Abstract
The legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is an insect pest species of crops grown by subsistence farmers in tropical regions of Africa. We present the de novo assembly of 3729 contigs from 454- and Sanger-derived sequencing reads for midgut, salivary, and whole adult tissues of this non-model species. Functional annotation predicted that 1320 M. vitrata protein coding genes are present, of which 631 have orthologs within the Bombyx mori gene model. A homology-based analysis assigned M. vitrata genes into a group of paralogs, but these were subsequently partitioned into putative orthologs following phylogenetic analyses. Following sequence quality filtering, a total of 1542 putative single nucleotide polymorphisms (SNPs) were predicted within M. vitrata contig assemblies. Seventy one of 1078 designed molecular genetic markers were used to screen M. vitrata samples from five collection sites in West Africa. Population substructure may be present with significant implications in the insect resistance management recommendations pertaining to the release of biological control agents or transgenic cowpea that express Bacillus thuringiensis crystal toxins. Mutation data derived from transcriptome sequencing is an expeditious and economical source for genetic markers that allow evaluation of ecological differentiation.
Highlights
The legume pod borer (LPB), Maruca vitrata (Lepidoptera: Crambidae) occurs throughout tropical and subtropical regions of the world
The derived protein products from gene model v. 2.3 of the model lepidopteran species B. mori in the file silkworm_glean_pep.fa.tar.gz from http://www.silkdb.org/silkdb/doc/ download.html were downloaded. These sequences represented all predicted genes from the whole genome assembly of B. mori. This GLEAN-predicted protein sequence data was imported into a local database using the program BioEdit [52], queried with M. vitrata Expressed sequence tags (ESTs) using the tblastx algorithm, and the results were filtered for E-values#1610240
Each single nucleotide polymorphisms (SNPs) detection assay consists of an initial multiplex PCR step that amplifies genome regions containing mutations, followed by a single base extension reaction that incorporates mass-modified dideoxynucleotides complementary to the allele at each polymorphic locus using the iPLEX-Gold mastermix (Sequenom; [58])
Summary
The legume pod borer (LPB), Maruca vitrata (Lepidoptera: Crambidae) occurs throughout tropical and subtropical regions of the world. ‘‘ Generation’’ sequencing (NGS) technologies are based on microscale pyrosequencing reactions [21,22] carried out in parallel on a PicoTitrePlateTM [23] or flow cell [24] These advances have resulted in the high throughput and low cost acquisition of EST reads from understudied species [25]. Functional annotation of EST-derived gene sequences is dependent upon their assignment to biochemical pathways using homology-based predictions with those of evolutionarily proximal model organisms This approach has identified target molecules of biological insecticides in crop pest species [18,20]. We demonstrate that EST assemblies are a source of mutation information from which high throughput SNP-based molecular genetic markers can readily be developed to assess population genetic structure and gene flow
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