Molecular Mapping Of Moltinism Allele (M) And Expression Analysis Of Prediction Genes In Silkworm, Bombyx Mori

In silkworm, Bombyx mori, molting is an important physiological phenomenon.Moltinism character of the silkworm is mainly controlled by M allele which on6th linkagegroup, as well as under the regulation of many minor genes and environmental conditions.In this research, High-resolution melting （HRM） analysis technique and simple sequencerepeat （SSR） molecular markers are used for mapping and lingkage analysis. Candidategenes are cloned by RACE, and in order to find out the major gene controlling moltinism insilkworm, the expression of all candidate genes are detectedThe progress of the study are as follows:（1） Discussion on the possibility of genotypic identification through SSR molecularmarker in silkworm based on HRM technologyTheoretically, Single-base genotype polymorphism can be identified by HRMtechnology. In order to determine the optimum technology parameters of HRM analysis forSSR molecular markers in Silkworm genome, and verify the production mechanism ofmelting curve polymorphism, we choosed3typical melting curve polymorphism which hada higher frequency in the experiment to be recovered, cloned, sequenced and CLUSTALWanalysised.The results showed that the production mechanism of marker polymorphisms wereformed due to comprehensive effect of sequence length polymorphism and single basepolymorphism using HRM analysis techniques. The study proved that SSR molecularmarkers were genotyped by HRM analysis technique was reliability and feasibility.（2） Molecular mapping of moltinism allele （M） in silkworm, Bombyx moriIn this study, we used a tetramolter race, Dazao （p50,+M/+M）, and a trimolter raceC3white （M3/M3）as parental strains to prepare BC1M population [p50×（C3white×p50）],598trimolter individuals of BC1M were employed as mapping population to fine map the Mallele, while SSR molecular markers were genotyped by HRM analysis technique.Polymorphic SSR markers linked with M allele were found by HRM, and we futureconstructed the fine molecular linkage map of M allele. The map covered a total geneticdistance of2.4cM. Marker S2853-2527and S2853-2843was located on the two sides of Mlocus, and the genetic distances between M and the marker was0.2cM and0.7cM,respectively. The M allele was located on the nscaf2853:2,527,929-2,843,864of the silkworm genome, and in the region there were4prediction genes that all belonged tosequence-specific DNA binding transcription factor. We guessed the action mode of Mallele might be related with it.（3）3’RACE of candidate genesBased on the fine mapping of M allele and the silkworm genome database, we designedspecific primers according to the CDS sequence of the candidate genes.3’-end unknownsequence of4genes were cloned using RACE technology.We obtained one sequence from p50strains （p50₆391） and two sequences fromC3white strains （C3₆391-1, C₆391-2） by3’RACE for gene BGIBMGA006391-TA.Among them, C₆391-1was the right product of BGIBMGA006391-TA with perfect3’terminal. C3white₆391-2might be the product of BGIBMGA006392-TA with perfect3’terminal.We obtained one sequence from p50strains （p50₆393） by for geneBGIBMGA006393-TA, which the length is523bp with perfect3’ terminal.We also obtained two sequences from p50strains（p50₆394-1,p50₆394-2）and onesequence from C3white strains （C3₆391-1, C₆391-2） for gene BGIBMGA006394-TA.p50₆394-1was the correct product of BGIBMGA006394-TA. p50₆394-2wasn’t locate inthe positioning interval through BC1M backcross mapping population using HRMgenotyping verification. C3white₆394wasn’t BGIBMGA006394-TA, it was the otherhomologous gene with conserved sequence. And the distance betweenBGIBMGA006394-TA and C3white₆394was about80K.（4） Expression analysis of candidate genesAccording to the results of3’RACE, the expression levels in different stage for5genesin larvae of tetramolter race p50and trimolter race C3white were detected and analyzed byfluorescent quantitative PCR technology.From the analysis between the change of expression of each gene and molting in p50and C3white, We could see that the dynamic change of p50₆391, C3white₆391-1, andC3white₆391-2in the larval stage were not relation to molting and dormancy, and wecould think thar candidate gene BGIBMGA0006391-TA had nothing to dormancy trait.The expression pattern of p50₆393were similar between1instar in trimolter and1-2instar in tetramolter,2-3instar in trimolter and3-4instar in tetramolter. There seems to besome relationship between the high expression of3-4instar in trimolter,3-4instar intetramolter and the decision of last instar. Differences and conversion of expression pattern were very similar in p50and C3white for C3white₆394, suggested a correlation between changes in expression levels ofC3white₆394and molting cycle and dormancy traits.The p50₆394gene only detected in p50race, and failed in all developmental stages inC3white. we guessed that p50₆394gene might have nothing to do with major gene ofmoltinism and dormancy traits.The results obtained in this research provided basic information to reveal the complexpattern regulation for molting in silkworm, Bombyx mori.