Development of Type I Genetic Markers from Express

Authors: WooJin Kim Hyungtaek Jung Patrick M Gaffney

Publish Date: 2010/03/23

Volume: 13, Issue: 2, Pages: 127-132

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Abstract

Expressed sequence tag EST databases provide a primary source of nuclear DNA sequences for genetic marker development in nonmodel 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 intronrich species such as vertebrates and lophotrochozoans We present an approach to marker development in the Pacific oyster Crassostrea gigas a highly polymorphic and intronrich species which minimizes these problems and should be applicable to other nonmodel 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 oystersThis project was supported by National Research Initiative Grant no 20043520514195 to PMG from the USDA Cooperative State Research Education and Extension Service Animal Genome Program WJK was supported in part by the Postdoctoral Fellowship Program of the Korea Science Engineering Foundation KOSEF and HTJ was supported in part by KOSEF grant 2005215F00009

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