Screening And Preliminary Application Of EST-SSR In The Silkworm

Bombyx mori as a model organism in genetics plays an important role, It is also identified as the representative of Lepidopterathe by the International Lepidopteran Genome Association. Using SSR (Simple Sequence Repeat) markers to study genetic diversity of silkworm to search economically valuable genetic resources, especially to build the silkworm genetic linkage map of high-density and position of genes with important economic value, is helpful On the breeding of high yield varieties of silkworm. In 1999, Reddy et al reported that 28 microsatellite loci was isolated from the silkworm genome and 15 of those microsatellite markers was used to analyze the Polymorphism of 13 silkworm varieties. Nagaraju et al By comparing the use of RFLP, RAPD, SSR, ISSR in the Silkworm, draw a conclusion that SSR is mostly suitable for linkage map of the silkworm. Presently,the chain map of the silkworm has been constructed, including 518 SSR markers and covering 3431.9cM. it is necessary to construct genomic libraries, probe preparation and molecular hybridization for conventional SSR markers development, the process is cumbersome and development costs is higher, so that its development and application have been greatly restricted. In recent years, EST has been widely used to study different species and the study gradually deepening,a large number of EST sequences of the economic value in many important biological have been accumulated, using SSR markers to develop EST sequences has become a new hot spot. Because of that SSR is as part of transcription gene and closely linked with the functional gene, EST-SSR markers have the advantages of genomic SSR markers and can directly identificate the important alleles which determine the important phenotypic, EST-SSR markers also have high common characteristics in species and can be applied to a series of related species Now EST-SSR markers have been established and carried out various analysis in the grapes, sugar cane, wheat, rye, barley, soybean, rice, pine trees and other plants of tall fescue and Chinese shrimp, martensii, bees, sheep, catfish and other animals in theof.As of December 26,2007, NCBI database has included approximately 245 000 silkworm EST sequences, but these sequences in the EST-SSR is yet clear, even without the report about the use of these sequences to build SSR markers. In this study, we have done a comprehensive analysis of the existing 12 silkworm linkage groups in the EST-SSR, in order to understand the characteristics of silkworm EST resources and lay the foundation of establishing the silkworm SSR markers and exploreing its application in silkworm breeding. The results are as follows:1) 4465 ESTs sequence associated with 12 silkworm linkage groups was btained from the NCBI public database, after clustering and splicing treatment,581 non-redundant ESTs sequences received.154 EST-SSR markers had been Detected in total in 122 Sequences, account for 2.73%, with an average of 3.12 kb EST-SSR; The repeats of Trinucleotide and tetranucleotide are the dominant types, the majority of those are Perfect,accounting for the total number of EST-SSR 36.36% and 28.57%; The average repeat length of nucleotides that are marker by EST-SSR is about 16.2bp and the longest is up to 30 bp; By homology comparison,we did not find any homologous sequences with 86 SSR-EST,we also fond that there are 10of thoes do not meet the screening criteria and the remaining 26 SSR-EST contain a total of 40 SSR,14 (35.0%) in the 5'-UTR,1 1 (27.5%) in the 3'-UTR and 15 (37.5%) in the CDS.2) In order to establish a stable silkworm EST-SSR marker system, L16 (45) orthogonal design was used to optimize the major PCR factors affecting the silkworm EST-SSR and established the best reaction system. In 15μL reaction system, containing 2.0 mmol/L Mg2+,60ng template DNA,0.30mmol/ LdNTPs,0.50μmol/L primer and 1.0U Taq enzyme, The optimal annealing temperature was determined by gradient PCR. Based on this system,we applied four pairs of EST-SSR primer amplify the silkworm genomic DNA of eight strains, and got clear and credible amplification bands, cluster analysis can reflect the geographical origin of the tested the silkworm. Using 0.5 similarities as a cut-off point,8 silkworm can be divided into two major groups, Group I mainly composed by the Chinese system of silkworm, Group II formed by the Japanese system. This study laid a foundation for application of SSR marker to molecular biology study of Lentinula edodes.3) The results show that importing the major dominant fluoride-resistant genes into silkworm Fang Xia by backcross method can improve the preparation of the hybrid generation of fluoride-resistance. Using the Obtained NS1201,pre-EST-SSR markers, which is closely linked with fluoride-resistant genes in Silkworm, to assisted select the target gene in the Backcross breeding process, can greatly reduce the linkage drag and speed up the genetic background of choice backcross speed, and then greatly short the breeding cycle.