Fine Mapping And Cloning Of DE9,A Dominant Epistatic Gene Of Bph9 Conferring Brown Planthopper(BPH) Resistance In Rice

The brown planthopper（Nilaparvata lugens St（?）l,BPH）is a migratory and sucking mouthpart-contained pest which is one of the most destructive pests to rice.BPH sucks juice from rice phloem by mouthparts,which results in rice withering and even death.Therefore it causes serious loss of rice yield.Traditional methods of pesticide control not only threaten the environment,but also affect the nutrient content of agricultural products;Besides,the long-term use of pesticides also causes occurance of vesistant pests.At present,the most cost-effective and environment-friendly way to control pests is to integrate host resistance genes into susceptible varieties.So far,38 rice BPH-resistant genes have been identified from cultivated rice and wild rice resources,of which 14 were successfully cloned.Both genetic background and some minor BPH resistance quantitative trait locus（QTLs）can considerably affect the resistance level or durability of those major BPH-resistance genes.Previous work in our laboratory found that the BPH-resistant gene Bph9 is resistant to BPH in indica cultivar 9311 but susceptible to BPH in japonica cultivar Nipponbare（Nip）.Therefore,we proposed that there must exist a gene loci that is required for the function of Bph9 in indica 9311,and this locus is absent or nonfunctional in Nipponbare genetic background.This locus was roughly mapped within 8.9 Mb-16.4 Mb on chromosom 8 by map-based cloning and molecular marker-assisted breeding（MAS）.In this study,we developed a series of markers and screened out 40 recombinants from 5970 BC₆F₃individuals.In Del markers were used to further confirm these recombinants affer BPH resistance evaluation at the seedling stage.The causal locus was fine mapped to a 20 kb region between In Del marker8D2-23 and 8D2-27 of chromosome 8.It contains a single candidate gene and is named as DE9（Dominant epistatic Gene of Bph9）.This gene is absent in japonica Nip cultivar.A 27 kb genome sequence covering this candidate gene was obtained by screening the indica cultivar Pokkali genomic library using Fosmid genomic library screening method.Then,complementation lines carying DE9 was obtained by introducing the DE9 genomic DNA into the susceptible rice variety NIL-Bph9-Nip.For further study of the expression pattern of DE9,the GUS report vector with DE9promoter sequence was also introduced into Nip.The transgenic introgression of DE9into the susceptible rice variety NIL-Bph9-Nip significantly increases resistance to BPH.Through Rapid Amplification of c DNA Ends（RACE）experiments,DE9 c DNA was cloned.The genomic sequence of DE9 contains two introns and three exons.DE9protein localizes to the nucleus in Rice.Here,we finely mapped and cloned DE9,a dominant epistatic gene of Bph9conferring brown planthopper resistance in rice.We further confired this function by genetic complementation assay.We also determined its subcellular location in the nucleus of rice cells.It provides solid genetic evidence for further elucidating the interaction mechanism between genetic background or QTL and major resistance genes.It also highlights the significance of combining both majoy resisitance gene with the vognate helper QTL for breeding stable broad-spectrum insect-resistant rice varieties.