Identification Of Candidate Gene And Development Of CAPS Markers Conferring Resistance To Soybean Cyst Nematode

Soybean cyst nematode(SCN, Heterodera glycines Ichinohe) is one of the most economically destructive pathogens of soybean [Glycine max(L.) Merr.], resulting in substantial yield losses worldwide. Planting resistant cultivars with crop rotation system is the most effective and economical way of controlling the damage caused by SCN in soybean production. Several of resistant accessions have been widely used in the improvement of modern elite accessions with SCN resistance, such as Huipizhiheidou or Peking. Substantial efforts have been made in the last two decades to clarify the genetic bases of resistance to SCN using linkage mapping approach. As a result, more than 160 QTL have been identified(www.soybase.org). Owing to the complex genetic architecture of resistance to SCN, including two major loci(rhg1-b and Rhg4) cloned recently. Pyramiding different resistance genes is necessary for increasing the durability of resistance to soybean cyst nematode(SCN, Heterodera glycines Ichinohe), the most destructive pathogen in soybean. To understand the genetic basis of resistant accessions and provide the information for developing elite resistant accessions using marker assistant selection(MAS) strategies, a panel of 768 SNP loci, selected based on resequencing genotypic data within the previously reported QTL regions, were used to genotype one diversity panel with 209 resistant or susceptible accessions, and one RIL population with 242 lines in this study. The objectives were(1) to detect quantitative trait nucleotide(s)(QTN) and putatively favorable alleles associated with the resistance to SCN race 3 by lingkage mapping and association mapping approaches;(2) to confirm the QTL previously identified in high mapping resolution;(3) to find novel QTNs controlling resistance to SCN race 3; and(4) to develop CAPS markers/genes for improving resistant cultivars and broadening the genetic base of soybean breeding.The major results were:(1) SCN race 3(SCN3) resistance was screened in an association mapping panel comprising 209 accessions and a recombination inbred lines(RIL) population of 242 lines derived from the cross Zhongpin 03-5373(ZP) × Zhonghuang 13(ZH). Parents of the RILs showed significant differences with respect to their resistance to SCN3. ZP was extremely resistant(immune, female index(FI) = 0) and ZH was highly susceptible(FI=239). FI(%) values of the association panel and the RILs showed large genetic variations with continuous distributions. The frequency(11.6%) of resistant RILs(FI<10, 28 lines) was lower than that(78.1%) of susceptible RILs(FI≥60, 189 lines), whereas most of accessions in the association panel(63.2%, 132 accessions) were resistant to SCN race 3. Many of the resistant accessions were landraces suggesting a broad genetic base of the resistance within the association mapping panel.(2) In total 39 SSR and 14 SNPs distributed across 20 soybean chromosomes were selected to infer the population structure in the association panel. The ad-hoc quantity based on the second order rate of change in the log probability(ΔK) indicated the presence of two genetically distinct subpopulations with a limited evidence of admixture among them. This finding is in accordance with the clusters displayed in the neighbor-joining tree. The two subpopulations corresponded largely to the geographical distribution of the accessions. Marker-trait(FI) association was analyzed for 585 SNP loci using TASSEL 4.0(http://www.maizegenetics.net). We applied a mixed linear model(MLM) method correcting for population stratification with the membership(Q matrix) and kinship(K matrix). A total of 28 significant(p<0.001) marker-trait associations were observed. Each marker-trait association explained 4.8-10.6% of the variance of SCN resistance(FI). All of these QTNs were nonsynonymous substitutions from 23 annotated genes, except for Map-5030 which is located in an intergenic region. In regions of the Rhg4 and a paralog of rhg1-b in Gm11 we detect the clustering of QTNs. The three strongest QTNs identified by association mapping involved the genes Glyma18g02590(a component of the multigene locus rhg1-b), Glyma11g35820 and Glyma11g35810(an rhg1-b paralog).(3) A new soybean linkage map was constructed using 1,062 molecular markers, including 556 newly developed SNPs and 506 published markers(312 SNPs, 166 SSRs, 4 EST-SST and 24 InDel). The linkage map spanned 2,990.7 cM across 20 soybean chromosomes with an average interval of 2.97 cM between mapped adjacent markers. A total of 11 gaps spanning more than 20 cM and rearrangements of marker orders were detected in some genomic regions. Among the 1,062 markers, 317 markers in 69 genetic loci were co-segregating. The number of markers co-segregating in each locus ranged from 2 to 42 with an average of 4.6. The genome-wide composite interval mapping(CIM) revealed the presence of two putative QTL(SCN3-18 and SCN3-11) underlying SCN3 resistances. Both of these two QTLs, one mapping to rhg1-b and the other to an rhg1-b paralog were positioned at known QTL regions, GR18 a and GR11 respectively; both of these loci were also identified by the association mapping. The former locus explained 25.5% of the phenotypic variance for SCN resistance and the latter 5.8%. The resistant alleles for QTLs SCN3-11 and SCN3-18 were both from parent ZP. The SCN3-18 was a major QTL which explained the largest proportion of phenotypic variation of SCN resistance(25.5%) with the largest logarithm of the odds(LOD) score of 15.87. The additive effect of SCN3-18 was 28.97%. The additive by additive effect is 36.86%, which indicated that with the presence of both resistant alleles, the SCN can be further resisted.(4) The above identification and previous reported SNPs confer SCN resistance or susceptibility to develop molecular marker which have the advantages of a simple and easy operation such as CAPS, dCAPS. One CAPS marker(Rhg4-389) and one dCAPS marker(Rhg4-1165) were developed based on the polymophism on these two SNPs(Rhg4-389-G/C and Rhg4-1165-T/A) of GmSHMT respectively. Based on the QTN(Map-5428) of rhg1-b, one CAPS marker(rhg1-2590) were developed. Then, 193 soybean cultivars were genotyped by Rhg4-389 marker, Rhg4-1165 marker, rhg1-2590 and Satt309 for MAS. Rhg4-389-G, Rhg4-1165-T and rhg1-2590-G alleles mainly occurred in resistant cultivars, and almost coincided with Rhg4-389-G/Rhg4-1165-T haplotype, screened out 88.2% of the resistant genotypes and rhg1-2590-G were 92.9%. Genetic basis of 193 soybean cultivars can be distinguished in the rhg1-b and Rhg4 by using the four markers, meanwhile, 6 germplasm can be detected with new resistant gene.