Fine Mapping And Transcriptome Analysis Of Brown Planthopper Resistance Gene Bph15in Rice

The brown planthopper (BPH; Nilaparvata lugens Stal) is a typical phloem-feeding insects and a major pest of rice. The most economical and environment-friendly strategy to control BPHs is to plant resistant rice varieties. At this time,27BPH-resistance genes have been identified in rice. Resistance gene Bph15was introgressed from wild rice Oryza officinalis. The gene showed significant resistance to BPH and have been broadly employed in rice breeding programs.Previously, we mapped Bph15on the short arm of chromosome4between marker RG1and RG2using RI93/TN1F2populations. To clone Bph15, we backcrossed the resistance rice (YHY15) that carries the Bph15locus from the F2populations to the susceptible recurrent parent (TNI) and developed the backcrossing populations. The Bph15was first mapped to markers between RM261and S16using random BC1F2populations and then further between markers g12140-2and T6using54recombinants from13,000BC2F2populations, but we did not further narrow the region of Bph15using61recombinants from10,000BC4F2individuals. The physical distance between these two markers was about210-kb according to Nipponbare genome sequences. Bph15located in a recombination coldspot region. Most of crossovers located outside of these two markers.To assemble physical map spanning the Bph15chromosome region, we built long insert (130-kb) genomic library of rice B5, which has about10-fold redundancy. The BAC DNA pools were prepared and candidate clones were screened by PCR based analysis. Then, we assembled a physical map of the Bph15region with a gap, and the selected BAC clones were sequenced. Finally, we mapped the gene between g12140-2and new developed marker T12using above mentioned recombinants, and the physical distance was580kb according to sequenced BAC clones. Eighty-seven genes within this580-kb region were annotated using online FGENESH software. The sequence of this region in the resistant rice genome differed significantly from the corresponding region in the reference genome of Nipponbare. The high level of sequence diversity in the Bph15region explained the heavy suppression of recombination in this region.Determining which candidate gene was responsible for resistance in the580-kb chromosome region was difficult. We applied deep RNA sequencing to investigate transcriptomes of the Bph15introgression line and susceptible receptor. In total,2,914differentially expressed genes (DEGs) were identified. We found that BPH-responsive transcript profiles were distinct between resistant and susceptible plants and between the early stage (6h after infestation, HAI) and late stage (48HAI). The key defense mechanism in resistant Bph15and susceptible receptor rice was related to jasmonate signaling, ethylene signaling, receptor kinase, MAPK cascades, Ca2+signaling, PR genes, transcription factors and protein posttranslational modifications.We searched for expressed genes within the87genes predicted in the580-kb Bph15region using RNA sequencing data of resistant rice. Four jacalin-related lectins and one LRR family proteins showed higher FPKM values. We found that they were expressed using RT-PCR and RACE. We inferred that the four jacalin-related lectin genes originated from the same ancestral gene. We then detected all potentially expressed genes and found that no other genes were expressed. The candidate gene Bph15represents one or a few of these expressed genes. The five candidate genes were transferred into the BPH-susceptible variety Kasalath and Nipponbare. In addition, we used RNA interference (RNAi) to suppress the expression of every candidate gene in the XF07-151rice plants. Then we examined the T2families of all transgenic plants for BPH-resistance using the bulked seeding test and found that all of the T2transgenic lines did not change resistance levels.