Cloning And Mapping Of NBS-LRR Type Resistance Gene Analogs In Cotton (Gossypium Barbadense L.)

In this research, two degenerate primers were designed based on the NBS (Nucleotide Binding Site) conserved motifs of the genes of N, L6, and RPS2. Specific fragments of 517bp were amplified from genomic DNA of cotton (Gossypium barbadense). Eight hundreds of clones were obtained by inserting the target PCR products into pGEM-T easy vector and transforming into E. coli DH5 a compatible cell. These candidate RGAs (Resistance Gene Analogs) were analysed by endonulease digestion and a pair of test/reverse primer identification, the clones were classified into five groups according to the result of four base endonulease digestion. There are 11 desired RGAs obtained after sequencing, which the length are from 500bp to 527bp, three of these RGAs contained 1 or 3 terminate codon. Sequence analysis showed that all RGAs contain four conserved motifs of NBS, which include kinase-la domain, kinase-2 domain, kinase-3a domain and transmembrance domain and are closely identical to those known NBS type R gene, such as N, Xal, L6 et al..The cloned RGAs in this research have high similarity to those R gene analogs cloned from Glycine max, Gossypium barbadense, Gossypium. hirsutum, Phaseolus. vulgaris. Putative amino acid sequences deduced from eight RGAs have from 25% to 58% homology compared to the Xal, N, RPS2, L6 , RPM1, Pib, I2C-2, Prf.In this research, F2 populations derived from G. barbardens var. X G. hirsutum were used for mapping those RGAs by RFLP (Restriction Fragment Length Polymorphisms) method. The results showed 7 RGAs have been located in cotton genome. Gbrga2 and Gbrga8 were mapped in linkage group D02 of homology 11 at the start point, which is the same site of maker pAR286E4C and is 5.6cM away from Unig22d03bE6D marker. In addition, Gbrga2 and Gbrga8 were mapped to linkage group A03 at the start point, which is the same site of maker pAR111E3C. Gbrga2 and Gbrga8 were also mapped to linkage group A03 at 59.2cM from the start point and is 1.7cM and 8.6cM away from Unig26B04E5D(22D) and Gate4DB08bE3D(14D) marker, respectively. Others of 5 RGAs were mapped in homology 4. Gbrga508 and Gbrga535 were mapped to chromosome 20(D) at 175.3cM from start point and are 2.7cM and 3.8cM away from Unig27A04E5C and M16-118E1C marker, respectively. Gbrga508 and Gbrga535 are also mapped in chromosome 05(A) at 142.9cM from start point and are 2.6cM and 1.2cM away from G1025aE5C and Gate2CFllE3C marker, respectively. Gbrga522, Gbrga568 and Gbrga39 were mapped in chromosome 20(D) at 5.5cM from start point and were 1.0cM and 2.0cM away from Unig24H03E6C and P13-6E3D marker, respectively.It is more benefit to understand the molecular evolution and origin of NBS type R genes, to develop new maker for MAS and to isolate the R related genes based on these mapped RGAs from cotton.