Development of novel polymorphic microsatellite markers by technology of next generation sequencing in western white pin
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Development of novel polymorphic microsatellite markers by technology of next generation sequencing in western white pine Jun-Jun Liu • Craig Hammett
Received: 25 January 2014 / Accepted: 24 March 2014 Ó Her Majesty the Queen in Right of Canada 2014
Abstract The invasive fungal pathogen Cronartium ribicola has seriously damaged five-needle pines in North America. Genetic diversity assessment using DNA markers among natural populations will benefit conservation and restoration programs. This report presents characterization of the first set of microsatellite markers developed by analyzing a reference transcriptome de novo assembled from RNA-seq data and expressed sequence tags in western white pine (Pinus monticola). A total of 1,948 microsatellite loci were detected in 1,876 contigs. Forty-six loci were verified to be polymorphic, with allele number per locus ranging from 2 to 12. The mean observed and expected heterozygosity per locus was measured as 0.671 and 0.485, respectively. These microsatellite markers will be useful for characterizing genetic diversity and gene-flow among natural stands and population structure among seed families for conservation of this conifer species across western North America. Keywords Expressed sequence tag (EST) Microsatellites Next generation sequencing Pinus monticola White pine blister rust, caused by the fungal pathogen Cronartium ribicola (J. C. Fisch), is a very important exotic forest disease that kills five-needle pines at all
Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0168-x) contains supplementary material, which is available to authorized users. J.-J. Liu (&) C. Hammett Pacific Forestry Centre, Canadian Forest Service, Natural Resources Canada, 506 West Burnside Road, Victoria, BC V8Z 1M5, Canada e-mail: [email protected]
development stages. About 90 % of natural stands of western white pine (Pinus monticola Douglas ex D. Don) have been damaged by WPBR since this rust was accidently introduced to North America in early 1900’s. The rust has also killed much of the Whitebark pine (P. albicaulis Engelm) and limber pine (P. flexilis E. James), both are very important to the forest ecosystems of high elevations (Sniezko 2006). Currently there are few DNA markers of microsatellite or simple sequence repeats (SSR) available for these five-needle pines, limiting efforts to understand conservation genetics, especially for comparative study of genetic resistance against C. ribicola amongst five-needle pines (Liu et al. 2013a). The present study developed a set of microsatellite markers from next generation sequencing and expressed sequence tag (EST) sequencing and examined key population parameters by genotyping these EST-SSR loci in western white pine population. A reference transcriptome of P. monticola primary needles was generated by RNA-seq analysis with 43,890 unique contigs (Liu et al. 2013b) and was used for in silico discovery of microsatellite markers using software SSR Locator w
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