Quantitative Trait Loci Analysis And The Major Gene Identification Of Silk Yield Related Traits In Silkworm,Bombyx Mori

Silkworm is an economic insect with great importance.Its powerful ability to synthesize silk protein laid the foundation for sericulture.However,the breeding of silkworm has stepped into the platform with no obvious elevation in silk production since the 70s of the last century.Thus,the breeders are eager to utilize the modern molecular technology with increasing development to promote the elevation of the economic traits of silkworm,such as the silk production.Besides,the silk proteins,including sericin and fibroin,have been shown to have a broad prospect of application in other emerging areas,such as the bio-medicine and functional bio-materials,etc.And for the powerful ability to synthesis silk protein,silk gland has been considered to be an ideal bioreactor to produce exogenous proteins.Note that understanding of the genetic basis underlying silk production and the regulatory mechanism of silk protein synthesis is indispensable for the molecular breeding and the new application of silk proteins and silk gland.The silk production related traits or the silk yield related traits,such as cocoon weight?CW?,cocoon shell weight?CSW?,cocoon shell ratio?CSR?and pupal weight,varied quantitatively and have complex genetic inheritance.Accordingly,quantitative genetic analysis is necessary to conduct to decipher their genetic basis.Even though several quantitative trait loci?QTL?analysis have been performed to analyze the genetic architecture of the silk yield related traits,there still has no successful case to identify genes controlling them.And the genetic characteristics of silkworm,such as the gender effect and female achiasmatic,may be the main factors hindering the identification of the controlling genes by traditional QTL mapping methods.Considering the interference of the genetic characteristics,we herein developed a methodology which is capable of mapping the QTL loci efficiently in silkworm.And by this method,we analyzed the genetic architecture of CSW,performed the fine mapping of the locus with main effect and identified the major gene.The follows list the main results in this research:1.Investigation of the genetic characteristics of the silk production related traits.For further understanding on the genetic characteristics of silk production,we investigated the silk production related traits of 98 silkworm germlines and of the artificial produced population.The investigation of the germline strains showed that these traits varied greatly among different strains;take the male as example,the CW,CSW,CSR and PW varied from 0.579g to 1.488g,0.074g to 0.375g,9.7%to 29.1%and from 0.505g to 1.147g,respectively.Except the CSW,the other traits exhibit obvious gender effect;the gender difference of CSW has a varied performance in different lines.Analysis of the trait correlation showed that there has a high correlation among these traits and out of them,the correlation between CSW and CSR,the two important traits related with silk production,is high up to 0.94.This indicates that these traits experienced a strong co-selection during domestication.Simultaneously,we made a serious of artificial population,including F₁ and the backcross population?BC₁?,and surveyed the silk production related traits in these population.And the results showed that the higher performance of each trait is dominant over the lower performance to some extent.Among them,the dominant degree of CW and PW is higher and that of CSW and CSR is a slightly lower which exhibit an incomplete dominance.It is noteworthy that the investigated traits showed a typical patrolinal inheritance.Besides,in light on the frequency distributed characteristics of these traits,we speculated that the genetic architecture underlying these traits harbors significant major gene effect.Compared with the correlation level among different traits in germline strains,this level in BC1 population is significantly lower,such as the correlation between CSW and CSR is 0.65;Combined with the frequency distributed characteristics of them in germlines,we concluded that the domestication and the genetic breeding in past century are the two distinct selection process and simultaneously indicative of the selective difference in the genomic level.2.Quantitative trait loci analysis of cocoon shell weight?CSW?Given the genetic characteristics of silk production related traits,we developed a methodology suitable for the efficient mapping of silkworm quantitative traits.We fit in this methodology the gender effect and female achiasmetic by the combination of suitable population selection and QTL analysis model.And it can be divided into two steps.The first step is the linkage analysis by selective genotyping to screen the chromosomes that harbor QTL loci controlling the trait of interest.Based on the female achiasmatic of silkworm,the linkage analysis population?BC₁F?was produced and from this population,individuals with the extreme high and low trait performance were selected to form two subpopulations contrasting for the trait of interest.Then,one or several polymorphic markers on each chromosome were developed and genotyped in the extreme population to identify the chromosome with QTL loci which was selected as the targets for the following mapping analysis.In the second step,more polymorphic markers were developed on the target chromosomes to make a genetic map with high density.Meanwhile,the segregation population?BC₁M?by backcrossing the male F₁ with two parents and then the comparable number of individuals were randomly selected in the two BC₁M population to form the mimic F2population?miF₂?.By this population and the genetic map,the genetic architecture of the trait of interest was analyzed including the QTL loci and the corresponding effects.Note that individuals used in the whole analysis were male to avoid the inference of gender effect.Through this methodology,we conducted the QTL analysis of CSW of silkworm.By linkage analysis,the chromosomes harboring QTL loci were screened which includes the 8^th,11^th,22^nd and the 23^rd chromosome.Among them,the linkage level between markers on the 11^th and the 22^nd chromosome and CSW is the most significant;furthermore,considering the effect of the 1^st chromosome on the economic traits,we select the 1^st,22^nd and 23^rd chromosome as the targets for mapping analysis.The results showed that each chromosome contains one QTL locus,and we named it csw1,csw2 and csw3;out of these,the csw1 and csw2 explain 34.1%and 15%of the phenotype variation and are the main effect loci;the csw3 explains 4.7%of the genetic variation which is a minor effect locus.Besides,we also identified one pair of epistasis between the locus on the 11^th and the 22^nd chromosome,and we named it cswE.These results laid the foundation for the following fine mapping.3.Fine mapping of csw2 and screening of candidatesTo confirm the accuracy of the QTL mapping result,we performed a BSA-sequencing experiment to screen the loci linked with CSW.Combined with the primary mapping result,csw2,the locus on the 11^th chromosome with a high linkage significance and genetic effect was selected as the target for the fine mapping.And in this mapping period,selective genotyping was utilized;thus we firstly genotyped the markers used in the extreme subpopulations which are phenotypically contrasting with respect to CSW.The result showed a high consistence with BSA-sequencing and primary QTL mapping result which proved the accuracy of mapping result.Afterwards,a larger backcross segregation population was produced and another and larger set of extreme subpopulations was selected and formed.By this population,the csw2 was narrowed into a locus covering 270kb genome region.This region contains 17predicted genes and then through the spatial and temporal expression pattern analysis,we identified a silk gland development associated gene,BMgn011646.Determination of the expression level in the silk gland of different silkworm with varied silk production showed that this gene expressed a pattern associated with CSW which indicates it a plausible candidate.This gene encodes the?-N-acetylglucosamine glucosidase 1,thus we termed it BmGlcNase1.4.Functional and domestication characteristic analysis of BmGlcNase1.By cloning sequencing,we surveyed the sequence variation in the cDNA and genomic region between parents.The result showed 34 SNPs in the full cDNA,out of them,30 locate in the CDS region and another 4 locate in the 5'UTR.Except for the SNP?G-C?at the 13^rd nucleotide of ORF,which transformed the Gly to Arg,all of other SNPs belong to synonymous mutation.But the amino acid substitution locates in the signal peptide region and it has no effect on the signal peptide characteristic.Investigation of the genomic sequence of candidate showed that there are multiple variation in the upstream and the introns,including SNPs,Indels and sequence substitution,in the genomic region.Based on the sequence variation,we developed markers and genotyped them in 98 surveyed germline strains.By association analysis between these variation and CSW,we detected a high association between CSW and the variants at the upstream and the first intron.Among them,the Indel at the 1,200bp upstream of the transcription start site showed the highest association significance with CSW.Besides,we verified the function of BmGlcNase1 by a series of transgenic experiments.The expression level of the candidate was specifically inhibited in the middle silk gland and it caused a significant decrease of CSW in the RNAi line.Furthermore,elevation of its expression in the middle silk gland increased the target trait,which combined with the RNAi experiment proved the function of BmGlcNase1in the regulation of CSW.Additionally,we also ectopic expressed this gene in the posterior silk gland but it has no effect on CSW.These result suggested that BmGlcNase1 may involve in the regulation of the sericin synthesis in middle silk gland but has no effect on the fibroin synthesis.We also determined the heterozygosity of the sequence variation of the genomic region in the wild silkworm,the varieties and the breed population of silkworm.By so doing,we found that this associated Indel experienced the different selection patterns in the domestication and the genetic breeding process;during the long-term domestication process,the allele with negative effect on CSW was weekly selected,while in the following breeding process based on the modern genetic theory,the allele with positive effect on CSW was strongly selected.Collectively,we developed an efficient QTL mapping methodology suitable for silkworm quantitative traits analysis.By this methodology and the following fine mapping,BmGlcNase1 was identified to be capable of regulating the synthesis of the sericin in middle silk gland.As we know,this is the first gene controlling silk protein synthesis identified by forward genetics method.Simultaneously,through the domestication characteristic analysis on the associated variation at the upstream of BmGlcNase1,we found the different selection pattern of this variation in the domestication and the genetic breeding process.These results can not only facilitate our understanding of the genetic basis underlying the silk protein synthesis regulation and the variation of the cocoon shell weight in different silkworm strains,but also contribute to our knowledge of the domestication and breeding history of silkworm.