Molecular Detection Of Resistance To Cyst Nematode Race 3 In Soybean

Soybean cyst Nematode (SCN) (Heterodera glycines Ichinohe) is a kind of soilborn sedentary endoparasite nematode and the pathogen of soybean chlorsis. It has over ten physiological races. It is economically the most destructive pathogen affecting soybean culture. SCN Race 3. 4 are the predominant races that are the most damaging pathogens in ChinaJn this study, eight conventional identified soybean varieties (including 6 nematode resistant varieties to to SCN race3, 2 susceptible varieties to SCN race3) and thirty-two soybean varieties (30 cultivars and 2 wild soybean varieties) without conventional identification were tested. According to the declared sequences of cyst nematode resistant gene in sugar beet and soybean, four forward primers and four reverse primers were designed, 16 pairs of primers were composed, and a pair of primers was selected among them by PCR special amplification. Amplification conditions for PCR in soybean were optimized, the results showed that a special fragment of about 600bp was amplified, and PCR-Southern blotting was further made in order to affirm the similarity and veracity. Then, the special fragment were cloned, sequenced and compared in GenBank by Blast software. Finally, 30 commerical cultivars of soybean and two wild soybean varieties were primarily detected by PCR. The results were summarized as followed:1. An optimal reaction system for PCR in soybean was established: the PCR volume was 25祃 and contained the optimal composition included SOng of template DNA, 10mMTris-HCl(pH8.3), 50mM KC1, 2.0mM MgCl2+, ISOuMdNTPs, 0.4礛 primers, 1.5U Taq polymerase. The PCR reaction program was devised for 1 cycle of pre-denaturation at 94 C for 5min, 35 cycles of 25s at 94C (denaturation), 30s at 59C (annealing) and 60s at 72C(extension).in the last cycle,the reaction was kept for an additional 10 min at 72 C then cooled to 4 C.2. A pair of primers, P1P5, were obtained for molecular detection of cyst nematodeRace3 in soybean. The results of PCR detection in soybean generally verified these of conventional identification, identify proportion was 100% by the pair primers. The feasibility of PCR detection was further testified by PCR-Southem blotting.3. Among 32 soybean varieties without conventional identification, five varieties appeared a special fragment of about 600bp: Dongnong 43,Dongnong40S67, Dongnong-410, Youwudou and wild soybean variety II. The results need be tested further by field identification.4. Cloned and sequenced special fragment, compared similarity with all the sequences in GenBank, EMBL, DDBJ, PDB by Blast. The result showed that it has 98% similarity with Glycine max receptor-like kinase RHG4 (Rhg4) gene (GenBank accession NO. AF506518) and 96% similarity with O.sativa mRNA for leucine rich repeat receptor-like kinase (GenBank accession NO. Y07748). At last, direct submission of sequence data to GenBank, GenBank accession number for the nucleotide sequence is AY580161.