Screening And Whole Genome Sequencing Of The Mutants Of PI437654 Conferring Resistance To Race 4 Of Soybean Cyst Nematode

Soybean cyst nematode(SCN,Heterodera glycines Ichinohe)is a devastating pathogen in the production of soybean and causes a large quantity of annual yield loss worldwide.Three races of SCN,races 1,3,4 of SCN are the dominant races in China which often burst out in the soybean production areas.Races 1 and 3 of SCN are mainly distributed in the Northeast soybean-production areas,while race 4 of SCN majorly occurs in the Huanghuaihai soybean-production areas.Two major genes(rhg1 and Rhg4)conferring resistance to race 3 of SCN have been identified in soybean.However,no progresses have yet been achieved in the soybean genes underlying resistance to race 4 of SCN,to our best knowledge.In this study,we obtained 13 mutants exbibiting alteration of resistance to race 4 of SCN from an ethane methylsulfonate(EMS)-mutagenesis mutants population of soybean PI437654,which shows a broad resistance to almost all of SCN races.Then we sequenced three resistance genes of these 13 mutants and conducted whole genome re-sequencing subsequently for the purpose of identification of new gene(s)conferring resistance to race 4 of SCN in these PI437654 mutants.According to the obtained results,the optimized EMS concentration is 0.75% for PI437654.From the mutants population,many types of significant morphological phenotypes such as partial or entire chlorosis leaves,four-or five-leaflet,wrinkled leaves,color change of seeds,short internode of stem,late maturing were observed.The PI437654 mutant population was inoculated with race 4 of SCN for phenotype identification.We finally forward genetically screened and confirmed 13 mutants from the PI437654 population of 400 mutant lines,the rate of mutants with alteration of SCN-infection phenotype is 3.25%.Compared to the wild-type soybean whose female index is 5.8%,the maximum female index of mutants is up to 16.9%.These identified mutants were further verified that there were no changes in the genomic sequences of the three known SCN resistant genes,GmSHMT08,GmSNAP18 and GmSANP11 by enzyme digestion and sequencing.In addition,all of them were still resistant to race 3 of SCN similar to the wild-type soybean.Taken together,we can conclude that absolutely the novel gene(s)conferring resistance to race 4 of SCN has/have been mutated in these mutants.The whole genome re-sequencing of some of the mutants were performed subsequently.The primary anlyses showed that the SNPs causing nonsynonymous mutations in exons were about 4% while the InDels mainly occurred in intergenic regions in the pattern of single base insertion or deletion.We are continuing to mine the whole genome re-sequencing data and the results will contribute to the identification of soybean resistance gene against race 4 of SCN.Identifying the SCN resistant genes and understanding the molecular nature of soybean's resistance to SCN is increasingly important for the long-term management of this devastating nematode disease and can contribute to high throughput screening and molecular maker assistant breeding.The PI437654 mutants population developed in this study will be used as the new genetic soybean germplasm resource.