The Positioning And Genetic Analysis Of Diapause And Voltinism Genes In The Silkworm, Bombyx Mori

Silkworm is a representative insect in lepidoptera. Voltinism is an importantphysiological character of silkworm. Voltinism refers to the amount of insect generationshappening per year, diapause is the phenomenon of stagnation growth in order to adaptadverse environment. Silkworm is egg diapause insect, it can be divided into univoltine race,bivoltine race and multivoltine race. Diapause hormone is the main control factor ofsilkworm diapause, in addition, the juvenile hormone and molting hormone has a certaininfluence on silkworm diapause. Silkworm diapause is also affected by some environmentalfactors, such as temperature, humidity, photoperiod and food. Traditional silkworm geneticsconsidered that silkworm voltinism regulation genes are associated with Lm and V. Lm geneis located on the linkage group1, Lm>+Lm>Lme; V gene is on the chromosome6, V1>+V>V3.The existence of Lmegene can promote silkworm voltinism migration in the direction ofmultivoltine. On the basis of Lmegene genetic analysis on nistari’s Z chromosome, weconstructed Lmegene positioning group with nistari and p50. The bivoltine race silkwormp50incubates non-diapause eggs under low temperature,and silkworm race54A is notsensitive to environmental factors.Taking advantage of the characteristics of p50and54A,we built the+Vgene positioning analysis group.Early in this study, the survey ofmultivoltine race nistari quoted from India autosome genetic separation materials bybiological statistical analysis found that the controlling patterns of nistari was complicated.Therefore, we used genetic breeding technology, molecular marker technology and theHRM analysis technology respectively on Lmegene and+Vgene positioning analysis; Inaddition, this study would focus on building nistari genetic patterns. The main researchresults were as follows:(1) Sex-linkage precocious gene LmepositioningChoosing multivoltine silkworm race nistari as female parent (P1) and bivoltinesilkworm race p50as male parent (P2), we utilized nistari and p50to build F1generationgroup; F1male moths backcross female parent p50, building BC1M group. P1, F1and P2were used for selecting the polymorphic SSR markers, BC1M female progeny which laynon-diapause eggs were used for positioning analysis. Based on this result,10polymorphicSSR markers which linked with the Lmegene were developed, and734BC1M femaleindividuals which laid non-diapause eggs were used as experiment material for fine mapping. Based on genotype classification results, we drew Lmegene genetic linkage mapby Mapmker3.0. The general genetic distance of linkage map was12.91cM. The resultpresented that a region of~226kb tightly linked to the Lmegene between the markersS2734-299and S2734-525was identified.(2) Silkworm voltinism major gene+Vpositioning analysisThe female p50parent (P1) and the male54A parent (P2) were selected from thehomozygous recessive individuals and dominant individuals, respectively. Both of themwere used as parent strains for building F1.The female moths of F1offspring were used forback-crossing with P2to produce BC1F progeny. The male moths of BC1F offspring wereused for back-crossing with P1to produce BC2M progeny. P1, P2and F1were used forselecting the polymorphic SSR markers, and BC2M female progeny which lay non-diapauseeggs were used for positioning analysis. BC2M group was incubated in low temperature, andraised in suitable temperature. Offspring produced diapause eggs and non-diapause eggsratio is3:1. Based on this result,12polymorphic SSR markers which linked with the+Vgene were developed, and310BC2M female individuals which laid non-diapause eggs wereused as experiment material for fine mapping. Based on genotype classification results, wedrew+Vgene genetic linkage map by Mapmker3.0. The general genetic distance of linkagemap was52.44cM.+Vgene positioning analysis was finished preliminary.(3) Linkage analysis of multivoltine race nistariIn this experiment, we tried to use nistari and p50respectively to build F2, F3generation, since multivoltine race nistari regulation pattern was very complicated. Thefemale nistari parent and the male p50parent were selected from the homozygous recessiveindividuals and dominant individuals, respectively. We concluded an interesting result fromthe analysis of F2, F3generation. The multivoltine character of nistari was not associatedwith V3gene.The female moths of F1offspring were used for back-crossing with P2to produce BC1Fprogeny. The moths of BC1F offspring were used for self-crossing to produce F3progeny.Primarily,5polymorphic SSR markers from silkworm chromosome27used in F3inividualslaying non-diapause eggs female moths.The results showed that it was tightly linked to thetwo markers S2836-2and S3072-3on chromosome27. About100polymorphic SSRmarkers from silkworm autosomes were ued to analyze F3individuals which layingnon-diapause eggs female moths. The result of other chromosomes’ polymorphic SSRmarkers showed that some individuals were linked to the S2209on chromosome22,andsome were linked to the two markers S1907and S2601on chromosome26.Besides,about80 F3laying non-diapause eggs female moths were tightly linked to the S2529-4onchromosome5. Thus, there might be others gene on chromosome5associated with nistarimultivoltine.