| Coloration is one of the critical attributes of organisms,which is involved in numerous biological behaviors such as concealment,camouflage,courtship,and signaling,and is crucial to the survival and reproduction of populations.In the life sciences,the study of coloration has played a vital role in constructing genetic,developmental,and evolutionary theories.The domestic silkworm was domesticated from the wild mulberry silkworm,whose cocoons are homogenized brown-yellow,while the cocoons of the domestic silkworm are incredibly diverse in color.According to the type and content of pigments,the cocoon of the domestic silkworm can be divided into three categories:white cocoons without or containing traces of pigments;yellow-red cocoons caused by the accumulation of carotenoids,such as golden cocoons,flesh-colored cocoons,pink cocoons,and rust-colored cocoons;green cocoons formed by the accumulation of flavonoids.The silkworm cocoon color system provides excellent material for studying the physiological and genetic related to biological coloration,as well as phenotypic evolution.The silkworm is an essential economic insect,and the silk it produces is a textile material with a long history of application.Many studies have shown that due to flavonoids,carotenoids,and other natural pigments,colored silkworm silk has stronger antibacterial,antioxidant and UV radiation resistance properties than white silkworm silk and harbors more excellent development value.Elucidating the genetic mechanism of the formation of colored cocoons of the silkworm is of great significance for the in-depth development of colored cocoon resources.The international sericulture community has previously revealed the genetic basis for the formation of the yellow-red cocoon of the silkworm,and the proteins responsible for carotenoid transport within the cell and on the cell membrane are critical to yellow-red cocoon formation.Classical genetic studies have identified at least seven loci involved in forming green cocoons,including Ga,Gb,Gc,Grc,Gre,Lg,and Yf.Currently,the Gb and Lg have been elucidated.The Gb encodes a uridine diphosphate glucosyltransferase（Bm UGT10286）,which is involved in the glycosylation of the 5-O position of quercetin,and the functional gene of Lg encodes a pyrroline-5-carboxylate reductase（Bm P5CR1）,the defect of Lg will lead to the accumulation of1-pyrroline-5-carboxylic acid and thus promote the production of proline flavonols.Metabolic modifications of flavonoids can significantly enhance their"permeability"in the silkworm,allowing them to be transported to the silk gland and to form green cocoons.However,the proteins responsible for the transport of flavonoids have not yet been identified.In addition,the evolution of cocoon colors from single to diverse cocoons,along with the domestication of the mulberry silkworm,is also remarkable,but little is known about the genetic mechanisms behind it.Our group discovered the New green cocoon（Gn）,and its cocoon color is bright and stable,and its genetic locus has been proved to be on chromosome 27,which is independently inherited from the reported green cocoon loci.In this study,the molecular mechanism of green cocoon formation was deeply revealed by taking Gn as the object of study.Meanwhile,the genetic mechanism of cocoon color evolution from wild mulberry silkworm to domestic silkworm was systematically analyzed based on the pangenome of the domestic silkworm.The main research results were obtained as follows:1.Fine mapping of the GnBased on the genetic law of complete linkage of chromosomes in female silkworms,males from the progeny of a cross between the New green cocoon strain G200 and the white cocoon strain 872B were backcrossed to 872B to obtain the BC1M population used for positional cloning of Gn.Genotyping of 1035 individuals building white cocoons in the BC1M population using 17 polymorphic molecular markers on chromosome 27 locked Gn to a 500 kb genomic region on chromosome 27 of the silkworm,which contained 17 protein-coding genes,including a sugar transporter gene cluster,Gn＿Str＿cluster;the Gn＿Str＿cluster consists of seven members,Bm Str Gn1 to Bm Str Gn7.2.Screening of candidate genes for GnTo identify the candidate genes of Gn among 17 genes within the Gn locus,we performed a joint analysis of gene expression patterns with the green cocoon formation process and an association analysis of gene sequence variation with green cocoon.Firstly,by dissecting and observing the silk glands of the New green cocoon strain,N100,at each developmental stage,it was determined that the fifth instar is a critical period for accumulating flavonoids in the silk glands.Consistent with the accumulation pattern of flavonoids in silk glands,the five members of the Gn＿Str＿cluster,Bm Str Gn3,Bm Str Gn4,Bm Str Gn5,Bm Str Gn6,and Bm Str Gn7（Gn＿Strs）,were specifically or highly expressed in the critical tissue（silk glands）and critical period（the fifth instar）of flavonoids uptake and were significantly more expressed in N100 than in 872B.Sequence investigation of Gn＿Strs in multiple strains revealed a dense distribution of sequence variants highly associated with the green cocoon in their coding and regulatory regions.In addition,one-way ANOVA indicated that the variation in the coding region of Bm Str Gn4 showed the most significant association signal with the green cocoon and the degree of green cocoon coloration.Based on the above findings,the Gn＿Strs were targeted as candidate genes for Gn.3.Functional study of Gn＿StrsSingle or multiple Gn＿Strs were knocked in the G200 using CRISPR/Cas9-mediated gene editing techniques based on the spatiotemporal expression characteristics of the genes:the genes with unique expression patterns（Bm Str Gn3,Bm Str Gn4,Bm Str Gn7）were knocked out individually;the genes with the similar expression patterns（Bm Str Gn5 and Bm Str Gn6）were co-knocked out;the large genomic regions where Gn＿Strs that were consistently highly expressed at the fifth instar（Bm Str Gn4 to Bm Str Gn7）were knocked out integrally.By investigating the cocoon color and the content of flavonoids in the silk glands and cocoons in each knockout line,it was found that the functional defects of different Gn＿Strs members would lead to the discoloration of cocoons and the reduction of flavonoids content in silk glands and cocoons to various degrees.The strongest effect of individual members on phenotype was found in the Bm Str Gn4.These results suggest that Gn＿Strs synergistically facilitates the uptake of flavonoids in the silkworm and ultimately affects the formation of green cocoons.4.Functional evolution of Gn＿StrsTo reveal the molecular mechanisms underlying the origin and functional evolution of Gn＿Strs,we performed a phylogenetic analysis of Strs encoded by the whole genome in several Lepidopteran insects and investigated the differentiation characteristics of gene structures and expression patterns of Gn＿Str＿cluster members.Gn＿Strs were found to be specific Strs in the Bombycidae,which may be produced by tandem duplication with the interspecies-conserved Gn＿Str＿cluster member,Bm Str Gn2,as the parental copy and have evolved a unique spatial expression pattern,i.e.,expressed mainly in the silk gland.In addition,they showed a complementary expression pattern in time,i.e.,when the expression of one Gn＿Str decreases,the expression level of the other Gn＿Str will rise to compensate,and vice versa.Overall,the expression of Gn＿Strs peaked at different stages of the fifth instar,which reflects Gn＿Strs undergoing subfunctionalization.These results suggested that Gn＿Strs underwent sub and neofunctionalization in the silkworm.Furthermore,it should be noted that even though Gn＿Strs share similar functions,they are not redundant in a strict sense,and Gn＿Strs achieve dosage-sharing through temporal"dosage-sharing",which is the key to the stable and simultaneous retention of Gn＿Strs in the genome and their synergistic functions.5.Screening of genome-wide green cocoon-related genesthe differentially expressed genes in the silk glands of green cocoon strains G200 and N100 versus white cocoon strain 872B on the third day of the fifth instar（GDEGs）were screened using the gene expression profiling microarrays of the silkworm.The sulfated and methylated metabolism of flavonoids is an essential factor affecting the bioavailability of flavonoids in animals.Gene microarray assays revealed that several GDEGs involved in molecular methylation and sulfation metabolism were up-regulated in G200 and N100.This result implies that flavonoids methylation and sulfation metabolism may occur in the silk glands of silkworms with green cocoons.In addition,among the unresolved green cocoon loci,Gc and Gre were known to be located on chromosomes 15 and 1,respectively;microarray results showed that 3 and 10 GDEGs are present on chromosomes 1 and 15,respectively,and several of these GDEGs are specifically or highly expressed in the midgut,hemolymph,or silk glands,which are vital tissues for flavonoids metabolism and uptake.In this study,we compiled the above GDEGs related to flavonoids metabolism and uptake or located on chromosomes with green cocoon loci,established a"genome-wide green cocoon candidate gene set",and mapped the molecular mechanism of green cocoon formation based on microarray results and related studies.This work provides important clues for the subsequent discovery of green cocoon genes and provides a reference for studying the mechanism of green cocoon formation.6.Genetic basis of cocoon color evolution from wild to domestic mulberry silkwormsBased on silkworm pan-genomic data,the genotypes of cocoon color-related loci in wild mulberry silkworm and domestic silkworm populations with different cocoon colors were investigated,and the loci significantly associated with cocoon color were identified by one-way ANOVA for each locus and Genome-wide association analysis.The results showed that the green cocoon-related loci Gn,Gb,and Lg,and the yellow-red cocoon-related loci Y,C,and F,which were resolved in the domestic silkworm,were all derived from the wild mulberry silkworm,among which Gn and Y were the key loci subject to selection during the evolution of cocoon color.The genotypes of cocoon color-related loci in the domestic silkworm population with yellow-red cocoons are similar to those of the wild mulberry silkworm.Therefore,the yellow-red cocoon may be a more ancient trait than the green and white ones.There are reciprocal,suppressive,and epistatic effects among cocoon color-related loci;during domestication,genetic segregation,recombination,and variation of these loci shaped a wide range of cocoon colors in silkworms.In summary,the present study has resolved the Gn and identified the transmembrane transporter related to flavonoids uptake in the silkworm for the first time.In combination with gene expression microarray analysis,we established a"genome-wide green cocoon candidate gene set"and proposed a model for green cocoon formation.Based on the silkworm pan-genomic data,the genetic basis of diverse cocoon color formation involving multiple loci was systematically studied,and a model for cocoon color evolution from wild to domestic silkworms was proposed for the first time.The systematic analysis of the genetic basis of silkworm cocoon color formation and evolution in this study has important reference values for our understanding of biological coloration,the emergence of phenotypic diversity and phenotypic variation or phenotypic innovation under artificial selection,and also has important guiding significance for the selection and creation of new colored cocoon domestic silkworm varieties with high quality and high added value. |
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