Utility of sequenced genomes for microsatellite marker development in non-model organisms: a case study of functionally important genes in nine-spined sticklebacks (Pungitius pungitius)

Takahito Shikano,Yukinori Shimada,Jetty Ramadevi,Juha Merilä

doi:10.1186/1471-2164-11-334

Takahito Shikano, Yukinori Shimada + Show 2 more

Open Access

https://doi.org/10.1186/1471-2164-11-334

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Journal: BMC Genomics	Publication Date: Jan 1, 2010
Citations: 114	License type: cc-by

Affiliation: University of Helsinki, University of Hyderabad

Abstract

BackgroundIdentification of genes involved in adaptation and speciation by targeting specific genes of interest has become a plausible strategy also for non-model organisms. We investigated the potential utility of available sequenced fish genomes to develop microsatellite (cf. simple sequence repeat, SSR) markers for functionally important genes in nine-spined sticklebacks (Pungitius pungitius), as well as cross-species transferability of SSR primers from three-spined (Gasterosteus aculeatus) to nine-spined sticklebacks. In addition, we examined the patterns and degree of SSR conservation between these species using their aligned sequences.ResultsCross-species amplification success was lower for SSR markers located in or around functionally important genes (27 out of 158) than for those randomly derived from genomic (35 out of 101) and cDNA (35 out of 87) libraries. Polymorphism was observed at a large proportion (65%) of the cross-amplified loci independently of SSR type. To develop SSR markers for functionally important genes in nine-spined sticklebacks, SSR locations were surveyed in or around 67 target genes based on the three-spined stickleback genome and these regions were sequenced with primers designed from conserved sequences in sequenced fish genomes. Out of the 81 SSRs identified in the sequenced regions (44,084 bp), 57 exhibited the same motifs at the same locations as in the three-spined stickleback. Di- and trinucleotide SSRs appeared to be highly conserved whereas mononucleotide SSRs were less so. Species-specific primers were designed to amplify 58 SSRs using the sequences of nine-spined sticklebacks.ConclusionsOur results demonstrated that a large proportion of SSRs are conserved in the species that have diverged more than 10 million years ago. Therefore, the three-spined stickleback genome can be used to predict SSR locations in the nine-spined stickleback genome. While cross-species utility of SSR primers is limited due to low amplification success, SSR markers can be developed for target genes and genomic regions using our approach, which should be also applicable to other non-model organisms. The SSR markers developed in this study should be useful for identification of genes responsible for phenotypic variation and adaptive divergence of nine-spined stickleback populations, as well as for constructing comparative gene maps of nine-spined and three-spined sticklebacks.

Highlights

Identification of genes involved in adaptation and speciation by targeting specific genes of interest has become a plausible strategy for non-model organisms
The tendency for higher amplification success with the ESTderived simple sequence repeats (SSRs) than with the genomic SSRs is in agreement with the results of previous studies [48,49,50,51] - finding which has been explained by high sequence conservation in coding regions [13,14,15]
Across the three SSR types, amplification success was high for the SSR markers in which both forward and reverse primers were located in exonic regions (62.5%, 15 out of 24), whereas it was lower if primers were located either in intronic (20.4%, 20 out of 98) or intergenic regions (26.3%, 42 out of 160; Table 1)

Summary

Introduction

Identification of genes involved in adaptation and speciation by targeting specific genes of interest has become a plausible strategy for non-model organisms. We investigated the potential utility of available sequenced fish genomes to develop microsatellite (cf simple sequence repeat, SSR) markers for functionally important genes in nine-spined sticklebacks (Pungitius pungitius), as well as cross-species transferability of SSR primers from three-spined (Gasterosteus aculeatus) to nine-spined sticklebacks. Targeting specific genes and genomic regions of Microsatellites or simple sequence repeats (SSRs) are highly abundant in eukaryotic genomes, accounting for 3-5% of the mammalian genomes [8,9]. Owing to their wide genomic distribution, codominant inheritance and hypervariability, they are widely recognized as one of the most powerful molecular markers in the field of genetics. Even if a large EST database is available for a target species, ESTs have limitations as a material for development of SSR markers for specific genes

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