| Verticillium wilt has been one of the most severe disease of cotton. Upland cotton is the main cotton variety cultivated in China, but poor resistance to Verticillium wilt. Application of molecular biology techniques on the study of Verticillium wilt play an imptortant role in improving resistance breeding of cotton. Cloning disease resistance gene is a significant way for resistance breeding and resistance mechanism. Cloning the resistance gene analogs from cotton using the homology-based candidate gene method which design primer and amplify the resistance gene analogs based on the conserved domain of resistance gene, and then adding them to linkage maps as molecular marker or making them probes to scanning genomic library in order to obtained the candidate disease resistance genes, is a shotcut for cloning disease resistance gene.In this study, we identify the resistance to Verticillium wilt of nine varieties of upland cotton. We identify the resistance to Verticillium wilt of the nine varieties of upland cotton with a susceptible control in disease nursery during three years. Three of them are high resistance, two are resistance, two are tolerance and two are susceptible. The three high resistance varieties have a very high degree of resistance to Verticillium wilt and the incidence is lower than 20%. The resistance of these varieties is in line with expectations. The two susceptible varieties have a very high degree of susceptible to Verticillium wilt comparing with the control and the incidence is higher than 80%. show There were no significant differences in the relative disease index of all the varieties of upland cotton between the three years. The resistance of them is steady.A specific fragment of about 500 bp was cloned from the genomic DNA of the nine identified upland cotton by PCR degenerate primer designed according to the conserved domains P-loop and GLPL in the NBS region of reported R genes. The fragments are recycled and inserted into pGM-T vector, and then transformed into E. coli DH5α. 350 recombinations were obtained through colony RPC and restriction digestion identification. The clones are sequenced and 74 RGAs with complete open reading frames (ORF) from eight varieties of cotton are finally obtained. Comparing with the reported R genes, the 74 RGAs all contain P-loop (Kinase-1a), Kinase-2, Kinase-3a, GLPL region, and motif RNBS-A, B, C defined by Meyers. Cluster analysis of their putative amino acid show that the RGAs sorted into two distinct types, TIR-NBS type and nonTIR-NBS type. The TIR type RGAs all contain the RNBS-A-TIR motif while the nonTIR type RGAs all contain RNBS-A-nonTIR motif. This result consists with the early report that NBS-LRR gene has two major groups in dicotyledon. The deduced amino acid of the RGAs have high similarity with the reported R genes. NonTIR type RGAs have a similarity of 32%～50% with L6,M,Gro1-4,N, while TIR type RGAs have a similarity of 23.2%～56.5% with I2C-1,Mi-1.1,RPM1,RPP8,RPS2,RPS5,XA1,PRf. By the intercomparison of some nonTIR type RGAs, we find that there are two site mutations between resistant cultivar and susceptible cultivar. The deduced amino acid of the two sites are both isoleucine (I) in resistant cultivar, while threonine in susceptible cultivar. The mutations of the two sites may be associated resistance and susceptible traits of Verticillium Wilt. Analysis of molecular variance (AMOVA) indicated that the genetic variation mainly exist within varieties rather than among varieties. Tajima's test show that both Tajima's D of the two populations don't reach significant level, and accord to the Neutral Theory.The research of these RGAs will provide with theories and experiment data for isolating related resistance gene of cotton and develop new markers for Marker Assistant Selection. And also, it will be benefit to understand the origin and evolution of NBS-LRR type R genes. |
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