Cloning And Analysis Of Resistance Gene Analog Sequences (RGAs) From Banana Germplasm Resistant To Fusarium Wilt

Banana fusarium wilt disease,also known as Panama disease,was a devastating disease caused by a soil fungus,which has been threatening banana production seriously around the world.The method of resistance gene analogs(RGAs) analysis based on sequence homology has been a quick and effective way to clone the R genes.Degenerate primers were designed based on conserved amino acid residues of plant resistance gene product and used to amplify genomic DNA and cDNA sequences.The obtained RGAs can be used to select the candidate resistance gene,and specific oligonucleotide primers can be designed to develop PCR-based markers such as CAPS or STS for evaluation of genetic linkage between RGAs and R gene loci.It is also of importance for the elucidation of the evolution relationship among the germplasm.In this study,the RGAs were isolated from genomic DNA and cDNA of ROSE and GCTCV-119 that are resistance to the fusarium wilt.The main results were as follows:1.Oligonucleotide primers of various degrees of degeneracy have been used to amplify RGAs.The majority of them were based on conserved motifs in the NBS domain of the NBS-LRR class R genes.Nineteen fragments of RGAs including 9 DNA sequences and 10 cDNA sequences were isolated from GCTCV-119,and their sizes were of expected size(about 500 bp).Analysis of the deduced amino acids of these RGAs showed that their domain structures were NB-ARC and belonged to NBS-class resistance gene candidates,containing 4 conservative amino acid domains of P-loop (GMGGVGKTT),Kinase-2(LLVLDDIW),RNBS-B(CKVLFTTRS) and hydrophobic amino acids(GLPLALKVL).These 19 sequences,designated as BR-1, BR-2,to BR-19,have been depositled in GenBank with accession numbers EF515833-EF515836,EU123871-EU123885;2.Four degenerate primers were designed according to 9 domains of PK-class resistance gene.Twenty fragments of PK-class RGAs including 14 DNA sequences and 6 cDNA sequences were isolated from ROSE and GCTCV-119,with sizes approximately 530 bp.Conservative domain analysis of their deduced amino acid sequences showed that the structure was the domain S_TKc,belonging to serine/tryptophan protein kinase family members.Four functional domains were present in the sequences,the ATP binding pocket,the substrate binding pocket,the catalytic loop and the activation loop. They contained nine highly conserved amino acid regionsâ… .FG(K/V/L/S)VY(K/R) G;â…¡.VAVK;â…¢.FxxE;â…£.(L/V/I)Vx(I/V/L);â…¤.ALV;â…¥.D(LI/V)K;â…¦.DFG; â…§.G(T/S)xGY(L/I/A)PE;â…¨.D(Y/I)YS(F/Y)G(V/I/M).These 20 sequences were named as Mu-PK1,Mu-PK2 to Mu-PK20,and have been deposited in GenBank with accession numbers EU293391-EU293404(DNA fragment),EU338457- EU338462 (cDNA fragment);3.According to the banana RGAs,seven pairs of NBS-class specific primers and two pairs of PK-class specific primers were designed.No significant difference was found in the genomic DNA sequences within 37 bananas.PCR-RFLP analysis of six resistance germplasms and five susceptible germplasms showed that the marker Mu-NBS5/Haeâ…¢was able to distinguish the genotypes among the eleven banana germplasms.4.In order to understand the difference in the RGAs between the germplasms resistant or susceptible to fusarium wilt,three pairs of NBS-class specific primers and one pair of PK-class specific primer were used to amplify the eleven banana genomic DNA.A lot of differences were identified on the sites of restriction endonuclease and changes of single nucleotide polymorphism(SNP) among the eleven banana germplasms.