Evolutionary Analysis And High-throughput Cloning Of Bacterial Blight Resistance Genes In Rice

Bacterial blight disease is the most devastating bacterial disease to rice worldwide.What's more,it could not be effectively defeated by traditional chemical methods.It is generally considered that using internal resistance genes in rice to resistance breeding could be the key.However,to defend the diversified and fast evolving pathogens,durable resistance,which could be produced by durable resistance genes or the combination of a set of resistance genes,is requested.Quick selection and cloning of a number of resistance genes underlies the realization of these strategies.Traditional methods to identify resistance genes are a time-consuming effort.Thus,try to decipher the molecular mechanism and evolutionary patterns of plant resistance genes and discover new efficient methods to identify resistance genes are urgently needed.Resistance genes of bacterial blight disease in rice exhibit diverse types,three of which are LRR-RLK,NBS-LRR and SWEET genes.LRR-RLK and NBS-LRR genes are large families in rice and drawn a lot of attentions.In previous studies,we had conveyed detailed analysis about NBS-LRR and LRR-RLK genes.As to another kind of resistance gene,SWEET genes,the evolution patterns underlying resistance remains unclear.In the present study,SWEET genes were identified in 28 well-sequenced plant genomes,which showed remarkable expansion and diversification from aquatic plants to land plants.Phylogenetic reconstruction of SWEET genes from 28 species was performed using maximum-likelihood(ML)method and obtained phylogenetic tree was diversified into four clades as previously reported.These clades were further classified into 9 monocot and 10 dicot SWEET gene families.Estimation of evolutionary rates within gene families illustrated two distinct types of SWEET members in monocots,namely,conserved and fast evolving SWEET genes.The conserved SWEET genes exhibited copy-number uniformity,clear orthologous relationship or low evolutionary rates,whereas the fast evolving genes showed an obscure orthologous relationship,with either highest nucleotide variation or positive selection.What's more,alleles of three rice SWEET homologs in one fast evolving monocot family,have been characterized to confer resistance to bacterial blight disease in rice.Finally,population analysis of 368 whole-genome sequenced rice accessions reveals that some SWEET loci were potentially involved in rice domestication process.Our research serves as the first comprehensive analysis of SWEET genes in various plant species,ranging from lower aquatic algae to higher terrestrial plants.The expansion and diversification of SWEET genes,as well as its distinct evolutionary patterns,adequately explain its functional diversity and redundancy.Besides,our results also lay the foundation for exploiting resistance(R)genes from transporters,indicating that large copy number variations,high genetic variations,and positive selection could be signature of functional R-genes.A new method,which called high-throughput cloning of resistance genes,was recently promoted,which based these chacteristics of resistance genes.We had applied this method in the cloning of NBS-LRR resistance genes and gained good achievement..In the present research,according to the high-throughput cloning method,we further selected 82 candidate LRR-RLK and 6 SWEET loci,cloned them from different rice accessions with diverse resistant to bacterial blight disease and linked them with binary vector.As to LRR-RLK genes,we finally completed 32 loci and constructed 119 recombinant plasmids.The cloning work of SWEET gene are going on and we had obtained 10 recombinant plasmids.Then we transferred these plasmids to susceptible rice cultivar,TP309,and at last 98 transgenic rice plants of LRR-RLK genes were obtained.What's more,in a previous reported research,we have successfully obtained 160 transgenic TP309 rice plants with different NBS-LRR genes applying the same method.Thus,we estimated the resistance of these transgenic TP309 rice of LRR-RLK and NBS-LRR genes with five diverse races of bacterial blight diseases,respecti vely.19 NBS-LRR lines showed obvious resistance,containing 17 loci.And 5 NBS-LRR lines exhibit obvious resistant to more than one races.In addition,out of the preliminary estimation of 70 surveyed LRR-RLK lines,30 lines showed the improvement of resistance to one or more races,and 12 lines showed resistance improvement to more than one races.These results again proved the efficiency of high-throughput cloning method.