Cloning And Functional Analysis Of A Soybean Cyst Nematode Race 4 Resistance Candidate Gene GmSNAP11 In Soybean

Soybean cyst nematode(SCN,Heterodera glycines Ichinohe)causes an estimated annual loss valued at billions of dollars worldwide.Among management strategies for SCN,host plant resistance is the most feasible option and will be easily accepted by farmers.Identification of gene or locus controlling SCN in soybean is of importance because of the devastating effect of the disease.Next-generation sequencing(NGS)-based bulked segregant analysis(BSA)approaches have dramatically accelerated the course of identifying causal genes and has been used for the discovery of novel disease resistance genomic regions or genes in some legumes.The development of methods that can be used for the rapid mapping of genes that contributes to SCN resistance is essential.The present study employed this approach for the discovery of novel genomic regions,candidate genes and diagnostic markers for SCN race 4(SCN4)resistance in soybean breeding programme.In the current study,we developed RIL populations from a cross between JD23 and HPD,with susceptibility and resistance to SCN race 4,respectively.The reaction of 145 RIL population to SCN was determined using a female index.The RILs were genotyped for rhg1 and Rhg4 and two bulks were generated from RILs with rhg1~+Rhg4~+background with 15 RILs each for susceptibility(S-bulk)and resistance(R-bulk)to SCN.About 5?g of DNA from the two bulks and the two parental lines were used to construct paired-end sequencing libraries,which were used for sequencing.The two bulks were re-sequenced separately and homozygous SNPs between parental lines and high-quality SNPs were selected for Euclidean distance and SNP-index analysis.We selected SNPs with non-synonymous splice site acceptor and donor,start and stop codon and gained a loss.By comparing the ED and?SNP-index results,an overlapped genomic region spanning a 5.08 Mb on chromosome 11 was identified underlying SCN4 resistance.Annotation was performed for the genomic region on chromosome 11.The nonsynonymous SNPs and the SNPs affecting splice sites and start or stop codons were taken to the subsequent screening in which the SNPs with different genotypes between resistant parent and bulk were filtered out.The 15SNPs belong to 12 annotated genes.Finally,two genes Glyma.11g234400 and Glyma.11g234500 with high similarity with Glyma.18G022600 and Glyma.18G022500 in rhg1 were found.Glyma.11g234400and Glyma.11g234500 were consistent with their paralogs,Glyma.18G022600 and Glyma.18G022500,and encodes(Z)-gamma-bisabolene synthase 1-related protein a homolog to PLAC8 family protein and?-SNAP,respectively.Moreover,the genomic region on which Glyma.11g234400 and Glyma.11g234500 were located showed high collinearity with rhg1,the major SCN resistant locus,therefore,this region was designated as rhg1-paralog and the genes were named GmPLAC8 and GmSNAP11,respectively.The identified candidate genes were further validated by overexpression and RNAi studies using hairy root transformation.The phenotypic analysis revealed significant differences(p<0.0001)between all the recombinant inbred lines(RILs)for their reaction to SCN4.Two putative candidate genes were identified in this region and only Gm SNAP11(Glyma.11g234500)showed a significant contribution to SCN4 resistance similar to GmSNAP18.The other gene,Glyma.11g234400(GmPLAC8)did not show a clear contribution to SCN4 resistance.The GmSNAP18 and GmSNAP11 genes are usually clustered together which suggests that they are highly related.However,the GmSNAP18 is more related to the ancestral SNAPs.Among the GmSNAPs,GmSNAP11 and GmSNAP18 are ubiquitously highly more expressed in most tissues and under SCN infection,GmSNAP18 transcripts are the most highly upregulated,followed by GmSNAP11.Furthermore,the GmSNAP18 and GmSNAP11?-SNAP are the major sources of total?-SNAP proteins in soybean.Taken together,considering the shared similar features between GmSNAP11 and GmSNAP18 we proposed that the mechanisms of action by which the GmSNAP11(rhg1-paralog)confers resistance to SCN maybe through similar action with Gm SNAP18(rhg1).The diagnostic marker developed for the identified gene(GmSNAP11),Rhg4 and rhg1 were applied to a set of 145 RILs and the results showed that the RILs having all three resistance loci had an elevated level of resistance than the RILs having a combination of any two resistance loci.Moreover,we have identified the resistance loci in 1600 soybean accessions based on KASP markers to explore their distribution characters in China.The identified gene and diagnostic markers can be used in genomic assisted breeding for the development of varieties with elevated resistance to SCN.