Cherdsak Liewlaksaneeyanawin

Single-copy, species-transferable microsatellite markers developed from loblolly pine ESTs

Microsatellites, or simple sequence repeats (SSRs), are usually regarded as the “markers of choice” in population genetics research because they exhibit high variability. The development cost of these markers is usually high. In addition, microsatellite primers developed for one species often do not cross-amplify in related species, requiring separate development for each species. However, microsatellites found in expressed sequence tags (ESTs) might better cross-amplify as they reside in or near conserved coding DNA. In this study, we identified 14 Pinus taeda (loblolly pine) EST-SSRs from public EST databases and tested for their cross-species transferability to P. contorta ssp. latifolia, P. ponderosa, and P. sylvestris. As part of our development of a P. contorta microsatellite set, we also compared their transferability to that of 99 traditional microsatellite markers developed in P. taeda and tested on P. contorta ssp. latifolia. Compared to traditional microsatellites, EST-SSRs had higher transfer rates across pine species; however, the level of polymorphism of microsatellites derived from ESTs was lower. Sequence analyses revealed that the frequencies of insertions/deletions and base substitutions were lower in EST-SSRs than in other types of microsatellites, confirming that EST-SSRs are more conserved than traditional SSRs. Our results also provide a battery of 23 polymorphic, robust microsatellite primer pairs for lodgepole pine.

Breeding without breeding: is a complete pedigree necessary for efficient breeding?

Complete pedigree information is a prerequisite for modern breeding and the ranking of parents and offspring for selection and deployment decisions. DNA fingerprinting and pedigree reconstruction can substitute for artificial matings, by allowing parentage delineation of naturally produced offspring. Here, we report on the efficacy of a breeding concept called “Breeding without Breeding” (BwB) that circumvents artificial matings, focusing instead on a subset of randomly sampled, maternally known but paternally unknown offspring to delineate their paternal parentage. We then generate the information needed to rank those offspring and their paternal parents, using a combination of complete (full-sib: FS) and incomplete (half-sib: HS) analyses of the constructed pedigrees. Using a random sample of wind-pollinated offspring from 15 females (seed donors), growing in a 41-parent western larch population, BwB is evaluated and compared to two commonly used testing methods that rely on either incomplete (maternal half-sib, open-pollinated: OP) or complete (FS) pedigree designs. BwB produced results superior to those from the incomplete design and virtually identical to those from the complete pedigree methods. The combined use of complete and incomplete pedigree information permitted evaluating all parents, both maternal and paternal, as well as all offspring, a result that could not have been accomplished with either the OP or FS methods alone. We also discuss the optimum experimental setting, in terms of the proportion of fingerprinted offspring, the size of the assembled maternal and paternal half-sib families, the role of external gene flow, and selfing, as well as the number of parents that could be realistically tested with BwB.

Pedigree and mating system analyses in a western larch (Larix occidentalis Nutt.) experimental population

Abstract•The mating pattern and gene flow in a western larch (Larix occidentalis Nutt.) experimental population was studied with the aid of microsatellite markers and a combination of paternity-mating system analysis. The commonly difficult to assess, male gametic contribution was determined with 95% confidence and its impact on genetic gain and diversity was determined.• Male fertility success rate ranged between 0 and 11%. Male reproductive output parental imbalance was observed with 50% of the pollen being produced by the top 5% of males while the lower 39% males only produced 10% of the pollen.• A significant difference was observed between male effective population size (genetic diversity) estimates from paternity assignment compared to those based on population’s census number (21 vs. 41); however, this difference did not affect estimates of genetic gain.• A total of 221 full-fib families were identified (sample size range: 1–8) and were nested among the studied 14 seed-donors.• A combination of paternity-mating system analysis is recommended to provide a better insight into seed orchards’ mating dynamics. While pollen flow tends to inflate mating system’s outcrossing rate, the paternity analysis effectively determined the rate and magnitude of contamination across receptive females.Résumé• Les modes de croisement et les flux de gènes dans une population expérimentale de mélèze occidental (Larix occidentalis Nutt.) ont été étudiés à l’aide de marqueurs microsatellites et d’une analyse combinée de paternité et du système de reproduction. La contribution gamétique mâle — communément difficile à estimer — a été déterminée avec un seuil de confiance de 95 % et son impact sur le gain génétique et la diversité a été déterminé.• Le taux de succès reproductif mâle était compris entre 0 et 11 %. Un déséquilibre dans la contribution des parents mâles a été observé avec la production de 50 % du pollen par 5 % des pères alors que 39 % d’entre eux ne contribuaient que pour seulement 10 % du pollen.• Une différence significative a été observée entre la taille efficace de la population mâle (diversité génétique) estimée par la recherche de paternité et celle basée sur les effectifs recensés de la population (21 vs. 41) ; cependant, cette différence n’affecte pas l’estimation du gain génétique.• 221 familles de plein-frères ont été identifiées (effectifs entre 1 et 8), regroupées parmi les 14 arbres-mères étudiés.• La combinaison d’une analyse de paternité et du système de reproduction est recommandée pour étudier de manière approfondie la dynamique de croisement en vergers à graines. Tandis que les flux de pollen tendent à augmenter le taux d’inter-croisements, l’analyse de paternité détermine de manière effective le taux et l’amplitude de contamination des arbres-mères.

Pollination dynamics in a Douglas-fir seed orchard as revealed by pedigree reconstruction

Abstract• Pollination dynamics was studied in a Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seed orchard using 8 nuclear microsatellite markers and pedigree reconstruction.• The seed orchard consisted of 49 parents (clones). Cone-crop management included bloom delay and supplemental mass pollination (SMP) using 12 internal and 4 external pollen donors.• A random sample of 801 bulk seeds was genotyped for both haploid megagametophyte and corresponding diploid embryo.• Using the parental population’s multilocus genotypes, full pedigree reconstruction generated all the information needed to estimate the maternal, paternal, and parental reproductive success, selfing, pollen contamination, and pollination success of the 4 external pollen donors.• Maternal, paternal, and parental reproductive success varied with 80% of gametes being produced by 23, 45, and 37% of the orchard’s parents, respectively, resulting in a drastically reduced effective population size as compared to the census number (14 vs. 53).• Selfing, pollen contamination, and aggregate SMP success (internal and external) were estimated to be 15.2, 10.4, and 15.0%, respectively.• Full pedigree reconstruction was effective in unraveling the orchard’s pollination dynamics and both female and male reproductive success.

Identification of COS markers in the Pinaceae.

Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were single-banded in all three species. Even though these COS markers were selected from large EST databases, a substantial proportion (20–30%) of amplicons displayed multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps are offset by their lower polymorphism.

Development and characterization of microsatellite loci in western larch (Larix occidentalis Nutt.)

Western larch (Larix occidentalis Nutt.) is an important ecological and commercial species in the Pacific Northwest. We isolated nine microsatellite loci with variable polymorphism ranging from five to 19 alleles per locus. Observed and expected heterozygosities averaged 0.42 and 0.64 and ranged from 0.11 to 0.83 and from 0.48 to 0.80, respectively. These markers, along with those already existing, will be useful for the species’ gene resource management activities.