Characterization of microsatellite markers in eastern white pine

Abstract

An enrichment cloning method was evaluated for the isolation of microsatellite loci from eastern white pine and the resulting markers were examined for polymorphisms. A 200-fold enrichment was achieved for highly abundant (AC)n repeats, but for much less abundant (ACAG)n repeats an enrichment of only 20-fold was obtained. Using a single set of PCR conditions, 19 microsatellite loci were identified from 77 primer pairs evaluated. Genotyping of 16 (AC)n loci in 16 unrelated white pines from the north-central United States revealed an average of 5.4 alleles per locus and an average observed heterozygosity of 0.515. Five loci were scored among megagametophytes from a single pine to obtain a haploid genotype of the segregating female meiotic products. All loci segregated according to Mendelian expectations and linkage was established for two of the loci. It was concluded that (AC)n loci are highly variable in this species and that SSR (simple sequence repeat) markers can be efficiently developed for genome mapping and population genetics studies.

Somatic stability of microsatellite loci in Eastern white pine, Pinus strobus L.

“…Moreover, each of the novel alleles observed by Fisher et al (1998) was at least four repeat units different from the parental allele, and thus contradicts the expectation that mutations at microsatellite loci should involve single repeat unit changes following slippage during DNA replication (Ellegren, 2000;Schlö tterer, 2000). Such hypermutable microsatellite loci seem to be the exception rather than the rule, since mutations have never been reported in any other study conducted in P. strobus (eg Echt et al, 1996), in other pine species (eg Smith and Devey, 1994;Hicks et al, 1998;Elsik et al, 2000), and in conifers in general (eg Ven et al, 1996;Pfeiffer et al, 1997;Khasa et al, 2000;Hodgetts et al, 2001;Rajora et al, 2001). Overall, these results suggest that the use of microsatellites as markers in longlived organisms (eg, in marker-assisted selection and population studies) should, in general, provide reliable results.…”

“…Cones were collected from three positions per ramet, and megagametophytes were extracted from seeds within these cones. The total vegetative growth separating all sampled cones was determined by summing tree segments a-h. Echt et al (1996). b Cato and Richardson (1996).…”

“…DNA samples were screened for variation at eight nuclear and four chloroplast microsatellite loci (Table 1). The choice of loci has been made on the basis of the level of polymorphism detected in previous studies (Echt et al, 1996). The more highly variables of these loci were selected since they are expected to have the highest mutation rate.…”

“…The more highly variables of these loci were selected since they are expected to have the highest mutation rate. DNA sequences of the PCR primers for the loci examined in this study have been published elsewhere (Cato and Richardson, 1996;Echt et al, 1996;Vendramin et al, 1996). The PCR reaction and amplification conditions were as described by Echt et al (1996Echt et al ( , 1999.…”

“…DNA sequences of the PCR primers for the loci examined in this study have been published elsewhere (Cato and Richardson, 1996;Echt et al, 1996;Vendramin et al, 1996). The PCR reaction and amplification conditions were as described by Echt et al (1996Echt et al ( , 1999. Fluorescently labeled amplified PCR products were separated by electrophoresis on a 41 cm polyacrylamide gel (Li-Cor model 4000L automatic DNA sequencer) (Li-Cor, Lincoln, NE, USA).…”