High-throughput Cloning Of Rice Blast Resistance Genes

Rice(Oryza sativa)is one of the most important food crops in the world.However,rice blast has been one of the most serious diseases,and leads to reduced yield in rice production.Because of the constantly emerging new physiological races,as well as the rapid speed of evolution and diversity in M.grisea,many rice cultivars may lose their resistance within a few years.Therefore,it is extremely difficult to control the disease effectively.Breeding the rice cultivars with resistance to the disease could be one of the safest and most effective ways to solve this plight,and it needs plenty of resistance genes as resources to defend against a variety of pathogens.Therefore,it is particularly important to develop a high-throughput cloning method.The major class of plant resistance genes is NBS-LRR genes.Most of the cloned rice blast resistance genes also encode NBS-LRR type proteins.NBS-LRR genes share the similar genetic characteristics,such as high diversity,clustered or within a gene family,under positive selection,frequent recombination and so on.The common features of the resistance genes provide the theoretical basis for the establishment of high-throughput cloning technology.Following this assumption and basis,we searched for the potential resistance genes by a genome-wide investigation of NBS-LRR genes and analyzing cloned rice blast resistance genes.Then,we chose the candidate resistance genes which were located in the specific regions on the phylogenetic tree,or in the gene families and gene clusters,or had a close relationship with the well-known functional resistance genes.The blast-resistant rice cultivars were used as resources to clone the candidate genes.These candidates were then introduced into the susceptible rice cultivars and the transformants were screened by the rice blast fungi to assess their resistance.We selected 147 candidate resistance gene loci,chose highly resistant rice cultivars as supplies,used long-PCR and other molecular techniques to clone the candidate genes and then linked them to the binary vector.We have completed 132 loci and gained 334 gene vectors.We then transferred the gene vectors to the susceptible rice cultivars by Agrobacterium-mediated transformation.All the 334 gene vectors were transferred to at least one receptor and 286 transgenic rice plants were obtained.The transformed rice plants were then inoculated with different rice blast fungi combining three inoculation methods,in vivo inoculation,in vitro leaf and panicle blast inoculation.Out of the 185 lines which have been screened,18 lines showed obvious and high resistance,containing 18 gene vectors and 16 gene loci,while 45 lines showed resistance,containing 41 gene vectors and 31 gene loci.In total,at least 9.7%transformed lines and 25.0%genes showed obvious resistance after blast screening.The results demonstrate the feasibility and efficiency of our high-throughput cloning technology.In this research,we successfully established a new technology of high-throughput cloning rice blast resistance genes.We found a group of rice blast resistance genes with important values and greatly enriched the resistance gene resources.Furthermore,our technology provides an example and guidance for cloning other plant disease resistance genes.