基于分子标记和高通量测序的基因精细定位

水稻斑点叶突变体Spl34是自然获得的突变体,在研究其形态学和生理生化等方面的基础上,利用粳稻Francis和Spl34杂交构建F_2基因定位群体进行精细定位研究,遗传分析表明Spl34的斑点叶表型受单个显性基因Spl34(t)调控;利用SSR/InDel标记对无斑点叶隐性单株进行连锁分析,将Spl34(t)精细定位于第11号染色体23.483～23.530 Mb之间,定位区间长度为46.99 kb,筛选出8个候选基因。另外,利用高通量测序技术对F_2(Francis/Spl34)有斑点个体混合池、无斑点个体混合池以及双亲进行重测序,利用基因组SNP对有斑点和无斑点混合池进行关联分析,将Spl34(t)定位于第11号染色体23.223～23.791 Mb之间,区间大小约568 kb;进一步利用单分子测序技术对Spl34突变体进行测序,通过从头组装、关联分析,在两个重叠群tig00001409和tig00003011检测到紧密连锁。通过对两个重叠群之间序列进行Sanger测序并产生的新重叠群长度达到233.95 kb,其中Spl34(t)定位区间大小约40 kb。将斑点叶基因Spl34(t)精细定位于第11号染色体约40 kb的区间,为进一步开展该基因的功能研究奠定了良好基础。

Fine mapping of genes based on molecular markers and high-throughput sequencing

The rice spotted leaf mutant Spl34 is a naturally obtained mutant. The mutant Spl34 had been studied in the morphological, physiological and biochemical aspects of the mutant Spl34. The F2 gene localization population was constructed by hybridization of indica rice Francis and Spl34. Genetic analysis showed that the spotted leaf phenotype of Spl34 was regulated by a single dominant gene Spl34(t). The SSR/InDel marker was used to link the leafless leaf recessive plants, and Spl34(t) was finely mapped on chromosome 11 between 23.483 and 23.530 Mb. The length of the localization interval was 46.99 kb, and 8 candidate genes were screened. In addition, high-throughput sequencing technology was used to re-sequence the F2 (Francis/Spl34) spotted individual pool, the spotless individual pool and the parents, and the genomic SNP was used to correlate the spotted and non-spotted pools. Positioned on the 11th chromosome between 23.223-23.791 Mb, the interval size is about 568 kb; further Spl34 mutants were sequenced by single-molecule sequencing technology, and detected by de novo assembly and correlation analysis in two overlapping groups tig00001409 and tig00003011 to the close chain. The length of the new contig generated by Sanger sequencing of the sequences between the two contigs reached 233.95 kb, and the Spl34(t) localization interval was about 40 kb. In this study, the spotted leaf gene Spl34(t) to the 40 kb region of chromosome 11, which laid a good foundation for the further functional study of this gene.