Fine mapping and genetic analysis of resistance genes,Rsc18,against soybean mosaic virus

LIU Sang-lin,CHENG Yan-bo,MA Qi-bin,LI Mu,,JIANG Ze,XIA Qiu-ju,NIAN Hai

1 The State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University,Guangzhou 510642,P.R.China

2 The Key Laboratory of Plant Molecular Breeding of Guangdong Province,College of Agriculture,South China Agricultural University,Guangzhou 510642,P.R.China

3 Maize Research Institute,Jilin Academy of Agricultural Sciences,Gongzhuling 130033,P.R.China4 Beijing Genomics Institute (BGI)-Shenzhen,Shenzhen 518086,P.R.China

Abstract Soybean mosaic virus (SMV) affects seed quality and production of soybean (Glycine max (L.) Merr.) worldwide. SC18 is one of the dominant SMV strains in South China,and accession Zhonghuang 24 displayed resistance to SC18. The F1,F2 and 168 F11 recombinant inbred lines (RILs) population derived from a hybridization between Zhonghuang 24 (resistant,R) and Huaxia 3 (susceptible,S) were used in this study. According to the segregation ratios of the F2 generation (3R:1S)and the recombinant inbred lines (RILs) population (1R:1S),one dominant locus may regulate the resistance to SC18 in Zhonghuang 24. By using composite interval mapping (CIM),Rsc18 was mapped to a 415.357-kb region on chromosome 13.Three candidate genes,including one NBS-LRR type gene and two serine/threonine protein type genes,were identified according to the genetic annotations,which may be related to the resistance to SC18. The qRT-PCR demonstrated that these genes were up-regulated in the R genotype compared to the control. In conclusion,the findings of this research enhanced the understanding about the R genes at the Rsc18 locus. Moreover,our results will provide insights for designing molecular markers to improve marker-assisted selection and developing new varieties with resistance to SC18.

Keywords:soybean mosaic virus (SMV),fine mapping,recombinant inbred lines (RILs),resistance gene

1.Introduction

Soybean (Glycine max(L.)Merr) is known as one of the most significant cultivated crops,as it is a fundamental source of oil and protein for human beings and animals.Nevertheless,soybean is susceptible (S) to many diseases,and soybean mosaic is a common disease caused by soybean mosaic virus (SMV). SMV is a serious disease in three primary soybean producing areas,including Northeast China,Huang-Huai Valleys and South China,which results in a characteristic mosaic form in necrosis of the top new leaves,plant dysplasia growth and seed spots (Li and Zhi 2016). Prevalent SMV reduces the soybean quality and causes a remarkable loss in yield,which can range from 50 to 80% (Hillet al.2001;Liet al.2017;Ruiet al.2017).

According to the responses of six resistant (R) cultivars(Buffalo,Marshall,York,Ogden,Kwanggyo,and Davis)and two S soybean cultivars (Rampage and Clark),98 SMV strains were isolated,and these SMV strains were classified into seven strains named as G1to G7in the United States (Cho and Goodman 1982). In China,based on the symptoms of 10 soybean cultivars (Nannong 1138-2,Youbian 30,8101,Tiefeng 25,Davis,Buffalo,Zaoshu 18,Kwanggyo,Qihuang 1,and Kefeng 1),a total of 22 SMV strains,which were named as SC1 to SC22,were verified country-wide (Zhanet al.2006;LiKet al.2010;Wanget al.2014). Of these strains,SC18 is not very toxic but widely distributed,and it appears in the main soybean producing parts of the Northeast and South China where it reduces the soybean production by approximately 25 and 33%,respectively (Zhiet al.2016).

The breeding of broad-spectrum and tolerant soybean cultivars is one of the most ecofriendly and effective methods to control SMV. However,breeding for resistance to SMV requires a large number of manual inoculation tests for assessing the R symptoms in greenhouses as well as in the fields. Hence the development of genetic linkage maps and quantitative trait loci (QTLs) is based on the application of markers,which have become quite accurate due to the availability of the whole soybean genome sequences and the site-specific molecular marker development (Liet al.2017).Molecular markers are widely used tool in mapping the key genes/QTLs in plants (Mohanet al.1997;Nadeemet al.2018). The verification of tightly linked markers contributes to developing marker-assisted selection (MAS) techniques,which are vital techniques to improve traditional breeding practices and resistant gene-mediated transgenic breeding methods to foster the resistance to SMV. By improving the identification of markers closely related to the R gene and markers derived from the cloned R gene,it is effective to use the MAS technique to transfer the R genes into varieties.Soybean has a compact molecular marker linkage map,and a number of researchers have studied the correlations between markers and genes (Songet al.2010). The soybean genome reference sequence has been published and served as a new tool for the identification of candidate genes. The resistance to SMV is mainly controlled by a dominant gene,and many different SMV strain-specific R genes were also detected (Karthikeyanet al.2018).

The R lociRsv1,Rsv3andRsv4had been located on chromosomes 13,14 and 2,respectively (Cheet al.2017).Rsv1was identified in PI96983,which was susceptible to G7but resistant to G1–G6(Cho and Goodman 1982);whileRsc-pmandRsc-psresistant to SC3,SC6,SC7,and SC17 have been mapped beside theRsv1locus (Yanget al.2013).Rsv3displayed resistance to G5–G7andRsv4was found to be resistant to the G1–G7strains (Hayeset al.2000;Jeonget al.2002). Recently,Rsv3andRsv4loci have been fine mapped to chromosomal regions of 154 and 100 kb on chromosomes 14 and 2,respectively. Suet al.(2011)implied thatRsv3gene most likely encodes a member of the CC-NB-LRR gene family based on sequence analysis ofRsv4andRsv3loci,and Maroofet al.(2010) consideredRsv4to be a novel R gene. Ilutet al.(2016) further narrowed the position ofRsv4locus down to a haplotype block of approximately 94 kb. Although studies have focused on the identification of R genes for decades,SMV-R genes have not yet been cloned. The relationship between Chinese SC1–SC22 and US G1–G7is unclear due to the various hosts and varieties used to identify the SMV strains. In China,many anti-SMV genes have been identified from various sources.For example,Rsc7,Rsc8,Rsc9,Rsc13,Rn1,Rn3,Rsa,andRsc18were identified on chromosome 2 (Wanget al.2011b;Yanet al.2015;Zhiet al.2016;Karthikeyanet al.2018).Rsc15was located on chromosome 6 from RN-9 (Yang and Gai 2011).Rsc-pmandRsc-psfrom PI96983,R genes to SC3Q,SC14Q,SC11,SC12,and SC18 from Qihuang 1 and Qihuang 22 were identified on chromosome 13 (Maet al.2011;Zhenget al.2014;Zhiet al.2016;Karthikeyanet al.2018). The R genes to SC3,SC6 and SC17 from PI96983 were all detected in a 345-kb region named asRsc-pmon chromosome 13,and the R gene to SC7 was localized to a 380-kb region denoted asRsc-psnear theRsc-pmin PI96983 (Yanget al.2013). In order to assist MSA and clone R genes,closely related molecular markers should be identified. The two simple sequence repeat (SSR) markers of BARCSOYSSR_14_1413 and BARCSOYSSR_14_ 1417 closely linked toRsc4were identified in the regions of 0.18 and 0.58 cM,respectively (Wanget al.2011a). TheRsc7in Kefeng 1 was fine-mapped to an area of about 158 kb between the two SSR markers BARCSOYSSR_02_0621 and BARCSOYSSR_02_0632 on chromosome 2,andRsc8was located between BARCSOYSSR_02_0610 and BARCSOYSSR _02_0616 at distances of 0.1 and 0.3 cM,respectively (Wanget al.2010,2011b).

Based on the conserved structure of the cloned plant R genes,the genes were divided into five types:(1)nucleotide-bind site and leucine-rich repeat (NBS-LRR)domain contained a lot of plant disease R genes. For instance,3Gg2(Hayeset al.2004) andXa1inOryza sativaand TMV R gene,N,in tobacco (Whithamet al.1994;Yoshimuraet al.1998) were classified into this type;(2)LRR transmembrane-protein kinase (LRR-TM-PK),such asPtoinSolanum lycopersicum(Martinet al.1993),Lrk10inTriticum aestivum(Feuilletet al.1997) andXa26andXa21inO.sativa(Songet al.1995;Sunet al.2004),where the structural features of these genes are LRR-TM-PK;(3)serine/threonine protein kinase (STK),likeRpg1inHordeum vulgare(Brueggemanet al.2002);(4) LRR-TM,for instance,Cf-9andCf-2genes inS.lycopersicum(Dixonet al.1996);and (5) others,such asXa5inO.sativa(Iyer and Mccouch 2005). The R mechanisms for pathogens between plant species are generally conserved (Ronald and Beutler 2010),and the cloned R genes in architectural characteristics could be applied for the detection of SMV R genes.

The present study includes three parts:(1) decipher the R mechanism of Zhonghuang 24 to the SMV strain SC18 by analyzing the phenotypic statistics;(2) detect genomic regions related withRsc18by high-throughput genome-wide resequencing;and (3) predict candidate genes forRsc18by combining qRT-PCR with sequence analysis.

2.Materials and methods

2.1.Cultivation of genetic materials

Soybean variety Zhonghuang 24 is immune to the strain SC18 of SMV,but Huaxia 3 is sensitive to the strain. In the present study,the F1,F2and recombinant inbred lines (RILs)populations acquired from the cross of Zhonghuang 24×Huaxia 3 were used to confirm the R inheritance. The F2population was generated by self-crossing of the F1plants,and the progeny of a single seed was selected from F3–F11to establish 168 F2:11derived lines as the RILs population.The two parents and their derived populations were planted in plastic basins (20 cm×20 cm) in a shed without aphids,and 25–30 seeds were planted in each pot. All the materials were taken from Guangdong Subcenter of National Center for Soybean Improvement (NCSI),South China Agricultural University (SCAU).

2.2.Inoculation,phenotypic assessments and genetic analysis of SC18

In this study,the strain SC18 provided by NCSI was used.The first leaf of plants at the the first trifoliate leaf (V1) stage was mechanically inoculated with SMV,and then inoculated again seven days later.The virus was collected from the tissue of the NN1138-2,which is known to be a susceptible variety,and the infected leaves of the S variety were ground with mortar and pestle in 0.01 mol L–1sodium phosphate buffer (approximately 3–5 mL g–1of leaf tissue,pH 7.2)while adding a small amount of 600 mesh emery powder to assist with grinding. The fully opened main leaves of the soybean plants were gently inoculated with a hand-brush.After inoculation,the inoculated leaves were sprayed with running water immediately. Insecticides were sprayed regularly on the plants to prevent cross-infection through aphids (LiDet al.2010).

The virus resistance of all the parents,F1,F2and RILs(each RIL and parents were duplicated three times) was evaluated weekly by visual inspection with scoring in detail within 40 days post-inoculation (dpi). Each inoculated plant was known as S if its upper leaves were lobular or had chlorosis symptoms,while asymptomatic plants were classified as R to the virus (Chenet al.1991). The Chisquare criterion was used to detect the segregation forms of phenotypes to conform to the Mendelian segregation ratio in the mapping population.

2.3.Genome-wide sequencing and analysis

This experiment used RAD sequencing technology to construct a concentrated genetic map (Appendix A) for proper mapping (sequencing and analysis of the 168 F11RILs of Zhonghuang 24×Huaxia 3 were performed in Beijing Genome Institute (BGI) Tech). Using soybean Williams 82 as the reference genome and performing genome-wide sequencing of Zhonghuang 24,Huaxia 3 and the RIL samples,a total of 47 472 high confidence SNPs were detected by whole genome sequencing. Bin maps were created through the sliding window which covered 15 SNPs,with each SNP sliding,the genotypes of the window and exchange sites of the test individual were confirmed.The same genotype across the whole RIL population was regarded as a single recombination bin (Huanget al.2009).Ultimately,the high-density genetic map which contained 2 639 bins was built in response to each individual bin genotype,covering the total genome length of 2 638.2 cM(Liuet al.2017).

2.4.Gene mapping

The 168 F11RILs of Zhonghuang 24×Huaxia 3 were used to construct a linkage map. In order to analyze QTL,the WinQTLCart 2.5 was used.Composite interval mapping(CIM) was introduced for the whole list of traits. Based on the phenotypic data of each recombinant inbred line for QTL mapping,the significance threshold value of LOD was confirmed by calculating the repeated sampling 1 000 times genome-wide and the permutation test was performed.At the 5% significance level,the threshold determination criterion was 3.0,and LOD≥3.0 was used as the basis for the existence of QTL (Liuet al.2017). According to the annotated soybean reference genome of SoyBase (http://www.soyba se.org/),we analyzed the candidate genes in the areas of QTL mapping results,and the functional prediction of genes could be manually confirmed through the Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html).

2.5.Candidate gene expression analysis

Seedlings of Zhonghuang 24 (R) and Huaxia 3 (S) were grown for 1 week. When the primary leaves were fully opened,they were infected with SC18. The inoculated plants were raised in a 25°C incubator with relative humidity of 75% and long daylight (16 h light/8 h dark). The extraction of total RNA from the top leaf occurred at 0,1,2,4,8,12,24,48,and 72 hours post-inoculation (hpi). Following the instructions of the kit,we used Trizol reagent (Invitrogen,USA) to extract total RNA from the plants. RNA was reversely transcribed into cDNA using PrimeScript RT Kit(TaKaRa,Japan). The experiment was repeated three times.

SoyBase (http://SoyBase.org),Williams 82 soybean reference genomes GlymaWm82.a1.v1.1 and GlymaWm82.a2.v1 were used to predict candidate genes from the localized area. The qRT-PCR was carried out for the expression profiles of candidate genes. The primers for qRT-PCR were designed using NCBI (https://www.ncbi.nlm.nih.gov/) (Appendix B). In addition,the reference geneActinwas regarded as an internal reference control,and qRT-PCR was completed on CFX96 Real-Time PCR Detection System Equipment (Bio-Rad,USA) according to the instructions of KAPA-SYBR?Rapid qPCR Kits (KAPA Biosystems,USA). The total reactions were carried out in 20 μL volumes including 1 μL cDNA as a template with three repetitions. The PCR reaction procedure was as follows:95°C for 3 min,95°C for 10 s (40 cycles) and 55°C for 30 s.The qRT-PCR statistics were calculated by the relative quantification (2?ΔΔCT) method (Ruiet al.2017).

3.Results

3.1.R mechanism of Zhonghuang 24 to SC18

Infection of SMV strain SC18 on Zhonghuang 24,Huaxia 3 and their progeny plants caused different symptoms(Appendix C). Zhonghuang 24 plants had no response to SC18,which were similar to the uninoculated plants(control),but plants from the sensitive male parent,Huaxia 3,displayed overall stunted growth and mosaic leaves within 30 dpi of SMV (Fig.1). Among the 168 RILs derived from the combination of Zhonghuang 24 and Huaxia 3,79 RILs were homozygous-S and 89 RILs were homozygous-R,so the segregation ratio fit well with the genotypic Mendelian 1R:1S ratio (χ21:1=0.595,P=0.4404;Table 1). All 11 F1plants showed high resistance to SC18 from the cross between Zhonghuang 24 and Huaxia 3,which indicated dominant resistance in Zhonghuang 24. Overall,the segregation ratio of a population of 268 F2plants for S and R progenies was in accordance with the Mendelian ratio (3:1),revealing that the R mechanism of SC18 is regulated by a single dominant gene in Zhonghuang 24.

Fig.1 Plant resistance phenotypes to soybean mosaic virus strain SC18. A and B represent the front view and top view of the parental phenotype,respectively. In the A and B panels,the left sides represent Zhonghuang 24,whose leaves were asymptomatic after SC18 inoculation,and the right sides are Huaxia 3,whose leaves shrunk after SC18 inoculation.

3.2.Fine mapping of the genomic region associated with resistance to SC18 by high-throughput genomewide resequencing

The high-density map of the SC18-R gene localization was marked with bins by using CIM with WinQTLCart 2.5(Appendix D). Here,a RIL population including 168 lines was inoculated to determine the genomic interval related to the resistance to SC18. Based on the phenotypic and genotypic data of the 168 RILs population,the SC18-R gene was detected on Gm13_bin65,in genomic regions of 70.2 cM on chromosome 13. The high-density linkage map ofRsc18is presented in Fig.2. The physical zone Gm13_bin65(i.e.,nucleotide positions of 24 850 727–25 266 083 bp on chromosome 13),was about 415.357 kb. According to the segregation ratio of the 268 F2population for S and R progenies,the R mechanism of SC18 is regulated by a single dominant gene in Zhonghuang 24. The SC18 R locus (Gm13_bin65) was detected on chromosome 13 in Zhonghuang 24,with LOD value (37.4336) higher than 3.0,which explained 62.01% of phenotypic variance (Table 2),indicating that it was closely linked to the SC18-R gene.

Fig.2 The quantitative trait loci (QTLs) identified for mosaic virus resistance traits on chromosome 13 in the high-density genetic map of the recombinant inbred lines (RILs) population.The bin markers and the locations are shown,but most of the bin marks in the middle are omitted and not shown. The QTL for mosaic virus resistance is highlighted in red.

Table 1 Genetic analysis of resistance to SC18 in Zhonghuang 24,Huaxia 3 and their progenies

Then we used BLAST searches and the TAIR protein datasets to screen for probable candidate genes against SC18. Based on the Williams soybean reference genome GlymaWm82.a1.v1 annotations,a total of 27 hypothetical genes were detected in the relevant genomic areas.Gene Ontology (GO) analysis revealed that 19 genes out of the 27 annotated genes were related to more than one GO term. These genes may play an auxiliary role in different biological processes,such as the regulation of biological and cellular processes,metabolic processes of macromolecular complexes,binding processes,catalytic activity,synthesis of organelles,and macromolecular complexes (Appendix E). These processes are involved in protein metabolism and are responsible for regulation of the metabolites and energy needed for life activities. According to the Phytozome v12.1 (https://phytozome.jgi.doe.gov/pz/portal.html) annotation,three candidate genes were selected as potential candidate genes for the resistance to SC18,namelyGlyma.13g150000,Glyma.13g151100andGlyma13g21640,which were associated with the resistance to SC18.Glyma.13g150000contains a LRR N-terminal domain (LRRNT_2) domain and is supposed to be involved in plant disease resistance.Glyma.13g151100contains serine/threonine-protein kinase andGlyma13g21640might be associated with serine/threonine specific protein phosphatase PP1,catalytic subunit (Table 3). These speculative R genes of Zhonghuang 24 might be related to the resistance to SC18.

Table 2 Identification of genomic regions of soybean mosaic virus resistant genes on the soybean chromosome

Table 3 Annotation of 22 candidate genes associated with SC18 resistance1)

3.3.Expression profiles of R genes

The expression levels of the three R genes were observed in Zhonghuang 24 (R) and Huaxia 3 (S) using qRT-PCRanalysis (Fig.3). All these genes,Glyma.13g150000,Glyma.13g151100andGlyma13g21640,were differentially expressed between Zhonghuang 24 and Huaxia 3. The expression patterns ofGlyma.13g150000were significantly up-regulated at 1 and 72 hpi after treatment in Zhonghuang 24.Similarly,the expression patterns ofGlyma.13g151100were found to be significantly up-regulated at 2 and 72 hpi in the Zhonghuang 24. The expression levels ofGlyma13g21640were significantly up-regulated at 24 hpi in the Zhonghuang 24 and displayed the highest expression at 72 hpi. In contrast,the expression levels of the genes were lower in Huaxia 3 than in Zhonghuang 24. Hence,it was speculated that these genes were induced by SC18under stress and might be involved in the disease defense mechanism.

Fig.3 Relative expression levels of Rsv18 candidate genes.Actin was used as an internal reference gene,and the expression of candidate genes of Rsv18 was evaluated by qRTPCR with the 2?ΔΔCT method. R,female parent Zhonghuang 24(resistant);S,male parent Huaxia 3 (susceptible). Values are mean±SD of three biological replicates.＊,significant difference at P<0.05.

4.Discussion

4.1.Comparison between Rsc18 and other reported anti-SMV genes on chromosome 13

Gene mapping is based on map-based cloning on a linkage map (Songet al.2010). With the development of soybean genome sequencing and molecular mapping,researchers can rapidly detect a vast number of molecular markers and recognize the chromosomal locations for specific genes.Genome sequencing and molecular mapping can also be used to identify the markers that are closely related to the desired locus. In this research,we investigated theRsc18of Zhonghuang 24 genetic analysis by segregation analyses of the F1and F2populations,and inferred that the SC18 R genes in Zhonghuang 24 were inherited independently.

In addition,we mapped a dominant gene locus of Zhonghuang 24 in the genomic region,Gm13_bin65,with a genetic distance of 70.2 cM by using the composite interval mapping. The LOD value of Gm13_bin65 was much higher than 3.0 (37.4336),which explained 62.01% of phenotypic variance,revealing that Gm13_bin65 is linked to the R gene.According to the Phytozome v12.1 (https://phytozome.jgi.doe.gov/pz/portal.html) annotation,there were 19 gene annotations in this interval. These genes participate in various biological processes,such as the regulation of biological and cellular processes,metabolic processes of macromolecular complexes,binding processes,catalytic activity,synthesis of organelles,and macromolecular complexes. In a previous report,the SC18 R locus of Qihuang 22 was mapped on chromosome 13,between Satt334 and SOYHSP176 with genetic distances of 5.0 and 6.7 cM,respectively (Zhiet al.2016). The gene locus detected in the present study was very close to this position. However,the fine mapping and precise candidate genes of SC18 have not been reported yet.Abundant R genes for viruses and bacteria were located on chromosome 13 where 10 mosaic virus R sites were detected(Fig.4). The US R variety PI96983,which carries theRsv1R locus,was first reported in the R gene cluster on chromosome 13 (Jeong and Maroof 2004). TheRsv1locus regulates the resistance to G1–G6strains in the US,and the loci were identified by SSR markers (between Sat_154 and Satt510)in China (Yanget al.2013;Zhenget al.2014). Moreover,many plant disease R genes are located on chromosome 13. For example,theRps3regulating Phytophthora root rot resistance was mapped between HSP176 and Satt114(Demirbaset al.2001);the peanut mottle virus R geneRpv1was located between Sat_154 and Satt510 (Goreet al.2002);and the SMV R genesRsc-psfor SC7 andRsc-pmfor SC3,SC6 and SC17 were detected on chromosome 13(Yanget al.2013). Karthikeyanet al.(2018) identified the SC20-R genesGlyma.13G194700andGlyma.13G195100,and classified the SMV-R genes on chromosome 13 into four groups (Fig.5-A). The results of this study did not overlap with the previous results,suggesting that this locus is a novel R site (Fig.5-B). Moreover,many anti-SMV genes,such asRsc3QandRsc14Qlocated on chromosome 13,were also found in China (Zhenget al.2014),but the R mechanism has not been documented.

Fig.4 Genetic and physical maps of soybean against soybean mosaic virus genes on chromosome 13. The QTL represents the gene locus identified in this study,and it is marked with light blue,the other loci reported by other studies are shown in different colors. F and R indicate the forward and reverse sequences,respectively.

Fig.5 Map of chromosome 13. The corresponding letters are Sct-033 (A),BARCSOYSSR-13-1185 (B),BARCSOYSSR-13-1155(C),BARCSOYSSR-13-1140 (D),BARCSOYSSR-13-1136 (E),BARCSOYSSR-13-1114 (F),BARCSOYSSR-13-1099 (G),and SOYHSP176 (H). A is adapted from Karthikeyan et al. (2018). B shows the relationship between the positioning interval of this study and the fourth group. F and R indicate the forward and reverse sequences,respectively.

4.2.The selection of potential SMV R genes

Referring to the Williams 82 Soybean Reference Genome(GlymaWm 82.a2.v1),30 genes within a genomic region of about 415 kb were detected,andGlyma13g150000might be a potential candidate R gene for SC18 with an LRR domain.LRR protein kinase family is homologous with members of the main disease-R families that have been reported (Michelmoreet al.2013). The three genes identified in this study could possibly be associated with a cluster of NBS-LRR genes.According to the parental haplotypes within the intervalbased on the re-sequencing results of the parents (Appendix F),the single base substitution was due to a single T to C transition in the exonic nonsynonymous SNV ofGlyma.13g150000.The gene function annotation is LRR protein kinase family protein,and it is generally known that the LRR receptorlike protein kinase (LRR-RLK) plays an important role in plant development and disease defence.Therefore,we preliminarily determined thatGlyma.13g150000may play a key role. In addition,several other types of genes within this interval might be related to the disease resistance.The serine carboxypeptidase (SCP1) has a strong effect on the formation of mycorrhizae and the development of twigs inMedicago truncatula(Rechet al.2013). The over expression ofOsBISCPL1improved the resistance of plants to pathogens (Liuet al.2008). Therefore,Glyma.13g151100andGlyma.13g21640identified in this study,which encoded serine/threonine-specific proteins,might be the candidate genes for the resistance to SC18. Other genes detected in this study were not found to be associated with the resistance to SC18,but might participate in the resistance to SC18.These results require further studies to identify their roles in the resistance to SC18.

5.Conclusion

We further fine-mapped theRsc18gene in a RIL population which includes 168 lines generated from the cross of Zhonghuang 24×Huaxia 3 using composite interval mapping(CIM). Three candidate genes related to SC18 resistance were identified by genetic annotation and expression analysis. Our results will provide insights for the designing of molecular markers,improving molecular assisted selection and developing new accessions with resistance to SC18.

Acknowledgements

This work was supported by the projects of the Key-Areas Research and Development Program of Guangdong Province,China (2020B020220008),the China Agriculture Research System of MOF and MARA (CARS-04-PSO9),the Major Project of New Varieties Cultivation of Genetically Modified Varieties,China (2016ZX08004002-007),the National Key R&D Program of China (2017FYD0101500),and the National Natural Science Foundation of China(31971966).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm