The Origin And Differentiation Of Silkworm, Bombyx Mori, Based On The Bmamy2 Gene

With important theory value and practice guidance to variety identification,resource using and hybrid vigour application,it is an important basic science subject about the sericulture history and sericultural industry production to study the origin and differentiation of silkworm,Bombyx mori.A lot of researchers from many countries have been attracted by the subject of the origin of silkworm since Indian entomologist,Hutton,pointed out it.1915,Japanese scholar,Qing mu qing,studied the relationship between B.mori.and Bombyx mandarina with blood serum-sediment reaction method, after that in about a centry,many researchers from internal and foreign,especially from China and Japan had made many efforts and obtained a lot of results by using various methods,such as bibliography,climatology,palaeobiology,bioecology,geography,archaeology,genetics,physiology, biochemics,cytology,molecular markers(RAPD,AFLP,SSR,etc),DNA sequence analysis,etc. They made a general approval that B.mori originated from Chinese Bombyx mandarina and were bred into different geography systems and ecological types by nature and artificial selection in the other world after spread from China,but there has long been dispute about where and how B.mori originated at the first time.There are three main hypotheses.The first is "univoltine and one centre" hypothesis pointed out by Jiwu based on the isoenzyme polymorphism of several enzymes.The second is the "multi-voltine and multi-centre" theory pointed out by Prof.Jiang based on the information of palaeobiology,climatology,and archaeology.The third is the "mix-voltine" theory pointed out by Prof.Lu based on the research using molecule markers.DNA is true genetic material which can provide more accurate genetic information than morphological and biochemical markers.Molecular phylogenetic analysis based on DNA sequences is the best effective and reliable method for systematics of organisms especially for those domesticated species.However,to investigate phylogenetic relationships among closely related species,it is necessary to look for a rapidly evolved gene.In this study,I sequencedα-amylase gene of different silkworm strains and different B.mandarina populations to study the origin and differentiation of domesticated silkworm,B.mori.The major results are as follows1.The bioinformatics analysis of theα-amylase geneα-amylase gene is a multi-gene family with improtant function for animals.Different species have differentα-amylase gene structures and corresponding distribution patterns in respective genomes.In some vertebrate,such as Gasterosteus aculeatus,Xenopus tropicalis,Gallus gallus and Homo sapiens,there are many overlapping genes and the a-amylase genes from different species have conservative structure,the different gene copies intra-species resemble in amino acid sequence extremely.But in some insects,such as Drosophilidae,Culicidae and B.mori,there is no overlapping gene and the genes have no conservative structure,the different gene copies within-species differ in amino acid sequence greatly.Theα-amylase genes differentiate extremely and display rich polymorphism in Culicidae and B.mori.There is only one copy ofα-amylase gene in C.elegans genome,while the gene family enlarged greatly in higher animal such as insect and mammal.The different gene copies intra-spesies cluster together respectively suggesting the gene copied after the species split in vertebrate,but in insect the clusters consist of gene copies from different species suggesting the gene copied before the species split.Further,theα-amylase gene families enlarged greatly after species split in Culicidae and B.mori.The gene copise from Culicidae and B.mori cluster into two clustes,one of which keeps close relationship with bacteria.Three of B.moriα-amylase genes with similar gene structure distribute in the same chromosome implying the copy relation.2.The cloning,expression pattern and sequence analysis of Bmamy2 gene in silkwom,B.mori.Bmamy2 CDS is 1752bp long and codes for 583 amino acids.There are 8 introns following the "GT-AG" rule in Bmamy2 gene and the first intron is 393bp long,the sixth 505 bp,the seventh 647bp,the eighth 1828bp,the others about 100bp;and the fourth exon is the longest,582bp,the others are short than 100bp.The Bmamy2 gene is expressed highly in midgut,lowly in fat body while not in head,epidermis,hemolymph,silk gland,testes,ovary inspecting by RT-PCR and microarray.It may lose the function to catalyze hydrolysis carbohydrate caused by the two key amino acid mutations in the catalysis centre(D258Y,D423P).The Bmamy2,which has orthologous gene in bacterium,is a fine marker to investigate polymorphism in silkworm.Our results suggested that nucleotide mutation rate is 5.6%in B.mori and 8.3%in B.mandarina.3.The population structure and genetic differentiation in B.mori.Although we initially sampled the silkworm according to ecological type classification,we failed to identify ecological type-specific clades in the study.Three clades were identified in 72 local strains,each of which consists of strains from different ecological types while the same ecology type strains distribute in different clades.There is great genetic diversity among clades and small diversity within clade among strains.It is presumed that several domestication events took place at different sites independently to make mix-voltine B.mandarina into mix-voltine B.mori.4.The geographic population structure and polymorphism in B.mandarinaThe B.mandarina is whidespread in Southeast Asia especially in China with multiple voltine of univoltine,divoltine and multi-voltine,and multiple molts of three molts,four molts and fewer five molts.B.mandarina has big polymorphism in morphological and ecological traits,development, food habit,resistance and molecular marker.Based on partial sequence of Bmamy2 gene,the nucleotide mutation rate is 8.3%in entire sequence and 10.8%in intron sequence,the haplotype diversity is 0.987±0.023,the nucleotide diversity is 0.02281±0.0034.Overlapping generation is one of the most popular ecology characteristic to B.mandarina for eggs hatching chronically and pupa development diversify,some have completed a generation while some did not hatch.It is a method to survive hard condition and keep up hybrid vigour avoiding sib mating.AMOVA indicates that being of mix-populiation of several ecological types and molts;there is no geographic population structure in B.mandarina anywhere.The genetic distance is not correlated with space distance.On the phylogenetics tree,the individuals from different regions clustered in the same clade,while the individuals from the same region distribute in different clades.5.The origin and differentiation of silkworm,B.mori.The same phylogenetic pattern is revealed by several inspections such as phylogenetics analysis, network analysis,AVOVA analysis,based on the nucleotide sequence polymorphism of Bmamy2 gene.90 samples of B.mori and B.mandarina clustered into four clades,each of which consists of B. mori from different ecological types and B.mandarina from different regions,with great genetic diversity between clades and small diversity between strains within clades.The domestication should start at multi-type B.mandarina because the B.mandarina are mix-typed populations anywhere and the silkworm strains in different ecological types might experience the same evolutionary process.The great genetic diversity between clades reveals the different geographic origin of silkworm in different clades.In summary,our results suggest that the silkworms originated from mix-typed B.mandarina in several different regions.