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Development of Type I Genetic Markers from Expressed Sequence Tags in Highly Polymorphic Species

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Abstract

Expressed sequence tag (EST) databases provide a primary source of nuclear DNA sequences for genetic marker development in non-model organisms. To date, the process has been relatively inefficient for several reasons: 1) priming site polymorphism in the template leads to inferior or erratic amplification; 2) introns in the target amplicon are too large and/or numerous to allow effective amplification under standard screening conditions; and 3) at least occasionally, a PCR primer straddles an exon–intron junction and is unable to bind to genomic DNA template. The first is only a minor issue for species or strains with low heterozygosity but becomes a significant problem for species with high genomic variation, such as marine organisms with extremely large effective population sizes. Problems arising from unanticipated introns are unavoidable but are most pronounced in intron-rich species, such as vertebrates and lophotrochozoans. We present an approach to marker development in the Pacific oyster Crassostrea gigas, a highly polymorphic and intron-rich species, which minimizes these problems, and should be applicable to other non-model species for which EST databases are available. Placement of PCR primers in the 3′ end of coding sequence and 3′ UTR improved PCR success rate from 51% to 97%. Almost all (37 of 39) markers developed for the Pacific oyster were polymorphic in a small test panel of wild and domesticated oysters.

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Acknowledgements

This project was supported by National Research Initiative Grant no. 2004-35205-14195 to PMG from the USDA Cooperative State Research, Education, and Extension Service Animal Genome Program. WJK was supported in part by the Post-doctoral Fellowship Program of the Korea Science & Engineering Foundation (KOSEF) and HTJ was supported in part by KOSEF grant 2005-215-F00009.

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Authors and Affiliations

  1. Biotechnology Research Institute, National Fisheries Research and Development Institute, 408-1 Sirang-ri, Gijang-eup, Gijang-gun, Busan, 619-705, South Korea

    Woo-Jin Kim

  2. College of Earth, Ocean, and Environment, University of Delaware, 700 Pilottown Road, Lewes, DE, 19958, USA

    Hyungtaek Jung & Patrick M. Gaffney

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  1. Woo-Jin Kim
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  2. Hyungtaek Jung
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  3. Patrick M. Gaffney
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Corresponding author

Correspondence to Patrick M. Gaffney.

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Table S1

Alignments of sequenced amplicons and ESTs showing representative genotypes and haplotypes. Sequenced amplicons represent individuals from several C. gigas accessions: cultured hatchery lines (2 × 10, 35 × 51, 41 × 35, and 46 × 10), Hokkaido (HO40, HO41, and HO52), Kyushu (KY31 and KY32), Korea (KO201 and KO202), and C. angulata from Portugal (PO83 and PO84). Majority consensus sequence shown on top includes coding sequence (capital letters), introns (lower case italic) and untranslated regions (lower case). For some loci, sequences from EST databases were available and are included. For each locus, only non-identical diploid sequences or haplotypes are presented. (DOC 288 kb)

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Kim, WJ., Jung, H. & Gaffney, P.M. Development of Type I Genetic Markers from Expressed Sequence Tags in Highly Polymorphic Species. Mar Biotechnol 13, 127–132 (2011). https://doi.org/10.1007/s10126-010-9280-4

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  • DOI: https://doi.org/10.1007/s10126-010-9280-4

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