Study On No.27, 28 Linkage Group Based On The Fine Map For The Silkworm (Bombyx Mori) Genome

Silkworm(Bombyx mori) is an insect whose genetics has been studied perfectly,but the 27 and 28 linkage groups have not been constructed till now.On the basis of silkworm fine genome sequence,we study deeply on chromosome 27 and 28,integrating map of linkage group,mending Gaps,gene prediction and annotation,screening and identification gene on chromosome 27 and 28. The results acquired can be used as providing important clues for constructing linkage groups 27 and 28.1.Integrating genetic map of chromosome 27 and 28 in Bombyx moriAn 9-fold coverage of fine genome data provided was used in the present study,which consists of 43622 scaffold sequences and 14632 predication genes.1577 SNP and 3000 SSR markers were organized and utilized.32 and 24 SNPs on chromosome 27 and 28 were screened respectively,and 9 and 5 genome segments were located on chromosome 27 and 28 respectively.The results show that the two chromosomes span 10.4Mb and 10.3Mb of genome sequences individually.2.Filling and verification of Gaps on chromosome 27,28In silkworm genome fine map there are remaining some gaps on chromosome 27 and 28. Chromosome 27 consists of 9 genome fragments in which exist 8 gaps,yet chromosome 28 includes 5 genome fragment and 4 gaps.We extracted end sequences derived from BAC clone in a BAC library using perl program,and aligned them against the 9-fold genome data with BLASTN program.The one with the highest score was defined as the expected result,and used to be candidate BAC to link Gaps.Possible "novel" fragments were found on the two chromosomes,such as the nscaf2803 and nscaf2804 covering approximately length 200kb on chromosome 27 and the nscaf3101 on chromosome 28.Two filled gaps to both chromosomes were selected randomly.The primers were designed on non-repeated genome sequence of length 10kb at the edge of the gaps,PCR experiment verified the bioinformatics results with BAC clones as a template.Filling the gaps of both chromosomes provided effective methods and data for perfecting DNA sequences of chromosome 27 and 28,and also added new contents in genome fine map of silkworm.3.Annotation of genes on chromosome 27 and 28Chromosome 27 and 28 were estimated to contain 241 and 288 prediction genes individually. The length of less 2000bp for putative protein coding sequence(CDS) are absolutely predominant, approximately 80.19%and 87.85%of gene total counts respectively.The enriched genes ranging in size from 400 bp to 800 bp in length,account for 22.82%and 26.39%among the total genes respectively,highly similarity for both chromosomes.The average presence of exon 7.7(>5.4) and exon 5.3(-5.4) of each gene on chromosome 27 and 28.Especially the presence of exon 102 of the gene BGIBMGA004547 located within chromosome 27,is the gene with the maximum exon count among silkworm genes.The intron length ranges from 49-9992bp in genes of chromosome 27,the average length is 1121bp(n=1619).Among them,the introns ranging in size from 500-999bp are most predominant, approximately 37.18%of gene total counts.The intron length ranges from 49-9919bp in genes of chromosome 28,the average length is 1354bp(n=1224).Among them,the introns ranging in size from 500-999bp are most prevalent,approximately 34.72%of gene total counts.This indicates that middle and small introns are predominant within the both chromosomes.And their average intron length both near the silkworm genome's average intron length(1289 bp).For some research object,its GC content is invariable.GC contents of CDS in chromosome 27 and 28 are 49.72%and 46.54%individually,and GC contents of whole chromosome sequence in the two chromosomes are 38.09%and 38.44%.GC content of CDS is more than that of chromosome sequence,which indicates that GC content of CDS is more than that of non-CDS.Putative protein domain analysis show that there are 76 kinds of domains,including Sugar_tr, MFS₁,zf-C2H2,Ion_trans,BESS,Trypsin and HSP20 etc.on chromosome 27,and 92 kinds of domains on chromosome 28,such as UDPGT,DUF,Serpin,Trypsin,Homeobox and ig etc..There are 195 and 227 microarray gene probes in chromosome 27 and 28 individually.The tissue-specific microarray data derived from this probes were preformed a clustering analysis.The results show that genes in 27 can be classified to 7 groups,and they exist obviously differences among tissue expression pattern.Genes in 28 also can be classified to many small groups and 4 groups are obvious. 4.Identification and analysis of Sugar Transporter gene family of silkwormDuring the process of gene prediction and annotation,a significant feature of chromosome 27 was found:existing a lot of sugar transporter genes(ST gene).To makes clear if the character is benefit to study the organization of 27 and find mutant genes,we analyzed the ST gene family.719 genes with ST or MFS annotation were extracted,and searched with BLAST,predicted transmembrane domain.98 ST genes were identified;about 0.7%of total genome genes.58 genes of 98 have EST evidence,approximately 57%of predicted ST genes counts.84 ST genes can be located on chromosome.The amounts located on chromosome 27 are 18 genes being largest.The clustering analysis indicate that two groups of ST genes have certain predicted function:one is transporter for glucose and trehalose another is for sugars and organic cations.The structure of ST genes in chromosome 27 was analysed.The results show that it is the largest tandem repeat gene cluster.7 ST genes range in size 680kb in nscaf2797,spanning 140kb in nscaf2800 containing 8 ST genes.The transmembrane structure varies from 2-12.Analysis about microarray expression indicate that 8 ST genes of 27 chromosome display upregulated expression in larvae silkgrand versus 5 in midgut which absorbs specifically nutrients.This may be relate to needing lots of energy materials for growth of silkgland.On development features of silkgland and midgut,we speculate upregulated expression of ST genes in midgut is a long-term feature during the whole growth except dormancy,versus a special phenomenon in special time in silkgland.5.Possible phenotype of mutant genes on chromosome 27 and 28Genes on chromosome 27 and 28 were searched homologous to Drosophila mutant gene sequences and got predicted genes in both chromosomes.26 predicted mutant genes were located on chromosome 27 through homologous alignment,16 of them being obviously related to reproduction, development,metamorphosis and morphologicalphenotypes.15 predicted genes on chromosome 28 are correspond to Drosophila mutant phenotype.This may provide important insights into discovering morphological markers on chromosome 27 and 28,and be benefit to collect various morphological mutants in a mutant resource pool which can be created through various mutagenesis approaches.A systematic study was preformed on chromosome 27 and 28 at present study.The analysis of existing possible phenotype mutants,all of these shed light on study of linkage group map of silkworm,Bombyx mori.