Prediction of genetic gain and gene diversity in seed orchard crops under alternative management strategies

Genetic gain and the gene diversity of seed crops from clonal seed orchards were formulated considering genetic selection, fertility variation and pollen contamination, and compared for five different management strategies. Genetic response was studied as a function of orchard management tactics. Management variables included the proportion of clones left after genetic thinning and/or selective seed harvesting. Formulae were derived to calculate gene diversity (expressed as group coancestry or status number) based on the sex ratio in an orchard population. The influence of having different sets of clones serving as seed parents, or pollen parents, or as both, was analysed. In addition, the impact on genetic gain and the gene diversity of seed crops was studied quantitatively as a function of the quantity and quality of gene flow from outside the orchard. The negative impact of fertility variation among orchard genotypes on the gene diversity of the seed crop was quantified. Numerical examples were given to illustrate the impact of orchard management alternatives on genetic gain and gene diversity. The formulae and results of this study can be used for identifying favourable alternatives for the management of seed orchards. Received: 16 December 2000 / Accepted: 13 March 2001     

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.     

Effects of male fecundity, interindividual distance and anisotropic pollen dispersal on mating success in a Scots pine (Pinus sylvestris) seed orchard

Quantifying the effect of pollen dispersal and flowering traits on mating success is essential for understanding evolutionary responses to changing environments and establishing strategies for forest tree breeding. This study examined, quantitatively, the effects of male fecundity, interindividual distance and anisotropic pollen dispersal on the mating success of Scots pine (Pinus sylvestris), utilizing a well-mapped Scots pine seed orchard. Paternity analysis of 1021 seeds sampled from 87 trees representing 28 clones showed that 53% of the seeds had at least one potential pollen parent within the orchard. Pronounced variation in paternal contribution was observed among clones. Variations in pollen production explained up to 78% of the variation in mating success, which was 11.2 times greater for clones producing the largest amount of pollen than for clones producing the least pollen. Mating success also varied with intertree distance and direction, which explained up to 28% of the variance. Fertilization between neighboring trees 2.3 m apart was 2.4 times more frequent than between trees 4.6 m apart, and up to 12.4 times higher for trees downwind of the presumed prevailing wind direction than for upwind trees. The effective number of pollen donors recorded in the seed orchard (12.2) was smaller than the theoretical expectation (19.7). Based on the empirical observations, a mating model that best describes the gene dispersal pattern in clonal seed orchards was constructed.     

Gene flow and mating system in five <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don seed orchards as revealed by analysis of microsatellite markers

We investigated gene flow in five Cryptomeria japonica D. Don seed orchards of two different types (common and miniature) at widely spaced locations using microsatellite markers. The quality of a seed crop is determined by many factors, including pollen contamination from outside sources, self-fertilization, and the proportion of contributions from constituent clones. Contamination rates were found to vary among ramets both within seed orchards (10.0–76.7% in the most variable seed orchard) and among seed orchards (35.0–65.8% on average). Among ramets, there were significant negative correlations between pollen contamination rate and their distance from the orchard edge; among seed orchards, there were significant positive correlations between the pollen contamination rate and the C. japonica forest area nearby. Some proportion of the pollen (10.7% of total contamination) also migrated from parts of the orchards that had not been treated with gibberellin to induce flowering. Self-fertilization rates varied among seed orchards (1.4–4.4% on average), and there were significant positive correlations between self-fertilization rate and the number of ramets per clone in both types of seed orchard. Contributions as pollen donors differed significantly among clones in all seed orchards. The distance between planted ramets, flowering phenology, and relative pollen fecundity may also have contributed to observed differences in paternal contribution. The influence of these factors on genetic potential did not differ greatly between the two types of orchards. This work was supported by grants from the Pioneer Special Studies Program of the Japanese Ministry of Agriculture, Fishery, and Forestry; the Program for Promotion of Basic Research Activities for Innovative Biosciences; and research fellowships from the Japan Society for the Promotion of Science for Young Scientists.     

林木种子园花粉污染与防治措施

林木种子园花粉污染是影响种子园种子遗传品质的重要因子,由于野生同种花粉进入种子园而增加遗传损失。本文综述了国内外林木种子园花粉污染研究的概况、污染水平、影响污染水平的因子和减少污染的措施。研究表明：DNA分子标记在分析种子园交配动态和种子园管理效果方面非常有效（例如,cpDNA、RAPD和SSR）;种子园污染水平比预期的更高;花粉产量好像对污染水平没有影响;空间和生殖隔离也没有产生预期的效果。目前,人工辅助授粉和疏伐是减少花粉污染的两种有效措施。为了改善种子园林木的亲本平衡,人工处理技术的使用,例如：赤霉素A4/7处理技术和无性繁殖技术在种子园很受欢迎。污染程度其他方面的研究工作仍在继续,本文提到的基因流的研究。并对我国种子园花粉污染研究和花粉管理提出建议：当分析种子园花粉污染时,涉及到花粉基因型自然的（花粉扩散）、生理（开花时间）和花粉生活力等因素都应该被考虑。     

Considerations of correlated fertility between genders on genetic diversity: the Pinus densiflora seed orchard as a model

The correlation between 99 clone female and male fertilities in a first generation seed orchard of Pinus densiflora was studied over 6 years. The effective number of the parent (N_p) and the variance effective population number [N_e^(v)] were used to assess the impact of total (O_T), female (N_f) and male (N_m) fertility variation. A theoretical framework was developed to account for female and male fertility correlations as well as the impact of possible pollen contamination. Total fertility variation was described by the sibling coefficient (O_T: the probability that two genes randomly chosen from the gamete gene pool originate from the same parent), which was further subdivided into N_f and N_m. These parameters were compared under various conditions including the total seed harvest, imposing on equal seed harvest among the orchard's clones and two contamination scenarios (M = 0 and 20%). Fertility variations among females, males and clones were observed within and among years. Sibling coefficients (O_T) were lower, but the effective number of parent (N_p) and variance effective population number (N_e^(v)) were higher in years with moderate female and good male strobilus production. N_p for female and male reproductive outputs varied from 49 to 82 and from 57 to 93, respectively. N_p was higher for males than females. When the crop of the 6 years was pooled, N_p for female, male and the clone were 73, 87 and 85, respectively. The impact of female-male fertility correlation for conditions with no-, positive- and negative-correlations were assessed and their impact on O_T, N_p and N_e^(v) was also evaluated. It was demonstrated that the practice of equal seed harvesting from every clone, or the mixing of seeds from several years, would substantially improve the genetic diversity and the genetic representation of the seed orchard population when a positive correlation between gender fertilities was observed. The relevance of these results to supplemental-mass-pollination was discussed under two cases where equal- and un-equal amounts of pollen from clones were included in the pollen mixes.     

Effective number of pollen parents in clonal seed orchards

Summary A method for quantifying mating behavior in clonal seed orchards of forest tree species is presented. It involves the estimation of effective numbers of pollen parents from seed samples collected from individual ramets in such orchards. These effective numbers are variance effective numbers for populations of male gametes that are successful in uniting with ovules to produce viable seed. Three such effective numbers are defined for clonal seed orchards:N _p (a) for male gamete populations for ramets within clones,N _p (b) for male gamete populations for clones, andN _p (c) for male gamete populations for entire orchards. Estimators for these effective numbers and for standardized variances of allele frequencies in the male gametic populations are presented. Expressions are also given for the confidence intervals for each of the three effective numbers. Estimates of these parameters and the corresponding confidence intervals for two seed orchards are presented and interpreted.     

Simultaneous estimation of pollen contamination and pollen fertilities of individual trees in conifer seed orchards using multilocus genetic data

Seed orchards of forest trees are established to produce genetically-improved seed for reforestation. Genetic efficiency requires seed orchards to be (1) reproductively isolated form surrounding trees, (2) that there be similar fertilities among all orchard trees, and (3) minimum inbreeding. Each aspect of seed orchard reproduction can be simultaneously estimated using the observed frequency of each multilocus gametic type contributed through fertilizing pollen and the expected multilocus gametic segregation frequencies of orchard tree and of the contaminating population. These genetic estimates are directly relevant to the genetics of the tree breeding program. The flexibility of sampling seed — the basic data for these techniques — allows great scope for hypothesis testing, including tests of the accuracy of predictions of biophysical models of pollen movement. A simple example and a white spruce seed orchard case study are presented to illustrate the estimation technique and to investigate its sensitivity.     

VARIATION IN MALE REPRODUCTIVE INVESTMENT AND MALE REPRODUCTIVE SUCCESS IN WHITE SPRUCE

An experimental population composed of ramets of white spruce clones was studied to determine the influence of different levels of clonal male reproductive investment on clonal male reproductive success, where the male reproductive success of different clones in the population was defined as the proportion of the seed crop sired by each clone. We present a multilocus estimation procedure which provides unbiased estimates of the proportion of seed sired by each parental genotypic class from pollen gamete frequency data, whenever gametic segregation frequencies are known. Significant linear and quadratic components were found for the relationship between the number of male cones produced and the estimated proportion of the seed crop sired. A clone that produced an exceptionally large number of male cones, however, did not have the greatest male reproductive success. In general, the results support an assumption made in theoretical models of selection on mating system modifiers regarding the relationship between male investment and male reproductive success. They also support the hypothesis that male reproductive success in wind-pollinated species continues to increase substantially with increasing male reproductive investment.     

Performance of microsatellites for parentage assignment following mass controlled pollination in a clonal seed orchard of loblolly pine (Pinus taeda L.)

Mass controlled pollination (MCP), involving large-scale application of pollen on physically isolated female reproductive organs, has been a lower cost alternative to controlled pollination for the commercial production of genetically improved seeds. Nevertheless, rare are the studies that examined the efficacy of operational MCP and no such assessment has been done in loblolly pine to date. The success of MCP was assessed by a microsatellite-based investigation of the realized versus expected parentage of a set of 300 Pinus taeda offspring in 19 families generated in two subsequent rounds of MCP in 2005 and 2006 in a clonal seed orchard in Brazil. Multi-locus combined probability of parentage exclusion both theoretical and realized from actual testing was >99 % for single parent and parent pair testing when using nine or ten markers. Parentage assignments carried out under a maximum likelihood framework revealed a significantly higher success rate of MCP in 2006 (84 %) following technical improvements adopted to minimize pollen contamination and maximize male reproductive success, although significant variability in the correct maternity and full parentage was seen among individual families. The observed patterns of unexpected parentage indicated that this variability likely resulted from mislabeling of clonal ramets of the parents used in the crosses which impacted both maternity and paternity. Preventing pollen contamination will not be sufficient for successful MCP if inaccuracies exist in the identity of the clonal plants that ultimately provide pollen and female strobili, showing that DNA marker auditing and correction of identity of all ramets in a clonal seed orchard should be a standard practice in the operational implementation of MCP.     

Differential male reproductive success in Douglas fir

Summary Differential male reproductive success was studied in clones at two seed orchards of Douglas fir, Pseudotsuga menziesii (Mirb.) Franco. The performance of tester pollen parents was compared in controlled pollinations with two-parent pollen mixes. Marker pollen homozygous for a rare IDH allele was the genetic marker in each pollen mix. The resulting seeds were analyzed electrophoretically. At both seed orchards, the proportion of seeds sired by tester pollen significantly varied among the tester pollen parents. Tester pollen parents did not perform the same across all seed parents. The significant interaction effect was evidence of male-female complementarity. These results suggest a genetic basis to differential male reproductive success in Douglas fir.     

Randomized,replicated, staggered clonal-row (R2SCR) seed orchard design

Spatial randomization of clones across a seed orchard’s grid is commonly applied to promote cross-fertilization and minimize selfing. The high selection differential attained from advanced-generation breeding programs sets high premier on the genetic gain and diversity delivery from seed orchards, thus clonal allocation is important and even more challenging when clones share common ancestry. Evidences of low selfing in many conifers’ seed orchards, as a result of their high genetic load, inbreeding depression, and polyembryony are abundant and call for orchards’ design re-evaluation, specifically when randomization is associated with added managerial burden. Clonal-rows represent a viable option for simplifying orchards management; however, they are often associated with elevated correlated matings between adjacent clones. Here, we propose a modified clonal-row design that replicates, staggers, and randomizes the rows, thus doubling the number of adjacent clones and providing different set of neighboring clones at each replication, thus allowing accommodating related parents more readily than any single-tree arrangement. We present a novel algorithm packaged in user-friendly software for executing various seed orchards’ designs. The developed program is interactive and suitable for any orchard size and configuration, accommodates any number of clones that are allocated to rows with variable length (ranging from a single tree to any even number) and pre-set separation zone between ramets of the same clone. The program offers three deployment modes (equal, linear, and custom) each with multiple layouts determined by the number of iterations requested. The resulting layouts are ranked based on four criteria including: (1) the number of empty positions, (2) deviation between expected and observed clone size, (3) minimum inbreeding, and (4) a neighborhood index that expresses the efficiency of clonal distribution.     

Gene flow estimation with microsatellites in a Malagasy seed orchard of Eucalyptus grandis

Eucalyptus grandis has a mixed-mating reproductive system. Malagasy Eucalyptus seed orchards were established 15 years ago with two aims both based on panmixia: open-pollinated seed production and genetic improvement. The panmixia hypothesis has never been confirmed in the seed orchard. From a seedling seed-orchard stand comprising 349 trees and using data obtained with six selected microsatellite markers, paternity analysis was performed for 724 offspring collected on 30 adult trees. Paternity assignment, based on exclusion procedures and likelihood-ratio method, was achieved with high accuracy; the exclusion probability value was 0.997. The outcrossing rate was very high (96.7%). More than 50% of potential male trees (199 out of 349) in the seed orchard contributed to pollination for 440 offspring of 30 progenies (8.6% of the basic population). The pollination rate from outside the seed orchard was high (39.2%), but might be due to the small size of this seed orchard. This study showed that "panmixia-like pollination" can be assumed.     

Optimum neighborhood seed orchard design

Original seed orchard algorithm “Optimum Neighborhood Seed Orchard Design” was developed as local heuristics to facilitate the establishment of advanced-generation seed orchards with complex configurations. The scheme leads to uniform spatial distribution among adjacent genetic entries, promoting panmixia. The resulting scheme is thus suitable to establishing both productive seed orchards as well as clonal archives of threatened species, etc. Further, it can accommodate variable clonal sizes, uneven grids, relatedness, assortative mating. It may supplement existing layouts (clonal rows, etc.). Layouts outperformed existing advanced-generation alternative schemes in most parameters and thus could be recommended to operational forestry and gene conservation management.     

马尾松二代无性系种子园子代父本分析及花粉散布

目前国内较早建立的马尾松(Pinus massoniana)二代种子园正陆续进入正常开花结实期。研究马尾松二代种子园花粉散布和自由授粉子代的父本组成, 可为生产上指导马尾松高世代种子园的规划设计和遗传管理提供理论依据。该文利用筛选的11对SSR引物, 对马尾松二代无性系种子园内8个无性系单株的320个自由授粉子代和48个候选父本进行了扩增, 并采用最大似然法对子代进行父本分析。结果表明: 11个位点共检测到61个等位基因, 每个位点的等位基因数在2-11之间, 平均为5.55个。试验亲本和子代群体的总平均观测杂合度(H_o)、期望杂合度(H_e)及多态信息含量(PIC)分别为0.428、0.433和0.387。在80%的可信度水平下可为232 (72.50%)个子代确定其父本。平均每个采种母树与19个父本产生子代。在自由授粉状态下, 马尾松二代种子园自交率为1.72%, 自交现象很弱, 其交配方式以异交为主。绝大多数亲本无性系的雄性繁殖适合度在1.00%-4.00%之间, 候选父本平均繁殖适合度为2.17%, 平均形成5个后代。马尾松有效花粉的散布距离和固定交配距离的父本繁殖适合度均符合正态分布, 两者呈极显著负相关, 其主要散布距离集中在0-100 m, 而检测到的最大散布距离为192 m。种子园花粉污染率较低, 仅为4.06%。总体看来, 树冠南面子代亲本交配距离较北面有增加的趋势, 但树冠南、北面子代父本组成数并未表现明显的规律。     

利用等位酶对油松无性系种子进行父本分析

利用水平淀粉凝胶电泳8种酶10个位点34个等位基因,对辽宁兴城油松无性系种子园中17个无性系20个单株的200粒种子作了父本分析。无性系雄花量的多少与授粉果率并不成正比;不同无性系对花粉的接受存在一定的选择性,分析试样单株与周围的无性系并没有构成固定的授粉组合,说明授粉期风向等气象因子,无性系的雌雄花器的构造,受精等对受粉的成败都有作用,种子园结实初期自交现象并不明显,在分析试样中只有16＃,17＃,19＃无性系存在自交现象;种子园内花偻的有效传播一般在半径30m范围内,同时,分析了种子园子代的等位酶变异和空间遗传结构,从另一侧面反映出采种母树周围花粉分布比较均匀,并说明散粉期间风向等气象因子对授粉成败影响较大。     

Masting in ponderosa pine: comparisons of pollen and seed over space and time

Many plant species exhibit variable and synchronized reproduction, or masting, but less is known of the spatial scale of synchrony, effects of climate, or differences between patterns of pollen and seed production. We monitored pollen and seed cone production for seven Pinus ponderosa populations (607 trees) separated by up to 28?km and 1,350?m in elevation in Boulder County, Colorado, USA for periods of 4?C31?years for a mean per site of 8.7?years for pollen and 12.1 for seed cone production. We also analyzed climate data and a published dataset on 21?years of seed production for an eighth population (Manitou) 100?km away. Individual trees showed high inter-annual variation in reproduction. Synchrony was high within populations, but quickly became asynchronous among populations with a combination of increasing distance and elevational difference. Inter-annual variation in temperature and precipitation had differing influences on seed production for Boulder County and Manitou. We speculate that geographically variable effects of climate on reproduction arise from environmental heterogeneity and population genetic differentiation, which in turn result in localized synchrony. Although individual pines produce pollen and seed, only one-third of the covariation within trees was shared. As compared to seed cones, pollen had lower inter-annual variation at the level of the individual tree and was more synchronous. However, pollen and seed production were similar with respect to inter-annual variation at the population level, spatial scales of synchrony and associations with climate. Our results show that strong masting can occur at a localized scale, and that reproductive patterns can differ between pollen and seed cone production in a hermaphroditic plant.     

Reproductive consequences of mate quantity versus mate diversity in a wind-pollinated plant

Since most pollen travels limited distances in wind-pollinated plants, both the local quantity and diversity of mates may limit female reproductive success. Yet little evidence exists on their relative contribution, despite the importance of viable seed production to population dynamics.To study how variation in female reproductive success is affected by the quantity versus the diversity of surrounding mates contributing pollen, we integrated pollination experiments, data on natural seed set and seed viability, and AFLP genetic marker data in the wind-pollinated dioecious clonal forest herb Mercurialis perennis.Pollination experiments indicated weak quantitative pollen limitation effects on seed set. Among-population crosses showed reduced seed viability, suggesting outbreeding depression due to genetic divergence. Pollination with pollen from a single source did not negatively affect reproductive success. These findings were consistent with results of the survey of natural female reproductive success. Seed set decreased with the distance to males in a female plants’ local neighborhood, suggesting a shortage of pollen in isolated female plants, and increased with the degree of local genetic diversity. Spatial isolation to other populations and population size did not affect seed set. None of these variables were related to seed viability.We conclude that pollen movement in M. perennis is likely very limited. Both male proximity and the local degree of genetic diversity influenced female reproductive success.     

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.     

Variation in Male Fertilities and Pairwise Mating Probabilities in Picea glauca

Frequencies of multilocus male gametes in seeds collected from clones in several blocks of a white spruce seed orchard were analyzed as part of a 2-yr study of mating system variation in this species. Observed frequencies of male gamete types departed significantly from those expected assuming equal male fertilities among clones. Male gamete frequencies in seed crops were significantly heterogeneous among clones within blocks, and among blocks within clones. Clonal male fertilities were estimated from male gamete frequency data. These estimates were highly skewed, with a small proportion of the clones contributing male gametes to the majority of the seed. The estimates were significantly heterogeneous among clones within blocks, and among blocks within clones. Between-year variation in clonal male fertilities was also pronounced, with male fertilities of some clones changing by as much as three orders of magnitude. Clonal male fertility was significantly correlated with clonal male cone production in both years. These results are important with regard to assumptions made for the estimation of general combining ability, average genetic correlation among progeny from single parents, and expected response to selection in open-pollinated plant populations.