title: EST sequencing and fosmid library construction in a non-model moth, Mamestra brassicae, for comparative mapping.
abstract: Genome data are useful for both basic and applied research; however, it is difficult to carry out large-scale genome analyses using species with limited genetic or genomic resources. Here, we describe a cost-effective method to analyze the genome of a non-model species, using the cabbage moth, Mamestra brassicae (Lepidoptera: Noctuidae). First, we conducted expression sequence tag (EST) analysis. In this analysis, we performed PCR-based prescreening of a non-normalized embryonic cDNA library to eliminate already sequenced cDNAs from further sequencing, which significantly increased the percentage of unique genes. Next, we constructed a fosmid library of M. brassicae and isolated 120 clones containing 119 putative single copy genes by PCR-based screening with primer sets designed from the ESTs. Finally, we showed that the isolated fosmid clones could be used as probes for multicolor fluorescence in situ hybridization (FISH) analysis against an M. brassicae chromosome and confirmed conserved gene order between M. brassicae and the silkworm, Bombyx mori. Thus, we developed new genomic resources for comparative genome analysis in M. brassicae using robust and relatively low cost methods that can be applied to any non-model organism.
authors.affiliation: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Kamimura
authors.forename: Ken
authors.initials: K
authors.lastname: Tateishi
authors.forename: Makiko
authors.initials: M
authors.lastname: Tanaka-Okuyama
authors.forename: Takuya
authors.initials: T
authors.lastname: Okabe
authors.forename: Fukashi
authors.initials: F
authors.lastname: Shibata
authors.forename: Ken
authors.initials: K
authors.lastname: Sahara
authors.forename: Yuji
authors.initials: Y
authors.lastname: Yasukochi
mesh_terms: D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D002875:Chromosomes / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D020224:Expressed Sequence Tags; D015723:Gene Library; D023281:Genomics / Q000191:economics / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D009036:Moths / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000191:economics / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger