The development of microsatellite DNA markers for genetic analysis in Douglas-fir

Abstract

The microsatellite motifs AG, AC, and ATG were found to be the most abundant in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and several other conifer tree species among di-, tri-, and tetra-nucleotide simple sequence repeats (SSR). Colonies containing AG, AC, and ATG repeats were selected from enriched genomic libraries of Douglas-fir, and 603 were sequenced. Polymerase chain reaction (PCR) primers were designed from flanking sequences in 102 of the SSR clones, of which 50 primer pairs (for 10 AC-repeat microsatellites and 40 AG-repeat microsatellites) produced robust amplification products. Variability was confirmed with 24 unrelated Douglas-fir trees and Medelian segregation with 33-66 progeny from 3 full-sib populations. Forty-eight of the 50 loci were polymorphic, with a mean of 7.5 alleles per locus. Allele sizes ranged from 73 to 292 base pairs. Allele frequencies for the 48 polymorphic loci varied from 0.017 to 0.906 with mean allele frequency of 0.250. Expected heterozygosities among the polymorphic loci varied from 0.174 to 0.926, with a mean of 0.673. Additional, high molecular weight PCR products were amplified by some of the primer pairs, but they did not interfere with the scoring of alleles. Most of the Douglasfir primer pairs also amplified SSR-containing loci in other conifer species.Résumé : Parmi les séquences simples répétées (SSR) montrant deux, trois ou quatre nucléotides, les motifs de microsatellite AG, AC et ATG ont été observés comme étant les plus abondants chez le sapin de Douglas (Pseudotsuga menziesii (Mirb.) Franco) et quelques autres espèces arborescentes de conifère. Les auteurs ont choisi des colonies arborant des motifs répétés AG, AC et ATG à partir de banques génomiques enrichies de sapin de Douglas et en ont séquencé 603. Des amorces PCR ont été élaborées à partir des séquences flanquant les régions répétées pour 102 des clones SSR, desquelles 50 paires d'amorces (pour 10 microsatellites arborant un motif répété AC et 40 microsatellites arborant un motif répété AG) ont résulté en des produits d'amplification robustes. La variabilité a été confirmée à l'aide de 24 sapins de Douglas non apparentés et la ségrégation mendélienne a été vérifiée avec 33 à 66 descendants issus de trois croisements biparentaux. Quarante-huit des 50 loci étaient polymorphes, avec une moyenne de 7,5 allèles par locus. La taille des allèles variait de 73 à 292 pb. Les fréquences alléliques pour les 48 loci polymorphes variaient de 0,017 à 0,906 avec une fréquence allélique moyenne de 0,250. Parmi les loci polymorphes, l'hétérozygotie espérée variait de 0,174 à 0,926, avec une moyenne de 0,673. Des produits PCR additionnels de haut poids moléculaire ont été amplifiés par certaines paires d'amorces, mais ils n'ont pas interféré avec la notation des allèles. La plupart des paires d'amorces de sapin de Douglas amplifiaient également des loci arborant des SSR chez les autres espèces conifériennes.[Traduit par la Rédaction] Amarasinghe and Carlson 1915

Development and characteristics of microsatellite markers for sugi (Cryptomeria japonica D. Don) derived from microsatellite-enriched libraries

“…Microsatellite markers have been developed for various coniferous species using microsatellite-enrichment methods (see, for instance, [1,10,37]. Our successful construction of microsatellite-enriched genomic libraries also showed that enrichment using magnetic particles can promote the efficiency of the development of large amounts of microsatellite markers for coniferous species.…”

“…When microsatellite markers for a particular species are developed, their capacity to be amplified by PCR allows large-scale genotyping on automated DNA analyzers for the construction of genetic linkage maps, and facilitates studies of population genetics and reproduction ecology. For these reasons, microsatellite markers have been developed for 570 N. Tani et al use in analyses of a number of coniferous species (see, for instance, [1,3,9,10,18,20,34,36]). …”

Breeding Douglas‐Fir

“…Allozymes were used throughout the 1980s and early 1990s to study Douglas-fir pollen contamination, seed orchard management, genotype mislabeling, population structure, genetic diversity, and mating systems (El-Kassaby et al 1981;Neale and Adams 1985;Merkle and Adams 1987;Li and Adams 1989;Wheeler and Jech 1992;El-Kassaby and Ritland 1996;Adams et al 1997). Since the mid-1980s, the use of DNAbased markers has steadily increased, including RFLPs (Neale et al 1986;Jermstad et al 1994), RAPDs (Krutovskii et al 1998), microsatellites (Amarasinghe and Carlson 2002;Slavov et al 2004), and single nucleotide polymorphisms (SNPs; Krutovskii et al 2004). RFLPs were first used to confirm the paternal inheritance of chloroplast DNA in Douglas-fir (Neale et al 1986) and to study the evolutionary relationships between Douglas-fir and its relatives (Strauss et al 1990).…”

Highly variable SSR markers in Douglas-fir: Mendelian inheritance and map locations

“…Minimizing the rate of mistyping and avoiding markers with high frequencies of null alleles may be crucial for obtaining unbiased estimates of gene flow and pollen contamination. Although markers which simultaneously detect two or more loci can be useful for some applications (Fisher et al 1998;Amarasinghe and Carlson 2002), their use in parentage analysis is likely to lead to increased rates of mistyping and false inferences. Therefore, our main goal was to develop SSR markers with strong and consistent single-locus banding patterns, and low frequencies of null alleles.…”

“…Fifty SSR markers for Douglas-fir were reported by Amarasinghe and Carlson (2002). We characterized 22 additional markers, 15 of which produce robust banding patterns and segregate as single codominant loci.…”