Identification and characterization of microsatellites in Norway spruce (Picea abiesK.)

Abstract

Norway spruce (Picea abies) genomic libraries were screened for presence of dinucleotide AC/GT and AG/CT microsatellites (or simple sequence repeats). On average, one (AG)n microsatellite every 194 kb and one (AC)n microsatellite every 406 kb were found. Forty-six positive clones were sequenced and primers flanking 24 AG microsatellites and 12 AC microsatellites diesigned. Only seven (20%) of them produced the expected single-locus polymorphic pattern when used to amplify Norway spruce DNAs. The other primer pairs gave either multiple bands or bad amplification, or a single monomorphic fragment. Such a small proportion of successful primer pairs was attributed to the high level of complexity of the Norway spruce genome. Dot blot analysis of the clones showed that many of them contained repetitive DNA and that those giving the single-locus polymorphic patterns usually corresponded to single-copy sequences. A family of repetitive DNA that contained AG repeats was identified and was present in about 40,000 copies per haploid genome. Simple Mendelian inheritance was observed for all the polymorphisms tested. The average number of alleles was 13, ranging from 6 to 22, and the expected heterozygosity was 0.79 when seven microsatellites were used to genotype a panel of 18 trees representing different populations. Compared with isozymes, microsatellites are about five times more informative and could provide an extremely valuable source of markers for genome mapping and genetic diversity studies.

High‐throughput sequencing and graph‐based cluster analysis facilitate microsatellite development from a highly complex genome

“…This supports a previous study of Norway spruce, which found that primer pairs amplifying a single polymorphic microsatellite were largely restricted to unique clone sequences that lacked repetitive DNA (Pfeiffer et al. 1997). Causes of microsatellite failure in our study included (1) PCR amplification failure, which resulted either in no discernible product or in a small number of nonspecific bands; (2) the amplification of monomorphic bands resembling microsatellite alleles; or (3) the amplification of more than one locus, indicated by the presence of up to four alleles within an individual.…”

“…All of these patterns have been observed in similar studies of other species with complex genomes (Pfeiffer et al. 1997; Bailie et al. 2010; McInerney et al.…”

“…This has been invoked as an explanation for the poor success rates (ranging from zero to around twenty percent) of efforts to develop microsatellites in species as diverse as Norway spruce (Pfeiffer et al. 1997), butterflies (Meglecz et al. 2004), squat lobsters (Bailie et al.…”

Genetic variability and health of Norway spruce stands in the Regional Directorate of the State Forests in Krosno

“…Microsatellite sequence analysis (SSR) was performed for 180 trees using the following 9 microsatellite loci: EATC1B02, EATC1E3, EATC2G05, SPAGC1, SPAGG3, EATC1G2, EATC2B02, G2 and SPAGC2 (Pfeiffer et al 1997;Scotti et al 2002). For tree genotyping, Genetic Analyzer 3500 (Life Technologies™) sequencer and Data Collection Software 3500 were used.…”

Differences in wood properties of Picea abies L. Karst. in relation to site of provenance and population genetics

“…Four nuclear primer pairs for SSR loci were used for this study: EAC1G05, EAC1D10 (Scotti et al 2002), SpAC1B8 (Pfeiffer et al 1997), and UAPCA24 (Hodgetts et al 2001). PCR amplifications were conducted by a 96-well MasterCycler EpGradient (Eppendorf, Hauppauge, NY, USA) using a total volume of 25 μl with 2.5 μl 10 × Dream Taq Buffer, 0.5 U of Dream Taq (Termo Scientific, Fermentas, Pittsburgh, PA, USA) 200 μM dNTPs, 0.2 μM of each primer, 50 ng of total genomic DNA.…”