A Novel Approach for Mining Polymorphic Microsatellite Markers In Silico

Abstract

An important emerging application of high-throughput 454 sequencing is the isolation of molecular markers such as microsatellites from genomic DNA. However, few studies have developed microsatellites from cDNA despite the added potential for targeting candidate genes. Moreover, to develop microsatellites usually requires the evaluation of numerous primer pairs for polymorphism in the focal species. This can be time-consuming and wasteful, particularly for taxa with low genetic diversity where the majority of primers often yield monomorphic polymerase chain reaction (PCR) products. Transcriptome assemblies provide a convenient solution, functional annotation of transcripts allowing markers to be targeted towards candidate genes, while high sequence coverage in principle permits the assessment of variability in silico. Consequently, we evaluated fifty primer pairs designed to amplify microsatellites, primarily residing within transcripts related to immunity and growth, identified from an Antarctic fur seal (Arctocephalus gazella) transcriptome assembly. In silico visualization was used to classify each microsatellite as being either polymorphic or monomorphic and to quantify the number of distinct length variants, each taken to represent a different allele. The majority of loci (n = 36, 76.0%) yielded interpretable PCR products, 23 of which were polymorphic in a sample of 24 fur seal individuals. Loci that appeared variable in silico were significantly more likely to yield polymorphic PCR products, even after controlling for microsatellite length measured in silico. We also found a significant positive relationship between inferred and observed allele number. This study not only demonstrates the feasibility of generating modest panels of microsatellites targeted towards specific classes of gene, but also suggests that in silico microsatellite variability may provide a useful proxy for PCR product polymorphism.

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

“…Newbler performed well at assembling full-length cDNA contigs, and our results are in line with Mundry et al’s [64] findings that Newbler outperformed other assembly programs in simulated experiments. The N50 value reported here is comparable to de novo Newbler cDNA assemblies for other organisms: N50 = 1,735 bp in Oncopeltus fasciatus , Ewen-Campen et al [65]; N50 = 1,333 bp in Silene vulgaris , Sloan et al [51]; N50 = 1,588 bp in Spalax galili , Malik et al [66]; and N50 = 854 bp in Arctocephalus gazella , Hoffman & Nichols [67]. …”

A combined strategy involving Sanger and 454 pyrosequencing increases genomic resources to aid in the management of reproduction, disease control and genetic selection in the turbot (Scophthalmus maximus)

“…In fact, recent studies have identified 26 polymorphic microsatellite by pyrosequencing in an endangered fish species of China [88] and 21 microsatellites loci from the threatened freshwater Yarra pygmy perch ( Nannoperca obscura ) [89]. However, few studies have been conducted to search for cDNA-associated microsatellites, like those identified in the Atlantic herring ( Clupea harengus ) [90], despite the potential for targeting candidate genes [91]. …”

Transcriptome of the dead: characterisation of immune genes and marker development from necropsy samples in a free-ranging marine mammal

“…In a subsequent pilot study, we showed that it was possible to obtain polymorphic microsatellites targeted towards candidate genes related to immunity and growth by selecting loci that appear variable in silico [30]. We also exploited homology to the dog ( Canis lupus familiaris ) genome to map transcripts to specific chromosomes, allowing the development of a genome-wide distributed panel of 104 validated, polymorphic SNPs [31].…”