Molecular Mapping Of Chocolate Mutants And The Primary Study Of Molecular Basis On Chocolate Purple (ch~p) In Silkworm, Bombyx Mori

From Bombyx mandarina Moore to Bombyx mori L, there are abundant genetic mutants by selection and artificial mutagenesis during thousands of year's artificial breeding. There are more than 100 mutants about body color. The coloration mutants of silkworm involve each periods including larval stage, pupal stage, adult stage, even the silkworm cocoons also have affluent colors. The body colors of silkworm are displayed by interactions of many pigments between epidermic cell and cuticle, and the main pigments are melanin, pteridines and ommochromes. There are many genes which control the pigment synthesis, that the variation of these genes can form different body mutants, different gene variations can form similar body colors, interactions of genes can also take on new colors. The generous and genetic stabile body color mutants make the silkworm be an important resource on researches of pigment synthesis. The chocolate mutate genes as marker genes in silkworm play important roles in genetic analysis, linkage search and gene mapping. The researches based on chocolate mutants can not only promote the utilizations of these mutants resources on sericulture, but also can improve the theory treasury of silkworm genetics.The publication of the SSR molecular linkage map of silkworm and the advantages of SSR molecular marker technique have created mature conditions for the integration of molecular linkage map and classical linkage map of silkworm. In this research, we located two chocolate mutate genes la and ch based on the SSR molecular linkage map, and had a primary study of molecular basis on chocolate purple mutant in silkworm, Bombyx mori. The research contents and results are as follows:(1) The populations used for mapping were backcross The backcross populations (BC₁F) of F₁ female backcrossed with recessive parents were used to screen the markers and determine the linkage. The backcross populations (BC₁M) of F₁ male backcrossed with recessive parents were used to analyze the genotype of the markers, calculate the genetic recombination rate between the marker gene and the SSR marker and ensure the genetic distance between them. We used the gene location system Near-isogenic line Dazao-Ia and inbred strain C108 to breed the materials for linkage and location combinations of gene Ia, and used Near-isogenic line Dazao-ch and inbred strain C108 to breed the materials for linkage and location combinations of gene ch. They were all maintained in the gene recourse library of silkworm in Southwest University.(2) Molecular mapping of gene IaTen pairs of SSR primers were found that had polymorphism between two parents, when we used 11 pairs of SSR primers which corresponding with the 9^th linkage group in the silkworm SSR linkage map to screen the polymorphism between parents Dazao-Ia and BmIS-C108. Then we performed PCR amplification and PAGE electrophoresis on a total of 23 individual genomic DNA including 2 parents,F₁ generation and 20 individuals (10 normal individuals and 10 individuals present chocolate phenotype) of linkage combination BC₁F by the ten pairs of primers. Five SSR markers were linked with the gene Ia:S0903, S0904, S0907, S0908 and S0910. Then based on the 5 SSR markers, we analyzed 100 individual genomic DNA of location combination BC₁M of Ia by SSR-PCR technique. Statistical analyzed the data; we got a linkage map between marker genes Ia and the 5 SSR markers which linked with them by mapping software. The order of linked SSR markers and mutate gene Ia on the linkage map was:S0907, Ia, S0904, S0903, S0908 and S0910. The total length of the linkage map was 44.4 cM, and the nearest marker to gene Ia was S0904 with 1.0cM.(3) Molecular mapping of chocolate gene chSimilarly,7 pairs of SSR primers were found that had polymorphism between two parents, when we used 11 pairs of SSR primers which corresponding with the 13^th linkage group in the silkworm SSR linkage map to screen the polymorphism between parents Dazao-ch and BmIS-C108. There were 5 SSR markers that linked with the marker genes ch by linkage testing used this 7 pairs of SSR primers in BC₁F.They were S1308, S1309, S1311, S1312 and S2213. Then we analyzed 166 individual genomic DNA of location combination BC₁M of ch based on SSR-PCR technique with the 5 SSR markers. Statistical analyzed the data; we located the gene ch between marker S1309 and S2213. The nearest marker to gene ch was S1309 with 7.3cM, and the genetic distance between ch and S2213 was 9.3cM.(4) Primary study of molecular basis on chocolate purple (ch^p)It is known that yellow was a responsible gene for larval color mutant (chocolate) of the silkworm. Based on the full CDS sequence of yellow gene downloaded from NCBI, we designed spanning-ORF primers to amplify the yellow gene of chocolate purple cDNA, and we got two different splicing. By cloning and sequenced the transcripts of the wild-type (Dazao) and ch^p mutant (ch^p), we found that two abnormal transcripts were present in the Dazao-ch^p compared with the WT (Dazao). ch^p typel inserted an rregularsequence before exon3, which had a premature codon by TAA before exon3. ch^p type2 spliced from exon2 to exon4 and deleted whole exon3 189 bp (the stop codon position is identical to that of wild type). Type1 of yellow gene was the specific spliced type in ch^p, analyzed the sequences of its coding amine acid, N-glycosylation sites, functional domain and higher structure, we suspected type 1 was the molecular basis which resultes to chocolate purple.The resrach results showed that the yellow gene expressed from the 12th hour in embryo. The yellow gene had a high expression in the head, which it's the most apparent place between chocolate purple and normal Dazao, showed that the yellow gene involved in melanize in the cuticle of silkworm. In addition to participating the pigment synthesis so as to affect body color, there needs a further study of how gene yellow effects in silkworm.