LTR retrotransposons from the Citrus x clementina genome: characterization and application

LTR retrotransposons from the Citrus x clementina genome: characterization and application Long terminal repeat retrotransposons (LTR-RTs) are a large portion of most plant genomes, and can be used as a powerful molecular marker system. The first citrus reference genome (Citrus x clementina) has been publicly available since 2011; however, previous studies in citrus have not utilized the whole genome for LTR-RT marker development. In this study, 3959 full-length LTR-RTs were identified in the C. x clementina genome using structure-based (LTR_FINDER) and homology-based (RepeatMasker) methods. LTR-RTs were first classified by protein domain into Gypsy and Copia superfamilies, and then clustered into 1074 families based on LTR sequence similarity. Three hundred fifty Copia families were grouped into four lineages: Retrofit, Tork, Sire,and Oryco. One hundred seventy-eight Gypsy families were sorted into six lineages: Athila, Tat, Renia, CRM, Galadriel,and Del. Most LTR-RTs (3218 or 81.3%) were anchored to the nine Clementine mandarin linkage groups, accounting for 9.74% of chromosomes currently assembled. Accessions of 25 Rutaceae species were genotyped using 17 inter-retrotransposon amplified polymorphism (IRAP) markers developed from conserved LTR regions. Sequence-specific amplified polymorphism (SSAP) makers were used to distinguish ‘Valencia’ and ‘Pineapple’ sweet oranges (C. x sinensis), and 24 sweet orange clones. LTR-RT markers developed from the Clementine genome can be transferred within the Rutaceae family demonstrating that they are an excellent tool for citrus and Rutaceae genetic analysis. . . . . . Keywords Citrus LTR retrotransposons Gypsy Copia IRAP SSAP Introduction long terminal repeat retrotransposons (LTR-RTs) and non- LTR retrotransposons, based on their structure and transposi- Retrotransposons (RT) are a type of transposable element (TE) tion mechanism (Todorovska 2007). A typical LTR-RT con- that moves through the genome via an RNA intermediate in a tains two highly similar long terminal repeats (LTRs), a process that resembles Bcopy and paste^ (Wicker et al. 2007). primer-binding site (PBS), a polypurine tract (PPT), and two Retrotransposons can be separated into two major subclasses, genes necessary for their retrotransposition, gag and pol (Du et al. 2010). The majority of LTR-RTs can be divided further into Copia and Gypsy superfamilies according to the order of Dongliang Du and Xiaoyun Du contributed equally to this work. proteinase (PR), integrase (IN), reverse transcriptase (RT), and Communicated by W.-W. Guo RNase H (RH) domains in Pol (Domingues et al. 2012). The Electronic supplementary material The online version of this article domains of Gypsy elements are arranged as LTR-GAG-PR- (https://doi.org/10.1007/s11295-018-1257-x) contains supplementary RT-RH-IN-LTR, whereas the Copia elements are organized as material, which is available to authorized users. LTR-GAG-PR-IN-RT-RH-LTR (Wicker et al. 2007). Gypsy and Copia superfamilies can be further classified into lineages * Fred G. Gmitter, Jr and families with phylogenetic analysis of protein domain fgmitter@ufl.edu sequences that are usually supported by differences in struc- ture (such as size of LTRs and elements) (Wicker et al. 2007). Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, FL 33850, In land plants, four Copia (Retrofit, Tork, Sire,and Oryco)and USA six Gypsy (Athila, Tat, Renia, CRM, Galadriel,and Del)lin- Yantai Academy of Agricultural Science, Yantai 265500, Shandong, eages were reported (Llorens et al. 2011). China 43 Page 2 of 14 Tree Genetics & Genomes (2018) 14:43 LTR-RTs can be used as a molecular marker system be- this is the first detailed report on high-throughput fluorescent cause of their high copy number, widespread distribution, SSAP in Citrus. and high heterogeneity (Kumar and Hirochika 2001). Several types of LTR-RT molecular markers have been devel- oped, such as inter-retrotransposon amplified polymorphism Material and methods (IRAP), sequence-specific amplified polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism Genomic sequences and plant materials (REMAP), insertion site-based polymorphism (ISBP), and retrotransposon-based insertion polymorphism (RBIP) The genomic sequences of C. x clementina (v1.0) and C. (Flavell et al. 1998; Kalendar et al. 1999;Paux etal. 2010; xsinensis were obtained from Phytozome v10 (http:// Waugh et al. 1997). Most notably, IRAP markers amplify the phytozome.jgi.doe.gov)(Wu et al. 2014) and the orange intervening region between two retrotransposons to show genome annotation project (Xu et al. 2013), respectively. polymorphisms (Kalendar et al. 2011; Kalendar et al. 1999). Twenty-five accessions fromthe genus Citrus (Table 1)and IRAP is used frequently because of the easy development of related genera in Rutaceae family (Table 2)wereusedfor one outward-facing LTR-derived primer and generation of IRAP analysis. Young leaves were collected at the Florida marker bands without digestion and ligation. SSAP exploits Citrus Arboretum (Winter Haven, Florida) and stored at − LTR-RT polymorphisms by amplifying the region between a 80 °C until used. For SSAP analysis, 27 sweet orange ac- retrotransposon and adjacent restriction site in the genome cessions (C. x sinensis Osb.) were used, including creating additional polymorphisms that can be used to differ- ‘Valencia’, ‘Pineapple’, and one irradiated bud mutant of entiate closely related accessions (Syed et al. 2005). SSAP is ‘Valencia’ or ‘Pineapple’ (denoted as OR); four bud- especially useful for clone identification (Bretó et al. 2001; derived clones generated from irradiated OR (denoted Venturi et al. 2006;Zhao etal. 2010). LTR-RT markers have as‘B-’ followed by a number that indicates a different been used widely for pedigree analysis, population structure, bud mutant); and 20 tissue culture-derived somaclones of fingerprinting, linkage, and genetic mapping in several plant OR (denoted as ‘OLL-’ followed by a number that indi- species (Branco et al. 2007; Farouji et al. 2015; Huo et al. cates a unique somaclone). Each OLL somaclone was an 2009; Jia et al. 2009; Kalendar et al. 2011;Mandoulakani et independent regeneration event from an embryogenic cal- al. 2015; Queen et al. 2004;Smykal 2006; Sun et al. 2015). lus line of the OR sweet orange. The development of next-generation sequencing technologies has allowed for huge numbers of retrotransposon sequences to LTR-RT identification be generated, providing new opportunities for molecular marker development (Barghini et al. 2014;Cossu et al. Considering that the primary objective of this study was to 2012;Duetal. 2010;Xuand Du 2013; Zhang et al. 2012). isolate LTR-RTs for molecular marker development, full- Many types of molecular markers have been used to length LTR-RTs were sufficient for this study. Solo LTR-RTs characterize the phylogenetic relationships of Citrus acces- and LTR-RT remnants containing only one complete or sions and relatives. Previously used markers include random fragmented LTR were not included, because they were con- amplified polymorphic DNA (RAPD), amplified fragment sidered to be derived from the recombination of full-length length polymorphism (AFLP), simple sequence repeat LTR-RTs (Ma and Bennetzen 2004). Full-length LTR-RTs (SSR), and sequence-related amplified polymorphism were limited to those that contain a pair of relatively identical (Barkley et al. 2006; Federici et al. 1998;Uzunetal. LTRs at both ends and at least one typical LTR-RT feature 2009; Yamamoto et al. 1993). These marker types could (PPT, PBS, and target site duplications (TSD)). LTR-RTs con- not fully reveal the origin and taxonomy of citrus. There taining two LTRs and all three features were defined as intact are few studies that use LTR-RT based makers in citrus LTR-RTs (Cossu et al. 2012). (Asins et al. 1999; Bernet and Asins 2003;Biswas etal. LTR_FINDER and RepeatMasker v-4.0.3 were used to 2010a;Biswas et al. 2010b;De Felice etal. 2009;Rico- identify the full-length LTR-RTs from C. x clementina Cabanas and Martinez-Izquierdo 2007). However, small (Tempel 2012; Xu and Wang 2007). LTR sequence lengths numbers of LTR-RT based primers were developed in these from 100 to 5000 bp and a maximum distance between studies. In this study, we identified and characterized full- LTRs of 25,000 bp were parameters used for length LTR-RTs in the C. x clementina genome for use as LTR_FINDER.RepeatMaskerv-4.0.3 wasusedtofindlo- molecular markers. The LTR-RT markers were tested for ci with high similarity to TEs in the Repbase Viridiplantae transferability among and differentiation between Rutaceae repeat library (Jurka et al. 2005). Crossmatch was used as species. The fluorescence-labeled SSAP system used in this search engine with a Smith-Waterman cutoff of 225. A perl study yields a high-throughput and highly efficient mutant tool, Bone code to find them all^ was used to annotate identification system for Citrus. To our knowledge, RepeatMaster output (Bailly-Bechet et al. 2014). Tree Genetics & Genomes (2018) 14:43 Page 3 of 14 43 Table 1 Citrus species and accessions used for IRAP analysis Groups Latin name Common name Accession No a b Tanaka Swingle and Reece Pummelo C. maxima (Burm.) Merr. C. maxima (Brum.) Merr. Pummelo Ling Ping Yau pummelo 1 C. maxima (Burm.) Merr. C. maxima (Brum.) Merr. Pummelo Siamese sweet 2 pummelo Grapefruit C. paradisi Macf. C. paradisi Macf. Grapefruit Duncan grapefruit 3 C. paradisi Macf. C. paradisi Macf. Grapefruit Ruby red grapefruit 4 Mandarin C. clementina Hort.ex Tanaka C. reticulata Blanco Clementine Nules Clementine 5 C. nobilis Lour. C. reticulata Blanco King King mandarin 6 Citron and its relatives C. medica L. C. medica L. Citron Citron 7 C. medica var. sarcodactylis Swing. C. medica var. sarcodactylis Swing. Citron Buddha’shand Citron 8 C. volkameriana Tan. C. limon (L.) Burm.f. Volkamer lemon Volkamer lemon 9 C. limon (L.) Burm.f. C. limon (L.) Burm.f. Lemon Eureka lemon 10 C. aurantifolia (Cristm.) Swingle C. aurantifolia (Cristm.) Swing. Lime Mexican lime 11 C. latifolia Tanaka C. aurantifolia (Cristm.) Swing. Bearss lime Persian lime 12 Sour and sweet oranges C. aurantium L. C. aurantium L. Sour orange Willowleaf SO 13 C. aurantium L. C. aurantium L. Sour orange Sour orange 14 C. sinensis (L.) Osbeck C. sinensis (L.) Osbeck Sweet orange Pineapple 15 C. sinensis (L.) Osbeck C. sinensis (L.) Osbeck Sweet orange Valencia 16 C. myrtifolia Raf. C. aurantium L. Myrtle-leaf Chinotto 17 orange Papeda C. hystrix DC. C. hystrix DC. Mauritius Papeda Kaffir lime 18 C. ichangensis Swingle C. ichangensis Swingle Ichang papeda Ichang Papeda 19 Latin name using Tanaka’ssystem Latin name using Swingle’ssystem Accession numberz Sequences that aligned with the query that had greater than respective length, and more than 99% identity, were con- 80 bp, 80% of their respective length, and 80% identity sidered a match to identified LTR-RTs. were used for downstream analysis. Redundant sequences and those containing N symbols were discarded. The LTR-RT classification boundaries and structures of all LTR-RTs were manually confirmed. To check the accuracy of the predicted LTR-RT, The identified LTR-RTs were classified according to the 46,339 BAC end sequences were downloaded from NCBI methods previously described by Wicker et al. (Wicker et al. (Terol et al. 2008)and alignedtothe identified LTR-RTs 2007). All putative LTRs were clustered into families using with BLASTN using default parameters. Sequences that the USEARCH program with a similarity cutoff of 80% aligned with the query with more than 99% of their (Edgar 2010). The sequences between two putative LTRs Table 2 Rutaceae Species (excluding Citrus)usedin this study Subfamily Tribe Subtribe Group Latin name Accession No Aurantioideae Citreae Citrinae True citrus fruit trees Poncirus trifoliata (L.) Raf. Trifoliate 20 Fortunella crassifolia Swing. Meiwa kumquat 21 Eremocitrus glauca Swing. Australian desert lime 22 Microcitrus australasica Swing. Australian finger lime 23 Balsamocitrinae Bael fruit group Afraegle gabonensis (Swing.) Engl. Gabon powder flask 24 Rutoideae Zanthoxylum fagara (L.) Sarg. Lime prickly ash 25 Accession number 43 Page 4 of 14 Tree Genetics & Genomes (2018) 14:43 were analyzed with BLASTN and BLASTX against Repbase DNA extraction and IRAP analysis (Camacho et al. 2009). Coding domains were located using hmmsearch with sequence threshold (-E): 10 and domain Total genomic DNA was extracted from leaves using the threshold (-domE): 10 (Bateman et al. 2004). The following CTAB method (Doyle 1991). DNA concentration was mea- pfam profiles were used: PF03732 and PF00098 for GAG; sured with a Nanodrop spectrophotometer (Biotek PF07727, PF05380 and PF00078 for RT; PF03078 for enve- Instruments, Winooski, VT). IRAP analysis was done as de- lope protein (ENV); PF02022, PF00665 and PF00552 for IN; scribed by Kalendar et al. (1999). Amplification reactions PF00077 and PF00026 for PR; PF00075 for RH (Wang and were done in a 20 μl solutioncontaining0.25mM dNTPs, Liu 2008). 0.4 μM primer, 100 ng genomic DNA, 2.0 mM MgCl ,1× PCR buffer, and 1 U Taq DNA polymerase (Promega, USA). Phylogenetic analysis The amplifications were carried out in a Bio-Rad thermal cy- cler using the following amplification profile: 1 cycle at 95 °C, The RT domains of 49 reference elements representing 5min;30 cyclesat95°C, 1min;55°C1 min; ramp+ −1 known Viridiplantae LTR-RT lineages were downloaded 0.5 °C s to 72 °C; 72 °C, 2 min + 5 s per cycle; 1 cycle at from the Gypsy Database (GyDB) (Llorens et al. 2011). 72 °C, 8 min. PCR products were separated on a 2% agarose Representative RT protein sequences of each citrus LTR- gel at 8 V/cm using 0.5X Tris-borate-EDTA buffer, stained RT family (for 350 Copia and 178 Gypsy families) were with ethidium bromide, and photographed using SYNGENE randomly selected and aligned with the RT domains men- Automated Gel Documentation System (Cambridge, USA). tioned above using default parameters in Clustal Omega At least two PCR amplifications were conducted for each (Sievers et al. 2011). Two neighbor-joining phylogenetic sample and only reproducible bands between 200 and trees were constructed separately for the Gypsy and Copia 2000 bp were used for downstream analysis. superfamilies using MEGA 5.05 with a p distance model and 100 bootstrap iterations (Tamura et al. 2007). SSAP analysis Estimation of insertion time Three SSAP forward primers were designed as mentioned above. Restriction, ligation, and pre-amplification reactions The two LTRs of a LTR-RT are considered to be identical at were done as described by Waugh et al. (1997). Selective the time of insertion. Therefore, the insertion time of intact amplification was conducted with a retrotransposon primer LTR-RTs can be estimated from the sequence divergence of 5′ in combination with either Mse I+3 or EcoR I + 3 (Online and 3′ LTRs (Ma and Jackson 2006). Two LTRs were aligned Resource 1). A total of 48 primer combinations were used (3 first using Clustal Omega, and the Jukes-Cantor distance (k) SSAP primers * 2 enzymes * 8 selective bases). SSAP anal- was calculated using the PHYLIP program dnadist (Retief ysis was repeated two times for each primer pair. Following 2000). The insertion time (T) of an intact LTR-RT was calcu- selective amplification, 0.5 μl of PCR amplicons was added to lated using the formula: T = k/2r. A substitution rate of 2.4 × a mixture with 9.25 μl Hi-Di formamide and 0.2 μlGenScan −9 10 mutations per site per year, 2-fold higher than determined 500 LIZ molecular weight markers (Applied Biosystems, for the genes in poplar, was used in this study (Ma and Foster City, CA) for 3 min at 94 °C and immediately placed Jackson 2006; Tuskan et al. 2006). on ice for 6 min. Samples were fluorescently labeled with an ABI PRISM 3130 xl Genetic Analyzer (Applied Biosystems, IRAP primers design Foster City, CA) as follows: POP-7™ polymer at 63 °C, sam- ple injection voltage was 1.6 kV with 12 s injection time, and To validate in silico analysis and to test the transferability of 10 kV run voltage for 7200 s. Clementine LTR-RT based markers, 17 IRAP primers were designed following the protocols published by Schulman AH Data analysis (Kalendar et al. 1999; Kalendar and Schulman 2006). Twenty- five LTR-RT families including at least two members were Raw fluorescent SSAP data were analyzed and visualized randomly selected, and their homologs in sweet orange were using Genemarker v.4.0 software (SoftGenetics LLC ,State identified using BLASTN (sequence identify > 90% and College, PA). A minimum fluorescence threshold value of 250 length > 90% of query LTRs). The LTRs within one family, was chosen. Peaks between 60 and 500 bp were included in along with their homologs from sweet orange, were aligned the analysis. The bin table output of peak areas called was using Clustal Omega. At least one conserved region for 17 transferred to an MS Excel spreadsheet. The peak areas were LTR-RT families was identified and used for primer design converted to 0 and 1 scores indicating peak/marker absence with Primer Premier 5.0 (Singh et al. 1998). Primer sequences and presence. For IRAP analysis, binary matrices (presence/ are listed in Online Resource 1. absence) were prepared from electrophoretic patterns. The Tree Genetics & Genomes (2018) 14:43 Page 5 of 14 43 simple matching (SM) similarity coefficient was calculated Three hundred fifty Copia families were grouped into four with the SIMQUAL module. Dendrograms were built by clus- previously defined lineages: Retrofit, Tork, Sire,and Oryco. ter analysis using the unweighted pair-group method with ar- The Sire lineage and Oryco lineages were first clustered to- ithmetic averages (UPGMA) and the SAHN clustering pro- gether, then grouped with Tork and Retrofit lineages. One gram. The FIND module was used to identify all trees that hundred seventy-eight Gypsy families fell into six lineages: resulted from different choices of tied similarity or dissimilar- Athila, Tat, Renia, CRM, Galadriel,and Del. The four line- ity values. The clustering goodness-of-fit to the data matrix ages-Renia, CRM, Galadriel,and Del of Branch 1, also called was calculated by the programs COPH and MXCOMP. chromoviruses, were first clustered together, then grouped Figures were generated with the PROJECTIONS module. with Athila and Tat, two lineages of Branch 2. Family num- All analyses were performed with the software NTSYS-pc bers of Retrofit and Tork lineages were almost equally repre- 2.10e (Rohlf 1992). sented within the Copia superfamily (Table 3). The Athila was the largest lineage and contained 814 LTR-RTs and was the most highly represented in Gypsy superfamily (Table 3). Results The size distributions of LTR-RTs and LTR length varied among lineages (Table 3).The average LTR-RT length of Sire Identification of full-length LTR-RTs lineage within the Copia superfamily was greater than that of other lineages. The average LTR length in the Sire lineage was A total of 3959 full-length LTR-RTs were identified in the over three times larger than LTRs in the Oryco and Retrofit Clementine genome (Online Resources 2 and 3). lineages. The LTR-RT sizes of Athila, Del, and Tat in the LTR_FINDER and RepeatMasker identified 3791 and 1099 Gypsy superfamily were larger than the other three lineages. full-length LTR-RTs (931 in common), respectively. Intact Del lineage was found to have the largest LTR length LTR-RTs containing two LTRs and the three features (PPT, variation. PBS, and TSD) comprised 40.7% (1612) of total LTR-RTs. Most of LTR-RTs in this study (3836) had at least two of the Distribution of full-length LTR-RTs on citrus three features. Most full-length LTR-RTs (3593) were termi- chromosomes nated by the highly conserved TG-CA boxes at both 5′ and 3′ ends of LTRs. The mean length of full-length LTR-RTs was Of the 3959 full-length LTR-RTs identified in this study, 3218 8.08 kb, with a standard deviation of 4.50 kb. For LTRs, the (81.3%) were anchored to nine Clementine linkage groups mean length was 781.69 bp, and the standard deviation was (Fig. 3). These LTR-RTs occupied 28.1 Mb of genome se- 572.57 bp. The accuracy of identified LTR-RTs was con- quence, accounting for 9.74% of the nine currently assembled firmed with the analysis of 28.6 Mb BAC end sequences, chromosomes (288.6 Mb) (Online Resource 4). LTR-RTs which corresponds to 8% of the Clementine genome (Terol were distributed throughout the genome and there was little et al. 2008). About half of full-length LTR-RTs (1738) variation in full-length LTR-RT density between the nine matched at least one BAC end sequence. The total length of chromosomes. LTR-RTs, especially in the Gypsy superfamily, matched region was 3.5 Mb, corresponding to 11% of the were more abundant in putative centromeric regions. identified LTR-RTs. Putative insertion time of intact LTR-RTs Classification of full-length LTR-RTs The insertion time distribution of LTR-RTs fits an exponential The 3959 full-length LTR-RTs were first classified into two decay curve (r square = 0.96, p < 0.01) (Fig. 4a). This pattern superfamilies (Gypsy, Copia, or unknown) according to their was expected, because intact LTRs were rapidly changed to protein domain organization. As shown in Table 3, 1285 and solo LTRs, truncated LTRs, or completely eliminated from the 1727 LTR-RTs were included in Copia and Gypsy superfam- genome. The half-life of intact LTR-RTs in citrus was estimat- ilies, respectively. Then, the full-length LTR-RTs were clus- ed to be 3.47 Myr. Most citrus LTR-RTs (73.6%) were ampli- tered into 1074 families based on LTR sequence similarity, fied in the last 10 Myr, and 519 LTR-RTs were inserted within including 386 Copia and 214 Gypsy families. The mean num- last 2.5 Myr. Significant Bpeaks^ representing the insertion ber of full-length LTR-RTs per family was 3.69, and the larg- time of different LTR-RTs families showed that these families est family had 282 LTR-RTs. The average size of the Gypsy were active over a short period of time, especially for recently families (8.07) was approximately two times higher than the amplified families (Fig. 4b). Almost all LTR-RTs in family Copia families (3.33). 271 were inserted within the last 2.5 Myr. Four of the most To understand the evolutionary relationships among LTR- recently inserted 271 family members (12.5%) were found to RT families, two phylogenetic trees were constructed sepa- have two identical LTRs, which suggests there was little time rately for Copia and Gypsy superfamilies (Figs. 1 and 2). to accumulate evolutionary mutations (Online Resource 3). 43 Page 6 of 14 Tree Genetics & Genomes (2018) 14:43 Table 3 General features of C. x Element Family clementina LTR-RT lineages a a Superfamily Lineage Size (kb) LTR-len (bp) No. % No. % Copia 7.02 (5.12–8.44) 625 (294–783) 1285 100 386 100 Oryco 6.32 (4.61–5.24) 312 (269–365) 57 4.44 14 3.63 Retrofit 5.87 (4.90–5.34) 309 (225–321) 380 29.57 165 42.75 Sire 9.63 (8.53–10.2) 1041 (966–1292) 207 16.11 30 7.77 Tork 6.5 (5.3–6.64) 669 (472–667) 525 40.86 141 36.53 Unknown 8.82 (5.94–10.5) 870 (424–1298) 116 9.03 36 9.33 Gypsy 9.54 (5.6–12.4) 940 (472–1352) 1727 100 214 100 Athila 10.91 (8.42–12.5) 1175 (1076–1383) 814 47.13 64 29.91 CRM 7.18 (5.43–6.83) 509 (379–678) 91 5.27 15 7.01 Del 9.31 (7.93–10) 1145 (303–2047) 41 2.37 15 7.01 Galadriel 5.79 (3.13–6.18) 836 (363–1082) 220 12.74 16 7.48 Renia 5.62 (5.07–5.56) 420 (342–473) 107 6.2 40 18.69 Tat 9.97 (7.99–11.13) 716 (664–795) 302 17.49 28 13.08 Unknown 10.99 (5.81–13.74) 838 (436–1312) 152 8.8 36 16.82 Unknown Unknown 6.85 (2.74–9.02) 707 (212–938) 947 474 Total 8.08 (5.02–11.28) 782 (329–1240) 3959 1074 Average, with 25th and 75th percentiles in parenthesis Polymorphisms of 25 Rutaceae accessions Citrus genus than Cluster 2–2. Therefore, if 0.587 was used as a cut-off value for defining the clusters, Cluster 2–1and Cluster1 All IRAP primers achieved successful amplification across the would be classified in the same group, clearly separated from 25 Rutaceae accessions. In total, 209 reproducible and unam- Cluster 2–2. The correlation between the similarity coefficient biguous bands were produced, ranging from 250 to 2000 bp in matrix and the cophenetic matrix derived from the UPGMA tree size. Most of these bands (205, 98.09%) were polymorphic. was 0.94, corresponding to a good fit. Six to 18 fragments were amplified from a single primer (Table 4). Representative patterns of four LTR primers are Polymorphisms of 27 sweet orange accessions shown in Online Resource 5. The genetic similarity coeffi- cients between the 25 accessions were calculated. The average For SSAP analysis, 24 out of 48 primer combinations generated similarity coefficients for the 25 accessions ranged from 0.493 easily readable patterns that were selected for the downstream to 0.997 with a mean of 0.650. analyses (Table 4). A single primer combination produced 17 to Using 0.659 as a threshold, the phylogenetic tree could be 198 bands. A total of 2156 amplification products were gener- split into two major clusters (Fig. 5). Cluster 1 included all of ated, of which 1518 (70%) were polymorphic. The primer com- accessions in Citrus except C. hystrix, indicating a greater ge- binations exhibited different levels of polymorphism ranging netic distance from the other Citrus species analyzed. All the from 23.86 to 98%. Genetic similarity coefficients among 27 other genera were included in Cluster 2. The two clusters could accessions were calculated using the SSAP analysis data. The be further divided into four and two groups, respectively. Within distribution of genetic similarity coefficients between Cluster 1, accessions from the same species were grouped to- ‘Pineapple’, ‘Valencia’, and 24 clones (including four ‘B-’ ac- gether. C. ichangensis and C. maxima alone formed two single cessions, and 20 ‘OLL-’) were compared in Fig. 6. The 24 clusters: Cluster 1–1 and Cluster 1–2. C. reticulata, C. sinensis, clones had a closer relationship to BValencia^ than C. paradisi,and C. aurantium were grouped in Cluster 1–3. C. BPineapple^ (p < 0.01, Student’s t test). The average genetic medica, C. limon,and C. aurantifolia shared Cluster 1–4. In similarity coefficients among the 24 clones was 0.796, implying Cluster 2, four of the six Citrus-related genera examined profound levels of genetic differentiation within these clones. (Eremocitrus, Microcitrus, Afraegle,and Zanthoxylum)were clustered together and formed an independent group (Cluster 2–2), which is in accordance with their remarkable phenotypic Discussion differences from Citrus. The other two genera, Poncirus and Fortunella, were grouped into Cluster 2–1. Comparatively Different lineage classification systems were used for LTR- speaking, Cluster 2–1 displayed a much closer relationship with RTs in different studies. We chose to use the classification Tree Genetics & Genomes (2018) 14:43 Page 7 of 14 43 Fig. 1 Neighbor-joining phylogeny of Copia families based on reverse sequences from GyDB are denoted with a plus symbol and shown in transcriptase. One representative element that contains a complete RT red. Bootstrap values below 60% are not shown domain was chosen for each of the 350 Copia families. Reference system from GyDB, a research project focused on the phylo- distinct LTR-RT compositions. A pattern similar to citrus genetic classification of transposable elements (Llorens et al. was found in Arabidopsis, where 59.3% of Gypsy elements 2011). Four and six lineages were reported in Copia and belonged to the Athila lineage (Du et al. 2010; Marco and Gypsy superfamilies of land plants, respectively. All these Marin 2008). However, only 0.1% of the Gypsy elements lineages were found in the C. x clementina genome, but the identified in rice were classified into the Athila lineage. The full-length LTR-RT numbers within each lineage varied great- largest lineage in rice was Tat, accounting for 55.8% of its ly. The two smallest lineages, Del and Oryco,werefound in Gypsy elements (Du et al. 2010). The ratio of Gypsy to the C. x clementina genome, containing 41 and 57 full-length Copia elements in Clementine is 1.34:1, and is much lower LTR-RTs, respectively. However, the largest lineage, Athila, than rice (4.9:1) (Tian et al. 2009) and sorghum (3.7:1) included 814 full-length LTR-RTs and accounted for 47.13% (Paterson et al. 2009), but is similar to soybean (1.4:1) (Du of the Gypsy elements identified. Different species have et al. 2010) and maize (1.6:1) (Baucom et al. 2009). 43 Page 8 of 14 Tree Genetics & Genomes (2018) 14:43 Fig. 2 Neighbor-joining phylogeny of Gypsy families based on reverse sequences from GyDB are denoted with a plus symbol and shown in transcriptase. One representative element that contains a complete RT red. Bootstrap values below 60% are not shown domain was chosen for each of the 178 Gypsy families. Reference Previous studies show that LTR-RT based primers can were reported in the Poaceae and Rosaceae species be used for closely related genera (Kalendar et al. 2011; (Mamaghani et al. 2015; Sun et al. 2015). One reason Kalendar et al. 1999). In this study, all designed IRAP for the high transferability of IRAP markers in this study primers can be transferred across the 25 Rutaceae species. may be that the primers were designed in conserved re- The transferability of pear IRAP to other Rosaceae spe- gions within LTR-RT families, not only in conserved re- cies ranged from 87.5 to 100% (Sun et al. 2015). The gions of orthologous LTR-RTs. We selected LTR-RT fam- transferability of IRAP markers is usually higher than ilies of different sizes for primer design contrary to an other makers. For SSR markers, approximately half of IRAP study in pear, where the largest sized LTR-RT fam- the primers developed in sweet orange can be used in ilies were selected for primer design (Sun et al. 2015). pummelo or lemon (Biswas et al. 2014). Similar results The largest sized full-length LTR-RT families are not Tree Genetics & Genomes (2018) 14:43 Page 9 of 14 43 scaffold_1 scaffold_2 scaffold_3 Gypsy Copia Unknown 0 5 10 15 20 25 30 010 20 30 0 1020304050 Mbp scaffold_4 scaffold_5 scaffold_6 0 5 10 15 20 25 010 20 30 40 0 5 10 15 20 25 Mbp scaffold_7 scaffold_8 scaffold_9 0 5 10 15 20 0 5 10 15 20 25 0 5 10 15 20 25 30 Mbp Fig. 3 Distribution of full-length Gypsy, Copia, and unknown LTR-RTs in the 9 linkage groups of C. x clementina. The putative positions of centromeres are based on the results of Wu et al., and shown by black boxes representative because they do not include homologous or divided into two subgenera Citrus and Papeda,according clustered LTR-RTs in the genome. to the classification system of Swingle and Reece According to the classification of Swingle and Reece (Swingle and Webber 1943). In the UPGMA phylogenetic (Swingle and Webber 1943), the Btrue citrus fruit trees^ tree, C. hystrix formed one cluster separated from other group (Citrinae) is divided into six genera: Citrus, Citrus species that suggests some Papeda species may be Fortunella, Poncirus, Clymenia, Eremocitrus,and the most primitive Citrus (Nicolosi et al. 2000). C. Microcitrus. Citrus is the most economically important, ichangnesis was first clustered with pummelo and manda- and is sexually compatible with all other genera. The rin, and then clustered with citron supporting the hypoth- UPGMA cluster analysis showed that Fortunella and esis that C. ichangnesis could be an ancestor of mandarin, Poncirus were closer to Citrus than were Eremocitrus and is supported by the results of Xie et al. (2008)and and Microcitrus. This result was consistent with the find- Pang et al. (2007). According to our results, the citron is ings by Pang et al. based on AFLP markers (Pang et al. the most distantly related species among them, which is 2007), but differ from the results obtained by Garcia-Lor confirmed by the sequencing of their chloroplast genomes et al. (2013) and Wu et al. (2018). The Citrus genus was (Carbonell-Caballero et al. 2015). (a) (b) family 28 family 98 family 326 family 271 010 20 30 40 0 5 10 15 20 25 30 35 Insertion time (mya) Insertion time (mya) Fig. 4 Insertion time of intact LTR-RTs in C. x clementina. a Insertion time of intact LTR-RTs (mya, million years ago). b Insertion time of intact LTR- RTs within four selected families Num/Mbp Num/Mbp Num/Mbp 05 10 15 20 25 0 510 15 20 25 0 5 10 15 20 25 No. of elements 0 100 200 300 400 500 600 No. of elements 0 5 10 15 20 25 30 43 Page 10 of 14 Tree Genetics & Genomes (2018) 14:43 Table 4 Summary of PCR amplification corresponding to individual primer in IRAP and SSAP analysis Primer Total Polymorphic Polymorphic Primer Total Polymorphic Polymorphic bands bands bands (%) bands bands bands (%) LTR03 11 11 100 SSAP1/E-AGC 198 163 82 LTR05 6 5 83.33 SSAP1/M-CAG 33 22 66.67 LTR06 12 12 100 SSAP1/M-CTA 17 9 53 LTR07 10 9 90.00 SSAP1/M-CTG 26 16 61.54 LTR08 10 10 100 SSAP1/M-CTT 42 29 69 LTR09 8 7 87.50 SSAP2/E-AAC 144 92 63.89 LTR10 11 11 100 SSAP2/E-AAG 135 80 59 LTR11 14 14 100 SSAP2/E-ACA 135 121 90 LTR12 13 13 100 SSAP2/E-ACG 159 153 96 LTR13 13 13 100 SSAP2/E-ACT 159 123 77 LTR14 18 18 100 SSAP2/E-AGC 141 138 98 LTR17 12 12 100 SSAP2/E-AGG 176 147 84 LTR19 13 13 100 SSAP2/M-CAA 77 45 58 LTR20 17 17 100 SSAP2/M-CAC 95 53 56 LTR21 13 13 100 SSAP2/M-CAG 31 25 81 LTR25 12 12 100 SSAP2/M-CAT 182 75 41 LTR29 16 15 93.75 SSAP2/M-CTA 95 73 76.84 SSAP2/M-CTG 88 21 23.86 SSAP2/M-CTT 27 14 52 SSAP3/E-ACA 19 11 58 SSAP3/E-AGC 44 34 77 SSAP3/M-CAA 65 46 70.77 SSAP3/M-CTG 34 17 50.00 SSAP3/M-CTT 34 11 32 Total 209 205 98.09 Total 2156 1518 70 The SSAP markers showed that the 24 sweet orange ‘Pineapple’, and this was supported by phenotypic traits clones had patterns that were closer to ‘Valencia’ than (data not shown). The SSAP markers also showed that the Fig. 5 Dendrogram of 25 accessions by the UPGMA cluster analysis based on IRAP analysis Tree Genetics & Genomes (2018) 14:43 Page 11 of 14 43 Fig. 6 The distribution of genetic Valencia similarity coefficients between Pineapple ‘Valencia’ or ‘Pineapple’ and 24 clones based on SSAP analysis 24 clones had abundant genetic variations which would be also found to contain an independent insertion of a similar helpful for future variety registration and protection of retrotransposon that confers tissue-specific red coloration intellectual property. Many widely grown citrus cultivars, also in response to cold conditions (Butelli et al. 2012). such as sweet orange, grapefruit, lemon, and various clon- al selections of Satsuma and Clementine mandarins, orig- inated as either bud sport or apomictic seedling mutations Conclusion (Rao et al. 2009). The use of traditional markers to dis- tinguish such mutant clones is difficult because of lower Full-length LTR-RTs were mined from the Clementine genome genetic variability associated with these maker systems. and classified based on structural details. Randomly selected The LTR-RT based SSAP markers were especially useful IRAP and SSAP markers were tested and showed that they could for distinguishing accessions with similar genetic back- differentiate citrus accessions and mutant clones. Our findings grounds, like bud sport mutations in citrus (Venturi et al. indicate that LTR-RTs are an excellent molecular marker re- 2006;Zhaoetal. 2010). In our previous study, 18 SSR source because they are easy to develop, polymorphic, widely primer sets were used to distinguish these 27 accessions. distributed, and transferable within the Rutaceae family. However, all accessions showed similar PCR amplifica- tion patterns and were indistinguishable (data now Funding information This work was supported by a grant from the Citrus shown). In C. x clementina, SSAP was successfully used Research and Development Foundation (CRDF-724) and the China to distinguish 24 accessions generated from bud muta- Scholarship Council. tions; other markers (ISSR, RAPD, AFLP, and SSR) test- ed could not (Bretó et al. 2001). Compared to other rou- Compliance with ethical standards tinely used molecular markers such as SSR, LTR-RT based SSAP markers are better suited for genetic relation- Conflict of interest The authors declare that they have no conflict of interest. ship analysis and phylogenetic analysis (Biswas et al. 2011;Schulmanet al. 2012). An advantage of SSAP is Open Access This article is distributed under the terms of the Creative that polymorphisms at multiple loci are detected in a sin- Commons Attribution 4.0 International License (http:// gle assay, while SSR usually detects polymorphisms at creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give one locus (Powell et al. 1996). Retrotransposons are im- appropriate credit to the original author(s) and the source, provide a link portant sources of variation in Citrus, especially in the to the Creative Commons license, and indicate if changes were made. species mentioned above (Bretó et al. 2001;Wangetal. 2017). Several Sicilian blood oranges arose by insertion of a Copia-like retrotransposon (Tcs-1) adjacent to a MYB transcriptional activator (named Ruby) for anthocy- References anin production. 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LTR retrotransposons from the Citrus x clementina genome: characterization and application

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Life Sciences; Forestry; Plant Genetics and Genomics; Plant Breeding/Biotechnology; Tree Biology; Biotechnology
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10.1007/s11295-018-1257-x
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Abstract

Long terminal repeat retrotransposons (LTR-RTs) are a large portion of most plant genomes, and can be used as a powerful molecular marker system. The first citrus reference genome (Citrus x clementina) has been publicly available since 2011; however, previous studies in citrus have not utilized the whole genome for LTR-RT marker development. In this study, 3959 full-length LTR-RTs were identified in the C. x clementina genome using structure-based (LTR_FINDER) and homology-based (RepeatMasker) methods. LTR-RTs were first classified by protein domain into Gypsy and Copia superfamilies, and then clustered into 1074 families based on LTR sequence similarity. Three hundred fifty Copia families were grouped into four lineages: Retrofit, Tork, Sire,and Oryco. One hundred seventy-eight Gypsy families were sorted into six lineages: Athila, Tat, Renia, CRM, Galadriel,and Del. Most LTR-RTs (3218 or 81.3%) were anchored to the nine Clementine mandarin linkage groups, accounting for 9.74% of chromosomes currently assembled. Accessions of 25 Rutaceae species were genotyped using 17 inter-retrotransposon amplified polymorphism (IRAP) markers developed from conserved LTR regions. Sequence-specific amplified polymorphism (SSAP) makers were used to distinguish ‘Valencia’ and ‘Pineapple’ sweet oranges (C. x sinensis), and 24 sweet orange clones. LTR-RT markers developed from the Clementine genome can be transferred within the Rutaceae family demonstrating that they are an excellent tool for citrus and Rutaceae genetic analysis. . . . . . Keywords Citrus LTR retrotransposons Gypsy Copia IRAP SSAP Introduction long terminal repeat retrotransposons (LTR-RTs) and non- LTR retrotransposons, based on their structure and transposi- Retrotransposons (RT) are a type of transposable element (TE) tion mechanism (Todorovska 2007). A typical LTR-RT con- that moves through the genome via an RNA intermediate in a tains two highly similar long terminal repeats (LTRs), a process that resembles Bcopy and paste^ (Wicker et al. 2007). primer-binding site (PBS), a polypurine tract (PPT), and two Retrotransposons can be separated into two major subclasses, genes necessary for their retrotransposition, gag and pol (Du et al. 2010). The majority of LTR-RTs can be divided further into Copia and Gypsy superfamilies according to the order of Dongliang Du and Xiaoyun Du contributed equally to this work. proteinase (PR), integrase (IN), reverse transcriptase (RT), and Communicated by W.-W. Guo RNase H (RH) domains in Pol (Domingues et al. 2012). The Electronic supplementary material The online version of this article domains of Gypsy elements are arranged as LTR-GAG-PR- (https://doi.org/10.1007/s11295-018-1257-x) contains supplementary RT-RH-IN-LTR, whereas the Copia elements are organized as material, which is available to authorized users. LTR-GAG-PR-IN-RT-RH-LTR (Wicker et al. 2007). Gypsy and Copia superfamilies can be further classified into lineages * Fred G. Gmitter, Jr and families with phylogenetic analysis of protein domain fgmitter@ufl.edu sequences that are usually supported by differences in struc- ture (such as size of LTRs and elements) (Wicker et al. 2007). Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Lake Alfred, FL 33850, In land plants, four Copia (Retrofit, Tork, Sire,and Oryco)and USA six Gypsy (Athila, Tat, Renia, CRM, Galadriel,and Del)lin- Yantai Academy of Agricultural Science, Yantai 265500, Shandong, eages were reported (Llorens et al. 2011). China 43 Page 2 of 14 Tree Genetics & Genomes (2018) 14:43 LTR-RTs can be used as a molecular marker system be- this is the first detailed report on high-throughput fluorescent cause of their high copy number, widespread distribution, SSAP in Citrus. and high heterogeneity (Kumar and Hirochika 2001). Several types of LTR-RT molecular markers have been devel- oped, such as inter-retrotransposon amplified polymorphism Material and methods (IRAP), sequence-specific amplified polymorphism (SSAP), retrotransposon-microsatellite amplified polymorphism Genomic sequences and plant materials (REMAP), insertion site-based polymorphism (ISBP), and retrotransposon-based insertion polymorphism (RBIP) The genomic sequences of C. x clementina (v1.0) and C. (Flavell et al. 1998; Kalendar et al. 1999;Paux etal. 2010; xsinensis were obtained from Phytozome v10 (http:// Waugh et al. 1997). Most notably, IRAP markers amplify the phytozome.jgi.doe.gov)(Wu et al. 2014) and the orange intervening region between two retrotransposons to show genome annotation project (Xu et al. 2013), respectively. polymorphisms (Kalendar et al. 2011; Kalendar et al. 1999). Twenty-five accessions fromthe genus Citrus (Table 1)and IRAP is used frequently because of the easy development of related genera in Rutaceae family (Table 2)wereusedfor one outward-facing LTR-derived primer and generation of IRAP analysis. Young leaves were collected at the Florida marker bands without digestion and ligation. SSAP exploits Citrus Arboretum (Winter Haven, Florida) and stored at − LTR-RT polymorphisms by amplifying the region between a 80 °C until used. For SSAP analysis, 27 sweet orange ac- retrotransposon and adjacent restriction site in the genome cessions (C. x sinensis Osb.) were used, including creating additional polymorphisms that can be used to differ- ‘Valencia’, ‘Pineapple’, and one irradiated bud mutant of entiate closely related accessions (Syed et al. 2005). SSAP is ‘Valencia’ or ‘Pineapple’ (denoted as OR); four bud- especially useful for clone identification (Bretó et al. 2001; derived clones generated from irradiated OR (denoted Venturi et al. 2006;Zhao etal. 2010). LTR-RT markers have as‘B-’ followed by a number that indicates a different been used widely for pedigree analysis, population structure, bud mutant); and 20 tissue culture-derived somaclones of fingerprinting, linkage, and genetic mapping in several plant OR (denoted as ‘OLL-’ followed by a number that indi- species (Branco et al. 2007; Farouji et al. 2015; Huo et al. cates a unique somaclone). Each OLL somaclone was an 2009; Jia et al. 2009; Kalendar et al. 2011;Mandoulakani et independent regeneration event from an embryogenic cal- al. 2015; Queen et al. 2004;Smykal 2006; Sun et al. 2015). lus line of the OR sweet orange. The development of next-generation sequencing technologies has allowed for huge numbers of retrotransposon sequences to LTR-RT identification be generated, providing new opportunities for molecular marker development (Barghini et al. 2014;Cossu et al. Considering that the primary objective of this study was to 2012;Duetal. 2010;Xuand Du 2013; Zhang et al. 2012). isolate LTR-RTs for molecular marker development, full- Many types of molecular markers have been used to length LTR-RTs were sufficient for this study. Solo LTR-RTs characterize the phylogenetic relationships of Citrus acces- and LTR-RT remnants containing only one complete or sions and relatives. Previously used markers include random fragmented LTR were not included, because they were con- amplified polymorphic DNA (RAPD), amplified fragment sidered to be derived from the recombination of full-length length polymorphism (AFLP), simple sequence repeat LTR-RTs (Ma and Bennetzen 2004). Full-length LTR-RTs (SSR), and sequence-related amplified polymorphism were limited to those that contain a pair of relatively identical (Barkley et al. 2006; Federici et al. 1998;Uzunetal. LTRs at both ends and at least one typical LTR-RT feature 2009; Yamamoto et al. 1993). These marker types could (PPT, PBS, and target site duplications (TSD)). LTR-RTs con- not fully reveal the origin and taxonomy of citrus. There taining two LTRs and all three features were defined as intact are few studies that use LTR-RT based makers in citrus LTR-RTs (Cossu et al. 2012). (Asins et al. 1999; Bernet and Asins 2003;Biswas etal. LTR_FINDER and RepeatMasker v-4.0.3 were used to 2010a;Biswas et al. 2010b;De Felice etal. 2009;Rico- identify the full-length LTR-RTs from C. x clementina Cabanas and Martinez-Izquierdo 2007). However, small (Tempel 2012; Xu and Wang 2007). LTR sequence lengths numbers of LTR-RT based primers were developed in these from 100 to 5000 bp and a maximum distance between studies. In this study, we identified and characterized full- LTRs of 25,000 bp were parameters used for length LTR-RTs in the C. x clementina genome for use as LTR_FINDER.RepeatMaskerv-4.0.3 wasusedtofindlo- molecular markers. The LTR-RT markers were tested for ci with high similarity to TEs in the Repbase Viridiplantae transferability among and differentiation between Rutaceae repeat library (Jurka et al. 2005). Crossmatch was used as species. The fluorescence-labeled SSAP system used in this search engine with a Smith-Waterman cutoff of 225. A perl study yields a high-throughput and highly efficient mutant tool, Bone code to find them all^ was used to annotate identification system for Citrus. To our knowledge, RepeatMaster output (Bailly-Bechet et al. 2014). Tree Genetics & Genomes (2018) 14:43 Page 3 of 14 43 Table 1 Citrus species and accessions used for IRAP analysis Groups Latin name Common name Accession No a b Tanaka Swingle and Reece Pummelo C. maxima (Burm.) Merr. C. maxima (Brum.) Merr. Pummelo Ling Ping Yau pummelo 1 C. maxima (Burm.) Merr. C. maxima (Brum.) Merr. Pummelo Siamese sweet 2 pummelo Grapefruit C. paradisi Macf. C. paradisi Macf. Grapefruit Duncan grapefruit 3 C. paradisi Macf. C. paradisi Macf. Grapefruit Ruby red grapefruit 4 Mandarin C. clementina Hort.ex Tanaka C. reticulata Blanco Clementine Nules Clementine 5 C. nobilis Lour. C. reticulata Blanco King King mandarin 6 Citron and its relatives C. medica L. C. medica L. Citron Citron 7 C. medica var. sarcodactylis Swing. C. medica var. sarcodactylis Swing. Citron Buddha’shand Citron 8 C. volkameriana Tan. C. limon (L.) Burm.f. Volkamer lemon Volkamer lemon 9 C. limon (L.) Burm.f. C. limon (L.) Burm.f. Lemon Eureka lemon 10 C. aurantifolia (Cristm.) Swingle C. aurantifolia (Cristm.) Swing. Lime Mexican lime 11 C. latifolia Tanaka C. aurantifolia (Cristm.) Swing. Bearss lime Persian lime 12 Sour and sweet oranges C. aurantium L. C. aurantium L. Sour orange Willowleaf SO 13 C. aurantium L. C. aurantium L. Sour orange Sour orange 14 C. sinensis (L.) Osbeck C. sinensis (L.) Osbeck Sweet orange Pineapple 15 C. sinensis (L.) Osbeck C. sinensis (L.) Osbeck Sweet orange Valencia 16 C. myrtifolia Raf. C. aurantium L. Myrtle-leaf Chinotto 17 orange Papeda C. hystrix DC. C. hystrix DC. Mauritius Papeda Kaffir lime 18 C. ichangensis Swingle C. ichangensis Swingle Ichang papeda Ichang Papeda 19 Latin name using Tanaka’ssystem Latin name using Swingle’ssystem Accession numberz Sequences that aligned with the query that had greater than respective length, and more than 99% identity, were con- 80 bp, 80% of their respective length, and 80% identity sidered a match to identified LTR-RTs. were used for downstream analysis. Redundant sequences and those containing N symbols were discarded. The LTR-RT classification boundaries and structures of all LTR-RTs were manually confirmed. To check the accuracy of the predicted LTR-RT, The identified LTR-RTs were classified according to the 46,339 BAC end sequences were downloaded from NCBI methods previously described by Wicker et al. (Wicker et al. (Terol et al. 2008)and alignedtothe identified LTR-RTs 2007). All putative LTRs were clustered into families using with BLASTN using default parameters. Sequences that the USEARCH program with a similarity cutoff of 80% aligned with the query with more than 99% of their (Edgar 2010). The sequences between two putative LTRs Table 2 Rutaceae Species (excluding Citrus)usedin this study Subfamily Tribe Subtribe Group Latin name Accession No Aurantioideae Citreae Citrinae True citrus fruit trees Poncirus trifoliata (L.) Raf. Trifoliate 20 Fortunella crassifolia Swing. Meiwa kumquat 21 Eremocitrus glauca Swing. Australian desert lime 22 Microcitrus australasica Swing. Australian finger lime 23 Balsamocitrinae Bael fruit group Afraegle gabonensis (Swing.) Engl. Gabon powder flask 24 Rutoideae Zanthoxylum fagara (L.) Sarg. Lime prickly ash 25 Accession number 43 Page 4 of 14 Tree Genetics & Genomes (2018) 14:43 were analyzed with BLASTN and BLASTX against Repbase DNA extraction and IRAP analysis (Camacho et al. 2009). Coding domains were located using hmmsearch with sequence threshold (-E): 10 and domain Total genomic DNA was extracted from leaves using the threshold (-domE): 10 (Bateman et al. 2004). The following CTAB method (Doyle 1991). DNA concentration was mea- pfam profiles were used: PF03732 and PF00098 for GAG; sured with a Nanodrop spectrophotometer (Biotek PF07727, PF05380 and PF00078 for RT; PF03078 for enve- Instruments, Winooski, VT). IRAP analysis was done as de- lope protein (ENV); PF02022, PF00665 and PF00552 for IN; scribed by Kalendar et al. (1999). Amplification reactions PF00077 and PF00026 for PR; PF00075 for RH (Wang and were done in a 20 μl solutioncontaining0.25mM dNTPs, Liu 2008). 0.4 μM primer, 100 ng genomic DNA, 2.0 mM MgCl ,1× PCR buffer, and 1 U Taq DNA polymerase (Promega, USA). Phylogenetic analysis The amplifications were carried out in a Bio-Rad thermal cy- cler using the following amplification profile: 1 cycle at 95 °C, The RT domains of 49 reference elements representing 5min;30 cyclesat95°C, 1min;55°C1 min; ramp+ −1 known Viridiplantae LTR-RT lineages were downloaded 0.5 °C s to 72 °C; 72 °C, 2 min + 5 s per cycle; 1 cycle at from the Gypsy Database (GyDB) (Llorens et al. 2011). 72 °C, 8 min. PCR products were separated on a 2% agarose Representative RT protein sequences of each citrus LTR- gel at 8 V/cm using 0.5X Tris-borate-EDTA buffer, stained RT family (for 350 Copia and 178 Gypsy families) were with ethidium bromide, and photographed using SYNGENE randomly selected and aligned with the RT domains men- Automated Gel Documentation System (Cambridge, USA). tioned above using default parameters in Clustal Omega At least two PCR amplifications were conducted for each (Sievers et al. 2011). Two neighbor-joining phylogenetic sample and only reproducible bands between 200 and trees were constructed separately for the Gypsy and Copia 2000 bp were used for downstream analysis. superfamilies using MEGA 5.05 with a p distance model and 100 bootstrap iterations (Tamura et al. 2007). SSAP analysis Estimation of insertion time Three SSAP forward primers were designed as mentioned above. Restriction, ligation, and pre-amplification reactions The two LTRs of a LTR-RT are considered to be identical at were done as described by Waugh et al. (1997). Selective the time of insertion. Therefore, the insertion time of intact amplification was conducted with a retrotransposon primer LTR-RTs can be estimated from the sequence divergence of 5′ in combination with either Mse I+3 or EcoR I + 3 (Online and 3′ LTRs (Ma and Jackson 2006). Two LTRs were aligned Resource 1). A total of 48 primer combinations were used (3 first using Clustal Omega, and the Jukes-Cantor distance (k) SSAP primers * 2 enzymes * 8 selective bases). SSAP anal- was calculated using the PHYLIP program dnadist (Retief ysis was repeated two times for each primer pair. Following 2000). The insertion time (T) of an intact LTR-RT was calcu- selective amplification, 0.5 μl of PCR amplicons was added to lated using the formula: T = k/2r. A substitution rate of 2.4 × a mixture with 9.25 μl Hi-Di formamide and 0.2 μlGenScan −9 10 mutations per site per year, 2-fold higher than determined 500 LIZ molecular weight markers (Applied Biosystems, for the genes in poplar, was used in this study (Ma and Foster City, CA) for 3 min at 94 °C and immediately placed Jackson 2006; Tuskan et al. 2006). on ice for 6 min. Samples were fluorescently labeled with an ABI PRISM 3130 xl Genetic Analyzer (Applied Biosystems, IRAP primers design Foster City, CA) as follows: POP-7™ polymer at 63 °C, sam- ple injection voltage was 1.6 kV with 12 s injection time, and To validate in silico analysis and to test the transferability of 10 kV run voltage for 7200 s. Clementine LTR-RT based markers, 17 IRAP primers were designed following the protocols published by Schulman AH Data analysis (Kalendar et al. 1999; Kalendar and Schulman 2006). Twenty- five LTR-RT families including at least two members were Raw fluorescent SSAP data were analyzed and visualized randomly selected, and their homologs in sweet orange were using Genemarker v.4.0 software (SoftGenetics LLC ,State identified using BLASTN (sequence identify > 90% and College, PA). A minimum fluorescence threshold value of 250 length > 90% of query LTRs). The LTRs within one family, was chosen. Peaks between 60 and 500 bp were included in along with their homologs from sweet orange, were aligned the analysis. The bin table output of peak areas called was using Clustal Omega. At least one conserved region for 17 transferred to an MS Excel spreadsheet. The peak areas were LTR-RT families was identified and used for primer design converted to 0 and 1 scores indicating peak/marker absence with Primer Premier 5.0 (Singh et al. 1998). Primer sequences and presence. For IRAP analysis, binary matrices (presence/ are listed in Online Resource 1. absence) were prepared from electrophoretic patterns. The Tree Genetics & Genomes (2018) 14:43 Page 5 of 14 43 simple matching (SM) similarity coefficient was calculated Three hundred fifty Copia families were grouped into four with the SIMQUAL module. Dendrograms were built by clus- previously defined lineages: Retrofit, Tork, Sire,and Oryco. ter analysis using the unweighted pair-group method with ar- The Sire lineage and Oryco lineages were first clustered to- ithmetic averages (UPGMA) and the SAHN clustering pro- gether, then grouped with Tork and Retrofit lineages. One gram. The FIND module was used to identify all trees that hundred seventy-eight Gypsy families fell into six lineages: resulted from different choices of tied similarity or dissimilar- Athila, Tat, Renia, CRM, Galadriel,and Del. The four line- ity values. The clustering goodness-of-fit to the data matrix ages-Renia, CRM, Galadriel,and Del of Branch 1, also called was calculated by the programs COPH and MXCOMP. chromoviruses, were first clustered together, then grouped Figures were generated with the PROJECTIONS module. with Athila and Tat, two lineages of Branch 2. Family num- All analyses were performed with the software NTSYS-pc bers of Retrofit and Tork lineages were almost equally repre- 2.10e (Rohlf 1992). sented within the Copia superfamily (Table 3). The Athila was the largest lineage and contained 814 LTR-RTs and was the most highly represented in Gypsy superfamily (Table 3). Results The size distributions of LTR-RTs and LTR length varied among lineages (Table 3).The average LTR-RT length of Sire Identification of full-length LTR-RTs lineage within the Copia superfamily was greater than that of other lineages. The average LTR length in the Sire lineage was A total of 3959 full-length LTR-RTs were identified in the over three times larger than LTRs in the Oryco and Retrofit Clementine genome (Online Resources 2 and 3). lineages. The LTR-RT sizes of Athila, Del, and Tat in the LTR_FINDER and RepeatMasker identified 3791 and 1099 Gypsy superfamily were larger than the other three lineages. full-length LTR-RTs (931 in common), respectively. Intact Del lineage was found to have the largest LTR length LTR-RTs containing two LTRs and the three features (PPT, variation. PBS, and TSD) comprised 40.7% (1612) of total LTR-RTs. Most of LTR-RTs in this study (3836) had at least two of the Distribution of full-length LTR-RTs on citrus three features. Most full-length LTR-RTs (3593) were termi- chromosomes nated by the highly conserved TG-CA boxes at both 5′ and 3′ ends of LTRs. The mean length of full-length LTR-RTs was Of the 3959 full-length LTR-RTs identified in this study, 3218 8.08 kb, with a standard deviation of 4.50 kb. For LTRs, the (81.3%) were anchored to nine Clementine linkage groups mean length was 781.69 bp, and the standard deviation was (Fig. 3). These LTR-RTs occupied 28.1 Mb of genome se- 572.57 bp. The accuracy of identified LTR-RTs was con- quence, accounting for 9.74% of the nine currently assembled firmed with the analysis of 28.6 Mb BAC end sequences, chromosomes (288.6 Mb) (Online Resource 4). LTR-RTs which corresponds to 8% of the Clementine genome (Terol were distributed throughout the genome and there was little et al. 2008). About half of full-length LTR-RTs (1738) variation in full-length LTR-RT density between the nine matched at least one BAC end sequence. The total length of chromosomes. LTR-RTs, especially in the Gypsy superfamily, matched region was 3.5 Mb, corresponding to 11% of the were more abundant in putative centromeric regions. identified LTR-RTs. Putative insertion time of intact LTR-RTs Classification of full-length LTR-RTs The insertion time distribution of LTR-RTs fits an exponential The 3959 full-length LTR-RTs were first classified into two decay curve (r square = 0.96, p < 0.01) (Fig. 4a). This pattern superfamilies (Gypsy, Copia, or unknown) according to their was expected, because intact LTRs were rapidly changed to protein domain organization. As shown in Table 3, 1285 and solo LTRs, truncated LTRs, or completely eliminated from the 1727 LTR-RTs were included in Copia and Gypsy superfam- genome. The half-life of intact LTR-RTs in citrus was estimat- ilies, respectively. Then, the full-length LTR-RTs were clus- ed to be 3.47 Myr. Most citrus LTR-RTs (73.6%) were ampli- tered into 1074 families based on LTR sequence similarity, fied in the last 10 Myr, and 519 LTR-RTs were inserted within including 386 Copia and 214 Gypsy families. The mean num- last 2.5 Myr. Significant Bpeaks^ representing the insertion ber of full-length LTR-RTs per family was 3.69, and the larg- time of different LTR-RTs families showed that these families est family had 282 LTR-RTs. The average size of the Gypsy were active over a short period of time, especially for recently families (8.07) was approximately two times higher than the amplified families (Fig. 4b). Almost all LTR-RTs in family Copia families (3.33). 271 were inserted within the last 2.5 Myr. Four of the most To understand the evolutionary relationships among LTR- recently inserted 271 family members (12.5%) were found to RT families, two phylogenetic trees were constructed sepa- have two identical LTRs, which suggests there was little time rately for Copia and Gypsy superfamilies (Figs. 1 and 2). to accumulate evolutionary mutations (Online Resource 3). 43 Page 6 of 14 Tree Genetics & Genomes (2018) 14:43 Table 3 General features of C. x Element Family clementina LTR-RT lineages a a Superfamily Lineage Size (kb) LTR-len (bp) No. % No. % Copia 7.02 (5.12–8.44) 625 (294–783) 1285 100 386 100 Oryco 6.32 (4.61–5.24) 312 (269–365) 57 4.44 14 3.63 Retrofit 5.87 (4.90–5.34) 309 (225–321) 380 29.57 165 42.75 Sire 9.63 (8.53–10.2) 1041 (966–1292) 207 16.11 30 7.77 Tork 6.5 (5.3–6.64) 669 (472–667) 525 40.86 141 36.53 Unknown 8.82 (5.94–10.5) 870 (424–1298) 116 9.03 36 9.33 Gypsy 9.54 (5.6–12.4) 940 (472–1352) 1727 100 214 100 Athila 10.91 (8.42–12.5) 1175 (1076–1383) 814 47.13 64 29.91 CRM 7.18 (5.43–6.83) 509 (379–678) 91 5.27 15 7.01 Del 9.31 (7.93–10) 1145 (303–2047) 41 2.37 15 7.01 Galadriel 5.79 (3.13–6.18) 836 (363–1082) 220 12.74 16 7.48 Renia 5.62 (5.07–5.56) 420 (342–473) 107 6.2 40 18.69 Tat 9.97 (7.99–11.13) 716 (664–795) 302 17.49 28 13.08 Unknown 10.99 (5.81–13.74) 838 (436–1312) 152 8.8 36 16.82 Unknown Unknown 6.85 (2.74–9.02) 707 (212–938) 947 474 Total 8.08 (5.02–11.28) 782 (329–1240) 3959 1074 Average, with 25th and 75th percentiles in parenthesis Polymorphisms of 25 Rutaceae accessions Citrus genus than Cluster 2–2. Therefore, if 0.587 was used as a cut-off value for defining the clusters, Cluster 2–1and Cluster1 All IRAP primers achieved successful amplification across the would be classified in the same group, clearly separated from 25 Rutaceae accessions. In total, 209 reproducible and unam- Cluster 2–2. The correlation between the similarity coefficient biguous bands were produced, ranging from 250 to 2000 bp in matrix and the cophenetic matrix derived from the UPGMA tree size. Most of these bands (205, 98.09%) were polymorphic. was 0.94, corresponding to a good fit. Six to 18 fragments were amplified from a single primer (Table 4). Representative patterns of four LTR primers are Polymorphisms of 27 sweet orange accessions shown in Online Resource 5. The genetic similarity coeffi- cients between the 25 accessions were calculated. The average For SSAP analysis, 24 out of 48 primer combinations generated similarity coefficients for the 25 accessions ranged from 0.493 easily readable patterns that were selected for the downstream to 0.997 with a mean of 0.650. analyses (Table 4). A single primer combination produced 17 to Using 0.659 as a threshold, the phylogenetic tree could be 198 bands. A total of 2156 amplification products were gener- split into two major clusters (Fig. 5). Cluster 1 included all of ated, of which 1518 (70%) were polymorphic. The primer com- accessions in Citrus except C. hystrix, indicating a greater ge- binations exhibited different levels of polymorphism ranging netic distance from the other Citrus species analyzed. All the from 23.86 to 98%. Genetic similarity coefficients among 27 other genera were included in Cluster 2. The two clusters could accessions were calculated using the SSAP analysis data. The be further divided into four and two groups, respectively. Within distribution of genetic similarity coefficients between Cluster 1, accessions from the same species were grouped to- ‘Pineapple’, ‘Valencia’, and 24 clones (including four ‘B-’ ac- gether. C. ichangensis and C. maxima alone formed two single cessions, and 20 ‘OLL-’) were compared in Fig. 6. The 24 clusters: Cluster 1–1 and Cluster 1–2. C. reticulata, C. sinensis, clones had a closer relationship to BValencia^ than C. paradisi,and C. aurantium were grouped in Cluster 1–3. C. BPineapple^ (p < 0.01, Student’s t test). The average genetic medica, C. limon,and C. aurantifolia shared Cluster 1–4. In similarity coefficients among the 24 clones was 0.796, implying Cluster 2, four of the six Citrus-related genera examined profound levels of genetic differentiation within these clones. (Eremocitrus, Microcitrus, Afraegle,and Zanthoxylum)were clustered together and formed an independent group (Cluster 2–2), which is in accordance with their remarkable phenotypic Discussion differences from Citrus. The other two genera, Poncirus and Fortunella, were grouped into Cluster 2–1. Comparatively Different lineage classification systems were used for LTR- speaking, Cluster 2–1 displayed a much closer relationship with RTs in different studies. We chose to use the classification Tree Genetics & Genomes (2018) 14:43 Page 7 of 14 43 Fig. 1 Neighbor-joining phylogeny of Copia families based on reverse sequences from GyDB are denoted with a plus symbol and shown in transcriptase. One representative element that contains a complete RT red. Bootstrap values below 60% are not shown domain was chosen for each of the 350 Copia families. Reference system from GyDB, a research project focused on the phylo- distinct LTR-RT compositions. A pattern similar to citrus genetic classification of transposable elements (Llorens et al. was found in Arabidopsis, where 59.3% of Gypsy elements 2011). Four and six lineages were reported in Copia and belonged to the Athila lineage (Du et al. 2010; Marco and Gypsy superfamilies of land plants, respectively. All these Marin 2008). However, only 0.1% of the Gypsy elements lineages were found in the C. x clementina genome, but the identified in rice were classified into the Athila lineage. The full-length LTR-RT numbers within each lineage varied great- largest lineage in rice was Tat, accounting for 55.8% of its ly. The two smallest lineages, Del and Oryco,werefound in Gypsy elements (Du et al. 2010). The ratio of Gypsy to the C. x clementina genome, containing 41 and 57 full-length Copia elements in Clementine is 1.34:1, and is much lower LTR-RTs, respectively. However, the largest lineage, Athila, than rice (4.9:1) (Tian et al. 2009) and sorghum (3.7:1) included 814 full-length LTR-RTs and accounted for 47.13% (Paterson et al. 2009), but is similar to soybean (1.4:1) (Du of the Gypsy elements identified. Different species have et al. 2010) and maize (1.6:1) (Baucom et al. 2009). 43 Page 8 of 14 Tree Genetics & Genomes (2018) 14:43 Fig. 2 Neighbor-joining phylogeny of Gypsy families based on reverse sequences from GyDB are denoted with a plus symbol and shown in transcriptase. One representative element that contains a complete RT red. Bootstrap values below 60% are not shown domain was chosen for each of the 178 Gypsy families. Reference Previous studies show that LTR-RT based primers can were reported in the Poaceae and Rosaceae species be used for closely related genera (Kalendar et al. 2011; (Mamaghani et al. 2015; Sun et al. 2015). One reason Kalendar et al. 1999). In this study, all designed IRAP for the high transferability of IRAP markers in this study primers can be transferred across the 25 Rutaceae species. may be that the primers were designed in conserved re- The transferability of pear IRAP to other Rosaceae spe- gions within LTR-RT families, not only in conserved re- cies ranged from 87.5 to 100% (Sun et al. 2015). The gions of orthologous LTR-RTs. We selected LTR-RT fam- transferability of IRAP markers is usually higher than ilies of different sizes for primer design contrary to an other makers. For SSR markers, approximately half of IRAP study in pear, where the largest sized LTR-RT fam- the primers developed in sweet orange can be used in ilies were selected for primer design (Sun et al. 2015). pummelo or lemon (Biswas et al. 2014). Similar results The largest sized full-length LTR-RT families are not Tree Genetics & Genomes (2018) 14:43 Page 9 of 14 43 scaffold_1 scaffold_2 scaffold_3 Gypsy Copia Unknown 0 5 10 15 20 25 30 010 20 30 0 1020304050 Mbp scaffold_4 scaffold_5 scaffold_6 0 5 10 15 20 25 010 20 30 40 0 5 10 15 20 25 Mbp scaffold_7 scaffold_8 scaffold_9 0 5 10 15 20 0 5 10 15 20 25 0 5 10 15 20 25 30 Mbp Fig. 3 Distribution of full-length Gypsy, Copia, and unknown LTR-RTs in the 9 linkage groups of C. x clementina. The putative positions of centromeres are based on the results of Wu et al., and shown by black boxes representative because they do not include homologous or divided into two subgenera Citrus and Papeda,according clustered LTR-RTs in the genome. to the classification system of Swingle and Reece According to the classification of Swingle and Reece (Swingle and Webber 1943). In the UPGMA phylogenetic (Swingle and Webber 1943), the Btrue citrus fruit trees^ tree, C. hystrix formed one cluster separated from other group (Citrinae) is divided into six genera: Citrus, Citrus species that suggests some Papeda species may be Fortunella, Poncirus, Clymenia, Eremocitrus,and the most primitive Citrus (Nicolosi et al. 2000). C. Microcitrus. Citrus is the most economically important, ichangnesis was first clustered with pummelo and manda- and is sexually compatible with all other genera. The rin, and then clustered with citron supporting the hypoth- UPGMA cluster analysis showed that Fortunella and esis that C. ichangnesis could be an ancestor of mandarin, Poncirus were closer to Citrus than were Eremocitrus and is supported by the results of Xie et al. (2008)and and Microcitrus. This result was consistent with the find- Pang et al. (2007). According to our results, the citron is ings by Pang et al. based on AFLP markers (Pang et al. the most distantly related species among them, which is 2007), but differ from the results obtained by Garcia-Lor confirmed by the sequencing of their chloroplast genomes et al. (2013) and Wu et al. (2018). The Citrus genus was (Carbonell-Caballero et al. 2015). (a) (b) family 28 family 98 family 326 family 271 010 20 30 40 0 5 10 15 20 25 30 35 Insertion time (mya) Insertion time (mya) Fig. 4 Insertion time of intact LTR-RTs in C. x clementina. a Insertion time of intact LTR-RTs (mya, million years ago). b Insertion time of intact LTR- RTs within four selected families Num/Mbp Num/Mbp Num/Mbp 05 10 15 20 25 0 510 15 20 25 0 5 10 15 20 25 No. of elements 0 100 200 300 400 500 600 No. of elements 0 5 10 15 20 25 30 43 Page 10 of 14 Tree Genetics & Genomes (2018) 14:43 Table 4 Summary of PCR amplification corresponding to individual primer in IRAP and SSAP analysis Primer Total Polymorphic Polymorphic Primer Total Polymorphic Polymorphic bands bands bands (%) bands bands bands (%) LTR03 11 11 100 SSAP1/E-AGC 198 163 82 LTR05 6 5 83.33 SSAP1/M-CAG 33 22 66.67 LTR06 12 12 100 SSAP1/M-CTA 17 9 53 LTR07 10 9 90.00 SSAP1/M-CTG 26 16 61.54 LTR08 10 10 100 SSAP1/M-CTT 42 29 69 LTR09 8 7 87.50 SSAP2/E-AAC 144 92 63.89 LTR10 11 11 100 SSAP2/E-AAG 135 80 59 LTR11 14 14 100 SSAP2/E-ACA 135 121 90 LTR12 13 13 100 SSAP2/E-ACG 159 153 96 LTR13 13 13 100 SSAP2/E-ACT 159 123 77 LTR14 18 18 100 SSAP2/E-AGC 141 138 98 LTR17 12 12 100 SSAP2/E-AGG 176 147 84 LTR19 13 13 100 SSAP2/M-CAA 77 45 58 LTR20 17 17 100 SSAP2/M-CAC 95 53 56 LTR21 13 13 100 SSAP2/M-CAG 31 25 81 LTR25 12 12 100 SSAP2/M-CAT 182 75 41 LTR29 16 15 93.75 SSAP2/M-CTA 95 73 76.84 SSAP2/M-CTG 88 21 23.86 SSAP2/M-CTT 27 14 52 SSAP3/E-ACA 19 11 58 SSAP3/E-AGC 44 34 77 SSAP3/M-CAA 65 46 70.77 SSAP3/M-CTG 34 17 50.00 SSAP3/M-CTT 34 11 32 Total 209 205 98.09 Total 2156 1518 70 The SSAP markers showed that the 24 sweet orange ‘Pineapple’, and this was supported by phenotypic traits clones had patterns that were closer to ‘Valencia’ than (data not shown). The SSAP markers also showed that the Fig. 5 Dendrogram of 25 accessions by the UPGMA cluster analysis based on IRAP analysis Tree Genetics & Genomes (2018) 14:43 Page 11 of 14 43 Fig. 6 The distribution of genetic Valencia similarity coefficients between Pineapple ‘Valencia’ or ‘Pineapple’ and 24 clones based on SSAP analysis 24 clones had abundant genetic variations which would be also found to contain an independent insertion of a similar helpful for future variety registration and protection of retrotransposon that confers tissue-specific red coloration intellectual property. Many widely grown citrus cultivars, also in response to cold conditions (Butelli et al. 2012). such as sweet orange, grapefruit, lemon, and various clon- al selections of Satsuma and Clementine mandarins, orig- inated as either bud sport or apomictic seedling mutations Conclusion (Rao et al. 2009). The use of traditional markers to dis- tinguish such mutant clones is difficult because of lower Full-length LTR-RTs were mined from the Clementine genome genetic variability associated with these maker systems. and classified based on structural details. Randomly selected The LTR-RT based SSAP markers were especially useful IRAP and SSAP markers were tested and showed that they could for distinguishing accessions with similar genetic back- differentiate citrus accessions and mutant clones. Our findings grounds, like bud sport mutations in citrus (Venturi et al. indicate that LTR-RTs are an excellent molecular marker re- 2006;Zhaoetal. 2010). In our previous study, 18 SSR source because they are easy to develop, polymorphic, widely primer sets were used to distinguish these 27 accessions. distributed, and transferable within the Rutaceae family. However, all accessions showed similar PCR amplifica- tion patterns and were indistinguishable (data now Funding information This work was supported by a grant from the Citrus shown). In C. x clementina, SSAP was successfully used Research and Development Foundation (CRDF-724) and the China to distinguish 24 accessions generated from bud muta- Scholarship Council. tions; other markers (ISSR, RAPD, AFLP, and SSR) test- ed could not (Bretó et al. 2001). Compared to other rou- Compliance with ethical standards tinely used molecular markers such as SSR, LTR-RT based SSAP markers are better suited for genetic relation- Conflict of interest The authors declare that they have no conflict of interest. ship analysis and phylogenetic analysis (Biswas et al. 2011;Schulmanet al. 2012). An advantage of SSAP is Open Access This article is distributed under the terms of the Creative that polymorphisms at multiple loci are detected in a sin- Commons Attribution 4.0 International License (http:// gle assay, while SSR usually detects polymorphisms at creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give one locus (Powell et al. 1996). Retrotransposons are im- appropriate credit to the original author(s) and the source, provide a link portant sources of variation in Citrus, especially in the to the Creative Commons license, and indicate if changes were made. species mentioned above (Bretó et al. 2001;Wangetal. 2017). Several Sicilian blood oranges arose by insertion of a Copia-like retrotransposon (Tcs-1) adjacent to a MYB transcriptional activator (named Ruby) for anthocy- References anin production. 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Tree Genetics & GenomesSpringer Journals

Published: Jun 1, 2018

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