Fine Mapping And Candidate Gene Analysis Of TAC8,A Major Quantitative Trait Locus Controlling Tiller Angle In Rice(Oryza Sativa L.)

The improvement of rice plant type is one of the most important method in increasing rice yields.food crops in China.Tiller angle,a significant component of ideotype of rice,plays an distinctly important role in rice breeding.Therefore,the study of tiller angle is very significantly.In this study,two rice varieties,glabrous tropical japonic rice D50,showing compact plant architecture and indica rice HB277,showing relatively spread plant architecture were employed to develop RIL population.Two major quantitative trait loci?QTLs?for tiller angle,TAC8 and TAC9,were consequently detected using RIL population.the QTL TAC8 were fine mapped.The main results are as following:1.Two major QTLs controlling tiller angle,TAC8 and TAC9,were mapped on chromosme 8 and 9,respectively.The TAC8 explained 33.4%of the total phenotypic varice with LOD of 12.87,and the additive allele came from D50,the TAC9 explained 17.4%of the phenotypic varience with LOD of 9.31,and the additive allele came from HB277.Four sets of NILs were established to analyse the genetic relationship between those two QTLs,the results showed that TAC8 took part in different genetic pathways with TAC9.Using the genotype and phenotype of BC₂F₂ popultaion and the phenotype of BC₂F_2:3 popultaion in Hainan and Hangzhou,the TAC8 was narrowed to a 16.5 cM region between RM7049 and RM23175.2.The dynamic change of tillering angle during the development of NIL?TAC8?and NIL?tac8?was studied.The tiller angle of NIL?TAC8?and NIL?tac8?both increased until 70 days after sowing?DAS?,NIL?TAC8?and NIL?tac8?both showed the biggest tiller angle and larger difference at 70 DAS.NIL?TAC8?and NIL?tac8?showed near the same tiller angle until 60 days after sowing and stable tiller angle from 80 DAS to 120 DAS.NIL?TAC8?and NIL?tac8?showed no significant difference in other agronomic traits.The plant type and yield related traits of NIL?TAC8?and NIL?tac8?were analyzed under two planting densities,that the results indicated that the tiller angle,plant height,Seed setting rate,Kernel weight,,and Spikelets per panicle of the TAC8 were affected by planting density,while the tiller number of the TAC8 was increasing along with the increase of distance between the plants.3.Two genetic segregation populations were developed for fine mapping of the TAC8.The TAC8locus was finally located in a 9.02 kb DNA region,BAC P0431A03,between the Indel markers In22and In36.Within this region,only one opening reading frame?ORF?was detected.Therefore,we tentatively designated this ORF as the TAC8 gene.TAC8 encoded a transcription factor that contained a bHLH domain.TAC8 gDNA and cDNA sequences showed that the gene contained two exons and one intron.The gDNA and cDNA of TAC8 stretched 2996 bp and 1059 bp,respectively.By sequencing the genomic DNA of this ORF,several SNPs changes were founded in the region between D50 and HB277,especially,a 24bp deletion in the promoter of this gene was founded in the parent HB277.In different tissus of the two groups of contrast,Real-Time qPCR analysis showed that there are large differences.The complementary vector,the over expression vector and the RNAi vector were constructed,and then introduced into rice calli by agrobacterium tumefaciens-mediated transformation,the transformation is under way.4.The expression patterns were conducted by real-time PCR analysis,which showed that TAC8expressed in young panicles leaves and less in leaf,sheaths,culms and root.At tillering stage,the TAC8was highly expressed in young panicles and expressed decreased systematically in root,culms,leaf and sheaths;At mature stage,TAC8 was highly expressed in the base of stem while low expressed in leaf sheaths,leaf and root.5.Based on RNA-seq analysis of NIL?TAC8?and NIL?tac8?,as a result,740 up-regulated genes and 1047 down-regulated genes were detected in NIL?TAC8?.These differentially expressed genes were widely involved in glycolysis,gluconeogenesis,carbon metabolism,synthesis and metabolism of amino acids.We found that Four auxin transport related differential expression genes in hormones signal transduction pathway?KO04075?,three of those genes down–regulated,one of them up-regulated.and the IAA content of NIL?TAC8?was significance lower than NIL?tac8?,this indicate that TAC8controlling tiller angle may be independent of auxin metabolism pathway.Real-time qPCR analysis part of differentially expressed genes showed an expression trend with RNA–seq,this indicates the consequence of RNA–seq is reliable.