Expressed sequence tags from Peromyscus testis and placenta tissue: Analysis, annotation, and utility for mapping

Abstract

BackgroundMice of the genus Peromyscus are found in nearly every habitat from Alaska to Central America and from the Atlantic to the Pacific. They provide an evolutionary outgroup to the Mus/Rattus lineage and serve as an intermediary between that lineage and humans. Although Peromyscus has been studied extensively under both field and laboratory conditions, research has been limited by the lack of molecular resources. Genes associated with reproduction typically evolve rapidly and thus are excellent sources of evolutionary information. In this study we describe the generation of two cDNA libraries, one from placenta and one from testis, characterize the resulting ESTs, and describe their utility for mapping the Peromyscus genome.ResultsThe 5' ends of 1,510 placenta and 4,798 testis clones were sequenced. Low quality sequences were removed and after clustering and contig assembly, 904 unique placenta and 2,002 unique testis sequences remained. Average lengths of placenta and testis ESTs were 711 bp and 826 bp, respectively. Approximately 82% of all ESTs were identified using the BLASTX algorithm to Mus and Rattus, and 34 – 54% of all ESTs could be assigned to a biological process gene ontology category in either Mus or Rattus. Because the Peromyscus genome organization resembles the Rattus genome more closely than Mus we examined the distribution of the Peromyscus ESTs across the rat genome finding markers on all rat chromosomes except the Y. Approximately 40% of all ESTs were specific to only one location in the Mus genome and spanned introns of an appropriate size for sequencing and SNP detection. Of the primers that were tried 54% provided useful assays for genotyping on interspecific backcross and whole-genome radiation hybrid cell panels.ConclusionThe 2,906 Peromyscus placenta and testis ESTs described here significantly expands the molecular resources available for the genus. These ESTs allow for specific PCR amplification and broad coverage across the genome, creating an excellent genetic marker resource for the generation of a medium-density genomic map. Thus, this resource will significantly aid research of a genus that is uniquely well-suited to both laboratory and field research.

Signatures of Rapid Evolution in Urban and Rural Transcriptomes of White-Footed Mice (Peromyscus leucopus) in the New York Metropolitan Area

“…UniGene [84] includes 8,946 genes for Mus testis, 5,285 for Mus ovaries, 4,355 for Rattus testis, and 5,093 for Rattus ovaries. The only cDNA library established in UniGene for Peromyscus spp. includes 635 putative genes from testis [85]. Our assembled libraries from gonad tissue fall within these ranges, and non-annotated transcripts could represent Peromyscus -specific genes.…”

A genetic map of Peromyscus with chromosomal assignment of linkage groups (a Peromyscus genetic map)

“…Primers were developed from P. maniculatus EST sequences (Glenn et al 2008) as previously described (Ramsdell et al 2008). Selected genes were also targeted by designing primers based on conserved mammalian sequences as previously described (Loschiavo et al 2007; Ramsdell et al 2008)…”

“…Approximately 79,000 P. maniculatus expressed sequence tags (ESTs) from various tissues have deposited in GenBank. Approximately 2900 ESTs from testes and placenta have been more thoroughly analyzed with many assigned to Mus or Rattus chromosomes or a biological function (Glenn et al 2008). Additional transcriptome sequencing has been conducted (e.g., Genbank accessions GH457086.1 - GH545901.1), but the summaries have not yet been published.…”

Five hundred microsatellite loci for Peromyscus

“…This large set of microsatellites contributes to a growing number of genomic resources (Mullen et al 2006; Glenn et al 2008) for studies of Peromyscus evolution.…”

“…An additional 78 primer pairs were developed from expressed sequence tags (EST libraries) from P. maniculatus (Glenn et al 2008). PCR primers were designed using Oligo 6.67 (Molecular Biology Insights) or Primer Premier 5 (Premier Biosoft International).…”