Mapping Brown Planthopper Resistance Genes And Development And Evaluation Of Near Isogenic Lines For Brown Planthopper Resistance In Rice

Rice(Oryza sativa L.)is a staple food for more than half of the population around the world.The brown planthopper(Nilaparvata lugens St?l,BPH)is one of the most devastating insect pests on rice.Breeding for host resistance is considered the most economical and environmentally friendly strategy for BPH control.Till now,at least 30 resistance genes have been identified in Oryza sativa spp.indica and wild rice species,of which 7 genes were cloned.The number of genes used for breeding is small and most were evaluated under different background,so it is hard to evaluate their effects and breeding prospects.Our study is to find new resistance genes by map-based cloning,construct near-isogenic lines(NILs)of 13 resistance genes by marker-assisted selection(MAS)system and backcross breeding,distinguish and evaluate their effects by combining genotype with phenotype.Our main results are as follows:We performed a QTL analysis using a F2:3 population derived from 9311 and AUS69(a resistant indcia line),and detected two major QTLs,which were located to the region flanking by marker J63 and RM252 on chromosome 4,and the region flanking by marker RM536 and RM1335 on chromosome 11,respectively.Their additive effect and LOD values were 1.56 and 1.14,9.46 and 6.18,explaining 11.65% and 5.95% of the variation for BPH resistance,respectively.We temporarily named these two QTLs Bph30 and Bph31.By analysing the phenotype and genotype of the recombinants,we fine-mapped Bph31 to a region of 400 Kb,between markers 11-165 and In88.According to the annotations of the reference sequence,we found two CC-NB-LRR genes in this region,namely LRR1 and LRR2,respectively.We sequenced and compared the sequence of the two genes of 9311 and AUS69,but only acquired sequence of the front part of LRR1 and full-length LRR2 of AUS69.We compared the amino acid sequences of LRR1 and LRR2 between AUS69 and Nipponbare,and found significant differences between them.The results showed LRR1 and LRR2 might play a role in the resistance phenotype,which needed a further validation of genetic transformation.In this study we individually transferred 13 genes or QTLs(Bph14,QBph3,QBph4,Bph17,Bph15,Bph20,Bph24,Bph6,Bph3,Bph9,Bph10,Bph18 and Bph21)into an elite indica restorer line 9311 by MAS from nine donors.Through positive,negative selection and RICE6 K gene chip,we got the high recurrent parent genome(RPG)near-isogenic lines(NILs),Bph14-NIL,QBph3-NIL,QBph4-NIL,Bph17-NIL,Bph15-NIL,Bph20-NIL,Bph24-NIL,Bph6-NIL,Bph3-NIL,Bph9-NIL,Bph10-NIL,Bph18-NIL and Bph21-NIL.All genes showed antibiotic responses in seedlings with reduced BPH survival rate and honeydew.For seedling responses,among all the NILs,Bph24-NIL and Bph6-NIL showed the highest resistance with scores 1.31 and 1.96,respectively;Bph3-NIL,QBph3-NIL,Bph15-NIL,Bph9-NIL and QBph4-NIL showed high resistance with scores 2.34,2.09,2.38,2.42 and 2.49,respectively;Bph17-NIL,Bph20-NIL and Bph14-NIL showed middle resistance with scores 2.74,3.16 and 3.37;Bph10-NIL,Bph21-NIL and Bph18-NIL showed the middle susceptible with scores 4.73,5.69 and 6.25.In short,the resistance genes from chromosome 4(QBph4,Bph17,Bph15,Bph20,Bph24 and Bph6)showed higher resistance than genes from chromosome 12(Bph10,Bph18 and Bph21),except for Bph9.The honeydew excretion areas and BPH survival rates on the NILs mostly paralleled the data for seedling response.Some of the 13 genes or QTLs used in this study were in clusters.There was an urgency to distinguish whether they were the same alleles or not.In these clusters,only Bph14,Bph17,Bph18 and Bph26 were cloned.Bph18 and Bph26 were different alleles.We sequenced and compared the amino acids of the clusters around the cloned genes.Results showed the proteins encoded by Bph26 alleles from the monogenic NILs carrying Bph10 and Bph21 were identical in amino acid sequence as the cloned Bph26.But a few nucleotide polymorphisms causing amino acid substitutions were detected in the proteins encoded by Bph9.Compared to Bph14,there were a number of amino acid substitutions in the LRR domain of the Bph14 allele from QBph3-NIL.Compared to the amino acids sequence of Bph17,Bph15-NIL had the same amino acids,while QBph4,Bph20 and Bph24 showed several substitutions.Considering the phenotypes(seedling response,honeydew and survival rate)and amino acids,we inferred that Bph10,Bph21 and Bph26,Bph15 and Bph17 might be the same genes;QBph3 and Bph14,QBph4 and Bph20 might be different alleles;Bph9and Bph18 might be multiple alleles to Bph26 and compared to Bph17,Bph24 might be a different gene.