Identification Of Genome-wide NBS-LRR Resistance Genes And Isolation Of NBS Resistance Gene Analogs In Radish(RAPHANUS SATIVUS L.)

Radish(Raphanus sativus L.,2n=2x=18)is annual or biennial root vegetable crop,belonging to the Brassicaceae family,which is widely cultivated in China.Radish plants are easy to be infected by various pathogenic bacteria in the growth and development process,resulting in low quality and deceased yield.Breeding new disease resistant varieties is an effective way to control the occurrence of radish diseases.In recent years,with the release of the whole genome sequences of radish,genome wide analysis of resistance genes has become a research hotspot.The disease-resistant genes containing the nucleotide binding site(NBS)are one of the most important disease-resistant gene familiesy,which play an important role in resisting pathogen infection.However,the genome-wide identification of NBS-LRR resistance genes and NBS resistance gene analogs remains to be explored in radish.In this study,the resistance gene candidate sequences of NBS-LRR class were identified on the whole-genome level in radish,and some candidate genes were cloned.Moreover,based on some previous studies,the degenerate primers of conserved domain of NBS were designed,which were used for isolation of radish resistance gene analogs.Furthermore,the expression pattern of some NBS resistance genes and homologous sequences were analyzed.The main results are as follows:1.A total of 188 NBS-containing disease-resistant candidate genes were identified in on the whole-genome level in radish.According to the fact that whether the C-terminal contains the TIR domain,188 genes could be divided into 128 TIR-NBS-LRR genes and 60 non-TIR-NBS-LRR(CC-NBS-LRR)genes.Among the 188 RsNBS genes,74(39.36%)were located on radish linkage group R1-R9.The distribution of RsNBS genes in the linkage group is relatively dispersed and the number is varied from each other.Some genes exist in the form of gene clusters.In this study,12 gene clusters were identified.R7 contains the largest number of genes(13 RsNBS genes)and R6 contains the largest number of gene clusters(3 gene clusters).The largest number of genes contained in one gene cluster existed in the R8(6 RsNBS genes).2.The evolutionary relationship study showed that the RsNBS genes have a close genetic relationship with AtNBS genes.The motif and gene structure analysis revealed that the NBS conserved domains were found in all the 188 radish resistance candidate genes,which also contained some conserved motifs such as P-loop,RNBS-A,Kinase-2,RNBS-B and GLPL.In addition,the conserved domains of TIR,CC and LRR were detected in the 188 candidate genes.The analysis of exon and intron structure revealed that the average number of exons of RsNBSs gene was 4.87,and the average number of exons of TNL and CNL RsNBSs was 5.59 and 3.33,respectively.A proportion of RsNBS genes(28.3%)of CNL class are encoded by an exon.In addition,the RsNBS genes of TNL class were composed of more than 2 exons,except for RsNBS 164.3.In this study,a total of 50 resistant gene analogs(RGAs)containing the complete open reading frame were isolated from radish using the degenerate primers,which were named as RsRGA01-RSRGA50.Phylogenetic analysis showed that 50 homologous sequences were divided into TNL and CNL classes,with 14 and 36 resistance gene analogs,respectively.They were further subdivided into RsRGA ?,RsRGA ?,RsRGA ?,RsRGA?,RsRGA ? and RsRGA ?.Sequence alignment and motif prediction showed that all sequences contain P-loop,RNBS-B and kinase-2 motifs.Among them,the similarity between RsRGA31 and AtRPMl was 89.77%,which inferred that the RsRGA31 might be homologous to RPM1 and has similar functions.Some RsNBSs genes identified in this study were cloned and verified the efficiency of the database.Further,three RsRGAs and four RsNBSs genes were selected for qRT-PCR analysis.The results showed that 7 sequences and genes(RsGA08?RsRGA34?RsRGA44?RsNBS021?RsNBS044?RsNBS062?RsNBS164)were differentially expressed in various radish varieties under different treatments.The qRT-PCR results indicated that these sequences and genes might play important roles in the resistant mechanism of defense signaling pathway in radish.