SilkMeta: a comprehensive platform for sharing and exploiting pan-genomic and multi-omic silkworm data.

The silkworm Bombyx mori is a domesticated insect that serves as an animal model for research and agriculture. The silkworm super-pan-genome dataset, which we published last year, is a unique resource for the study of global genomic diversity and phenotype-genotype association. Here we present SilkMeta (http://silkmeta.org.cn), a comprehensive database covering the available silkworm pan-genome and multi-omics data. The database contains 1082 short-read genomes, 546 long-read assembled genomes, 1168 transcriptomes, 294 phenotype characterizations (phenome), tens of millions of variations (variome), 7253 long non-coding RNAs (lncRNAs), 18 717 full length transcripts and a set of population statistics. We have compiled publications on functional genomics research and genetic stock deciphering (mutant map). A range of bioinformatics tools is also provided for data visualization and retrieval. The large batch of omics data and tools were integrated in twelve functional modules that provide useful strategies and data for comparative and functional genomics research. The interactive bioinformatics platform SilkMeta will benefit not only the silkworm but also the insect biology communities.

Identification, expression, and artificial selection of silkworm epigenetic modification enzymes.

BACKGROUND: Understanding the genetic basis of phenotype variations during domestication and breeding is of great interest. Epigenetics and epigenetic modification enzymes (EMEs) may play a role in phenotypic variations; however, no comprehensive study has been performed to date. Domesticated silkworm (Bombyx mori) may be utilized as a model in determining how EMEs influence domestication traits. RESULTS: We identified 44 EMEs in the genome of silkworm (Bombyx mori) using homology searching. Phylogenetic analysis showed that genes in a subfamily among different animals were well clustered, and the expression pattern of EMEs is constant among Bombyx mori, Drosophila melanogaster, and Mus musculus. These are most highly expressed in brain, early embryo, and internal genitalia. By gene-related selective sweeping, we identified five BmEMEs under artificial selection during the domestication and breeding of silkworm. Among these selected genes, BmSuv4-20 and BmDNMT2 harbor selective mutations in their upstream regions that alter transcription factor-binding sites. Furthermore, these two genes are expressed higher in the testis and ovary of domesticated silkworm compared to wild silkworms, and correlations between their expression pattern and meiosis of the sperm and ova were observed. CONCLUSIONS: The domestication of silkworm has induced artificial selection on epigenetic modification markers that may have led to phenotypic changes during domestication. We present a novel perspective to understand the genetic basis underlying animal domestication and breeding.

Horizontal transfer of a novel Helentron in insects.

Helentrons represent a novel subtype of Helitrons. However, the evolutionary history of Helentrons in organisms is not clearly understood. In this study, we performed structure and autonomous partner analyses, which revealed that bm_455, a TE obtained from the Bombyx mori TE database, BmTEdb, was a member of Helentrons but not a long-terminal repeat (LTR) retrotransposon. Further analyses showed that bm_455 was also present in a wide range of insects including lepidopterans, coleopterans and hymenopterans using a homology-based search strategy. Several lines of evidence (high sequence identity, discontinuous distribution and lack of intense purifying selection) suggested that these elements could have been transferred into these species in part by horizontal transfers (HTs). Because Helentrons can capture host gene fragments, HTs of Helentrons might have a huge impact on their host genome evolution.

An adaptive transposable element insertion in the regulatory region of the EO gene in the domesticated silkworm, Bombyx mori.

Although there are many studies to show a key role of transposable elements (TEs) in adaptive evolution of higher organisms, little is known about the molecular mechanisms. In this study, we found that a partial TE (Taguchi) inserted in the cis-regulatory region of the silkworm ecdysone oxidase (EO) gene, which encodes a crucial enzyme to reduce the titer of molting hormone (20-hydroxyecdysone, 20E). The TE insertion occurred during domestication of silkworm and the frequency of the TE insertion in the domesticated silkworm (Bombyx mori) is high, 54.24%. The linkage disequilibrium in the TE inserted strains of the domesticated silkworm was elevated. Molecular population genetics analyses suggest that this TE insertion is adaptive for the domesticated silkworm. Luminescent reporter assay shows that the TE inserted in the cis-regulatory region of the EO gene functions as a 20E-induced enhancer of the gene expression. Further, phenotypic bioassay indicates that the silkworm with the TE insertion exhibited more stable developmental phenotype than the silkworm without the TE insertion when suffering from food shortage. Thus, the inserted TE in the cis-regulatory region of the EO gene increased developmental uniformity of silkworm individuals through regulating 20E metabolism, partially explaining transformation of a domestication developmental trait in the domesticated silkworm. Our results emphasize the exceptional role of gene expression regulation in developmental transition of domesticated animals.

Demographic history and gene flow during silkworm domestication.

BACKGROUND: Gene flow plays an important role in domestication history of domesticated species. However, little is known about the demographic history of domesticated silkworm involving gene flow with its wild relative. RESULTS: In this study, four model-based evolutionary scenarios to describe the demographic history of B. mori were hypothesized. Using Approximate Bayesian Computation method and DNA sequence data from 29 nuclear loci, we found that the gene flow at bottleneck model is the most likely scenario for silkworm domestication. The starting time of silkworm domestication was estimated to be approximate 7,500 years ago; the time of domestication termination was 3,984 years ago. Using coalescent simulation analysis, we also found that bi-directional gene flow occurred during silkworm domestication. CONCLUSIONS: Estimates of silkworm domestication time are nearly consistent with the archeological evidence and our previous results. Importantly, we found that the bi-directional gene flow might occur during silkworm domestication. Our findings add a dimension to highlight the important role of gene flow in domestication of crops and animals.

Copy number variations among silkworms.

BACKGROUND: Copy number variations (CNVs), which are important source for genetic and phenotypic variation, have been shown to be associated with disease as well as important QTLs, especially in domesticated animals. However, little is known about the CNVs in silkworm. RESULTS: In this study, we have constructed the first CNVs map based on genome-wide analysis of CNVs in domesticated silkworm. Using next-generation sequencing as well as quantitative PCR (qPCR), we identified ~319 CNVs in total and almost half of them (~ 49%) were distributed on uncharacterized chromosome. The CNVs covered 10.8 Mb, which is about 2.3% of the entire silkworm genome. Furthermore, approximately 61% of CNVs directly overlapped with SDs in silkworm. The genes in CNVs are mainly related to reproduction, immunity, detoxification and signal recognition, which is consistent with the observations in mammals. CONCLUSIONS: An initial CNVs map for silkworm has been described in this study. And this map provides new information for genetic variations in silkworm. Furthermore, the silkworm CNVs may play important roles in reproduction, immunity, detoxification and signal recognition. This study provided insight into the evolution of the silkworm genome and an invaluable resource for insect genomics research.

Multiple independent origins of the female W chromosome in moths and butterflies.

Lepidoptera, the most diverse group of insects, exhibit female heterogamy (Z0 or ZW), which is different from most other insects (male heterogamy, XY). Previous studies suggest a single origin of the Z chromosome. However, the origin of the lepidopteran W chromosome remains poorly understood. Here, we assemble the genome from females down to the chromosome level of a model insect (Bombyx mori) and identify a W chromosome of approximately 10.1 megabase using a newly developed tool. In addition, we identify 3593 genes that were not previously annotated in the genomes of B. mori. Comparisons of 21 lepidopteran species (including 17 ZW and four Z0 systems) and three trichopteran species (Z0 system) reveal that the formation of Ditrysia W involves multiple mechanisms, including previously proposed canonical and noncanonical models, as well as a newly proposed mechanism called single-Z turnover. We conclude that there are multiple independent origins of the W chromosome in the Ditrysia (most moths and all butterflies) of Lepidoptera.

Maternal dominance contributes to subgenome differentiation in allopolyploid fishes.

Teleost fishes, which are the largest and most diverse group of living vertebrates, have a rich history of ancient and recent polyploidy. Previous studies of allotetraploid common carp and goldfish (cyprinids) reported a dominant subgenome, which is more expressed and exhibits biased gene retention. However, the underlying mechanisms contributing to observed 'subgenome dominance' remains poorly understood. Here we report high-quality genomes of twenty-one cyprinids to investigate the origin and subsequent subgenome evolution patterns following three independent allopolyploidy events. We identify the closest extant relatives of the diploid progenitor species, investigate genetic and epigenetic differences among subgenomes, and conclude that observed subgenome dominance patterns are likely due to a combination of maternal dominance and transposable element densities in each polyploid. These findings provide an important foundation to understanding subgenome dominance patterns observed in teleost fishes, and ultimately the role of polyploidy in contributing to evolutionary innovations.

[Computational approaches for identification and classification of transposable elements in eukaryotic genomes].

Repetitive sequences (repeats) represent a significant fraction of the eukaryotic genomes and can be divided into tandem repeats, segmental duplications, and interspersed repeats on the basis of their sequence characteristics and how they are formed. Most interspersed repeats are derived from transposable elements (TEs). Eukaryotic TEs have been subdivided into two major classes according to the intermediate they use to move. The transposition and amplification of TEs have a great impact on the evolution of genes and the stability of genomes. However, identification and classification of TEs are complex and difficult due to the fact that their structure and classification are complex and diverse compared with those of other types of repeats. Here, we briefly introduced the function and classification of TEs, and summarized three different steps for identification, classification and annotation of TEs in eukaryotic genomes: (1) assembly of a repeat library, (2) repeat correction and classification, and (3) genome annotation. The existing computational approaches for each step were summarized and the advantages and disadvantages of the approaches were also highlighted in this review. To accurately identify, classify, and annotate the TEs in eukaryotic genomes requires combined methods. This review provides useful information for biologists who are not familiar with these approaches to find their way through the forest of programs.

High-resolution silkworm pan-genome provides genetic insights into artificial selection and ecological adaptation.

The silkworm Bombyx mori is an important economic insect for producing silk, the "queen of fabrics". The currently available genomes limit the understanding of its genetic diversity and the discovery of valuable alleles for breeding. Here, we deeply re-sequence 1,078 silkworms and assemble long-read genomes for 545 representatives. We construct a high-resolution pan-genome dataset representing almost the entire genomic content in the silkworm. We find that the silkworm population harbors a high density of genomic variants and identify 7308 new genes, 4260 (22%) core genes, and 3,432,266 non-redundant structure variations (SVs). We reveal hundreds of genes and SVs that may contribute to the artificial selection (domestication and breeding) of silkworm. Further, we focus on four genes responsible, respectively, for two economic (silk yield and silk fineness) and two ecologically adaptive traits (egg diapause and aposematic coloration). Taken together, our population-scale genomic resources will promote functional genomics studies and breeding improvement for silkworm.

The Landscapes of Full-Length Transcripts and Splice Isoforms as Well as Transposons Exonization in the Lepidopteran Model System, Bombyx mori.

The domesticated silkworm, Bombyx mori, is an important model system for the order Lepidoptera. Currently, based on third-generation sequencing, the chromosome-level genome of Bombyx mori has been released. However, its transcripts were mainly assembled by using short reads of second-generation sequencing and expressed sequence tags which cannot explain the transcript profile accurately. Here, we used PacBio Iso-Seq technology to investigate the transcripts from 45 developmental stages of Bombyx mori. We obtained 25,970 non-redundant high-quality consensus isoforms capturing ∼60% of previous reported RNAs, 15,431 (∼47%) novel transcripts, and identified 7,253 long non-coding RNA (lncRNA) with a large proportion of novel lncRNA (∼56%). In addition, we found that transposable elements (TEs) exonization account for 11,671 (∼45%) transcripts including 5,980 protein-coding transcripts (∼32%) and 5,691 lncRNAs (∼79%). Overall, our results expand the silkworm transcripts and have general implications to understand the interaction between TEs and their host genes. These transcripts resource will promote functional studies of genes and lncRNAs as well as TEs in the silkworm.

Burst expansion, distribution and diversification of MITEs in the silkworm genome.

BACKGROUND: Miniature inverted-repeat transposable elements (MITEs) are widespread in plants and animals. Although silkworm (Bombyx mori) has a large amount of and a variety of transposable elements, the genome-wide information of the silkworm MITEs is unknown. RESULTS: We used structure-based and homology approaches to search for MITEs in the silkworm genome. We identified 17 MITE families with a total of 5785 members, accounting for ~0.4% of the genome. 7 of 17 MITE families are completely novel based on the nucleotide composition of target site duplication (TSD) and/or terminal inverted repeats (TIR). Silkworm MITEs were widely and nonrandom distributed in the genome. One family named BmMITE-2 might experience a recent burst expansion. Network and diversity analyses for each family revealed different diversification patterns of the silkworm MITEs, reflecting the signatures of genome-shocks that silkworm experienced. Most silkworm MITEs preferentially inserted into or near genes and BmMITE-11 that encodes a germline-restricted small RNA might silence its the closest genes in silkworm ovary through a small RNA pathway. CONCLUSIONS: Silkworm harbors 17 MITE families. The silkworm MITEs preferred to reside in or near genes and one MITE might be involved in gene silence. Our results emphasize the exceptional role of MITEs in transcriptional regulation of genes and have general implications to understand interaction between MITEs and their host genome.

Evolutionary dynamics of transposable elements during silkworm domestication.

Although there are some documented examples on population dynamics of transposable elements (TEs) in model organisms, the evolutionary dynamics of TEs in domesticated species has not been systematically investigated. The objective of this study is to understand population dynamics of TEs during silkworm domestication. In this work, using transposon-display we examined the polymorphism of seven TE families [they represent about 59% of silkworm (Bombyx mori) total TE content] in four domesticated silkworm populations and one wild silkworm population. Maximum likelihood (ML) was used to estimate selection pressure. Population differentiation and structure were performed by using AMOVA analysis and program DISTRUCT, respectively. The results of transposon-display showed that significant differentiation occurred between the domesticated silkworm and wild silkworm. These TEs have experienced expansions and fixation in the domesticated silkworm but not in wild silkworm. Furthermore, the ML results indicated that purifying selection of TEs in the domesticated silkworm were significantly weaker than that in the wild silkworm. Interestingly, an adaptation insertion induced by BmMITE-2 was found, and this insertion can reduce the polymorphism of the flanking regions of its neighboring COQ7 gene. Our results suggested that TEs expanded and were fixed in the domesticated silkworm might result from demographic effects and artificial selection during domestication. We concluded that the data presented in this study have general implication in animal and crop improvements as well as in domestication of new species.

Unexpected invasion of miniature inverted-repeat transposable elements in viral genomes.

BACKGROUND: Transposable elements (TEs) are common and often present with high copy numbers in cellular genomes. Unlike in cellular organisms, TEs were previously thought to be either rare or absent in viruses. Almost all reported TEs display only one or two copies per viral genome. In addition, the discovery of pandoraviruses with genomes up to 2.5-Mb emphasizes the need for biologists to rethink the fundamental nature of the relationship between viruses and cellular life. RESULTS: Herein, we performed the first comprehensive analysis of miniature inverted-repeat transposable elements (MITEs) in the 5170 viral genomes for which sequences are currently available. Four hundred and fifty one copies of ten miniature inverted-repeat transposable elements (MITEs) were found and each MITE had reached relatively large copy numbers (some up to 90) in viruses. Eight MITEs belonging to two DNA superfamilies (hobo/Activator/Tam3 and Chapaev-Mirage-CACTA) were for the first time identified in viruses, further expanding the organismal range of these two superfamilies. TEs may play important roles in shaping the evolution of pandoravirus genomes, which were here found to be very rich in MITEs. We also show that putative autonomous partners of seven MITEs are present in the genomes of viral hosts, suggesting that viruses may borrow the transpositional machinery of their cellular hosts' autonomous elements to spread MITEs and colonize their own genomes. The presence of seven similar MITEs in viral hosts, suggesting horizontal transfers (HTs) as the major mechanism for MITEs propagation. CONCLUSIONS: Our discovery highlights that TEs contribute to shape genome evolution of pandoraviruses. We concluded that as for cellular organisms, TEs are part of the pandoraviruses' diverse mobilome.

iMITEdb: the genome-wide landscape of miniature inverted-repeat transposable elements in insects.

Miniature inverted-repeat transposable elements (MITEs) have attracted much attention due to their widespread occurrence and high copy numbers in eukaryotic genomes. However, the systematic knowledge about MITEs in insects and other animals is still lacking. In this study, we identified 6012 MITE families from 98 insect species genomes. Comparison of these MITEs with known MITEs in the NCBI non-redundant database and Repbase showed that 5701(∼95%) of 6012 MITE families are novel. The abundance of MITEs varies drastically among different insect species, and significantly correlates with genome size. In general, larger genomes contain more MITEs than small genomes. Furthermore, all identified MITEs were included in a newly constructed database (iMITEdb) (http://gene.cqu.edu.cn/iMITEdb/), which has functions such as browse, search, BLAST and download. Overall, our results not only provide insight on insect MITEs but will also improve assembly and annotation of insect genomes. More importantly, the results presented in this study will promote studies of MITEs function, evolution and application in insects. DATABASE URL: http://gene.cqu.edu.cn/iMITEdb/.

Enhancer activity of Helitron in sericin-1 gene promoter from Bombyx mori.

Sericin is a kind of water-soluble protein expressed specifically in the middle silk gland of Bombyx mori. When the sericin-1 gene promoter was cloned and a transgenic vector was constructed to express a foreign protein, a specific Helitron, Bmhel-8, was identified in the sericin-1 gene promoter sequence in some genotypes of Bombyx mori and Bombyx mandarina. Given that the Bmhel-8 Helitron transposon was present only in some genotypes, it could be the source of allelic variation in the sericin-1 promoter. The length of the sericin-1 promoter sequence is approximately 1063 or 643 bp. The larger size of the sequence or allele is ascribed to the presence of Bmhel-8. Silkworm genotypes can be homozygous for either the shorter or larger promoter sequence or heterozygous, containing both alleles. Bmhel-8 in the sericin-1 promoter exhibits enhancer activity, as demonstrated by a dual-luciferase reporter system in BmE cell lines. Furthermore, Bmhel-8 displays enhancer activity in a sericin-1 promoter-driven gene expression system but does not regulate the tissue-specific expression of sericin-1.

TRT, a Vertebrate and Protozoan Tc1-Like Transposon: Current Activity and Horizontal Transfer.

We report a Danio rerio transposon named DrTRT, for D. rerio Transposon Related to Tc1 The complete sequence of the DrTRT transposon is 1,563 base pairs (bp) in length, and its transposase putatively encodes a 338-amino acid protein that harbors a DD37E motif in its catalytic domain. We present evidence based on searches of publicly available genomes that TRT elements commonly occur in vertebrates and protozoa. Phylogenetic and functional domain comparisons confirm that TRT constitutes a new subfamily within the Tc1 family. Hallmark features of having no premature termination codons within the transposase, the presence of all expected functional domains, and its occurrence in the bony fish transcriptome suggest that TRT might have current or recent activity in these species. Further analysis showed that the activity of TRT elements in these species might have arisen about between 4 and 19 Ma. Interestingly, our results also implied that the widespread distribution of TRT among fishes, frog, and snakes is the result of multiple independent HT events, probably from bony fishes to snakes or frog. Finally, the mechanisms underlying horizontal transfer of TRT elements are discussed.

Microarray analysis of New Green Cocoon associated genes in silkworm, Bombyx mori.

Green cocoons in silkworm, Bombyx mori, are caused by flavonoids accumulation in the silk proteins, fibroin and sericin. Despite the economic value of natural green cocoon and medical value of flavonoids, there is limited understanding of the molecular mechanism regulating flavonoids uptake in silkworm, which is tightly associated with the trait of green cocoon. The purpose of this study is to perform a comprehensive analysis to understand the molecular mechanisms of flavonoids uptake in silkworm based on microarray analyses. The study subject was the New Green Cocoon from the silkworm strains, G200 and N100, a new spontaneous dominant green cocoon trait identified in the 2000s. The genes regulating this trait are independent of other green cocoon genes previously reported. Genome-wide gene expression was compared between the New Green Cocoon producing silkworm strains, G200 and N100, and the control sample, which is the white cocoon producing strain 872B. Among these strains, N100 and 872B are near-isogenic lines. The results showed that 130 genes have consistently changing expression patterns in the green cocoon strains when compared with the white cocoon strain. Among these, we focused on the genes related to flavonoids metabolism and absorption, such as sugar transporter genes and UDP-glucosyltransferase genes. Based on our findings, we propose the potential mechanisms for flavonoids absorption and metabolism in silkworm. Our results imply that silkworm might be used as an underlying model for flavonoids in pharmaceutical research.

Identification of MBF2 family genes in Bombyx mori and their expression in different tissues and stages and in response to Bacillus bombysepticus infection and starvation.

The Multiprotein bridge factor 2 (MBF2) gene was first identified as a co-activator involved in BmFTZ-F1-mediated activation of the Fushi tarazu gene. Herein, nine homologous genes of MBF2 gene are identified. Evolutionary analysis showed that this gene family is insect-specific and that the family members are closely related to response to pathogens (REPAT) genes. Tissue distribution analysis revealed that these genes could be expressed in a tissue-specific manner. Developmental profiles analysis showed that the MBF2 gene family members were highly expressed in the different stages. Analysis of the expression patterns of nine MBF2 family genes showed that Bacillus bombysepticus treatment induced the up-regulation of several MBF2 family genes, including MBF2-4, -7, -9, -8. Furthermore, we found the MBF2 family genes were modulated by starvation and the expression of these genes recovered upon re-feeding, except for MBF2-5, -9. These findings suggested roles for these proteins in insect defense against pathogens and nutrient metabolism, which has an important guiding significance for designing pest control strategies.

Comparative Analysis of Transcriptomes among Bombyx mori Strains and Sexes Reveals the Genes Regulating Melanic Morph and the Related Phenotypes.

As a source of insect polymorphism, melanism plays an important role in ecological adaption and usually endows advantageous phenotypic-effects on insects. However, due to the mechanistic diversity, there are knowledge gaps in the molecular mechanisms underlying melanism and the related phenotypes. In silk moths, a recessive melanic mutant (sex-controlled melanism, sml) strain exhibits extended adult longevity. We took a transcriptome approach to perform a comparative analysis between this sml strain and a wild-type strain (Dazao). Our analysis resulted in the identification of 59 unique differentially expressed genes in the melanic mutant. Two key genes (laccase2 and yellow) involved in melanin formation were significantly up-regulated in melanic individuals. The laccase2 B-type isoform (BGIBMGA006746) was found to likely participate in the silkworm cuticular melanism process at late pupal stage. Moreover, we discovered 22 cuticular protein encoding genes with the possible function in melanin transport and/or maintenance. Based on our findings, we presume that the longer survival of the melanic sml male moths might be associated with the enhanced antioxidant defense systems and a reduction in the insulin/IGF-1 signaling pathway (IIS). These findings will facilitate the understanding of the molecular basis underlying melanism and the derived phenotypic-effects.