIP solution (Roche, Switzerland) for staining at room temperature for 10 h. The slides were dried and mounted with CC/mount medium (Sigma, USA). Samples were analyzed using the Zeiss Axio Imager A microscopy (Carl Zeiss, Germany). Recombinant Protein Production and EMSA A DNA fragment of 665-bp from the start codon of SsCYC or Sscyc was amplified from the WT-PF and MU-WB gDNA, respectively. The PCR product was digested with BamH I and Hind III and inserted into the pET30α vector (Merck, Germany). Constructed plasmids were verified by sequencing and then introduced into BL21 Escherichia coli cells. The His-tagged recombinant proteins were purified from the soluble fraction of the cell lysate using Ni sepharose (GE Healthcare, USA). For EMSA, the 20 bp biotin-labeled probes were generated by Sangon Company (Sangon, Shanghai). EMSA was performed using nonradioactive NF-κB EMSA Kit (Thermo, USA) following the manufacturer’s instructions. After the reaction, electrophoresis was conducted on a 6.5% nondenaturing polyacrylamide gel at 175 V in 0.25× TBE (22.25 mM Tris–HCl, 22.25 mM boric acid, and 5 mM EDTA, pH8.0) buffer at 4 °C for 1 h. The reaction products were transferred to the binding membrane at 394 mA in 0.5× TBE) at room temperature for 40 min. The probes were detected according to the manufacturer’s instructions using the Imager Apparatus (Alpha, Canada). Two independent experiments were carried out to ensure that probe–protein interactions were specific. Transgenic Analysis For dominant repression of the SsCYC protein, the full length 1,035-bp SsCYC-CDS was fused in-frame with the EAR repression domain from the SUPERMAN gene and inserted downstream of the CaMV 35S promoter of the pCAMBIA 1301 vector to construct the SsCYC-SRDX vector. The vector was verified by sequencing and introduced into the Agrobacterium tumefaciens strain LBA4404 by electroporation. The transformation of Sinningia speciosa followed the methods described in Li et al. (2013) and Liu et al. (2014) with minor modifications (Li et al. 2013; Liu et al. 2014). Leaf discs from 8-weeks-old plantlets were precultured on MS medium containing 2 mg l−1 2,4-dichlorophenoxy acetic acid (2,4-D) for 2 days. Discs were then subjected to infiltration with Agrobacterium tumefaciens (O.D. 600, 0.3) at room temperature for 15 min, transferred to the coculture MS medium containing 100 μM Acetosyringone (AS), and kept in the dark for 3 days. Resultant explants were transferred to selection MS medium containing 2 mg l−1 6-Benzylaminopurine, 0.2 mg l−1 Naphthylacetic acid, 5 mg l−1 Hygromycin (Hyg) and 200 mg l−1 Cefotaxime (Cef). After six 2-week rounds of selection, regenerated Hyg-resistant adventitious shoots were obtained. The shoots were then transferred to MS medium for root induction. Discs of untransformed cultures were carried through the regeneration process as the wild type control. Transgenic and control plants were transplanted into the glasshouse as described above. For SsCYC-SRDX, a total of seven individual transgenic lines were obtained. The flower morphology of the transgenic plants was recorded with a Nikon D7100 camera. For overexpression of SsCYC/Sscyc in Arabidopsis, the 1,267-bp of SsCYC/Sscyc g-DNA sequence encompassing the entire ORF was isolated from WT-PF and MU-WB, and inserted downstream of the CaMV 35S promoter of pCAMBIA 1301. Vectors were verified by sequencing and then introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation. The resultant Agrobacterium was infiltrated into Arabidopsis using the floral dipping method (Clough and Bent 1998). Positive primary transformants were selected on 1/2 MS medium containing 40 mg/ml Hyg and 250 mg/ml Cef. Among the 45 SsCYC-OE T1 transformants, 39 showed a retarded plant development phenotype, with smaller rosette leaves and reproductive organs when compared with WT plants, whilst all 36 Sscyc-OE T1 transformants exhibited wild-type characteristics without any phenotypic changes. For each transgene, we randomly chose at least ten independent T1 transgenic plants, and selfed them to generated T2 plants. The phenotypic analysis was performed on the T2 population. To record leaf and petal parameters, the seventh leaf and sixth flower were collected for measurement by using a vernier. The SEM of leaf and petal epidermal cells was conducted as mentioned above. Phylogenetic Analysis We isolated the ∼1,250-bp SsCYC from 23 wild gloxinia collections with WT phenotypes, 40 peloric gloxinias and two members of Sinningia (S. tubiflora and S. rupicola) which were used as an outgroup (supplementary table S2, Supplementary Material online). The sequences were aligned using Clustal X software and adjusted manually with the software Geneious version 7.1.4 (Kearse et al. 2012). Parsimony analysis was implemented in PAUP*4.0B10 (Swofford 2003). Bayesian inference analyses were carried out in MrBayes version 3.2.2 (Ronquist and Huelsenbeck 2003). Mrmodeltest version 2.3 (Nylander 2004) was used to select an appropriate model of sequence evolution for each DNA data set in Bayesian inference analyses. Bootstrap values of parsimony analysis and posterior probabilities (PP) obtained from the analysis were used to test the credibility of various branches. For the neutral marker ncpGS, we isolated a ∼710-bp sequence from 20 wild gloxinia collections, 19 peloric gloxinias, and two members of Sinningia (S. tubiflora and S. rupicola) which were used as outgroups (supplementary table S2, Supplementary Material online). The Phylogenetic analysis were conducted as aforementioned methods. For the phylogenetic analysis of CYC-like genes from Gesneriaceae species, the full-length protein sequences were downloaded from GenBank database and aligned with Clustal X software. The protein matrix was determined autoomatically in RAxML and 1000 bootstrap replicates were conducted in ML analysis. The Maximum likelihood (ML) tree with bootstrap support value was generated based on Protein sequence matrix by RAxML on the CIPRES Science Gateway Portal (Miller et al. 2010). Population Genetic Analysis We isolated the 3,111-bp gDNA sequence of SsCYC from 40 peloric gloxinia accessions from diverse locations around the globe and 23 wild gloxinia collections from Brazil (supplementary table S2, Supplementary Material online). Sequences were aligned by Clustal X software to generate the matrix (Thompson et al. 1997). Then the matrix was adjusted manually using BioEdit software and inputted into DnaSP 5.10 software (Hall 1999; Librado and Rozas 2009). Values of genetic diversity per base pair (π) were estimated for the domesticated peloric gloxinia and wild gloxinia groups. Sliding window analysis of genetic diversity was calculated using 100-bp window with a 25-bp step with average pairwise difference per base pair between sequences. Acknowledgments We thank André Kuhn, Feng-Xian Guo, Heather Bland, James F. Smith, Lukasz Langowski, Lars Østergaard, Pauline Stephenson, Rebecca Mosher, and three anonymous reviewers for their constructive comments. This study is funded by the National Natural Science Foundation of China (Grants 31470333, 31530003 to Yin-Zheng Wang and Grands 31400205 to Yang Dong) and the Financial Grant from the China Postdoctoral Science Foundation (Grants 2014M550878 and Grants 2015T80151 to Yang Dong). Author’s Contributions Y.Z.W. initiated, conceived, designed, supervised the research and wrote the article. Y.D. conceived, designed and performed all the research, analyzed the data, and wrote the article. 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Molecular Biology and EvolutionOxford University Press

Published: Apr 27, 2018

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