Seed Orchard Crops Research Articles

The effect of gene flow on frost tolerance in Scots pine – Latitudinal translocation of genetic material

Extensive gene flow can be detrimental to local adaptation and similarly, forestry seed sources such as seed orchards can be heavily influenced by external pollination, especially if the orchard material has been translocated a great distance. Here we conducted a coordinated genotyping-phenotyping study to examine how external pollination events and fecundity variation in a Pinus sylvestris seed orchard influence the genetic composition and the seed-lots’ autumn frost hardiness when genetic material had been translocated 630 km south. The results were then compared to those of a in situ established seed orchard. We genotyped and phenotype >1000 seedlings from these orchards, and constructed their pedigrees and scored their autumn frost tolerance in a controlled climate chamber environment. The hardiness scores were compared with a reference of nine natural stands along a latitudinal cline. We find substantial variation in fecundity and external pollination over crop years, thus unpredictable genetic composition because the contribution of some orchard clones is high in one crop but low in another. We observed that seedlings produced by mating among orchard genotypes were less hardy than expected (corresponding to an origin of −0.6°N) but the opposite in externally pollinated seedlings (+0.3 to +0.7°N). The freeze damage levels reflect the origin of parental genotypes, but to a smaller degree than expected (13% lower than expected damage levels for externally pollinate seedlings and 21% greater damage levels for internally pollinates seedlings). These results suggest that orchard parents’ origins, mating composition and orchard local environment could all affect the seed crops’ quality and their climate adaptation. Seed orchard crops are the key to realize the gain in forestry from breeding efforts. However, genetic monitoring of seed crops is necessary to improve the performance of seed orchards further and adjust deployment areas of seed crops in a timely manner for a more dynamic forestry, considering climate change and biodiversity demands.

Pedigree reconstruction and spatial analysis for genetic testing and selection in a Larix kaempferi (Lamb.) Carri\xe8re plantation

BackgroundLarix kaempferi is one of the major timber species in Northeast Asia. Demand for the reforestation of the species is rising in South Korea due to an increase in large timber production and utilization. However, progeny trials for the species have not been explored, making it challenging to foster advanced generations of tree improvement. In the present study, genetic testing and selection for diameter growth were conducted using pedigree reconstruction and phenotypic spatial distribution analysis in a plantation of L. kaempferi. The aim of the present study was to select the superior larch individuals using the pedigree reconstruction and phenotypic spatial distribution to substitute progeny trials. The plantation of seed orchard crops was established in 1990 and one-hundred and eighty-eight trees were selected as the study material. Genetic variation was investigated first to validate its adequacy as breeding material. Genetic testing was carried out using a model considering pedigree information and spatial autoregression of the phenotypes.ResultsThe expected heterozygosity of the mother trees and offspring were 0.672 and 0.681 presenting the corresponding level of genetic variation between two groups. The pedigree reconstruction using maternity analysis assigned one to six progenies to ninety-two candidate mothers. The accuracy of genetic testing was exceedingly increased with the animal model considering AR1 ⊗ AR1 structure compared to the animal model only. The estimated genetic variance of the former was 9.086 whereas that of the latter was 4.9E-5 for DBH. The predicted breeding values of the offspring for DBH were ranged from -5.937 cm to 5.655 cm and the estimated heritability of diameter growth was 0.344.ConclusionsThe genetic testing approach based on pedigree reconstruction and phenotypic spatial distribution analysis was considered a useful analytical scheme that could replace or supplement progeny trials.

Parentage and relatedness reconstruction in Pinus sylvestris using genotyping-by-sequencing

Estimating kinship is fundamental for studies of evolution, conservation, and breeding. Genotyping-by-sequencing (GBS) and other restriction based genotyping methods have become widely applied in these applications in non-model organisms. However, sequencing errors, depth, and reproducibility between library preps could potentially hinder accurate genetic inferences. In this study, we tested different sets of parameters in data filtering, different reference populations and eight estimation methods to obtain a robust procedure for relatedness estimation in Scots pine (Pinus sylvestris L.). We used a seed orchard as our study system, where candidate parents are known and pedigree reconstruction can be compared with theoretical expectations. We found that relatedness estimates were lower than expected for all categories of kinship estimated if the proportion of shared SNPs was low. However, estimates reached expected values if loci showing an excess of heterozygotes were removed and genotyping error rates were considered. The genetic variance-covariance matrix (G-matrix) estimation, however, performed poorly in kinship estimation. The reduced relatedness estimates are likely due to false heterozygosity calls. We analyzed the mating structure in the seed orchard and identified a selfing rate of 3% (including crosses between clone mates) and external pollen contamination of 33.6%. Little genetic structure was observed in the sampled Scots pine natural populations, and the degree of inbreeding in the orchard seed crop is comparable to natural stands. We illustrate that under our optimized data processing procedure, relatedness, and genetic composition, including level of pollen contamination within a seed orchard crop, can be established consistently by different estimators.

Mating patterns and pollen dispersal in a Japanese larch (Larix kaempferi) clonal seed orchard: a case study.

Pollination dynamics highly determines the genetic quality of seed orchard crops. However, there is less research about the effect of mating patterns on seed productivity of orchard crops. So far, clonal seed orchards have been producing genetically improved seedlings used for most Japanese larch (Larix kaempferi (Lamb.) Carr.) plantations in China. In the present study, a total of 17 highly variable simple sequence repeat (SSR) markers were used for genotyping a progeny trial population consisting of 647 open-pollinated progenies germinated from seeds which were collected from 63 maternal clones with 140 potential paternal clones in a Japanese larch clonal seed orchard in China. Paternity analysis was used in the present case study in order to evaluate the level of paternal gametic contribution, estimate pollen contamination and selfing rates, and investigate pollination patterns, pollen dispersal patterns and the impact of mating patterns on seed productivity of orchard crops. We observed 93.7% of the success rate of the parental assignment, unequal paternal gametic contribution (0-12.4%) with 6.3% of the progenies derived from pollen contamination or unsampled pollen donors, and absence of evidence for selfing. We also found that pollination rate highly depended on the distance between pollen donors and maternal parents, the majority of the identified crossing (65.7%) occurred between clones within a 150-m radius, and large variations in growth performance existed among the paternal half-siblings. Progeny growth performance (diameter at breast (DBH) and height (HGT)) was measured at Age-20 in order to investigate the impact of mating patterns on timber production of orchard crops. As either the paternal or maternal, two clones (i. e., clones Z38 and Z62) were identified to have produced progenies with higher average stem volume breeding values than that of all of the progenies. Specifically, the genetic gains for volume were 3.53% for the two clones as paternal parents, and 8.26% as the maternal parents at Age-20. Thus, both elite clones were ideal candidates for the construction of next-generation clonal seed orchards due to their synchronous reproductive phenology with greater crossing rate and higher genetic gain. These results improved the pedigree information to provide solid evidence of mating patterns for future design and effective management of seed orchards and for the development of viable long-term breeding strategies for other coniferous species.

Integrating fecundity variation and genetic relatedness in estimating the gene diversity of seed crops:Pinus koraiensisseed orchard as an example

The genetic gain and gene diversity of seed crops from a 1.5-generation clonal seed orchard of Pinus koraiensis Siebold & Zucc. were estimated under consideration of parental genetic values and fecundity variation. Fecundity variation among clones was estimated for 5 consecutive years (2010–2014) as the sibling coefficient, which was drawn from clonal contribution to the total production of seed conelet. To monitor gene diversity, status number was estimated by the integration of fecundity variation and group coancestry. Group coancestry was calculated as the average of genetic relatedness (coancestry) among orchard clones. The averages of conelet production were high in 2010 and 2011, moderate in 2013 and 2014, and poor in 2012 with a grand mean of 13.7. Correlation analysis showed that good conelet producers consistently gave good production. Cumulative distribution of clonal conelet production was presented as a function of the total conelet yield, and this distribution indicated deviation from the expected clonal equal production. Group coancesrtry was 0.0096, indicating minimal loss of gene diversity. Status number and genetic gain were higher in good than in poor conelet production years, highlighting the importance of fecundity variation in determining the genetic gain and gene diversity of seed orchard crops.

Mating dynamics of Scots pine in isolation tents

Seed orchards are forest tree production populations for supplying the forest industry with consistent and abundant seed crops of superior genetic quality. However, genetic quality can be severely affected by non-random mating among parents and the occurrence of background pollination. This study analyzed mating structure and background pollination in six large isolation tents established in a clonal Scots pine seed orchard in northern Sweden. The isolation tents were intended to form a physical barrier against background pollen and induce earlier flowering relative to the surrounding trees. We scored flowering phenology inside and outside the tents and tracked airborne pollen density inside and outside the seed orchard in three consecutive pollination seasons. We genotyped 5683 offspring collected from the tents and open controls using nine microsatellite loci, and assigned paternity using simple exclusion method. We found that tent trees shed pollen and exhibited maximum female receptivity approximately 1 week earlier than trees in open control. The majority of matings in tents (78.3 %) occurred at distances within two trees apart (about 5 m). Self-fertilization was relatively high (average 21.8 %) in tents without supplemental pollination (SP), but it was substantially reduced in tents with SP (average 7.7 %). Pollen contamination was low in open controls (4.8–7.1 %), and all tents remained entirely free of foreign pollen. Our study demonstrates that tent isolation is effective in blocking pollen immigration and in manipulating flowering phenology. When complimented with supplemental pollination, it could become a useful seed orchard management practice to optimize the gain and diversity of seed orchard crops.

Chloroplast DNA variation and pollen contamination in a Pinus brutia Ten. clonal seed orchard: implication for progeny performance in plantations

Pollen contamination is one of the important factors affecting the yield, adaptability, and genetic quality of the seed produced from seed orchards in forest tree breeding programs. Incoming pollen from the forests surrounding the seed orchard is a major concern in tree breeding because it contributes to losses in the expected genetic gains from seed orchard crops. The genetic variation and the level of pollen contamination in a 16-year-old Pinus brutia Ten. first-generation clonal seed orchard was studied using chloroplast microsatellite markers (cpSSRs). In total, 23 alleles and 36 unique allelic combinations (haplotypes) were detected based on the 6 cpSSR loci analyzed. The haplotypic diversity of the clones in the seed orchard was found to be 0.849. Out of 300 embryos analyzed, 87 were not compatible with any male parent within the seed orchard. Thus, 29% of the embryos were sired by pollen sources outside the orchard (i.e. apparent contamination). Microsatellite-based analysis revealed that the estimated contamination rate was 39.3%. Background pollination at this level will cause losses of 20% in the expected genetic gains. Our findings are valuable for the assessment of the intended seed orchard function, i.e. provision of genetically improved seed. It may be worthwhile to use pollen management strategies like strobilus stimulation, controlled pollination, and supplemental mass pollination to decrease pollen contamination and increase the genetic quality of the seeds produced.

Optimization of genetic gain and diversity in seed orchard crops considering variation in seed germination

Balancing genetic gain and diversity in seed orchard crops is of a major regeneration importance. Here we extend our earlier work on the development of an optimization protocol that maximizes crops' genetic gain at any predefined diversity level, considering parental reproductive output, co-ancestry, and inbreeding, by incorporating variation in seed germination as an input. Variation in seed germination capacity substantially affected seedlots' genetic diversity by either under- or overestimating their effective population size; however, genetic gains were robust and their differences were negligible. The contrasting results of gain and diversity support the inclusion of the germination capacity, when available, in the optimization protocol.

Genetic worth and diversity across 18 years in a Nordmann fir clonal seed orchard

Many tree breeding programmes rely entirely on clonal seed orchards as the link between breeding activities and forestry, and are thereby also exposed to yearly fluctuations in the orchard crops. The main objective was to study the impact of year and clone fluctuations in strobili production on genetic worth, as well as on genetic diversity, of 18 consecutive commercial seed orchard crops. In a mature Abies nordmanniana clonal seed orchard, a yearly visual assessment of female and male strobili was carried out on a total of 18 clones (260 ramets), and coupled with clone breeding values. Despite large annual and clone variation, and year-by-clone interaction in strobili production, the orchard’s genetic worth was somewhat uniform when considering four evaluated traits in the 11 years of superior or medium crop size. These years accounted for 95% of the accumulated cone crop over the whole period (18 years). Noteworthy deviations were only seen in the seven poorest seed years, comprising the remaining 5% of the total cone crop, where genetic diversity measures were also low. Avoiding small crop years seems to be a reasonable way to avoid fluctuations in the commercial seed orchard product.

Congruence between parental reproductive investment and success determined by DNA-based pedigree reconstruction in conifer seed orchards

Seed orchards’ parental reproductive success is often assessed through surveys of seed- and pollen-cone production (reproductive investment). Although the reliability of this approach has been questioned, its speed and ease made it the method of choice for providing approximate estimates for the genetic quality and quantity of seed orchard crops. In this study, reproductive investment data from various female and male phenotypic assessment methods from three seed orchards (lodgepole pine ( Pinus contorta Dougl. ex. Loud. var. latifolia Engelm.), Douglas-fir ( Pseudotsuga menziesii (Mirb.) Franco), and western larch ( Larix occidentalis Nutt.)) were compared with reproductive success estimates based on DNA fingerprinting and parentage analyses. The use of reproductive investment as a proxy to reproductive success on estimating seed crop’s volume genetic worth and diversity (measured by effective number of parents) was evaluated. Regression analyses indicated that the phenotypic assessment methods reflect the true parental reproductive success and that estimates of seed- and pollen-cone volume production appear to be the most appropriate indicators of female and male reproductive success, respectively. Although the best combination of female–male survey methods produced inflated effective number of parents estimates, the genetic worth estimates were similar to those based on DNA, highlighting their robustness to fertility variation assessment.

Marker-based parentage analysis facilitates low input ‘breeding without breeding’ strategies for forest trees

Controlled crosses and progeny testing are important components of tree breeding programs. Recently, a new approach, ‘breeding without breeding’ (BWB), has been proposed to obtain structured pedigrees for the breeding and testing from wind-pollinated progenies; the technique uses high-resolution markers instead of conventional approaches. The BWB approach is attractive for outcrossing conifers where the breeding cycle is long and reproductive maturity is often delayed. For the application of the BWB approach, the accurate assignment of parents is essential. The first step in the application of the BWB approach is to conduct tests into the reliability of paternity and parent-pair exclusion procedures in complex situations. In this study we conducted multi-locus-based empirical and simulation analyses for a Scots pine seed orchard crop, in order to develop the appropriate parentage assignment techniques for BWB and to investigate whether pedigrees from a seed orchard crop can be determined with sufficient accuracy. The results were promising, as 93–97% of the seeds generated by mating among the seed orchard clones were correctly assigned to a parent-pair. When mating occurred with foreign pollen, the success rate of identifying mother clones was 60–78%. Based on these results, we propose three novel low input breeding strategies for forest trees. The principles of the different options and their potential benefits and costs are described and discussed using Scots pine as a case study.

Optimization of combined genetic gain and diversity for collection and deployment of seed orchard crops

Genetic gain and diversity of seed orchards’ crops are determined by the number of parents, their breeding values and relatedness, within-orchard pollination efficiency, and level of pollen contamination. These parameters can be manipulated at establishment by varying clonal representation (e.g., linear deployment), during orchard development by genetic thinning, or by selective harvesting. Since clonal fecundities are known to vary both within and among years, then each seed crop has a unique genetic composition and, therefore, crops should be treated on a yearly basis. Here we present an optimization protocol that maximizes crop’s genetic gain at any desired genetic diversity through the selection of a subset of the crop that meets both parameters. The genetic gain is maximized within the biological limit set by each clone’s seed-cone production and effective population size is used as a proxy to genetic diversity whereby any relationship among clones is considered. The optimization was illustrated using 3 years’ reproductive output data from a first-generation western larch seed orchard and was tested under various scenarios including actual male and female reproductive output and male reproductive output assumed to be either equal to that of female or a function of clonal representation. Furthermore, various levels of co-ancestry were assigned to the orchard’s clones in supplementary simulations. Following the optimization, all solutions were effective in creating custom seedlots with different gain and diversity levels and provided the means to estimate the genetic properties of composite seedlots encompassing the remaining “unused” seed from a number of years.

Effective Number of Clones in Seed Orchards of Cedrus libani A. Rich.

Numbers of clone and ramet/clone were studied and discussed based on effective number of clones and relative effective number of clones in seed orchards of Taurus cedar (Cedrus libani A. Rich.). While, seed orchards had similar number of clones (15, 30 and 33), there were large differences for number of graft/clone within and among orchards. Number of ramets per clone varied between 4 and 43 in an orchard and between 4 and 106 in all orchards considered. Effective number of clones ranged from 24.6-31.4 and relative effective number from 0.82 to 0.99. The results of the study showed that ramet number had moderate variation among clones. It was important that establishment and thinning of seed orchards and estimation of gene diversity in seed orchard crop.

Female fertility variation in mature Pinus sylvestris clonal seed orchards

Female fertility is the basis for the output of seeds from clonal orchards and its variation is of major interest for the economics and biology of seed orchards, especially for the efficiency and diversity of seed orchard crops. Assessments of female fertility variation in 10 mature (>15 years old) seed orchards of Scots pine (Pinus sylvestris L.) were evaluated and compared. Depending on the individual orchard, fertility variation for each clone was assessed in slightly different ways, e.g. number of strobili, cones, seeds or litre of cones per ramet. Assessments in five of the orchards were made over consecutive years. The main result was that the clonal variation in mean female fertility per surviving ramet was lower than expected from the literature; the Kang–Lindgren sibling coefficient (Ψ) within individual years averaged 1.35. The variation between ramets within clones and years was of similar magnitude as that between clones. Clone by year interactions were noticeable, but were slightly lower than the variation between as well as that within clones in individual years. There was considerable variation in the variance components between years. The limited variation in female fertility indicated that it should not be a selection criterion when selecting clones for a seed orchard. Furthermore, it will not result in large differences between clones in seed set or large reductions in gene diversity in productive Scots pine seed orchards.

Pollen contamination and it’s control in the management of forestry seed orchard

The pollen contamination in a seed orchard is a principal factor influencing the genetical quality of a seed orchard. Genetic losses arise from the influx of 'wild' conspecific pollen into seed orchards.In this paper a review was made of the general researches on pollen contamination, contamination level, the extent of its influence and the measures to reduce it in the orchards at home and abroad. Researches showed that the DNA marker proved to be very useful in assessing seed orchard mating dynamics and orchard management efficacies for conifer seed orchards(e.g.,chloroplast DNA(cpDNA),RAPD,SSR);in a seed orchard the contamination level is higher than that formerly estimated ;pollen yield seems to have no influence on contamination level;space and reproductive isolation are not so effective as to be expected. At present time, supplemental mass pollination(SMP)and thinning are two effective measures to reduce pollen contamination. To improve the parental balance of seed orchard crops, the use of artificial management techniques such as gibberellin A4/7 treatment, rearrangement of clonal ramets is recommended in seed orchards. Work on the other facets of contamination measurement,gene-flow studies, are listed. The authors made some recommendations about research on and management of pollen contamination in seed orchards in our country. Mechanical(pollen dispersal),phenological(the timing of flowering) and viability factors related to the pollen genotype should all be considered when assessing pollen contamination in seed orchards.

Paternity analysis with microsatellites in a Danish Abies nordmanniana clonal seed orchard reveals dysfunctions

A paternity analysis using five microsatellite markers was conducted in a Danish clonal seed orchard with 13 Abies nordmanniana (Stev.) Spach clones. The purpose was to investigate potential seed-orchard dysfunctions, with special emphasis on nonequal pollen contributions and selfing. Male paternity was found for 232 seedlings germinated from seeds collected on three ramets, each of eight clones, and the relative contribution of each clone to the gene pool of male gametes was calculated. Furthermore, 49 ramets were genotyped to check for erroneous grafting. The effect of an unbalanced male contribution was quantified by means of two measures: (1) the status number (NS), which reflects buildup of coancestry in the seed-orchard crop as a result of a low number of clones and an unequal male contribution, and (2) the asymptotic variance effective population number (Ne(v)). The contributions by pollen donors from the 13 clones were highly skewed. Three clones were fathers to more than 75% of the progenies, while making up only 24% of the ramets in the seed orchard. Four clones sired no progenies at all. The unequal contribution on the male side corresponded to NS = 4.2 and Ne(v) = 5.8. Some selfing was observed, which may give rise to concern if clonal seed orchards with few clones are established. The estimated maximum pollen contamination from outside the seed orchard was 4.3%. No grafting–labelling errors were identified.

Pollen contamination and mating patterns in a Douglas-fir seed orchard as measured by simple sequence repeat markers

Pollen contamination is detrimental to the genetic quality of seed orchard crops. Highly variable simple sequence repeat (SSR) markers make it possible to accurately measure pollen contamination and characterize patterns of within-orchard mating by directly identifying the male and female parent of each seed produced in the orchard. We used nine SSR markers to measure pollen contamination and characterize mating patterns based on seed samples collected in 3 years (1999, 2000, and 2003) from one block of a nonisolated, open-pollinated, clonal seed orchard of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) in western Oregon. Pollen contamination was consistently high across the 3 years (mean = 35.3%, range = 31.0%-41.3%) and appeared to result primarily from cross-pollination among the orchard blocks. Levels of pollen contamination varied substantially among clones and were higher in clones with early female receptivity (mean = 55.5%) than in those with either mid (mean = 36.4%) or late (mean = 28.3%) female receptivity. We detected low rates of self-pollination (mean = 1.8% per clone) and over 10-fold differences in the relative paternal contributions of the clones. There was a clear pattern of positive assortative mating with respect to floral phenology. This study illustrates that SSR markers are powerful tools for characterizing seed lots and improving the design and management of Douglas-fir seed orchards.

Balancing seed yield and breeding value in clonal seed orchards

Seed orchards should produce seeds that are both abundant and of high genetic value. This study suggests methods to achieve such a compromise and study their efficiency. The methods were applied on data obtained from 41 seed orchard clones of Scots pine from mid-Sweden. The value of the seed orchard crop was set as a function of its breeding value, the amount of seeds produced and their gene diversity, measured as the effective number of clones. The proportion of ramets of different clones that maximized this value was regarded as the optimum for deployment of the clones in a seed orchard. The results were compared with truncation selection for breeding value, truncation selection for clone benefit (the product of seed production and breeding value) and linear deployment (where ramets are deployed linearly in relation to breeding value). The influence of two parameters was studied: the relative importance of breeding value for seed value and the size of the penalty for reducing the value of the seed crop with respect to lost gene diversity. The conventional wisdom is to select the clones with the highest breeding values, but that turned out to be the most inferior alternative studied. Clone benefit truncation provided a good approximation to optimal benefit for cases, where the effective number was low and dependence of breeding value limited.

Monitoring male reproductive success in a Japanese black pine clonal seed orchard with RAPD markers

Overall male reproductive success was investigated in a Japanese black pine (Pinus thunbergii Parl.) clonal seed orchard consisting of 16 nematode-resistant clones, using random amplified polymorphic DNA (RAPD) markers. We genotyped all 16 of the orchard clones at selected RAPD loci by analyzing diploid needles and 10 haploid megagametophytes from each of them. In addition, the male parent of open-pollinated seedlings was identified by comparing their phenotypes with the genotypes of the orchard clones. In this way the male parents of 559 seedlings of 648 seedlings of progenies from 14 parental (orchard) clones were determined. The level of external pollen contamination in the seed orchard was estimated to be 2.2% for the total population. The effective mean selfing rate observed was 1.5%. Male reproductive success among clones varied widely, from 0.8 to 24.4%, for the total population. Our data suggest that the significant deviation in male reproductive success of individual clones from panmictic ratios may be a problem for the genetic quality in seed orchard crops. To improve the parental balance of seed orchard crops, the use of artificial management techniques such as gibberellin A4/7 treatment, supplemental mass pollination, and rearrangement of clonal ramets is recommended in seed orchards.

Fertility variation and flowering asynchrony in Pinus sylvestris: consequences for the genetic structure of progeny in seed orchards

Effects of the variation in male and female fertilities of clones, flowering phenology, and spatial distribution of clones on the genetic structure of progenies of three Scots pine seed orchards were investigated using isozyme gene markers. A pronounced variation of male and female gametic contributions was found. Most clones appear to be phenologically synchronized, but up to 15% of early or late flowering clones were found in all seed orchards. The presence of ramets, which do not belong to any seed orchard clone, does not affect allelic or genotypic diversity of the seed orchard crop. Gene-pool genetic distances based on allelic frequencies between the parental and the progeny generations are not very high, but there are certain loci exhibiting a considerable differentiation. On the other hand, variance effective population sizes indicate a substantial drift of gene frequencies between seed orchard itself and its offspring. The necessity of a cautious use of the reproductive material originating from seed orchards is emphasized.