Study On The Heredity Of Mutants And Near Isogenic Lines In Silkworm, Bombyx Mori

Silkworm(Bombyx mori)is the important economic insect and satisfactory experimental animals on genetics research.In the early genetics and molecular biology research,there are nearly comparative position between silkworm and Drosophila.Silkworm has several merits,such as:the convenient of operation,the suitable size of individual,the short growth and development cycle,the high of propagation coefficient,and so on.Moreover,its genetic resources are abundant,and it is satisfactory materials for the gene mutant's research.The research of silkworm genetic resources was constantly a significant territory,it provide significant support for related research territory. Now,it becomes the fundament of the researches on silkworm functional genome.The expanding of the basic of silkworm researching,specially,the accomplished of the silkworm whole genome project,not only improved the researches on silkworm in the silk territory,but also made the silkworm attracting more attentions on the research of the mode of Lepidoptera,bioreactor, developmental biology,ect.It is a necessary tendency that silkworm become a mode organism.So, the innovative genetic resources such as new mutants,NILs and inbred strains,were the important experimental materials in silkworm basic research.So,on the background of constructing the biggest silkworm genetic resource in the world,and establishing the genetic resource system for the functional genome research,we systematically performed the discovery of new mutants,phenotype research,gene location and genetic analysis.We bred the chromosomal marker Near-isogenic lines and inbred strains of some important genetic strain,and preliminarily utilized them to locate the mutant genes,and integrate silkworm classical genetic linkage map and molecular marker linkage map.The results we obtained are as the follows:1.Discovery of new mutant genes and important genetic resources in silkwormThe silkworm mutant materials were obtained through extensively and systematically collected diversity resources,especially the rich germplasm resources in China,and physicochemical inductions,or the mutant characters fixed in the separation when wild mulberry silkworm crossed with the domestic silkworm.Then,many new mutants and some important genetic resources were found by character survey and genetic identification.In our research,we obtained many original and innovative results:we found 29 new visible mutation genes;bred the euryphagous genetic systems, such as GS01;found 11 mutant types of ovariole number in silkworm.The 29 new mutants were introduced as follow:(1)8 mutants about shape and colour of eggs:new small egg(sm-n),pink egg(re^p),new maternity white egg(w-1ⁿ),Chinese white egg 3(w-3^C),white egg BT924(w-3^bt),white egg BH863 (w-3^bh),new brown egg 2(b-2ⁿ).(2)7 mutants about body color and stripes of larva:small multistars(ms^s),multistars x(mst^x), wedge eye-spot(Wes),cordiform eye-spot(ces),blind 2(bl-2),red molting(Rm),Xanthous C(Xan^c).(3)4 translucent mutants:sex linked translucent 2(os²),mottled translucent(oh^m),mottled translucent h2(oh^m2),⁶⁰Co-γ-ray-induced translucent(oc^co).(4)3 larva shape mutants:moniliform 2(mr-2),new constricted(co-n),squab silkworm (Sq).(5)2 body color of pupa mutants:black pupa 2(bp²),sooty 2(so²).(6)2 body color of moth mutants:sex-conditioned melanism(sml),black abdomen(Ba).(7)2 mutants in color of cocoon:green d(Gd),new green(Gn).(8)1 mutants in synthesis of silk protein:flimsy cocoon c(flc-c).This mutants covered more than 90%of the total mutants found in China,and covered more than 70%of the total found in the world at the same time.It greatly increased the number of silkworm mutant resources,also improved and completed the Bombyx mori Gene Bank.2.Genetic analysis of new mutants in silkworm(1)In the study,we analyzed the basic genetic regularity of the 30 mutants by meticulously planned and large genetic analysis.Among them,21 were recessive mutant genes and 9 were dominant,sm-n and Get are found to be pseudo-maternally inherited,w-1ⁿ and b-2ⁿ are maternal influence inherited,Get and os² are sex-linked inheritance,sml is typical sex-influrenced inheritance, Wes and Sq are lethal of the homologue mutant.(2)Linkage analyses were extensively performed between the new mutants and the known genes,we fixed 18 of the above-mentioned new mutants on the corresponding linkage group,15of them were determined their locuses.We found a new group of multiple alleles,the relationship of dominance-recessiveness among the genes is+＞oh^m＞oh^m2＞oh. (3)The researches of the genetic law in silkworm have offered some precious new findings. Mainly about:The sex-controlled melanism discovered in our study was the fast inheritance character found in silkworm which showed typic sex-controlled inheritance;we revealed that the epistasis of white egg mutants to the dark color egg(eg:red egg)in Bombyx mori was universal;The outer coating yellow cocoon(C)showed dominant epistasis to the greed cocoon(Gn),the oh^m displayed recessive epistasis to the oc,we also found that a dominancy inhibiting gene to be present to influence the ingestion of cabbage in the inbreeding lines—Gs01;We found the interaction: elations between the brown egg 4(b-4)and the red egg(re);The colour of the egg in double recessive homozygote b-4/b-4 re/re displayed a new phenotype—light orange egg,etc.This discovers made the silkworm genetics more plentiful.3.Identify the genotypes of silkworm mutation systemsThough cis-trans test,we identified the genotypes or genetic models of more than 20 white egg mutant lines,as well as more than 20 red egg mutant lines,and that of some brown egg mutation lines,and so on.We could systematically trim the silkworm mutation genes resource library and utilize these mutation lines based on the informations.4.Linkage localization of mutant genesWe performed the linkage analysis of new mutant genes.Based on the determination of linkage relationship,we applied classic three-point test or two-point test to locate new mutant genes and added them to the silkworm linkage genetic map.In this study we accomplished the linkage localization of 5 new mutant genes.They are new constricted(co-n:2-5.5),wedge eye-spot(Wes: 6-24.3),non-lepis wings(nlw:13-28.6),cordiform eye-spot(ces:14-50.7)and Sq(14-34.6).In addition,we accomplished linkage tests of mf-2,and found that it belong to the 15^thlinkage group.But the locus of mf-2 gene needs to be determined in the future.So,we had added the 5 above-mentioned new located genes into the silkworm linkage genetic map,and revised it.We determined the linkage groups of 24 new mutants and locus of 20 mutant genes.5.The revise of the 27^thlinkage groupThe result of linkage analysis of new green cocoon(Gn)showed that Gn present independent heredity with all of the marker genes in every linkage group of the silkworm linkage genetic map.It seems to mean that Gn is not belonging to any existent linkage group in the silkworm linkage genetic map.According to the recent studies,the 24^thand 27^thlinkage group shoud merge as a new 24^thlinkage group.So,we took the new green cocoon(Gn)gene as unique represent in the 27^th linkage group according to our study,thus,the silkworm linkage genetic map still include complete 28 linkage groups.This is the linkage group which first discovered and determined by Chinese researchers on the research of the silkworm linkage genetic map.6.Construction of linkage localization linesThe classics gene localization analyses need the parent systems contains two or more marker genes which located on the same linkage group,they are named linkage localization lines.We bred and constructed several important linkage localization lines in our study.They were including co-n-Y on 2th linkage group,E^Kp-Wes on the 6th linkage group,b-t-ch-nlw,ch-nlw,b-t-nlw on the 13th linkage group,oa-ces on the 14th linkage group,bl-mf-2 on the 15th linkage group,and nb-ki-2 on the 19th linkage group.Construction of these heredity materials were usefully for the gene location on the same linkage group and integrate these genes with molecular linkage map.It is very difficult to construct the linkage localization lines containing the maternal influence inherited genes.We established an effective program to construct this linkage localization lines in the study.The most important strategy was made the maternal influence inherited genes homozygosis at first.This is a universal problem of biological heredity;the construction program could be used in other organism.7.Construction of Near Isogenic LinesWe used Dazao as donor parental strains to establish Near Isogenic Lines(NILs),which contained one important marker in 28 chromosomes each.The recurrent parents and donor parents had breed crossed 20～25 times.Based on SSR-PCR,it showed the genetic similarity index between the 28 NILs and recurrent parents reached 98.3%～100%.In the breeding,we breed the recurrent parents Dazao simultaneously to ensure the genetic purity of the NILs.Used SSR-PCR to test one of the NILs Dazao-K,the population genetic purity was 99.9%.All the results showed this group of NILs reached a high degree of genetic standard.It was the first time to establish NILs of silkworm on a large scale in history;it's also the most complete suit of NILs in the current world.8.Breeding of inbred strainsUsed the main genetic strain Dazao and C108 in silkworm used in the basic research,we bred corresponding silkworm inbred strains by full-sib mating and choose upwards of 20 generations,and at the same time we also bred inbred strain of euryphagous genetic system GS01.They were named as BmlS-Dazao,BmlS-C108 and BmlS-GS01.SSR-PCR was used to test the individual DNA of BmIS-Dazao₂₀,BmIS-C108₂₀,the population genetic purities of each were 99.17%,99.87%; The population genetic purity of BmlS-GS01₁₀was 88.31%(the genetic purity detect of the 20_th generations was in progress).All the results showed that the genetic purity of bombyx mori inbred strains through 20 generations full-sib mating could come up to the criterion that the genetic purity of mammalian inbred strains must reach 99%above.The three inbred strains had made a foundation of standardization of experimental materials in silkworm basic research,and the inbred strains of euryphagous system GS01 was fitter to be an experimental animal strain.This research was an initiate for the silkworm fuU-sib inbred strains project.9.Molecular location of mutant genes utilized by near isogenic lines and inbred strains(1)Crossed the NILs Dazao-Ze,Dazao-L,Dazao-pe,Dazao-ch with the inbred strains BmIS-C108,we located the SSR linked markets of genes Ze,L,pe,ch in the 3rd,4th,5th and 13th chromosomes and got the SSR linkage map of the fours genes.It integrated the classical linkage genetic map of silkworm and the SSR linkage map primary.It also made an important base for the corresponding integration of the aforementioned linkages with the genome map.(2)Constructed the crossing combined between the inbred strains and the marker genes U,Nd-s,Di,oa in 14th linkage,we located the genes,and integrated the SSR linkage map of the fours genes though mapping software joinmap.The location of the 4 genes and the genetic distance between them were consistent with the 14th linkage chromosome in the classical linkage genetic map of silkworm.The research showed that we can integrate the SSR linkage map with the classical linkage genetic map totally by use standard genetic materials with many experiments.