Detection Of Genome-wide Variations In Common Wild Rice And Its Inbred Lines To Reveal Resistant Genes

Common wild rice(Oryza rufipogon Griff.)is a valuable genetic variation resource for rice genetic improvement,which can provide various resistant gene alleles.However,their high genome heterozygosity limited sequencing precision for using elite genes in molecular breeding.Hence,we obtained two stable inbred lines from common wild rice S24,namely S18 and 11352.We firstly observed the agronomic trait and evaluated the resistance to rice blast and brown planthopper,and then we re-sequenced the genome of S24,S18 and 11352 to reveal the genome variation characteristics and potential resistant gene alleles.The results are as follows:(1)Observation of agronomic traits and assessment of resistance.We observed the agronomic traits in mature stage,S18 and 11352 showed significant differences in plant height,flag leaf length/width,main panicle length,productive panicle number,seed setting,seed length/width,total grain weight and 1000-grain weight.The plant and productive panicle number of S18 were 60.24 cm and 15.08,while thar were121.15 cm and 6.65 in 11352.Resistance evaluation indicated S18 and 11352 showed high resistant levels to rice blast(resistant level 2.0 in S18 and 2.7 in 11352);Brown planthopper resistance evaluation indicated that S18 were highly(resistant level 3)a nd 11352(resistant level 3)was moderately resistant.(2)Evaluation and filteration of re-sequencing data and variation distribution.After quality assessment of the re-sequencing data and filteration of the low quality data,we obtained 77907543,95453868 and 75256129 high quality reads covered(coverage depth >= 5X)93.57%,92.76% and 89.99% of the reference N ipponbare genome with 44 X,65X and 42 X average coverage depth,respectively.Four categories genome-wide variations were detected in S24,S18 and 11352,namely single nucleotide polymorphisms(SNPs),insertions and deletions(InDels),structure variations(SVs)and copy number variations(CNVs).The numbers of SNPs were 1804889,1349511 and 1686727,SNP heterozygosity were 54.71%,11.64% and 11.35%,respectively,the genome hetrozygosity were significantly decreased in S18 and 11352.The numbers of InDels were 444968,333914 and 391597,majority of the insertions and deletions were in the length of 1-9bp.The numbers of SVs were 6489,9728 and 7863,which spanning 1990,3189 and 2115 variant genes,and 66.13%,56.16% and 64.82% of these genes were belonging to transposon element.The numbers of CNVs were 90,99 and 109,which spanning 4076,2535 and 3258 genes,and 58.56%,53.53% and 53.13% of these genes were belonging to transposon element.(3)Detection of resistance genes in S18 and 11352.16692,12248 and 14434 large-effect variant genes in S24,S18 and 11352 were annotated by the GO?COG?KEGG and Pfam database.In S18 and 11352,we identified 194 and 245 NBS-LRR variant genes,including 6 blast resistance genes(Pit,Pi9,Pi36,Pi56(t),Pia,Pi5),2 BPH resistance genes(Bph14 and Bph26),2 bacterial blight resistance genes(Xa-1 and Os RP1L1)and 1 defence response gene(NLS1).Further,4583 variant genes with the same genotype in two inbred lines S18 and 11352 were retained to eliminate the variations associated other agronomic traits.GO analysis indicated that 67 variant genes were involved in cell death,including a brown planthopper resistant gene LOC_Os12g37280(Bph26);82 variant genes were involved in response to stress.58 variant genes were involved in both GO terms.(4)Protein interaction and phylogenetic relationship analysis of candidate genes.The protein-protein interaction analysis indicated that 58 candidate genes were putatively interactive with 8 BLAST resistant genes(Pi-d3,Pi5,Pi56(t),Pi9,Pia,Pish,Pit,Pita)and 3 BPH resistant genes(Bph14,Bph26,BPH18).Candidates and its homologs in S18 and 11352 belong to NB-ARC domain containing gene family.Phylogenetic and MEME analysis indicated that these genes were divided into four different groups,Group?,Group?,Group? and Group? contain 69,32,67 and 6 genes respectively,which may involve in different function.CD-search analysis indicated conserved domain in 28 candidate genes and its homologs were changed,the domain changed homologs maybe new resitance alleles.In summary,two resistant inbred lines and its common wild rice progenitor were re-sequenced in the present study.Detection of the genome variations will further enrich the rice variation resource database,and the new putative resistant alleles will facilitate the usage of common wild rice in resistance molecular breeding and functional genomics.