家蚕非滞育红卵突变体Re-nd突变基因的定位克隆

Positional cloning of the mutant gene of the non-diapause red egg mutant Re-nd in the silkworm,Bombyx mori

【目的】家蚕Bombyx mori非滞育红卵突变体Re-nd是唯一在非滞育状态下卵色呈现鲜红色的突变品种。本研究通过基因连锁分析和定位克隆的方法确定Re-nd的突变基因所在的染色体及紧密连锁位置,为后续Re-nd的功能研究及应用奠定基础。【方法】以家蚕卵色突变体Re-nd和野生型大造进行杂交,配制基因连锁分析群体材料和定位克隆群体材料;针对家蚕全染色体进行SNP标记开发,利用BC_(1)代群体材料进行基因连锁分析,确定Re-nd的突变基因所在的染色体;针对定位的Re-nd的突变基因所在染色体进行SNP标记开发,利用BC_(1)群体材料对Re-nd的突变基因进行定位克隆。【结果】基因连锁分析结果显示Re-nd的突变表型与第6号染色体上的SNP标记完全连锁;初步定位克隆结果显示Re-nd的突变基因位于SNP标记SNP7和SNP17之间,物理距离4.04 Mb;以SNP7和SNP17之间筛选出的6个SNP标记和25个重组个体进行精细定位克隆,结果显示Re-nd的突变基因所在的区域位于SNP10和SNP12两个SNP标记之间的nscaf2853上,物理距离949.3 kb左右。【结论】将Re-nd的突变基因定位于第6号染色体的2个SNP标记SNP10和SNP12之间,物理距离约949.3 kb。本研究为后续Re-nd突变基因的精细定位及功能应用研究奠定了基础。

【Aim】Re-nd,a non-diapause red egg mutant of Bombyx mori,is the only mutant with bright red egg color in non-diapause.This study aims to determine the chromosome and tightly linked location of the mutant gene of Re-nd by genetic linkage analysis and positional cloning,so as to lay a foundation for functional research and application of the Re-nd mutant.【Methods】The experimental materials for genetic linkage analysis and positional cloning were prepared by hybridization of the mutant Re-nd and the wild-type Dazao of B.mori.The SNP markers were developed for the whole chromosomes of B.mori and used for genetic linkage analysis with the BC_(1) generation population materials to determine the chromosome where the mutant gene of Re-nd is located.Then the SNP markers were developed for this chromosome and used for positional cloning of the mutant gene of Re-nd with the BC_(1) generation population materials.【Results】The genetic linkage analysis results showed that the mutation phenotype of Re-nd was completely linked to the SNP markers on chromosome 6.The preliminary positional cloning results showed that the mutant gene of Re-nd was located between the SNP markers SNP7 and SNP17,with a physical distance of 4.04 Mb.The fine positional cloning was performed with six SNP markers between the SNP markers SNP7 and SNP17 and 25 recombined individuals screened,and the results showed that the region of the mutant gene of Re-nd was located on nscaf2853 between the SNP markers SNP10 and SNP12,with a physical distance of about 949.3 kb.【Conclusion】The mutant gene of Re-nd is located between the SNP markers SNP10 and SNP12 on chromosome 6,with a physical distance of 949.3 kb.This study lays the foundation for the fine positioning and the functional research and application of the Re-nd mutant genes.