End-sequencing and characterization of silkworm (Bombyx mori) bacterial artificial chromosome libraries

<p>Abstract</p> <p>Background</p> <p>We performed large-scale bacterial artificial chromosome (BAC) end-sequencing of two BAC libraries (an <it>Eco</it>RI- and a <it>Bam</it>HI-digested library) and conducted an <it>in silico </it>analysis to characterize the obtained sequence data, to make them a useful resource for genomic research on the silkworm (<it>Bombyx mori</it>).</p> <p>Results</p> <p>More than 94000 BAC end sequences (BESs), comprising more than 55 Mbp and covering about 10.4% of the silkworm genome, were sequenced. Repeat-sequence analysis with known repeat sequences indicated that the long interspersed nuclear elements (LINEs) were abundant in <it>Bam</it>HI BESs, whereas DNA-type elements were abundant in <it>Eco</it>RI BESs. Repeat-sequence analysis revealed that the abundance of LINEs might be due to a GC bias of the restriction sites and that the GC content of silkworm LINEs was higher than that of mammalian LINEs. In a BLAST-based sequence analysis of the BESs against two available whole-genome shotgun sequence data sets, more than 70% of the BESs had a BLAST hit with an identity of ≥ 99%. About 14% of <it>Eco</it>RI BESs and about 8% of <it>Bam</it>HI BESs were paired-end clones with unique sequences at both ends. Cluster analysis of the BESs clarified the proportion of BESs containing protein-coding regions.</p> <p>Conclusion</p> <p>As a result of this characterization, the identified BESs will be a valuable resource for genomic research on <it>Bombyx mori</it>, for example, as a base for construction of a BAC-based physical map. The use of multiple complementary BAC libraries constructed with different restriction enzymes also makes the BESs a more valuable genomic resource. The GenBank accession numbers of the obtained end sequences are <ext-link ext-link-type="gen" ext-link-id="DE283657">DE283657</ext-link>–<ext-link ext-link-type="gen" ext-link-id="DE378560">DE378560</ext-link>.</p

KAIKObase: An integrated silkworm genome database and data mining tool

<p>Abstract</p> <p>Background</p> <p>The silkworm, <it>Bombyx mori</it>, is one of the most economically important insects in many developing countries owing to its large-scale cultivation for silk production. With the development of genomic and biotechnological tools, <it>B. mori </it>has also become an important bioreactor for production of various recombinant proteins of biomedical interest. In 2004, two genome sequencing projects for <it>B. mori </it>were reported independently by Chinese and Japanese teams; however, the datasets were insufficient for building long genomic scaffolds which are essential for unambiguous annotation of the genome. Now, both the datasets have been merged and assembled through a joint collaboration between the two groups.</p> <p>Description</p> <p>Integration of the two data sets of silkworm whole-genome-shotgun sequencing by the Japanese and Chinese groups together with newly obtained fosmid- and BAC-end sequences produced the best continuity (~3.7 Mb in N50 scaffold size) among the sequenced insect genomes and provided a high degree of nucleotide coverage (88%) of all 28 chromosomes. In addition, a physical map of BAC contigs constructed by fingerprinting BAC clones and a SNP linkage map constructed using BAC-end sequences were available. In parallel, proteomic data from two-dimensional polyacrylamide gel electrophoresis in various tissues and developmental stages were compiled into a silkworm proteome database. Finally, a <it>Bombyx </it>trap database was constructed for documenting insertion positions and expression data of transposon insertion lines.</p> <p>Conclusion</p> <p>For efficient usage of genome information for functional studies, genomic sequences, physical and genetic map information and EST data were compiled into KAIKObase, an integrated silkworm genome database which consists of 4 map viewers, a gene viewer, and sequence, keyword and position search systems to display results and data at the level of nucleotide sequence, gene, scaffold and chromosome. Integration of the silkworm proteome database and the <it>Bombyx </it>trap database with KAIKObase led to a high-grade, user-friendly, and comprehensive silkworm genome database which is now available from URL: <url>http://sgp.dna.affrc.go.jp/KAIKObase/</url>.</p

A BAC-Based Integrated Linkage Map of the Silkworm \u3cem\u3eBombyx mori\u3c/em\u3e

Background: In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps. Results: We mapped 1,755 single nucleotide polymorphism (SNP) markers from bacterial artificial chromosome (BAC) end sequences onto 28 linkage groups using a recombining male backcross population, yielding an average inter-SNP distance of 0.81 cM (about 270 kilobases). We constructed 6,221 contigs by fingerprinting clones from three BAC libraries digested with different restriction enzymes, and assigned a total of 724 single copy genes to them by BLAST (basic local alignment search tool) search of the BAC end sequences and high-density BAC filter hybridization using expressed sequence tags as probes. We assigned 964 additional expressed sequence tags to linkage groups by restriction fragment length polymorphism analysis of a nonrecombining female backcross population. Altogether, 361.1 megabases of BAC contigs and singletons were integrated with a map containing 1,688 independent genes. A test of synteny using Oxford grid analysis with more than 500 silkworm genes revealed six versus 20 silkworm linkage groups containing eight or more orthologs of Apis versus Tribolium, respectively. Conclusion: The integrated map contains approximately 10% of predicted silkworm genes and has an estimated 76% genome coverage by BACs. This provides a new resource for improved assembly of whole-genome shotgun data, gene annotation and positional cloning, and will serve as a platform for comparative genomics and gene discovery in Lepidoptera and other insects

Genome-wide Identification of Tebufenozide Resistant Genes in the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae)

The smaller tea tortrix, Adoxophyes honmai, has developed strong resistance to tebufenozide, a diacylhydrazine-type (DAH) insecticide. Here, we investigated its mechanism by identifying genes responsible for the tebufenozide resistance using various next generation sequencing techniques. First, double-digest restriction site-associated DNA sequencing (ddRAD-seq) identified two candidate loci. Then, synteny analyses using A. honmai draft genome sequences revealed that one locus contained the ecdysone receptor gene (EcR) and the other multiple CYP9A subfamily P450 genes. RNA-seq and direct sequencing of EcR cDNAs found a single nucleotide polymorphism (SNP), which was tightly linked to tebufenozide resistance and generated an amino acid substitution in the ligand-binding domain. The binding affinity to tebufenozide was about 4 times lower in in vitro translated EcR of the resistant strain than in the susceptible strain. RNA-seq analyses identified commonly up-regulated genes in resistant strains, including CYP9A and choline/carboxylesterase (CCE) genes. RT-qPCR analysis and bioassays showed that the expression levels of several CYP9A and CCE genes were moderately correlated with tebufenozide resistance. Collectively, these results suggest that the reduced binding affinity of EcR is the main factor and the enhanced detoxification activity by some CYP9As and CCEs plays a supplementary role in tebufenozide resistance in A. honmai

Construction of a Single Nucleotide Polymorphism Linkage Map for the Silkworm, Bombyx mori, Based on Bacterial Artificial Chromosome End Sequences

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female × an F(1) (p50T × C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest