Mapping Of Minute Wing (mw)Gene And Analysis Of Candidate Genes In Silkworm,Bombyx Mori

The wings of insects have an important role in predation,mate choice,avoidingpredators and expanding distribution,and so on.Under natural selection, insects usuallyevolve in the way that most conducive to their survival and reproduction, so the wings of theinsects come in various forms. Silkworm is the lepidoptera insects as well as model insectfor the genetic research.Through artificial selection, silkworm wings have lost the ability offly, wings also have degenerated severely. Genetic mutations associated with wings cancause different silkworm adult wing shapes or abnormal wing color.At present, the datarecorded in the wing mutant has more than20, it has been reported that some genes whichparticipate in wing forming as well as play an important role in the process of its entiredevelopment, such as embryonic development, somite forming, development of legs, silkgland development, etc. So, researching for the function of these genes in wing formationcan give reference to the research on the development of the organism. In addition, theability of flying are necessary to pests’ spread and spawning, so the research of developmentand mutation mechanism in insects wings provides a very good reference for the preventionand control of the pests in genetics.The silkworm minute wing gene(mw) mutation is one of mutations of silkworm wings,controlled by recessive gene mw, the mutant wings are much smaller than the normal wingsin pupal stage and adult stage. mw gene has been proved in silkworm22thlinkage group. Inthis research, the STS molecular marker technology and CRISPR/Cas system was used tomake gene mapping and analyze the function of the candidate gene respectively. thecontents are as follows:1. Initial Linkage Analysis of Gene mw and Molecular MappingWe used silkworm strain P50as wild type and U11(mw/mw) mutation variety asparents, both of which are preserved in Sericulture Research Institute, Chinese Academy ofAgricultural Sciences. In this research, silkworm variety P50which has the normal wingformation and silkworm variety U11which has the minute wing (mw) were used as parentalstrains to prepare reciprocal populations. After back-crossing，two back crossed populations,(P50×U11)♀×U11♂and U11♀×(P50×U11)♂, were obtained and designated asBC1F and BC1M,respectively. Owing to the lack of crossing over between chromosomes Zand W of silkworm females, reciprocal backcrossed BC1F progeny were used for linkageanalysis and mapping of the mw gene in chr22using backcrossed BC1M.The STS molecular marker linkage are used to make linkage map.We found five STS markersincluding T2210, T2238, T2269, T2272and T2298, which were identified to be linked tothe mw gene. All the5primers have the same homozygous profile as the parental mw in theminute wing individuals of BC1F, and heterozygous in the wild type individuals as inF1.Using MAPMAKER3.0at a LOD score of5.0for further confirmation using BC1M, weconstructed a linkage map of12.4cM, with mw mapped at the end of nscaf3056, T2272isnext to it,which has1.1cM.2. The analysis of candidate genesT2272located at the site of1.265Mb in nscaf3056after blasting the Bombyx morigenome database, the mw gene is located between the end of the nscaf3056and T2272,which has275kb. In this region,there are15candidate genes totally, in these genes thehomologous gene of BGIBMGA012725-TA in Drosophila is relate to the development ofwing disc.But after cloning and sequencing, no differential expression was found in thewing disc between P50and U11. RT-PCR primers were designed based on the EST of thesegenes,we found a gene named BGIBMGA012754-TA expression different between P50and U11, therefore BGIBMGA012754-TA is likely the key gene in minute wing mutation.As the important candidate gene, we had a5’and3’ RACE-cDNA amplification of P50wing disc,but have not get the full sequence of it owing to the unqualified primer or thefailing to predict the complete sequence of the gene.3. analyze the function of the candidate geneGenome editing by CRISPR/Cas system is to use the sg RNA carrying Cas9endonuclease to cut the specific pieces of DNA sequences (PAM),then repaired by thehomologous recombination which will appear gene mutations during its gene expression.We used the CRISPR/Cas9system interfere with the expression of gene BGIBMGA012754-TA to observe whether it is the key gene of minute wing. Results show that afterinterference BGIBMGA012754-TA expression in normal wing strain Nistari, we foundminute wing mutation rate is50%.