Reconstruction of an SSR-based Magnaporthe oryzae physical map to locate avirulence gene AvrPi12

Reconstruction of an SSR-based Magnaporthe oryzae physical map to locate avirulence gene AvrPi12 Background: Pathogen avirulence (Avr) genes can evolve rapidly when challenged by the widespread deployment of host genes for resistance. They can be effectively isolated by positional cloning provided a robust and well-populated genetic map is available. Results: An updated, SSR-based physical map of the rice blast pathogen Magnaporthe oryzae (Mo) has been constructed based on 116 of the 120 SSRs used to assemble the last map, along with 18 newly developed ones. A comparison between the two versions of the map has revealed an altered marker content and order within most of the Mo chromosomes. The avirulence gene AvrPi12 was mapped in a population of 219 progeny derived from a cross between the two Mo isolates CHL42 and CHL357. A bulked segregant analysis indicated that the gene was located on chromosome 6, a conclusion borne out by an analysis of the pattern of segregation shown by individual isolates. Six additional PCR-based markers were developed to improve the map resolution in the key region. AvrPi12 was finally located within the sub-telomeric region of chromosome 6, distal to the SSR locus LSM6–5. Conclusions: The improved SSR-based linkage map should be useful as a platform for gene mapping and isolation in Mo. It was used to establish the location of AvrPi12, thereby providing a starting point for its positional cloning. Keywords: Magnaporthe oryzae, SSR physical map, Avirulence gene, AvrPi12, Genetic and physical mapping Background Positional cloning has proven to be an effective The pathogen responsible for the highly damaging dis- means of isolating both host resistance and pathogen ease of rice known as blast is the filamentous asco- Avr genes [10–14]. The method relies on the prior es- mycete Magnaporthe oryzae (Mo)[1, 2]. Deployment of tablishment of a comprehensive linkage map [11, 15]. host genes conferring resistance is widely recognized as The effort to develop such a linkage map for Mo, the most environmentally benign and cost-effective begun in the 1990s, and by 2007 had delivered one means of its control [3–5]. However, the effectiveness of based on SSR (simple sequence repeats; also called most blast resistance genes ceases after a few seasons, as microsatellite) markers, taking advantage of the acqui- a result of the emergence of pathogen races in which the sition of thegenomesequenceof Mo isolate 70–15 matching avirulence (Avr) gene has mutated to virulence (MG5; http://www.ncbi.nlm.nih.gov/bioproject/13840; [6–10]. A better understanding of the mode of evolution [15, 16]). In the meantime, improvements have been of pathogen Avr genes should aid in forming an effective made to this genome sequence (MG8; www.ncbi.nlm.- strategy for resistance gene deployment. nih.gov/assembly/GCF_000002495.2/); [17, 18]), which now allows for an update of the Mo linkage map. * Correspondence: panqh@scau.edu.cn Pi12, a gene which conditions blast resistance, was ini- Tonghui Li, Jianqiang Wen and Yaling Zhang contributed equally to this tially identified in the African cultivar Moroberekan [19, work. 20], but is also effective in parts of East Asia [21–23]. State Key laboratory for Conservation and Utilization of Subtropic Agrobioresurces, Guangdong Provincial Key Laboratory for Crop Molecular Here, the updated Mo physical map was used to identify Breeding, College of Agriculture, South China Agricultural University, the genomic location of the Avr gene matching Pi12, the Guangzhou 510642, China most important blast resistance gene harbored by Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. BMC Microbiology (2018) 18:47 Page 2 of 8 Moroberekan, as a prelude to undertaking its positional 25], and the observed ratio was tested against this ex- cloning. pectation using a standard χ test. Methods Linkage mapping The Mo mapping population Initially, the bulked segregant analysis (BSA) [28] ap- Isolates CHL357 and CHL42 were collected from dis- proach was taken to identify the genetic location of eased plants in, respectively, Jiangsu and Yunnan prov- AvrPi12. The two necessary contrasting bulk DNAs were inces (China). The two isolates were crossed with one formed by creating an equimolar mixture of DNA ex- another in vitro using a published protocol [24, 25], and tracted from either ten isolates which were all avirulent the resulting single-spored progeny were incubated for or ten which were all virulent when inoculated on the first 7 days at 25 °C and thereafter at 20 °C under IRBL12-M. The two bulk DNAs were tested with 132 continuous illumination provided by dark-blue fluores- genomically well dispersed SSR assays. SSRs identified as cent light. Single ascospores were randomly selected being potentially linked to AvrPi12 according to the BSA [24], generating a population of 219 viable isolates, assay were then used to genotype individual members of which were stored on dry filter papers [25]. This set of the mapping population. To refine the map location, this progeny represented the mapping population used to de- step was repeated using a number of newly developed termine the genetic location of AvrPi12. PCR-based markers (single nucleotide polymorphisms and indels) known to lie within the critical genomic re- Reconstruction of SSR physical map for Mo genome gion. Since Mo is a haploid organism, the recombination The SSR loci previously developed based on the genome frequency between adjacent markers is given by the ratio of the version MG5 [15] were reassembled onto the ver- between the number of recombinants and the total sion MG8 by aligning their primer sequences using the number of individuals tested [10, 15, 24]. As the relevant Blast algorithm (http://www.ncbi.nlm.nih.gov/BLAST), recombination frequencies were all below 4.5%, they even though some primer sequences were slightly im- were directly equivalent to cM [24]. Since the markers proved for better detection. A number of additional SSR used for mapping had been placed by inspection of the assays were subsequently developed to fill gaps resulting genome sequence of Mo isolate 70–15, it was possible to from monomorphism between the mapping population convert the genetic map into a physical one. The disor- parents. The old markers (prefixing with MS) integrated dered genomic region between the donor isolate and the into the new version of the SSR physical map were reference isolate 70–15, if any, was adjusted by the ac- re-prefixed with SM, and the new ones further post fixed tual recombinants detected at the respective loci. The with A and B, if any. The Avr genes isolated were also methods used to extract DNA from the Mo isolates, to included in the SSR physical map with their sequence develop the new markers and to perform genotyping information. The methods used to identify SSRs, to de- followed those given by Ma et al. [24]. sign primers and to deploy the PCR assays followed those described by Feng et al. [15]. Results The updated SSR-based Mo physical map Gene analysis Of the 120 SSR markers used to develop the original A monogenic line with resistance gene Pi12, linkage map, 116 were retained in the updated ver- IRBL12-M, the universal susceptible cultivar Lijiang- sion (Fig. 1; Additional file 1:Table S1;also see [15]). xintuanheigu (LTH; also the recipient for a set of The four old markers, MS4–6, 4–7, 6–15, and 7–2, monogenic lines;), and other 10 monogenic cultivars/ were ruled out, as their positions were missing in the lines with the respective resistance genes, Pia, Pii, MG8 (MS4–6, 7–2), or their assays proved to be Pik, Pik-p, Pit, Pi2, Pi11, Pib, Pita, Pita-2 [26](data non-robust (MS4–7, 6–15). A further 18 SSR loci will be shown elsewhere), were used as the host culti- were added to the set of 116. A comparison between vars in this study. Seeds of 12 cultivars were separ- the two versions of the physical map showed that just ately sown in a plastic pot (diameter 17 cm, height two of chromosomes (3 and 7) were almost main- 9.5 cm). Three to five plants per cultivar were used tained with respect to their SSR content, although for inoculation. The methods used for Mo inoculation their marker order was altered in both cases (see SM and disease scoring followed those described else- and MS marker codes in Additional file 1:Table S1). where [27]. Each isolate was tested at least twice, and The marker content of the updated version of the highest disease score recorded was adopted as the chromosome 1 was almost the same as that of the true score. The pattern of segregation for Avr (disease previous version of chromosome 2, and similarly for rating of 0–2) to Pi12 across the mapping population the new chromosome 5/previous chromosome 6 and was expected to be 1:1 since Mo is haploid [10, 24, the new chromosome 6/previous chromosome 4. The Li et al. BMC Microbiology (2018) 18:47 Page 3 of 8 Chr 1 Chr 3 Chr 5 Chr 2 Chr 4 Chr 6 Chr 7 TEL 1 TEL 3 TEL 5 TEL 7 TEL 9 TEL 11 TEL 13 SM1-1 SM7-1 Avr1-CO39 SM6-1 SM4-1 SM3-1 SM7-2 SM5-1 SM4-2 SM7-3 PWL3/4 SM6-2 SM1-2 SM7-4 SM2-1 SM3-2 AvrPita SM5-2 SM4-3 SM7-5 SM6-3 SM1-3 SM3-3 SM4-4 SM5-3 SM6-4 SM7-6 SM1-4 SM2-1A SM4-5 SM2-2 PWL2 * SM1-5 SM7-7 SM3-4 AvrPi54 SM5-4 SM2-3A SM6-5 SM7-8 SM4-6 SM5-5 SM1-6 SM6-6 SM2-3 SM7-8A SM5-6 SM2-4 SM7-9 SM3-5 SM6-7 SM1-7 SM7-10 SM1-7A SM2-4A AvrPi9 SM4-7 SM5-7 SM6-8 SM1-8 SM7-11 SM3-6 AvrPiz-t SM4-8 SM7-11A SM6-9 SM1-9 SM2-5A SM7-12 SM4-9 SM5-8 SM6-10 SM3-7 SM2-5 SM7-13 SM1-10 SM4-10 SM6-11 SM5-9 SM7-14 SM2-6 SM1-11 SM6-12 SM1-12 SM6-13 SM3-8A SM7-15 SM5-10 SM4-11 SM6-14 SM1-13 SM2-7A SM5-11 SM3-8 SM6-15 TEL 14 SM1-14 SM6-16 SM3-9 SM4-12 SM5-12 AvrPib SM1-15 SM2-7 SM6-17 SM5-13 SM1-16 Supercontig 8.8 SM5-14 SM4-13 TEL 12 SM2-8 SM3-10 SM4-14 PWL1 SMSC-2 SM2-9 TEL 10 SM1-16A SM4-15 SMSC-1 SM4-16 SMSC-3 SM1-17 SM4-17 SM2-10 SMSC-4 SM3-11 SM4-18 SM1-18 SM3-11A SM4-19 SM1-18A TEL 8 SM3-11B SM2-11 SM3-12 PWL2 * SM1-19 SM3-13 SM1-20 AvrPii * SM2-11A AvrPi15 * SM1-20A TEL 6 SM2-12 SM2-13A SM2-13 SM1-21 SM2-14 AvrPia AvrPii * ACE1 AvrPi15 * 1 Mb TEL 2 SM2-15 AvrPi7 AvrPik/kp/k TEL 4 m Fig. 1 Updating the Mo SSR-based linkage map, based on the current version of the reference genome sequence of isolate 70–15 (MG8; https:// www.ncbi.nlm.nih.gov/assembly/GCF_000002495.2/). The map was constructed using 116 of the 120 markers reported in [15], along with 18 newly developed ones (see also Additional file 1: Table S1). The telomeres were indicated by boldface, and the cloned Avr genes were integrated into the map with their sequences. PWL1, and PWL3/4 [35], PWL2 [36], AvrPita [31], ACE1 [37], AvrPiz-t [43], AvrPia [33, 38], AvrPii and AvrPik/kp/km [33], AvrPi54 [44], AvrPi15 [24], AvrPi7 [25]. AvrPi15, AvrPii, Avr1-CO39 and PWL2 located on double positions were marked by *. AvrPia, AvrPii and Avr1-CO39, which were absent on 70–15, were landed by their flanking sequences updated version of the chromosome 2 map incorpo- Chromosome landing rated SSRs previously allocated to chromosomes 1 To search for the chromosomal location for the AvrPi12 and 4; while the new version of chromosome 4 combined locus, a total of 132 SSR markers covering the whole markers previously associated with chromosomes 1, 3 and genome of Mo, were screened by BSA assay, of which 90 5. An additional chromosome, referred to here as “Super- showed a polymorphism between two parental isolates contig 8.8”, harbored markers previously assigned to (Fig. 2 and Additional file 3: Figure. S2). The results chromosome 7. Thus the updated version of the linkage showed that markers on chromosomes 1–5 and 7 as well map comprised eight chromosomes, according to the as Supercontig 8.8 were unlikely as candidate ones MG8 genome (Fig. 1). linked to the locus (Additional file 3: Figure. S2). In con- trast, a series of markers located on chromosome 6 Avirulence inheritance showed concurrent polymorphisms for both parental The parental isolates CHL42 and CHL357 were, respect- and bulk pairs (Fig. 2). Three polymorphic markers, ively, avirulent and virulent on IRBL12-M, while they SM6–12, SM6–16, and SM6–17, were selected for tenta- were both virulent on LTH (Additional file 2: Figure S1). tive validation with the progeny isolates ranged from When the 219 progeny isolates were tested individually #103 to #135. The results showed that all the three for their avirulence/virulence on IRBL12-M, avirulence markers were linked to the AvrPi12 locus (Fig. 3). Taking and virulence segregated in a 1:1 ratio (111:108, χ = into a consideration that the AvrPi12 locus might be 0.02, P < 0.10). This result further suggested that the rearranged by some segments from other chromosomes progeny population tested consisted of random asco- [10], four putative polymorphic markers, SM2–1, SM2– spore isolates. The progeny population was, therefore, 15, SM4–2, and SM7–14, were tested with the progeny recognized as an appropriate mapping population for isolates ranged from #1 to #035, individually. The results AvrPi12, which is responsible to the resistance gene Pi12 showed that all the four markers were not linked to the carried by IRBL12-M. AvrPi12 locus (Additional file 4: Figure. S3). Taken Li et al. BMC Microbiology (2018) 18:47 Page 4 of 8 Fig. 2 PCR profiles of chromosome 6 markers putatively linked to AvrPi12 as a result of applying the bulked segregant analysis assay (also see Additional file 2: Figure S1). In each panel, lanes #1 through #4 represent, respectively, the isolate CHL42 (avirulent against Pi12), the isolate CHL357 (virulent), a bulk formed by ten avirulent progeny isolates and a bulk formed by ten virulent progeny isolates. Informative markers are shown in blue. *One of two possible genomic locations for PWL2 [36] was suggested together, the data supported a location for AvrPi12 on to an interval between markers SM6–16 and SM6–17. chromosome 6. As to marker LSM6–2, there were 23 recombinants, suggesting a genetic inversion between markers Gene location SM6–16 (22 recombinants) and LSM6–2 was occur- Three above-mentioned markers were further tested ring in the parental genome (Fig. 4). The six recom- with the rest progeny isolates in the mapping popula- binants detected between AvrPi12 and LSM6–5were tion. The results showed that 47, 22, and 6 recombi- the same as those detected between AvrPi12 and nants, respectively, detected at markers SM6–12, SM6– SM6–17, thereby narrowing the genetic window har- 16, and SM6–17 in the first round of linkage analysis boring AvrPi12 to the interval between LSM6–5and (Fig. 4). Because all the recombinants from SM6–12, TEL12. Each genetic distance was determined by through SM6–16, and to SM6–17 overlapped, and the recombinants occurred in the region between adja- last one is close to the telomere of Mo, TEL12, the cent markers, which was shown above the map in AvrPi12 locus was defined between SM6–17 and TEL12 cM. The estimated physical distances between the (Fig. 4). To construct a fine map of the locus, six add- various linked markers were mostly derived from the itional markers were de novo developed for the second isolate 70–15 genome sequence; the exceptions in- round of linkage analysis (Table 1). The results showed volved the inferred inversion between SM6–16 and that there were 20, 16, 13, and 9 recombinants, detected LSM6–2, and the interval between LSM6–5and at markers, LSM6–4, LSM6–6, LSM6–9, and LSM6–1, TEL12, which is of uncertain length, given that telo- respectively, indicating that these markers were located meric regions of many plant pathogens appear to be Li et al. BMC Microbiology (2018) 18:47 Page 5 of 8 SM6-12 Phenotype AV VV VVVVA V VAV V VVVAV V V A V VA VVA A AVV Genotype AV VV VVVVA A VAV A VVVAV A V V V AV VVA V AVV Isolate SM6-16 AV VV VVVVAVVAV V VVVAV V V A VV A VVAAAVV Phenotype AV VV VVVVAVVAV A VVVAV A V V VV V VVAAAVV Genotype Isolate SM6-17 Phenotype AV VV VVVVAVVAVVVVVAVVV A VV A VVAAAVV Genotype AV VV VVVVAVVAVVVVVAVVV V VV V VVAAAVV Isolate Fig. 3 PCR profiles of 30 progeny isolates and their parental isolates derived from three linked markers. Phenotypes: A, avirulent; V, virulent. Genotypes: A, the same with A parent (Ap); V, the same with V parent (Vp). The recombinants were in red hypervariable [24, 29, 30]. As per the reference gen- Discussion omic sequence of 70–15, in this region, there were The updated Mo physical map can serve as a platform for 12 candidate genes predicted for AvrPi12 (Add- gene mapping and cloning itional file 5: Table S2). Intriguingly, there was not The first whole genome sequence of Mo was released by any one meeting criterions for both secretion and ef- IRGBC in 2003 (MG5; [16]). Since then, a critical effort fector, indicating that AvrPi12 might be located on a has been made by the IRGBC in collaboration with the specific interval, which was absent in the genome of Broad Institute, USA, to improve the accuracy and in- 70–15. tegrity of the reference sequence that led to the release AvrPi12 10 kb TEL12 70-15 542 35 76 65 27 25 20 9.3 37.5 (Physic distance, kb) (47/219) (22) (23) (20) (16) (13) (9) (6) (6/219) (recombinants/gametes) (Genetic distance, cM) 11.0 0.5 0.9 1.8 1.4 1.8 0.9 0.0 2.7 CHL42/CHL357 542 35? 76 65 27 25 20 9.3 37.5? (Physic distance, kb) Fig. 4 Genetic and physical maps of the AvrPi12 locus. a A physical map of markers used for chromosome walking to AvrPi12 locus based on the reference genome of isolate 70–15. b Genetic and physical map of the AvrPi12 locus. The numbers shown below the map indicate distance between adjacent markers. Recombinants detected at each marker was shown in parenthesis, and the respective genetic distance between adjacent markers was shown above the map in cM (not shown to scale). The physical distances in the parental genomes of both isolates CHL42 and CHL357 were generally referred to those of 70–15, where the physical distances of two intervals were questioned, one was in an inversion between markers SM6–16 and LSM6–2, and another in the telomeric region between markers LSM6–5 and TEL12 SM6-12 Ap Ap Ap Vp Vp Vp SM6-16 103 103 103 104 104 LSM6-2 105 105 106 106 106 107 107 107 108 108 LSM6-4 108 109 109 109 110 110 110 111 111 LSM6-6 112 112 112 114 114 114 116 116 116 LSM6-9 117 117 118 118 118 119 119 119 LSM6-1 120 120 121 121 121 122 122 122 SM6-17 123 123 124 124 124 LSM6-5 125 125 125 126 126 126 127 127 128 128 128 129 129 129 130 130 131 131 131 132 132 132 133 133 133 135 135 135 Li et al. BMC Microbiology (2018) 18:47 Page 6 of 8 Table 1 PCR-based markers linked to the AvrPi12 locus mapping to Mo chromosome 6 a b c d e f Marker Type Primer sequence (5′→ 3′) Genomic position (bp) Tm (°C) Size (bp) SM6–12 SSR F: CGTATTCTTGGCTGAGTGGC 6: 3283269 60 293 R: GCCGACGACCTGTGTGATAC LSM6–2 InDel F: GCGAGAGTTTGACTGATGTTTG 6: 3857026 60 86 R: ATCCACCCAAGCTTTCGTTTTAG SM6–16 SSR F:TGATACTAACTCCTCCTCCCAAAAC 6: 3826452 57 159 R: CAGTCACGGTCTCCTAAGCC LSM6–4 InDel F: GCAGTAGGTGAATTGTCTCGGT 6: 3932760 58 124 R: AGTCACCCCTACTCTGTTGTTGT LSM6–6 SNP F: ACCGAGTTTAGTGTTTTGGATGA 6: 4011510 58 165 R: TCGAAGGTTTATGGTGCCAAT LSM6–9 SNP F: CCACCCTGTGTCGTACTTCAATT 6: 4040268 58 112 R: AAGATTGGTGGCCTGTCGTT LSM6–1 InDel F: ATACCAGAACAAATTGCAAACAGC 6: 4065773 60 46 R: GTTACACCGAGGAACTTGCTTG SM6–17 SSR F: GGCGGCAAGTCGCTGAAGG 6: 4086125 58 330 R: GAGTTTGAGACCTTGCGATT LSM6–5 SSR F: GAGACGATGGGCCTCTAGCA 6: 4096513 59 143 R: CTCCACGACGGTATGTTTGC SM markers were the basic markers as shown in Fig. 1 and Additional file 1: Table S1, and LSM markers were de novo developed markers specific for the AvrPi12 locus SSR, simple sequence repeat; SNP, single nucleotide polymorphism; InDel, insertion-deletion F, forward; R, reverse Genomic positions were based on the latest version of the reference genome sequence of isolate 70–15 (MG8; https://www.ncbi.nlm.nih.gov/assembly/GCF_000002495.2/) All runs began with one cycle at 94°C for 3 min, followed by 30 cycles at 94°C for 30 s, 55–62°C (annealing temperature, Tm) for 30 s, and 72°C for 1–1.5 min; with a final extension at 72°C for 7 min Amplicons obtained in the mapping population were separated by electrophoresis on 8% or 10% polyacrylamide gels of the latest version of the reference genome sequence The physical maps of AvrPi12 can serve as a start point (MG8; [17, 18]). Comparison of the two versions of the for deciphering molecular mechanism underlying durable SSR physical maps established based on MG5 and MG8 resistance of Moroberekan revealed that there were certain changes in both the In the updated SSR physical map, some 14 Avr gene loci content and the order of each chromosomal marker set including three PWL loci, where some target genes have (Fig. 1; Additional file 1: Table S1; also see [15]). Such been isolated, have been mapped based on their se- dramatic changes between the two versions of the SSR quence information (Fig. 1). Ten loci were located in the physical maps that, in turn, indicated that the new one sub-telomeric and telomeric regions: PWL1,and PWL3/ would serve as a more robust platform for gene mapping 4 [35], PWL2 [36], AvrPita [31], ACE1 [37], AvrPia [33, and cloning. It is, however, necessary to take into ac- 38], AvrPii [33], AvrPik/kp/km [33], Avr1-CO39 [39], count a careful consideration for the specificities of the and AvrPib [10]. A similar situation was also identified pathogen genomes, such as variability and plasticity, in other pathosystems [29, 30, 40]. It was well recog- when adopted any reference sequence and linkage/phys- nized that the allocation of Avr genes in the dynamic ical map for individual studies [10, 14, 25, 31–34]. As to telomeric regions was indeed one of the most forceful the linkage/physical maps of the AvrPi12 locus, there strategies for the pathogens to confront resistance were two genomic intervals being evaluted in the gene-driven positive selection [7, 10, 30, 31, 41]. As a re- current study, one for genetic inversion between sult, it is in turn a bigger challenge for chromosome markers SM6–16 and LSM6–2, and another for the telo- walking to the target locus in the hypervariable region meric region between LSM6–5 and TEL12. A close un- [10, 24, 25, 41, 42]. However, the substantial genomic se- derstanding of the dynamics of genomic structure quence resources developed for species belonging to the surrounding the target locus is crucial for chromosome Magnaporthaceae family can facilitate an in silico recon- walking to the target region [10, 14, 25, 32, 34]. struction of the telomeric region of a given isolate Li et al. BMC Microbiology (2018) 18:47 Page 7 of 8 (www.ncbi.nlm.nih.gov/assembly/organism/318829/lat- Mo: Magnaporthe oryzae; SNP: Single nucleotide polymorphism; SSR: Simple sequence repeats est/;[10, 24, 25]). The blast resistance of Moroberekan appears to have Acknowledgments help up very well despite its cultivation over many years We thank Profs. Y. Zhu at Yunnan Agricultural University, and X. Zhen at Nanjing Agricultural University for providing the parental isolates, CHL42 and across a large area of West Africa [19]. Its durability is CHL357, respectively. thought to be due its harboring of the three major genes Pi12, Pi5 and Pi7, in combination with an unknown Funding This work was supported by the National Key R&D Project number of minor genes [19, 20]. Consideration of its re- (2016YDF0100601), the National Transgenic Research Project (2016ZX08001– sistance spectra has concluded that the key determinant 002), the National Natural Science Foundation of China (U1131003), and the of its high level of blast resistance across Africa has been Natural Science Foundation of Heilongjiang Province (QC2011C046). Each funding body has not role in the design of the study and collection, analysis, Pi12, while its interaction with the other two major and interpretation of data and in writing the manuscript. genes may be as important in East Asia [21–23]. Because AvrPi5 and AvrPi7 are known to represent alleles of, re- Availability of data and materials All data generated or analysed during this study are included in this spectively, AvrPii and AvrPik (unpublished data), the iso- published article and its supplementary information files. Collection of both lation of the AvrPi12 should allow for studies on the plant and pathogen materials were complied with institutional, national, and gene-for-gene network underlying the durable resistance international guidelings. of Moroberakan. Based on the genetic and physical maps Author’s contributions of the AvrPi12 locus reported here, the next step for- Project conception (QP), mapping population construction (YZ, LW), ward will be to initiate a chromosome walk from phenotyping (JW, TL, LW, QP), genotyping (TL, JW, YZ), data analysis (TL, YZ, JW, QP), map reconstruction (TL, JW, QP), manuscript preparation (QP, JC). All LSM6–5 to TEL12. authors read and approved the final manuscript. Ethics approval and consent to participate Conclusions Not applicable. The current version of the Mo genome sequence has Competing interests been exploited to update its SSR-based physical map. A The authors declare that they have no competing interests. comparison between the updated and the original ver- sions of the physical maps has revealed alterations with Publisher’sNote respect to both marker content and marker order within Springer Nature remains neutral with regard to jurisdictional claims in most of the chromosomes. AvrPi12 was mapped to published maps and institutional affiliations. chromosome 6 using a population of 219 progeny iso- Author details lates derived from the cross CHL42 x CHL357. More State Key laboratory for Conservation and Utilization of Subtropic detailed mapping showed that the gene lays in a telo- Agrobioresurces, Guangdong Provincial Key Laboratory for Crop Molecular Breeding, College of Agriculture, South China Agricultural University, meric region distal to the SSR locus LSM6–5; the latter Guangzhou 510642, China. College of Agronomy, Heilongjiang Bayi marker could serve as the starting point for a chromo- Agricultural University, Daqing 163319, China. Department of Plant some walk to the target locus. Pathology, University of Arkansas, Fayetteville, AR 72701, USA. Received: 21 February 2018 Accepted: 21 May 2018 Additional files References Additional file 1: Table S1 Primer pair sequences, SSR motifs, genomic 1. Kiyosawa S. 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Reconstruction of an SSR-based Magnaporthe oryzae physical map to locate avirulence gene AvrPi12

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Abstract

Background: Pathogen avirulence (Avr) genes can evolve rapidly when challenged by the widespread deployment of host genes for resistance. They can be effectively isolated by positional cloning provided a robust and well-populated genetic map is available. Results: An updated, SSR-based physical map of the rice blast pathogen Magnaporthe oryzae (Mo) has been constructed based on 116 of the 120 SSRs used to assemble the last map, along with 18 newly developed ones. A comparison between the two versions of the map has revealed an altered marker content and order within most of the Mo chromosomes. The avirulence gene AvrPi12 was mapped in a population of 219 progeny derived from a cross between the two Mo isolates CHL42 and CHL357. A bulked segregant analysis indicated that the gene was located on chromosome 6, a conclusion borne out by an analysis of the pattern of segregation shown by individual isolates. Six additional PCR-based markers were developed to improve the map resolution in the key region. AvrPi12 was finally located within the sub-telomeric region of chromosome 6, distal to the SSR locus LSM6–5. Conclusions: The improved SSR-based linkage map should be useful as a platform for gene mapping and isolation in Mo. It was used to establish the location of AvrPi12, thereby providing a starting point for its positional cloning. Keywords: Magnaporthe oryzae, SSR physical map, Avirulence gene, AvrPi12, Genetic and physical mapping Background Positional cloning has proven to be an effective The pathogen responsible for the highly damaging dis- means of isolating both host resistance and pathogen ease of rice known as blast is the filamentous asco- Avr genes [10–14]. The method relies on the prior es- mycete Magnaporthe oryzae (Mo)[1, 2]. Deployment of tablishment of a comprehensive linkage map [11, 15]. host genes conferring resistance is widely recognized as The effort to develop such a linkage map for Mo, the most environmentally benign and cost-effective begun in the 1990s, and by 2007 had delivered one means of its control [3–5]. However, the effectiveness of based on SSR (simple sequence repeats; also called most blast resistance genes ceases after a few seasons, as microsatellite) markers, taking advantage of the acqui- a result of the emergence of pathogen races in which the sition of thegenomesequenceof Mo isolate 70–15 matching avirulence (Avr) gene has mutated to virulence (MG5; http://www.ncbi.nlm.nih.gov/bioproject/13840; [6–10]. A better understanding of the mode of evolution [15, 16]). In the meantime, improvements have been of pathogen Avr genes should aid in forming an effective made to this genome sequence (MG8; www.ncbi.nlm.- strategy for resistance gene deployment. nih.gov/assembly/GCF_000002495.2/); [17, 18]), which now allows for an update of the Mo linkage map. * Correspondence: panqh@scau.edu.cn Pi12, a gene which conditions blast resistance, was ini- Tonghui Li, Jianqiang Wen and Yaling Zhang contributed equally to this tially identified in the African cultivar Moroberekan [19, work. 20], but is also effective in parts of East Asia [21–23]. State Key laboratory for Conservation and Utilization of Subtropic Agrobioresurces, Guangdong Provincial Key Laboratory for Crop Molecular Here, the updated Mo physical map was used to identify Breeding, College of Agriculture, South China Agricultural University, the genomic location of the Avr gene matching Pi12, the Guangzhou 510642, China most important blast resistance gene harbored by Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. BMC Microbiology (2018) 18:47 Page 2 of 8 Moroberekan, as a prelude to undertaking its positional 25], and the observed ratio was tested against this ex- cloning. pectation using a standard χ test. Methods Linkage mapping The Mo mapping population Initially, the bulked segregant analysis (BSA) [28] ap- Isolates CHL357 and CHL42 were collected from dis- proach was taken to identify the genetic location of eased plants in, respectively, Jiangsu and Yunnan prov- AvrPi12. The two necessary contrasting bulk DNAs were inces (China). The two isolates were crossed with one formed by creating an equimolar mixture of DNA ex- another in vitro using a published protocol [24, 25], and tracted from either ten isolates which were all avirulent the resulting single-spored progeny were incubated for or ten which were all virulent when inoculated on the first 7 days at 25 °C and thereafter at 20 °C under IRBL12-M. The two bulk DNAs were tested with 132 continuous illumination provided by dark-blue fluores- genomically well dispersed SSR assays. SSRs identified as cent light. Single ascospores were randomly selected being potentially linked to AvrPi12 according to the BSA [24], generating a population of 219 viable isolates, assay were then used to genotype individual members of which were stored on dry filter papers [25]. This set of the mapping population. To refine the map location, this progeny represented the mapping population used to de- step was repeated using a number of newly developed termine the genetic location of AvrPi12. PCR-based markers (single nucleotide polymorphisms and indels) known to lie within the critical genomic re- Reconstruction of SSR physical map for Mo genome gion. Since Mo is a haploid organism, the recombination The SSR loci previously developed based on the genome frequency between adjacent markers is given by the ratio of the version MG5 [15] were reassembled onto the ver- between the number of recombinants and the total sion MG8 by aligning their primer sequences using the number of individuals tested [10, 15, 24]. As the relevant Blast algorithm (http://www.ncbi.nlm.nih.gov/BLAST), recombination frequencies were all below 4.5%, they even though some primer sequences were slightly im- were directly equivalent to cM [24]. Since the markers proved for better detection. A number of additional SSR used for mapping had been placed by inspection of the assays were subsequently developed to fill gaps resulting genome sequence of Mo isolate 70–15, it was possible to from monomorphism between the mapping population convert the genetic map into a physical one. The disor- parents. The old markers (prefixing with MS) integrated dered genomic region between the donor isolate and the into the new version of the SSR physical map were reference isolate 70–15, if any, was adjusted by the ac- re-prefixed with SM, and the new ones further post fixed tual recombinants detected at the respective loci. The with A and B, if any. The Avr genes isolated were also methods used to extract DNA from the Mo isolates, to included in the SSR physical map with their sequence develop the new markers and to perform genotyping information. The methods used to identify SSRs, to de- followed those given by Ma et al. [24]. sign primers and to deploy the PCR assays followed those described by Feng et al. [15]. Results The updated SSR-based Mo physical map Gene analysis Of the 120 SSR markers used to develop the original A monogenic line with resistance gene Pi12, linkage map, 116 were retained in the updated ver- IRBL12-M, the universal susceptible cultivar Lijiang- sion (Fig. 1; Additional file 1:Table S1;also see [15]). xintuanheigu (LTH; also the recipient for a set of The four old markers, MS4–6, 4–7, 6–15, and 7–2, monogenic lines;), and other 10 monogenic cultivars/ were ruled out, as their positions were missing in the lines with the respective resistance genes, Pia, Pii, MG8 (MS4–6, 7–2), or their assays proved to be Pik, Pik-p, Pit, Pi2, Pi11, Pib, Pita, Pita-2 [26](data non-robust (MS4–7, 6–15). A further 18 SSR loci will be shown elsewhere), were used as the host culti- were added to the set of 116. A comparison between vars in this study. Seeds of 12 cultivars were separ- the two versions of the physical map showed that just ately sown in a plastic pot (diameter 17 cm, height two of chromosomes (3 and 7) were almost main- 9.5 cm). Three to five plants per cultivar were used tained with respect to their SSR content, although for inoculation. The methods used for Mo inoculation their marker order was altered in both cases (see SM and disease scoring followed those described else- and MS marker codes in Additional file 1:Table S1). where [27]. Each isolate was tested at least twice, and The marker content of the updated version of the highest disease score recorded was adopted as the chromosome 1 was almost the same as that of the true score. The pattern of segregation for Avr (disease previous version of chromosome 2, and similarly for rating of 0–2) to Pi12 across the mapping population the new chromosome 5/previous chromosome 6 and was expected to be 1:1 since Mo is haploid [10, 24, the new chromosome 6/previous chromosome 4. The Li et al. BMC Microbiology (2018) 18:47 Page 3 of 8 Chr 1 Chr 3 Chr 5 Chr 2 Chr 4 Chr 6 Chr 7 TEL 1 TEL 3 TEL 5 TEL 7 TEL 9 TEL 11 TEL 13 SM1-1 SM7-1 Avr1-CO39 SM6-1 SM4-1 SM3-1 SM7-2 SM5-1 SM4-2 SM7-3 PWL3/4 SM6-2 SM1-2 SM7-4 SM2-1 SM3-2 AvrPita SM5-2 SM4-3 SM7-5 SM6-3 SM1-3 SM3-3 SM4-4 SM5-3 SM6-4 SM7-6 SM1-4 SM2-1A SM4-5 SM2-2 PWL2 * SM1-5 SM7-7 SM3-4 AvrPi54 SM5-4 SM2-3A SM6-5 SM7-8 SM4-6 SM5-5 SM1-6 SM6-6 SM2-3 SM7-8A SM5-6 SM2-4 SM7-9 SM3-5 SM6-7 SM1-7 SM7-10 SM1-7A SM2-4A AvrPi9 SM4-7 SM5-7 SM6-8 SM1-8 SM7-11 SM3-6 AvrPiz-t SM4-8 SM7-11A SM6-9 SM1-9 SM2-5A SM7-12 SM4-9 SM5-8 SM6-10 SM3-7 SM2-5 SM7-13 SM1-10 SM4-10 SM6-11 SM5-9 SM7-14 SM2-6 SM1-11 SM6-12 SM1-12 SM6-13 SM3-8A SM7-15 SM5-10 SM4-11 SM6-14 SM1-13 SM2-7A SM5-11 SM3-8 SM6-15 TEL 14 SM1-14 SM6-16 SM3-9 SM4-12 SM5-12 AvrPib SM1-15 SM2-7 SM6-17 SM5-13 SM1-16 Supercontig 8.8 SM5-14 SM4-13 TEL 12 SM2-8 SM3-10 SM4-14 PWL1 SMSC-2 SM2-9 TEL 10 SM1-16A SM4-15 SMSC-1 SM4-16 SMSC-3 SM1-17 SM4-17 SM2-10 SMSC-4 SM3-11 SM4-18 SM1-18 SM3-11A SM4-19 SM1-18A TEL 8 SM3-11B SM2-11 SM3-12 PWL2 * SM1-19 SM3-13 SM1-20 AvrPii * SM2-11A AvrPi15 * SM1-20A TEL 6 SM2-12 SM2-13A SM2-13 SM1-21 SM2-14 AvrPia AvrPii * ACE1 AvrPi15 * 1 Mb TEL 2 SM2-15 AvrPi7 AvrPik/kp/k TEL 4 m Fig. 1 Updating the Mo SSR-based linkage map, based on the current version of the reference genome sequence of isolate 70–15 (MG8; https:// www.ncbi.nlm.nih.gov/assembly/GCF_000002495.2/). The map was constructed using 116 of the 120 markers reported in [15], along with 18 newly developed ones (see also Additional file 1: Table S1). The telomeres were indicated by boldface, and the cloned Avr genes were integrated into the map with their sequences. PWL1, and PWL3/4 [35], PWL2 [36], AvrPita [31], ACE1 [37], AvrPiz-t [43], AvrPia [33, 38], AvrPii and AvrPik/kp/km [33], AvrPi54 [44], AvrPi15 [24], AvrPi7 [25]. AvrPi15, AvrPii, Avr1-CO39 and PWL2 located on double positions were marked by *. AvrPia, AvrPii and Avr1-CO39, which were absent on 70–15, were landed by their flanking sequences updated version of the chromosome 2 map incorpo- Chromosome landing rated SSRs previously allocated to chromosomes 1 To search for the chromosomal location for the AvrPi12 and 4; while the new version of chromosome 4 combined locus, a total of 132 SSR markers covering the whole markers previously associated with chromosomes 1, 3 and genome of Mo, were screened by BSA assay, of which 90 5. An additional chromosome, referred to here as “Super- showed a polymorphism between two parental isolates contig 8.8”, harbored markers previously assigned to (Fig. 2 and Additional file 3: Figure. S2). The results chromosome 7. Thus the updated version of the linkage showed that markers on chromosomes 1–5 and 7 as well map comprised eight chromosomes, according to the as Supercontig 8.8 were unlikely as candidate ones MG8 genome (Fig. 1). linked to the locus (Additional file 3: Figure. S2). In con- trast, a series of markers located on chromosome 6 Avirulence inheritance showed concurrent polymorphisms for both parental The parental isolates CHL42 and CHL357 were, respect- and bulk pairs (Fig. 2). Three polymorphic markers, ively, avirulent and virulent on IRBL12-M, while they SM6–12, SM6–16, and SM6–17, were selected for tenta- were both virulent on LTH (Additional file 2: Figure S1). tive validation with the progeny isolates ranged from When the 219 progeny isolates were tested individually #103 to #135. The results showed that all the three for their avirulence/virulence on IRBL12-M, avirulence markers were linked to the AvrPi12 locus (Fig. 3). Taking and virulence segregated in a 1:1 ratio (111:108, χ = into a consideration that the AvrPi12 locus might be 0.02, P < 0.10). This result further suggested that the rearranged by some segments from other chromosomes progeny population tested consisted of random asco- [10], four putative polymorphic markers, SM2–1, SM2– spore isolates. The progeny population was, therefore, 15, SM4–2, and SM7–14, were tested with the progeny recognized as an appropriate mapping population for isolates ranged from #1 to #035, individually. The results AvrPi12, which is responsible to the resistance gene Pi12 showed that all the four markers were not linked to the carried by IRBL12-M. AvrPi12 locus (Additional file 4: Figure. S3). Taken Li et al. BMC Microbiology (2018) 18:47 Page 4 of 8 Fig. 2 PCR profiles of chromosome 6 markers putatively linked to AvrPi12 as a result of applying the bulked segregant analysis assay (also see Additional file 2: Figure S1). In each panel, lanes #1 through #4 represent, respectively, the isolate CHL42 (avirulent against Pi12), the isolate CHL357 (virulent), a bulk formed by ten avirulent progeny isolates and a bulk formed by ten virulent progeny isolates. Informative markers are shown in blue. *One of two possible genomic locations for PWL2 [36] was suggested together, the data supported a location for AvrPi12 on to an interval between markers SM6–16 and SM6–17. chromosome 6. As to marker LSM6–2, there were 23 recombinants, suggesting a genetic inversion between markers Gene location SM6–16 (22 recombinants) and LSM6–2 was occur- Three above-mentioned markers were further tested ring in the parental genome (Fig. 4). The six recom- with the rest progeny isolates in the mapping popula- binants detected between AvrPi12 and LSM6–5were tion. The results showed that 47, 22, and 6 recombi- the same as those detected between AvrPi12 and nants, respectively, detected at markers SM6–12, SM6– SM6–17, thereby narrowing the genetic window har- 16, and SM6–17 in the first round of linkage analysis boring AvrPi12 to the interval between LSM6–5and (Fig. 4). Because all the recombinants from SM6–12, TEL12. Each genetic distance was determined by through SM6–16, and to SM6–17 overlapped, and the recombinants occurred in the region between adja- last one is close to the telomere of Mo, TEL12, the cent markers, which was shown above the map in AvrPi12 locus was defined between SM6–17 and TEL12 cM. The estimated physical distances between the (Fig. 4). To construct a fine map of the locus, six add- various linked markers were mostly derived from the itional markers were de novo developed for the second isolate 70–15 genome sequence; the exceptions in- round of linkage analysis (Table 1). The results showed volved the inferred inversion between SM6–16 and that there were 20, 16, 13, and 9 recombinants, detected LSM6–2, and the interval between LSM6–5and at markers, LSM6–4, LSM6–6, LSM6–9, and LSM6–1, TEL12, which is of uncertain length, given that telo- respectively, indicating that these markers were located meric regions of many plant pathogens appear to be Li et al. BMC Microbiology (2018) 18:47 Page 5 of 8 SM6-12 Phenotype AV VV VVVVA V VAV V VVVAV V V A V VA VVA A AVV Genotype AV VV VVVVA A VAV A VVVAV A V V V AV VVA V AVV Isolate SM6-16 AV VV VVVVAVVAV V VVVAV V V A VV A VVAAAVV Phenotype AV VV VVVVAVVAV A VVVAV A V V VV V VVAAAVV Genotype Isolate SM6-17 Phenotype AV VV VVVVAVVAVVVVVAVVV A VV A VVAAAVV Genotype AV VV VVVVAVVAVVVVVAVVV V VV V VVAAAVV Isolate Fig. 3 PCR profiles of 30 progeny isolates and their parental isolates derived from three linked markers. Phenotypes: A, avirulent; V, virulent. Genotypes: A, the same with A parent (Ap); V, the same with V parent (Vp). The recombinants were in red hypervariable [24, 29, 30]. As per the reference gen- Discussion omic sequence of 70–15, in this region, there were The updated Mo physical map can serve as a platform for 12 candidate genes predicted for AvrPi12 (Add- gene mapping and cloning itional file 5: Table S2). Intriguingly, there was not The first whole genome sequence of Mo was released by any one meeting criterions for both secretion and ef- IRGBC in 2003 (MG5; [16]). Since then, a critical effort fector, indicating that AvrPi12 might be located on a has been made by the IRGBC in collaboration with the specific interval, which was absent in the genome of Broad Institute, USA, to improve the accuracy and in- 70–15. tegrity of the reference sequence that led to the release AvrPi12 10 kb TEL12 70-15 542 35 76 65 27 25 20 9.3 37.5 (Physic distance, kb) (47/219) (22) (23) (20) (16) (13) (9) (6) (6/219) (recombinants/gametes) (Genetic distance, cM) 11.0 0.5 0.9 1.8 1.4 1.8 0.9 0.0 2.7 CHL42/CHL357 542 35? 76 65 27 25 20 9.3 37.5? (Physic distance, kb) Fig. 4 Genetic and physical maps of the AvrPi12 locus. a A physical map of markers used for chromosome walking to AvrPi12 locus based on the reference genome of isolate 70–15. b Genetic and physical map of the AvrPi12 locus. The numbers shown below the map indicate distance between adjacent markers. Recombinants detected at each marker was shown in parenthesis, and the respective genetic distance between adjacent markers was shown above the map in cM (not shown to scale). The physical distances in the parental genomes of both isolates CHL42 and CHL357 were generally referred to those of 70–15, where the physical distances of two intervals were questioned, one was in an inversion between markers SM6–16 and LSM6–2, and another in the telomeric region between markers LSM6–5 and TEL12 SM6-12 Ap Ap Ap Vp Vp Vp SM6-16 103 103 103 104 104 LSM6-2 105 105 106 106 106 107 107 107 108 108 LSM6-4 108 109 109 109 110 110 110 111 111 LSM6-6 112 112 112 114 114 114 116 116 116 LSM6-9 117 117 118 118 118 119 119 119 LSM6-1 120 120 121 121 121 122 122 122 SM6-17 123 123 124 124 124 LSM6-5 125 125 125 126 126 126 127 127 128 128 128 129 129 129 130 130 131 131 131 132 132 132 133 133 133 135 135 135 Li et al. BMC Microbiology (2018) 18:47 Page 6 of 8 Table 1 PCR-based markers linked to the AvrPi12 locus mapping to Mo chromosome 6 a b c d e f Marker Type Primer sequence (5′→ 3′) Genomic position (bp) Tm (°C) Size (bp) SM6–12 SSR F: CGTATTCTTGGCTGAGTGGC 6: 3283269 60 293 R: GCCGACGACCTGTGTGATAC LSM6–2 InDel F: GCGAGAGTTTGACTGATGTTTG 6: 3857026 60 86 R: ATCCACCCAAGCTTTCGTTTTAG SM6–16 SSR F:TGATACTAACTCCTCCTCCCAAAAC 6: 3826452 57 159 R: CAGTCACGGTCTCCTAAGCC LSM6–4 InDel F: GCAGTAGGTGAATTGTCTCGGT 6: 3932760 58 124 R: AGTCACCCCTACTCTGTTGTTGT LSM6–6 SNP F: ACCGAGTTTAGTGTTTTGGATGA 6: 4011510 58 165 R: TCGAAGGTTTATGGTGCCAAT LSM6–9 SNP F: CCACCCTGTGTCGTACTTCAATT 6: 4040268 58 112 R: AAGATTGGTGGCCTGTCGTT LSM6–1 InDel F: ATACCAGAACAAATTGCAAACAGC 6: 4065773 60 46 R: GTTACACCGAGGAACTTGCTTG SM6–17 SSR F: GGCGGCAAGTCGCTGAAGG 6: 4086125 58 330 R: GAGTTTGAGACCTTGCGATT LSM6–5 SSR F: GAGACGATGGGCCTCTAGCA 6: 4096513 59 143 R: CTCCACGACGGTATGTTTGC SM markers were the basic markers as shown in Fig. 1 and Additional file 1: Table S1, and LSM markers were de novo developed markers specific for the AvrPi12 locus SSR, simple sequence repeat; SNP, single nucleotide polymorphism; InDel, insertion-deletion F, forward; R, reverse Genomic positions were based on the latest version of the reference genome sequence of isolate 70–15 (MG8; https://www.ncbi.nlm.nih.gov/assembly/GCF_000002495.2/) All runs began with one cycle at 94°C for 3 min, followed by 30 cycles at 94°C for 30 s, 55–62°C (annealing temperature, Tm) for 30 s, and 72°C for 1–1.5 min; with a final extension at 72°C for 7 min Amplicons obtained in the mapping population were separated by electrophoresis on 8% or 10% polyacrylamide gels of the latest version of the reference genome sequence The physical maps of AvrPi12 can serve as a start point (MG8; [17, 18]). Comparison of the two versions of the for deciphering molecular mechanism underlying durable SSR physical maps established based on MG5 and MG8 resistance of Moroberekan revealed that there were certain changes in both the In the updated SSR physical map, some 14 Avr gene loci content and the order of each chromosomal marker set including three PWL loci, where some target genes have (Fig. 1; Additional file 1: Table S1; also see [15]). Such been isolated, have been mapped based on their se- dramatic changes between the two versions of the SSR quence information (Fig. 1). Ten loci were located in the physical maps that, in turn, indicated that the new one sub-telomeric and telomeric regions: PWL1,and PWL3/ would serve as a more robust platform for gene mapping 4 [35], PWL2 [36], AvrPita [31], ACE1 [37], AvrPia [33, and cloning. It is, however, necessary to take into ac- 38], AvrPii [33], AvrPik/kp/km [33], Avr1-CO39 [39], count a careful consideration for the specificities of the and AvrPib [10]. A similar situation was also identified pathogen genomes, such as variability and plasticity, in other pathosystems [29, 30, 40]. It was well recog- when adopted any reference sequence and linkage/phys- nized that the allocation of Avr genes in the dynamic ical map for individual studies [10, 14, 25, 31–34]. As to telomeric regions was indeed one of the most forceful the linkage/physical maps of the AvrPi12 locus, there strategies for the pathogens to confront resistance were two genomic intervals being evaluted in the gene-driven positive selection [7, 10, 30, 31, 41]. As a re- current study, one for genetic inversion between sult, it is in turn a bigger challenge for chromosome markers SM6–16 and LSM6–2, and another for the telo- walking to the target locus in the hypervariable region meric region between LSM6–5 and TEL12. A close un- [10, 24, 25, 41, 42]. However, the substantial genomic se- derstanding of the dynamics of genomic structure quence resources developed for species belonging to the surrounding the target locus is crucial for chromosome Magnaporthaceae family can facilitate an in silico recon- walking to the target region [10, 14, 25, 32, 34]. struction of the telomeric region of a given isolate Li et al. BMC Microbiology (2018) 18:47 Page 7 of 8 (www.ncbi.nlm.nih.gov/assembly/organism/318829/lat- Mo: Magnaporthe oryzae; SNP: Single nucleotide polymorphism; SSR: Simple sequence repeats est/;[10, 24, 25]). The blast resistance of Moroberekan appears to have Acknowledgments help up very well despite its cultivation over many years We thank Profs. Y. Zhu at Yunnan Agricultural University, and X. Zhen at Nanjing Agricultural University for providing the parental isolates, CHL42 and across a large area of West Africa [19]. Its durability is CHL357, respectively. thought to be due its harboring of the three major genes Pi12, Pi5 and Pi7, in combination with an unknown Funding This work was supported by the National Key R&D Project number of minor genes [19, 20]. Consideration of its re- (2016YDF0100601), the National Transgenic Research Project (2016ZX08001– sistance spectra has concluded that the key determinant 002), the National Natural Science Foundation of China (U1131003), and the of its high level of blast resistance across Africa has been Natural Science Foundation of Heilongjiang Province (QC2011C046). Each funding body has not role in the design of the study and collection, analysis, Pi12, while its interaction with the other two major and interpretation of data and in writing the manuscript. genes may be as important in East Asia [21–23]. Because AvrPi5 and AvrPi7 are known to represent alleles of, re- Availability of data and materials All data generated or analysed during this study are included in this spectively, AvrPii and AvrPik (unpublished data), the iso- published article and its supplementary information files. Collection of both lation of the AvrPi12 should allow for studies on the plant and pathogen materials were complied with institutional, national, and gene-for-gene network underlying the durable resistance international guidelings. of Moroberakan. Based on the genetic and physical maps Author’s contributions of the AvrPi12 locus reported here, the next step for- Project conception (QP), mapping population construction (YZ, LW), ward will be to initiate a chromosome walk from phenotyping (JW, TL, LW, QP), genotyping (TL, JW, YZ), data analysis (TL, YZ, JW, QP), map reconstruction (TL, JW, QP), manuscript preparation (QP, JC). All LSM6–5 to TEL12. authors read and approved the final manuscript. Ethics approval and consent to participate Conclusions Not applicable. The current version of the Mo genome sequence has Competing interests been exploited to update its SSR-based physical map. A The authors declare that they have no competing interests. comparison between the updated and the original ver- sions of the physical maps has revealed alterations with Publisher’sNote respect to both marker content and marker order within Springer Nature remains neutral with regard to jurisdictional claims in most of the chromosomes. AvrPi12 was mapped to published maps and institutional affiliations. chromosome 6 using a population of 219 progeny iso- Author details lates derived from the cross CHL42 x CHL357. More State Key laboratory for Conservation and Utilization of Subtropic detailed mapping showed that the gene lays in a telo- Agrobioresurces, Guangdong Provincial Key Laboratory for Crop Molecular Breeding, College of Agriculture, South China Agricultural University, meric region distal to the SSR locus LSM6–5; the latter Guangzhou 510642, China. College of Agronomy, Heilongjiang Bayi marker could serve as the starting point for a chromo- Agricultural University, Daqing 163319, China. Department of Plant some walk to the target locus. Pathology, University of Arkansas, Fayetteville, AR 72701, USA. Received: 21 February 2018 Accepted: 21 May 2018 Additional files References Additional file 1: Table S1 Primer pair sequences, SSR motifs, genomic 1. Kiyosawa S. 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BMC MicrobiologySpringer Journals

Published: May 31, 2018

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