| Soybean cyst nematode(Heterodera glycines, Soybean cyst nematode, SCN), a worldwide soybean disease, caused a severe damage on the yield and quality of soybean all over the world. The resistance mechanism of soybean cyst nematode is complex. Therefore, it is becoming a new direction using genetic engineering and molecular marker-assisted selection in SCN-resistance breeing. In this study, we analyzed the secondary metabolites in roots and the distribution of resisitance loci Rhg1 and Rhg4 in susceptible and resistant soybean varieties using the predominant soybean cyst nematode race(SCN 4) in the Huang-Huai-Hai region, as well as the function analysis of resistant candidate genes selected from microarray analysis, in order to illuminate the mechanism of soybean SCN resistance.The main results are as follows:1. The isoflavone content variation in the roots of the susceptible and resistantsoybean varities inoculated the SCN4Five soybean cultivars, including the resistant soybean varieties(HPZ and WZH), the susceptible varieties(ZH13 and ZH35), and the standard susceptiable variety Lee, were used in SCN4 inoculation assay. The results showed that the isoflavone contents were induced to increase after SCN4 inoculation in the roots of all varieties, but the patterns were different.Isoflavone conents have been significantly increased in the roots of all varieties except for HPZ after SCN4 inoculation. Specially, the both resistant varieties HPZ and WZH showed the different inducing patterns, indicating that there were multiple resistant mechanisms to SCN4,and isoflavones induction may be involved in the partly resistance mechanisms in soybean.2.Resistant function of the allelic loci Rhg1 and Rhg4 to SCN4 in soybeanIn this study, twenty-two varieties with susceptible and resistant to SCN4, as well as a RILpopulation including 150 lines derived from the cross between HPZ(resistant cultivar) and JD23(susceptible cultivar) were used to determine the copy number of Rhg1 and haplotype of Rhg4 loci. The results showed that all resistant varieties and lines to SCN4 have multi-copy number in the locus Rhg1. Otherwise, all susceptible varieties from the 22 soybean cultivars were single copy in the locus Rhg1, while the copy number from the susceptible lines from RILs were different. In the locus Rhg4, we detected two novel SNP loci at 114 bp and 1165 bp from SHMT coding region in some resisitant varieties originated from China. Based on the results from both Rhg1 and Rhg4 loci to SCN4 resistance, we concluded that the loci of Rhg1 and Rhg4 are necessary, but not sufficient for SCN4 resistance, therefore, there maybe exist other important resistance genes to SCN4 in soybean.3.Preliminary analysis of candidate resistance genes to SCN4 in soybeanTo further verify the function of Rhg1 and Rhg4 genes and other candidate resistant genes screened from microarray analysis, we constructed nine RNAi constructs for candidate genes, respectively. Among them, genes Gm18g02590, Gm18g02580 and Gm18g02610 belong to the Rhg1 loci genes, GmSHMT is the encoding gene for Rhg4, while Gm06g14090,Gm09g08830, Gm05g28280, Gm18g44560 and Gm03g02870 are candidate resistant genes screened from microarray analysis. In this study, five genes were silenced using the RNAi method and transformed in soybean hairy roots, and the female index was also analyzed after SCN4 inocμLation. To date, the function of five genes(i.e. Gm18g02610, GmSHMT,Gm06g14090、Gm09g08830、Gm05g28280) has been performed. According to the results, the five candidate resistance genes can parcipitate in the resistance to SCN4. Especially, the silencing of GmSHMT caused significantly decreased the resistance to SCN4, indicating that Rhg 4 play an important role in the resistance to SCN4 in soybean resistant cv. HPZ. |
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