Fine Mapping And Breeding Application Of Brown Planthopper Resistance Gene Bph9 And Bioinformatics Analysis Of ALOG Domain Genes In Rice

The brown planthopper(Nilaparvata lugens Stal,BPH)is one of the most destructive insect pests world-wide to the rice production and breeding application.To prevent and cure the harmfulness of the brown planthopper to the rice under the background of implementing environmental-friendly agriculture effectively,exploring novel Bph-resistant genes from diverse rice germplasms and transferring them into cultivated varieties for new ones with resistance are critical for controlling harmfulness of this insect.Previous studies indicated the rice variety of Sri Lanka Pokkali carries a resistance gene Bph9 and has a high resistance level to the local population of the brown planthopper.On the basis of the primary mapping results that Bph9 is located in the regions between the SSR markers RM28438 and RM28486 on the long arm of No.12 chromosome of rice genome,the backcross population of BC3F2 and advanced backcross population of BC5F2 were used for screening the recombinants by such two flanked marker and fine mapping of the Bph9 gene.The Bph9 was fine-mapped to the 68kb regions between Indel marker InD2 and Rsal.Then,the MAS(marker-assisted-selection)of resistance gene by InD2/IR2 and the purification of genetic background by polymorphic markers genome-widely between pokkali and 9311/N were carry out for constructing the NIL-Bph9 in different genetic background of 9311 and Nipponbare.In characterization of Bph9-mediated resistance by NIL-Bph9 between some phases in the vegetative period and reproductive period,combined with settling of BPH in a choice test,BPH survival and the increased weight of BPH after ingestion,we found the allele from Pokkali significantly increased resistance to the BPH in the genetic background of 9311,Whereas,the NIL-Bph9 in that of Nipponbare exhibited an obviously low resistance level to the BPH comparing with the resistance parent Pokkali.The agronomic traits related to grain yield of rice such as length of panicles,number of panicles,filled grain per panicle,were investigated.No significant differences of such traits were found between NIL-Bph9 and 9311.Therefore,the variety of NIL-Bph9 in genetic background of 9311 can be used in the practical production and breeding application of rice.We applied for the variety rights and renamed it as Luo Yang 9.Lately research of our laboratory have also shown that the resistance gene Bph6 carried by the rice cultivated variety Swarnalata was located in the region of 25kb between two markers of Y9 and Y19 on No.4 chromosome of rice genome.The NIL-Bph6(BC5F2)was obtained by us.The hybridization of NIL-BphP and NIL-Bph6 was carried out for multiple BPH-resistant genes pyramiding.The homozygous Bph6 and Bph9 were screened by marker H and InD2 in F2 population.Subsequent BPH-resistance evaluation of such resistant line referred a fairly higher resistant level of(Bph9+Bph6)to three different biotypies of BPH.Moreover,a stronger resistance effect was observed comparing with the Near-isogenic line only contains one BPH resistance gene that the BPH prefers to select NIL-Bph9 or NIL-Bph6 for settling in the choice tests rather than the gene pyramiding line after feeding for 24h,though the survival rates of BPHs on all these three resistant lines were very low,especially the Bph6&9 after feeding for 9 days.All the 10 yield-related agronomic traits were investigated that no significant differences were observed between Bph6&9 and 9311,with exception of the weight of one thousand grains.We applied for the variety rights of Bph6&9 line and renamed it as LuoYang69.The variety of LuoYang69 may provide us another choice for preventing the harmfulness of BPH besides LuoYang9.The LuoYang69 may retain the resistance to BPH longer during the process of co-evolution between BPH and rice.The ALOG domain is named after the Arabidopsis LSH1 and Oryza sativa G1 genes.OsG1/G1L genes represent the ALOG domain genes in rice.Only three members of OsG1/G1L genes have been identified and characterized in previous studies.These OsGl/GIL genes all participate in the development of spikelet organs and the regulation of spikelet meristem specification,which both have tight relationships to the grain yield of rice.Here,a comprehensive bioinformatics analysis was performed that revealed the presence of 84 ALOG domain genes,including at least 14 OsG1/G1L genes,with 4 newly discovered genes in rice and 10 in Arabidopsis,compared with the 60 ALOG domain genes from 5 other representative plant species.A phylogenetic analysis indicated that the expansion of this land-specific gene family mainly occurred after the divergence of dicotyledonous and monocotyledonous plants.Chromosomal segmental duplications primarily contributed to the expansion of the OsG1/G1L subfamily.The transcripts of the ALOG domain genes were differentially accumulated during the 33 tissues and developmental stages in rice,as demonstrated by a microarray analysis from CREP database.On the basis of hierarchical clustering of expression pattern,some co-expression patterns were observed in the manner of tissue specificity.The potential functions of OsGl/GIL genes were speculated according to the microarray data and quantitative RT-PCR.The OsMADS,a former well-studied subfamily for the identities of the floral organs in rice was taken for microarray analysis of temporal and spatial co-expression with OsG1/G1L genes.Our results indicated that some OsG1/G1L genes f were primitively recruited for development of some non-floral organs in rice as seeds and roots.These genes share either the similar structures and the similar expression pattern in some tissues or same conserved motifs in the corresponding proteins with those from the Arabidopsis.Then some genes obtained novel functions after the gene segmental duplication events as for regulation of panicles,spikelet and plant growth regulated by ABA.Meantime,some genes became the nonfunctional pseudogenes for lacking some necessary motifs in their corresponding protein sequences.Some certain gene became the house-keeping gene for expressing in almost all tissues and developmental stages.Interestingly,the OsMADS and OsG1/GIL genes may be recruited together to coordinate the developmental regulation of floral and non-floral organs in rice.Additionally,the phylogenetic analysis and expression profiles cross the species will provide some clues for the expression pattern and potential functions of ALOG orthologs in other species.