Genetic Mapping Of Silkworm Black Moth (Bm) Mutant And Screening Of The Candidate Genes

Silkworm, Bombyx mori, is the most thoroughly domesticated insect and a model for Lepidoptera in basic research. During the period of nearly 100 years of research on silkworm linkage genetic map. more than 300 morphological mutant characters have been mapped at 230 loci, most of them are important biochemistry traits. With the development of DNA molecular marker techniques and their application in silkworm, we can map the mutants on chromosomes at level of fine scale by DNA molecular markers and further clone the genes underlying these mutants. Silkworm genetic map and physical map with high resolution have been successfully constructed, and EST databases and gene chip as well as expression profiles of the whole gene set are available. Especially the new assembly of silkworm genome sequence has been completed recently. These advances provide a good opportunity for fine mapping of silkworm mutants and map-based gene cloning. Because of these developments, some of the silkworm mutant genes were identified. Therefore. positioning cloning work has become an important topic in silkworm genetics.There are more than 600 mutant and inbred lines in the silkworm genetic resource bank at Southwest University, China. Black moth (Bm) is a mutant about body color of silkworm adult. The character is that adult wings and body are dark brown. But larvae body color of the mutant silkworm larvae is normal. Bm was mapped at O.OcM of 17th linkage group on classical linkage map. In this study. we searched the silkworm genome sequence by using sequence of a conserved supergene locus controlling color pattern diversity in butterflies as query and found the syntenic region in the silkworm. Focusing on this syntenic region, we developed molecular markers for'linkage analysis and used BC1 segregation population to map Bm. The results are as follows:(1) Development of molecular markers:We searched the silkworm genome sequence by using sequence of a conserved supergene locus controlling color pattern diversity in butterflies as query and found the svntenic region in the silkworm. According to this svntenic region. we designed primers to screen the polymorphism between parents C108 and black moth as well as dazao-Bm. We found two available polymorphism regions. Then we used SSR primers of 17th linkage group to screen polymorphism and found that one pair of SSR primers is suit for linkage analysis.(2) Testifying molecular markers:We used 20 individual genomes of BC1F to testify the 3 molecular markers and sequenced the molecular markers HD2 and M-7. The result indicated that 3 molecular markers are correct and linked to Bm gene.(3) Linkage analysis of mutant Bm gene:We genotyped 417 individuals of BC1 derived from a cross between parents C108 and Dazao-Bm at 3 molecular marker loci. The results showed that 1 of 417 individuals underwent crossing-over between M-7 and Bm, the recombination frequency is 0.24%;6 of 417 individuals experienced crossing-over between HD2 and Bm, the recombination frequency is 1.44%;9 of 417 individuals experienced crossing-over between S1702 and Bm. the recombination frequency is 2.16%. A linkage group including Bm and 3 molecular markers was constructed by the program JoinMap 4.0. Total length of the linkage group is 3.8 cM. Bm is located between M-7 and HD2. The shortest genetic distance of Bm to M-7 is 0.2 cM.(4) Screening of candidate gene:We used a microarray to screen significantly different expression levels of genes between Dazao and near isogenic line Dazao-Bm. and found that there are two differentially expressed genes between molecular markers M-7 and HD2. These two genes may be candidates of Bm.The results presented in this study indicated that svntenic analysis of distantly related species can provide useful information for mutant gene mapping. Through the development of molecular markers and linkage analysis of backcross population. we finished initial mapping of target gene Bm and screening of candidate genes. This study represents the first step for further cloning Bm gene.