Based On High-throughput Sequencing Fine Mapping Of Spl34(t) Gene Spotted Leaf In Rice

The rice leaf spot mutant is a kind of special genetic material,which is of great significance to explore the programmed cell death,allergic reaction and resistance mechanism of rice.Dotted leaves Rice Mutant Spl34（Spotted Leaf 34）from two normal leafy material Guangdong crystal Mew 2 and H4 of F₂ Natural mutants in the population obtained.Predecessors have mutant Spl34 were preliminary genetic analysis and gene targeting,showed Spl34 spotted leaf phenotype by a dominant gene Spl34（t）regulation,using F₂（02428/Spl34）group which is positio ned in the initial section.No11.chromosome19.84-22.48 Mb interval.On the basis of this study,the agronomic traits and physicochemical indexes of mutant Spl34 were further identified,and the spotted leaf genes were fine-mapped by SSR/InDel and high-throughput sequencing,and the sequences of the loci were compared.The main results achieved are as follows:1.Mutant Spl34 major agronomic and quality traits were not adversely a ffected by spot traits.In F₈ substituting Spl34 no spots isolated plant plants as control（CK）,field phenotype found Spl34 and CK premature aging phenomenon does not occur as compared to no significant difference in both agronomic traits.Analysis of rice quality showed that the whole milled rice rate of mutant Spl34 and CK was significantly different at 0.01 level,and there was no significant difference between the other indexes.Sprouting experiments showed that the germination rate and germination potential of mutant Spl34 and CK were different.2.Mutant Spl34Effects of Spot on Physiological and Biochemical Indexes.The UV light test showed that the spots of Spl34 may be caused by the accumulation of some unknown pigment produced by UV light.The SPAD and soluble protein of Spl34 at flowering stage were lower than those of control,and the rate of decline was faster.The results of field photosynthesis showed that the net photosynthetic rate,stomatal conductance and transpiration rate of flowering mutant Spl34 were lower than those of control,indicating that the emergence of mutant Spl34 plant spots could affect the photosynthesis of rice leaves.The staining results of N BT and DAB showed that the spots of the mutant Spl34 leaves were still brown,so it was speculated that Spl34 had stronger antioxidant ability than the control.3.Mutant Spl34 spotted leaf traits controlled by a dominant gene,using SSR/InDel mark the group due to the fine positioning of the 11 chromosomes 46.99 kb within the range.Preliminary study were F₂（02428/Spl34）small groups,and some late-maturing plant leaf spots phenotype is not obvious,unable to meet the fine mapping Spl34（t）needs.Thus,this study tropical japonica Francis and Spl34 hybridization constructed F₂ gene mapping population thereof,fine localization of genes for leaf spot.Genetic analysis showed that the proportion of F₂（Francis/Spl34）population with or without spots was 3:1,it was further confirmed that the leaf phenotype of Spl34 was regulated by a single dominant gene Spl34（t）.In N ipponbare genomic refe rence sequence,using the SSR/InDel of recessive plant leaves no spots linkage analysis labeling,Spl34（t）Fine mapping to the first 11chromosome 23.483-23.530Mb,with a locating length of 46.99 kb,containing 8 annotated genes,N ipponbare is sunny in this interval.The sequences of the eight genes were amplified by reference design primers,and the amplification products were not obtained in Spl34 and control,while Japanese and Francis were able to obtain specific products.Therefore,it is speculated that the sequence of Spl34 and Japanese clear in the locus is very different,and the candidate gene of Spl34（t）can not be screened by reference sequence of N ipponbare.4.The use of BSA-seq and single molecule sequencing of the mutant genome de novo assembly,further refine the positioning interval to 40 kb.Using Illumina high-throughput sequencing technology,F₂（Francis/Spl34）spotted individual pool,spotless individual pool and parents were resequenced.Using Genomic SNP of spots and no spots mixing tank correlation analysis,the Spl34（t）is positioned in the first 11 chromosome 23,223,221 bp to23,791,314 bp between the size range of about 568 kb.Further use of single-molecule sequencing technology Spl34 mutants were sequenced,is produced by de novo assembly4290 th greater than 10 kb contig,the average length 61.97 kb,accumulating a total length of 265.86 Mb.As a reference,the BSA correlation analysis was performed on the mixing pool,and the tight chain was detected in the two overlapping gro ups tig00001409 and tig00003011.Two further partial sequence contigs were on N ipponbare genomic Blast,two contigs were found N ipponbare chr11:23.46-23.51 Mb flanking sequences highly consistent.The length of the new overlapping group is 233.95 kb by Sanger sequenc ing fill gap between the two overlapping groups,where the Spl34（t）locating interval is about 40kb.The positioning section,mutant Spl34 amplification product of a control member,there are obvious differences in fragment length.95 kb,where Spl34（t）positioning interval size is about 40 kb.The positioning section,Spl34 mutant amplification product of a control member,there are obvious differences in fragment length.95 kb,where Spl34（t）positioning interval size is about 40 kb.The positioning section,Spl34 mutant amplification product of a control member,there are obvious differences in fragment length.Based on previous studies,the present study further clarified leaf spot mutant Spl34phenotypic and biochemical characteristics,while using high throughput sequencing-based gene targeting method of the leaf spots gene Spl34（t）finely positioned on the first 11chromosome from about 40 kb interval,which laid a good foundation for further functional study of this gene.