Development And Application Of SNP Markers Of Candidate Resistance Gene Rhg4 And GmHs1^pro-1 And Discovery Of New Resistance Gene For Soybean Cyst Nematode In Soybean

Soybean cyst nematode（SCN）,Heterodera glycines,is a seriously destructive pest in soybean production worldwide and causes great yield loss every year.Planting resistant soybean cultivars has been an economical and effective method to decrease or avoid its damage.However,narrow genetic vulnerability emerged due to incorporating resistance genes from only a few resistant materials into soybean cultivars.Therefore mining novel resistant gene or novel resistant germplasm is an effective strategy to increase genetic diversity for SCN resistance.In this study,we evaluated genetic diversity of some domesticated soybean materials mainly from mini core collection,applied core collection resistant to SCN and new released varieties and some wild soybean at two candidate resistant genes of Rhg4 and GmHs1^pro-1,in order to properly utilize of soybean germplasm in soybean improvement.Bioassay of SCN resistance is an important procedure in genetic analysis or breeding for SCN resistance,but it is labor intensive and time costly.Relatively molecular marker assisted selection（MAS）has several advantages such as rapid,less costly,less environmental variation,and high efficiency,however molecular markers associated with SCN resistance recently reported have low accuracy,strong dependence on genetic background in MAS.Markers in genes conferring resistant to SCN maybe have greater accuracy,so we converted 8 SNPs in Rhg4 and GmHs1^pro-1into SNP markers,developed a rapid,efficient method for SNP genotyping,and even investigated their relationship with SCN resistance,meanwhile 41 new soybean lines were genotyped with the 8 SNP markers as well as 130 SSR ones.The main results were as follows:1.Development of a new method for SNP genotyping in soybean,named fragment length discrepant allele specific PCR（FLDAS-PCR）.Two allele-specific primers which had 4⁵bp length difference,and a common primer were necessary in the method for typing one SNP site,as a result PCR products of different alleles（with about 100bp or 150bp）had 4⁵bp difference in length,which could be separated by 6%denature polyacrylamide gel.An additional base mismatch was introduced at the 3^rdd or 4^th nucleotide closest to the 3’-end of each of the specific primers and multiplex PCR was employed in order to improve PCR specificity and data accuracy.The genotypes of SNPs by the FLDAS-PCR agreed with the results by PCR sequencing for 24 soybean genotypes.It was approved that the method was simple,rapid and effective in SNP genotyping in soybean,and we had genotyped 8 SNP loci of Rhg4 and GmHs1^pro-1 of the soybean germplasm.2.Illustrating of DNA diversity of Rhg4 and its distribution in soybean germplasm.Rhg4 had high diversity,and higher diversity was found in domesticated soybean than that in wild one.Significant difference for distribution of Rhg4 at the SNP site 1592,1724,1760,2120 and 2387 was tested among wild materials,landrace and varieties（lines）or between varieties and core collection,which will be useful for adjustment of core collection and increasing genetic diversity with wild germplasm or landrace.Distribution of the 5 SNP sites mentioned above between resistant materials and susceptible ones to SCN1,SCN3 and SCN4 indicated that Rhg4 takes a role in soybean resistance to the 3 races of SCN.3.Illustrating of DNA diversity of GmHs1^pro-1 and its distribution in soybean germplasm.GmHs1^pro-1ro-1 was obtained with the complete gene sequence of 1477bp.Compared with Rhg4,it had slightly lower DNA diversity and obviously lower diversity in domesticated soybeans than wild ones was found,which was probably caused by selection.Significant difference was also discovered for distribution of GmHs1^pro-1ro-1 at SNP site 177,1036 and 1380,which was helpful for incorporated wild genotypes into soybean breeding.However,we didn’t find significant difference for distribution of the 3SNPs between resistant germplasm and susceptible one to SCN1,SCN3 and SCN4,which suggested that GmHs1^pro-1ro-1 maybe not responsible for soybean resistance to SCN.4.Discovery of a new locus and two SSR markers associated with SCN resistance for MAS of SCN resistance.41 new soybean lines from the cross of Hartwig by Jin1261,Jin1265 or Jin1267 were genotyped with 8 SNP markers developed in the dissertation from Rhg4 and GmHs1^pro-1 and 130 SSR markers.We found that Hartwig had different resistant gene,comparing with the Jin1261,Jin1265 or Jin1267.23 SSR loci were conferring soybean resistance to SCN4,resistant allele at 6 SSR loci was from Hartwig,at the other 17 SSR loci,resistant allele was from Jin strains.3 SSR loci on LG D1b was detected associated with resistance to SCN4,there was no report about QTL for SCN resistance on LG D1b by now,therefore we supposed that Jin strains had at least a new resistant gene on LG D1b.We could differentiate all susceptible lines from resistant ones with the two SSR markers Satt684 and Sat₄00,and had 96.8%accuracy for identification of resistant lines.