Study On SSR Molecular Marker Mapping Of Four Marker Gene On 14th Linkage Group In Silkworm, Bombyx Mori

Silkworm is a representative insect in Lepidoptera,and also an excellent material for genetic research.It has rich genetic mutants by natural selection and artificial mutagenesis after more than 5,000 years artificial breeding.After nearly 100 years of research on silkworm linkage genetic map, more than 300 mutated genes have been located in 220 loci covenng all 28 linkage groups.With the development of DNA molecular marker technique and their application in silkworm,the researches on molecular genetic map of silkworm have obtained plentiful and substantial achievements.RFLP,RAPD,AFLP,SSR,SNP molecular linkage maps have been constructed successively.Because of the isolation between the silkworm classics linkage map with molecular linkage map,the application of these achievements are restricted.Therefore,the work of integrating classical map with molecular linkage map becomes a hot focus on silkworm genetic research.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 four mutant genes including U,Nd-s,Di,oa on the 14th linkage group of classical linkage map based on the SSR molecular linkage map in silkworm.The research contents and results are as follows:(1)The populations used for mapping were backcrosses:The backcross population(BC₁ F)of F₁ female backcrossed with recessive parents and the backcross population(BC₁M)of F₁ male backcrossed with recessive parents.The BC₁F were used to screen the markers and determine the linkage;The BC₁M 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 17-141 U·Nd-s and inbred strain "Dazao" to breed the materials for linkage and location combinations of gene U and Nd-s,and used 17-142 oa and 09-500 Di to breed the materials for linkage and location combinations of gene Di and oa.They were all maintained in the gene recourse library of silkworm in southwest university.(2)Nine pairs of SSR primers were found that had polymorphism between two parents,when we used 26 pairs of SSR primers which corresponding with the 14th linkage group in the silkworm SSR linkage map to screen the polymorphism between parents 17-141 U·Nd-s and "Dazao".Then we performed PCR amplification and PAGE electrophoresis on a total of 24 individual genomic DNA including 22 individuals(11 individuals present sericin cocoon phenotype and 11 normal individuals)of linkage combination BC₁F and 2 parents by the nine pairs of primers.Four SSR markers were linked with marker genes U and Nd-s:S1401,S1411,S1419,S1426.Then based on the 4 SSR markers,we analyzed 110 individual genomic DNA of location combination BC₁M of U and Nd-s based on SSR-PCR technique.Statistical analyzed the data,we got a linkage map between marker genes U,Nd-s and the 4 SSR markers which linked with them by mapping software.The total length of the linkage map was 50.4 cM.The nearest marker to gene Uwas S1411 with 0.9 cM, and the nearest marker to gene Nd-s was S1419 with 0.0cM.(3)Similarly,eight pairs of SSR primers were found that had polymorphism between two parents,when the same 26 pairs of SSR primers were used to screen the polymorphism between 17-142 oa and 09-500 Di.There were also four SSR markers that linked with the marker genes Di and oa by linkage testing used this eight pairs of SSR primers in BC₁F.They were S1414,S1418, S1419 and S1426(Marker S1419 and S1426 were also linked with marker genes Uand Nd-s).Then we analyzed 145 individual genomic DNA of location combination BC₁M of Di and oa based on SSR-PCR technique with the 4 SSR markers.Statistical analyzed the data,we got a linkage map between marker genes Di,oa and the 4 SSR markers by mapping software.The total length of the linkage map was 55.8 cM.The nearest marker to gene oa was S1414 with 13.1cM.(4)We integrated the two linkage maps by mapping software,and integrated the 4 marker genes with the 6 SSR markers that linked with them to one linkage map.The distance from SSR marker S1411 to marker gene U was 0.9 cM.The distance from SSR marker S1419 to marker gene Nd-s was 0.6 cM.The order of the 4 genes in this integrative map was exactly consistent with the 14th linkage group in classic linkage map,with nearly the same genetic distance.The results showed that it is entirely possible to integrate the classic linkage map with molecular linkage map in silkworm through repeated experiments.