Mating patterns,genetic composition and diversity levels in two seed orchards with few clones—Impact on planting crop

Paternity analyses using microsatellites were conducted in two conifer clonal seed orchards (CSOs). The aim was to study mating patterns and potential dysfunctions such as unbalanced male contribution, and how these factors influence genetic diversity. The effect of unequal male contribution was quantified by the status number (N_S), which reflects build up of coancestry in the seed orchard crop due to low number of clones and unequal male contribution. This approach was also used to quantify the effect of clonal differences in germination percentages on effective population sizes. Furthermore the genetic diversity of the Abies alba and Larix kaempferi CSOs was compared to other alternative seed sources via observed heterozygosity and allelic richness.     

Impact of Fertility Variation on Gene Diversity and Drift in two Clonal Seed Orchards of Teak (Tectona grandis Linn. f.)

Two 25 year old teak clonal seed orchards comprising 15 (CSO-I) and 20 clones (CSO-II), respectively, selected mostly from moist forests of Western Ghats (latitude 10° N) in southern India, were evaluated for fertility, offspring diversity, and genetic drift. The orchards differed in fertility of clones as well as flower and fruit production per ramet. Fertility was highly skewed in CSO-II, where one clone (originating from higher latitude −17° N, in Eastern Ghats of peninsular India) produced 55% of the fruits and 68% of the flowers in the orchard, in contrast to a similar contribution from four most fertile clones in CSO-I. Fertility variation, measured as `sibling coefficient' (1.7 in CSO-I and 8.3 in CSO-II), was high in CSO-II resulting in high coancestry and low effective population size (3 times lower than CSO-I) in the seed crop. In CSO-I, 58% of the clones contributed effectively to seed production compared to only 12% effective contribution resulting in eight times higher genetic drift in CSO-II. Placing limits on how much seed can be collected per clone might be useful in restricting over representation of highly reproductive clones thereby increasing genetic diversity in the seed crop.     

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

?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.

     

Pollen dispersal,mating patterns and pollen contamination in an insect-pollinated seed orchard of <Emphasis Type="Italic">Schima superba</Emphasis> Gardn. et Champ.

Pollen flow and mating patterns are the most important factors influencing the genetic structure of insect-pollinated forest trees and are essential parts of genetic management in seed orchards. We investigated pollen flow, the mating system and the level of pollen contamination in a clonal seed orchard of Schima superba Gardn. et Champ. In total, 328 open-pollination progenies coming from 11 mother trees were identified using 13 polymorphic microsatellite loci. A total of 203 full-sib families were identified and were nested among the 11 studied seed donors. The male reproductive success rate from 0.49 to 7.77% for most male parents, with an average of 2.44%. More than 80% of the crosses were found within a distance of 60 m, and the most frequent pollination distance between female parent and male parents was approximately 20 m. Mating system analysis showed that the outcrossing level was very high (t _m  = 1.000, outcrossing rate = 98.5%) in the seed orchard and that there was an average of 2.3 effective pollen donors (N _ep ) per female parent. In addition, the mating success of individual males within neighborhoods was moderately influenced by their fecundity and the direction of their location relative to mother trees. The pollen contamination from outside the seed orchard was high (7.01%). Our findings are valuable for the assessment of seed orchards, and it may be worthwhile to use pollen management strategies to decrease pollen contamination and increase the genetic quality of the seeds produced.     

Reduction of pollen contamination in Scots pine seed orchard crop by tent isolation

This study examined the differences in mating system between indoor versus outdoor Scots pine (Pinus sylvestris L.) seed orchard using DNA markers. By analyzing the paternities in seed crops from isolation tents, we found a significant reduction in contamination rate, from 23% in the outdoor open blocks to 0% inside the tents. Increase in selfing rate and a decrease in genetic diversity in the crops were also observed inside the tents. These undesirable effects can be mitigated to some extent by the supplementation of extra pollen genotypes, e.g. selfing rate decreased from 14.4% to 6.6% and the effective number of fathers increased from 6 to 11.4 in the tents without and with supplemental mass pollination, respectively. Our study showed that tent isolation may become an effective rescuer for seed orchards where pollen contamination is severe.     

Juvenile radiata pine clonal seed orchard management in Galicia (NW Spain)

A juvenile radiata pine clonal seed orchard (Pinus radiata D. Don) in Sergude (northwestern Spain), comprising 57 clones, was evaluated for fertility, offspring diversity and genetic drift during 4 years. Flower and fruit production was used to asses clone fertility in the orchard. Fertility variation measured as “sibling coefficient” was found to be an average of 1.5, having high genetic diversity (0.99) and low coancestry (0.013). The clones varied in fertility from year to year. Effective population size in the orchard was between 35 and 40, indicating that between 60.30 and 69 %, the clones contributed effectively to seed yield. One way to improve seed yield in the orchard is by increasing the effective population size. Thinning and pruning highly reproductive clones of gibberellin application might be useful in increasing effective population size. The orchard’s parental balance showed consistent improvement over time. Cone and seed characteristics were also investigated. The results showed that significant genetic variation exists among clones for cone width and length; total, sound and empty seed number; as well as 100 seed weight. The seed orchard is still in a juvenile phase; moreover, the broad-sense heritability and variation data obtained in this study could be very valuable in the management practices of the existing radiata pine clonal seed orchard and can be also used for better planning of advanced generation of clonal seed orchards in Spain.     

Estimates of pollen contamination and selfing in a coastal Douglas-fir seed orchard

Seed orchards are the link from tree breeding to reforestation programs and are theoretically expected to function as closed, perfect populations, ensuring gain and diversity are consistently and predictively delivered as improved seed and seedlings. Seed orchard populations often deviate from panmixia due to fertility variation, reproductive asynchrony, and gene flow, leading to reduced seed crops genetic quality. Here, as a part of multiyear monitoring study, we used DNA fingerprinting (simple sequence repeat markers) to assess a Douglas-fir (Pseudotsuga menziesii) seed orchard's seed crop genetic quality (2009 seed crop). The studied seed crop was produced under ambient temperature (i.e. no reproductive phenology manipulation) and pollination was augmented by pollen from within orchard's pollen donors. DNA fingerprinting of the parental population (66 parents) along with 207 gametophyte (1n) – embryo (2n) pairs of random bulk sample of seed allowed parentage (maternal and paternal) assignment and the direct assessment of pollen contamination (0.18 ± 0.027) and selfing (0.17 ± 0.025) rates as well as parental (pollen, ovule and individual parent) gametic contribution was compared to a previous year's crop (2005). The extended reproductive phenology coupled with variable within-orchard pollen availability has created opportunities for both self and foreign pollen to be successful at various times resulting in the seemingly paradoxical scenario of high selfing and gene flow. These results (2005 and 2009) allowed comparison of seed orchard's crop management practices and are expected to provide scientific foundations to effective seed crops genetic quality improvement.     

Magnitude and efficiency of genetic diversity captured from seed stands of <Emphasis Type="Italic">Pinus nigra</Emphasis> (Arnold) subsp. <Emphasis Type="Italic">pallasiana</Emphasis> in established seed orchards and plantations

To test how efficiently plantations and seed orchards captured genetic diversity from natural Anatolian black pine (Pinus nigra Arnold subspecies pallasiana Holmboe) seed stands, seed sources were chosen from 3 different categories (seed stands (SS), seed orchards (SO) and plantations (P)) comprising 4 different breeding zones of the species in Turkey. Twenty-five trees (mother trees) were selected from each SS, SO and P seed sources and were screened with 11 Random Amplified Polymorphic DNA (RAPD) markers. Estimated genetic diversity parameters were found to be generally high in all Anatolian black pine seed sources and the majority of genetic diversity is contained within seed sources (94%). No significant difference in genetic diversity parameters (numbers of effective alleles, % of polymorphic loci and heterozygosity) among seed source categories was found, except for a slight increase in observed heterozygosities in seed orchards. For all seed source categories, observed heterozygosity values were higher (H_o = 0.49 for SS, 0.55 for SO and 0.49 for P) than expected ones (H_e = 0.40 for SS, 0.39 for SO and 0.38 for P) indicating the excess of heterozygotes. In general, genetic diversity in seed stands has been transferred successfully into seed orchards and plantations. However, the use of seeds from seed orchards can increase the amount of genetic diversity in plantations further. The study also demonstrated that number of plus-tree clones (25–38) used in the establishment of seed orchards was adequate to capture the high level of diversity from natural stands.     

Some Genetic Aspects of Human Intervention in Forest Regeneration: Considerations Based on Examples from an Experiment in Northern Sweden

Some genetic consequences of forest regeneration methods arediscussed based on results from an 11-year-old experiment withScots pine in northern Sweden. The experiment comprises twoenvironments (seed-trees and clear-felling), two regenerationmethods (planting and sowing) and four seed sources (local seedsfrom the seed trees, northern stand seeds and two seed orchardcrops). Seed source was important compared with environmentand regeneration method for the occurrence of empty cultivationquadrats ( 2 x 2 m). In relative terms it was less importantfor growth. There was no indication that progenies of the localtrees were best suited for the locality. There was no evidentpositive effect of the higher selection caused by higher mortalityand release thinning in some entries compared with others. Hence,a higher selection after sowing than after planting could notbe shown to have positive effects. The variance between halfsib families was a small share of the total variance, and thusthe number of parents is probably unimportant for variance oftree height or seedling survival in a forest. Progenies fromthe seed orchards grew fast, but the mortality was higher thanfor the stand progenies. There was no significant genotype xenvironment interaction. There was a significant genotype xmethod interaction; seed orchard progeny were better for sowingthan for planting.     

Low rates of pollen contamination in a Scots pine seed orchard in Sweden: the exception or the norm?

We investigated mating structure and gene flow in a clonal seed orchard of Scots pine (Pinus sylvestris L.) over three consecutive pollination seasons (2010–2012) with nine nuclear microsatellite markers. The paternity of 1991 offspring from four maternal parents was assigned to 28 candidate fathers using an exclusion procedure and a likelihood-based method implemented in the program CERVUS. Relative reproductive success was highly variable among pollen parents but consistent across years and ranged from 0.1% to 18.3%. Consequently, the seed crops’ effective number of fathers was reduced to 52.9%, 48.8%, and 45.7% of the census in the three seasons, respectively. Self-fertilization fluctuated around the orchard's expected value of 5.1%, reaching 4.05%, 7.71%, and 6.61%, respectively. Pollen contamination was estimated to be 5.64%, 7.29%, and 4.89%, respectively, after correction for cryptic gene flow. CERVUS provided similar results as the exclusion method, but estimates greatly varied depending on the input parameters, mainly the proportion of fathers sampled. These results indicate the studied seed orchard is a well-functioning production population with only minor negative effects of self-fertilization and pollen contamination on the quality of seed crops. Genotyping issues associated with microsatellites as a potential source of false paternity assignment and exclusion are discussed.     

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.     

Narrow genetic base in forest restoration with holm oak (Quercus ilex L.) in Sicily

In order to empirically assess the effect of actual seed sampling strategy on genetic diversity of holm oak (Quercus ilex) forestations in Sicily, we have analysed the genetic composition of two seedling lots (nursery stock and plantation) and their known natural seed origin stand by means of six nuclear microsatellite loci. Significant reduction in genetic diversity and significant difference in genetic composition of the seedling lots compared to the seed origin stand were detected. The female and the total effective number of parents were quantified by means of maternity assignment of seedlings and temporal changes in allele frequencies. Extremely low effective maternity numbers were estimated (Nf _e ≈ 2–4) and estimates accounting for both seed and pollen donors gave also low values (N _e ≈ 35–50). These values can be explained by an inappropriate forestry seed harvest strategy limited to a small number of spatially close trees.     

Genetic variation in progenies of 23 relic trees of Metasequoia glyptostroboides in their original habitat

Seeds of 23 relic Metasequoia glyptostroboides trees were randomly collected from their original, natural populations found in Lichuan City, Hubei Province, China. The seed characteristics and growth of their three-year-old progenies were investigated. Results show that the average intra-family coefficient of variation (CV) in height growth of the 23 three-year-old families was 14.53%, slightly higher than that of the inter-family CV (14.02%). Correspondingly, these two parameters for root-collar diameter growth were 15.44% and 13.15%. The 23 relic mother trees were significantly differed in their general combining abilities (GCA) and relative effect values of GCA (RGCA) of height growth and root-collar diameter growth over the three years. Both height and root-collar diameter growth had high family heritability in this seedling stage. After eliminating two inferior families, i.e., 15 and 46, 2.21% of genetic gain in height growth was obtained and 1.50% in root-collar diameter growth. It was also significantly different in crown size and number of branches among the 23 three-year-old seedling families. From a cluster analysis we concluded that eight families, i.e., 23, 28, 29, 30, 31, 33, 35 and 44 displayed superior GCA and RGCA in both height and root-collar diameter growth. Therefore, these relic mother trees displayed their potential as parents in seed orchards. With the exception of family 23, the other seven superior families were all collected at Guihua Village, Zhonglu Town, Lichuan City, which has been identified as an optimum district of M. glyptostroboides for seed collection.     

Background pollination in Pinus sylvestris seed orchards

The proportion of background pollen grains in the seed crops of two Pinus sylvestris seed orchards in Central Finland was estimated with the aid of multilocus allozyme markers. The orchards studied were more than 30 years old and in full pollen production. For the bulked seed crops of the seed orchard with southern clones the estimated average of background pollination over four years was 26%. There were statistically significant differences between years. No significant heterogeneity in the degree of background pollination between clones was found. Among single ramets there was significant heterogeneity in the estimated contamination rates, but the variation was not related to position in the orchard. For the seed orchard with northern clones the bulked seed crop was studied only for one year and the level of background pollination was found to be 33 %. These estimates are fairly high, but lower than for many other orchards. Background pollination at this level will cause losses in expected genetic gains. Part of the seeds from northern orchards will not be adapted to the intended area of use.     

Genetic Diversity and Mating System Analysis of Pinus massoniana in Second-Generation Clonal Seed Orchard

A total 61 clone parents and 320 open-pollination progenies from 8 clone individuals were identified by 12 polymorphic SSR loci.The result showed that the level of genetic diversity in progeny population was the same as in maternal population, progeny population had all alleles detected in maternal population,and there were not obvious surplus phenomenon of homozygotes in progeny population (F= 0.046).The south crown had more strobili than the north crown,however the genetic diversity of the north crown strobili was not substantially reduced.The progeny of neutral trees had the same level of genetic diversity as in the progeny of partial female trees,with the fixation index tended to be zero,which was accorded with Hardy-Weinberg equilibrium.The multilocus outcrossing rate in the seed orchard was 1.097,and there was no significant inbreeding between parents(t_m-t_s= - 0.031).The multilocus outcrossing rate and singlelocus outcrossing rate in the south crown were higher than that in the north crown.The multilocus outcrossing rate of partial female trees was the same as in neutral trees,and the neutral tree had no obvious declining outcrossing rate with reduced proportion of female and male strobilus compared with partial female tree, with the fixation index tended to be zero.The progeny of the second generation clonal seed orchard still had rich genetic diversity,gene exchange among clones was relatively sufficient,and biparental inbreeding was not significant.     

马尾松二代无性系种子园遗传多样性和交配系统分析

运用12对SSR引物,对马尾松二代无性系种子园内61个亲本及其中8个无性系单株的320个子代进行研究。结果显示:子代群体包含亲本群体的所有等位基因,子代与亲本具有同样高的遗传多样性,子代群体的F为0.045,纯合子过剩的现象不明显;树冠北面子代遗传多样性并未因其雌、雄球花量较树冠南面减少而有明显的降低;雌雄均衡和偏雌型植株子代遗传多样性基本一致,以偏雌型植株子代略大,二者F趋于0,子代基本符合哈温平衡。种子园异交率较高,多位点异交率为1.098,子代亲本的近交现象不显著(tm-ts=-0.033);树冠南面多位点与单位点异交率均高于树冠北面;偏雌与雌雄均衡型植株的多位点异交率基本相当,雌雄均衡型植株并未因其雌、雄球花量比例较偏雌型植株减小而呈现异交率明显降低的现象,2种类型植株的近交指数均接近于0。整体而言,马尾松二代种子园子代仍具有丰富的遗传多样性,无性系间基因交流相对充分,子代亲本近交现象不明显。     

Gene flow pattern and mating system in a small population of Quercus semiserrata Roxb. (Fagaceae)

Pollen flow from external sources is important for the conservation of tree species in fragmented forests or small populations, because it can be sufficient to prevent differentiation among them, and appears to be able to prevent the loss of their genetic diversity through genetic drift. In this study, we examined the genetic heterogeneity of pollen pools accepted by each Quercus semiserrata seed parent at the Khun Wang Royal Agriculture Research Center, Thailand, both within and among two mast fruiting years (2005 and 2007), using paternity analysis and analysis of molecular variance (AMOVA). The mating systems of the trees were also examined using the multilocus mating system model (MLTR), after determining the genotypes at eight microsatellite loci of 26 seed-trees and 435 seeds from 8 seed-trees in the 2 mast fruiting years. The average distance of effective pollen flow within the plot was estimated to be 52.4 m, and 95% of effective pollen was dispersed within 200 m, indicating that effective pollen flow is highly localized and that most effective pollen is contributed by near-neighbor trees. The proportion of effective pollen that immigrated from external sources was estimated to be 26.2%. The AMOVA analysis based on the pollen haplotypes showed that the pollen pools, both total and for each reproductive year, significantly genetically differed among the seed parents. Using a mixed mating model, the estimate of biparental inbreeding for the total population (t_m − t_s) was 0.013, indicating that a low proportion of mating occurred among close relatives. The effective number of pollen donors (N_ep) was estimated to be 9.987 using the TwoGener model, or 10.989 using the mixed mating model. The effective number of pollen donors of seeds was higher in the mast fruiting year 2005 than in the other examined year, 2007. Consequently the allelic richness and genetic diversity of seeds produced in 2005 were higher than those produced in 2007. Overall, the results show that high outcrossing rates, high levels of gene flow from other populations and heterogeneity in the pollen received by an individual may enhance the ability of populations to maintain effective population sizes. Therefore, these processes may be sufficient to prevent loss of genetic diversity through genetic drift of Q. semiserrata at this study site.     

Genetic diversity analysis in a seed orchard of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis>

India is the largest grower of Eucalyptus with an area of 3.943 million hectares under plantations and E. tereticornis is the predominant species in the plains of southern India with an average productivity of 12–25 m³ ha⁻¹ year⁻¹. With the aim to establish seedling seed orchards of the species, seed lots of fifteen provenances were imported from Australia and a trial was laid. In the present study the genetic diversity existing in the seed orchard was estimated using ISSR–PCR. Seven ISSR primers amplified 663 amplicons in the size ranging from 255 to 2,711 bp. The total number of polymorphic bands varied from 59 to 123 with 100% polymorphic banding profiles. The average gene diversity (Hj) of all the provenances ranged from 0.0589 to 0.1109 and the total gene diversity estimated was low (H _T = 0.130) when compared to the earlier reports from other eucalypts species. Analysis of Molecular Variance partitioned the ISSR variation into inter and intra population components. The inter population component accounted for 55.2% of the variation and the intra population component accounted for 46.3% (P < 0.001). A neighbour-joining analysis was done using the dissimilarity matrix to determine the aggregation of the individuals into clusters. Existence of population structure among the populations was revealed in STRUCTURE analysis but geographical region based clustering was not observed. The assessment of intra and inter genetic variation documented in the present study suggests that, along with the phenotypic traits, knowledge about genetic diversity measured at the DNA level in individuals of seed orchards provide an objective guide for selective culling of trees for maintaining optimal diversity for enhanced genetic gains.     

Are Phenological Observations Sufficient to Estimate the Quality of Seed Crops from a <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> Open-pollinated Seed Orchard? Consequences for Seed Collections

Reproductive phenology was observed over three years in a Eucalyptus grandis seed orchard in Madagascar to determine the impact of geographic differences of parental selections on the expected genetic composition of seed crops. Pollen flow (observed pollen cloud) was deduced from a paternity assignment based on sampling from one year’s seed production. The two approaches were used to verify whether knowledge of phenology is sufficient to predict the genetic quality of the seeds collected. Despite the high flowering level, with a constant cycle over three years, the results demonstrated wide reproductive phenological differences associated with the parents’ origin, suggesting putative pollination disequilibrium. From both observed and expected pollen clouds, the results showed preferential mating among different provenances, which has consequences for seed crop composition. Phenological observations, which can be made inexpensively, can be used to promote methods of effective seed orchard management in order to improve the genetic quality of seeds.     

红豆树优树子代遗传多样性及与生长相关性分析

[目的]研究红豆树优树自由授粉子代遗传多样性及其对生长的影响,比较天然居群子代和孤立木子代遗传多样性差异,揭示子代遗传多样性的变化规律及天然居群在子代遗传多样性维持中的作用,为红豆树遗传保育和优异种质挖掘提供科学依据。[方法]以来自浙、闽、赣、川等26个红豆树优树自由授粉家系为研究对象,利用11对SSR引物对765个子代群体进行遗传多样性评价,同时分析子代遗传多样性参数与种子、生长性状的相关性。[结果](1)红豆树优树子代群体具有较高的遗传多样性,有效等位基因数为7.766个,观测杂合度(H_O)和期望杂合度(H_E)分别为0.469和0.865。(2)除SSR8外,其余位点的观测杂合度均小于期望杂合度,表明子代群体绝大多数位点处于杂合子缺失状态。(3)红豆树不同家系的遗传多样性存在明显差异,12号家系的遗传多样性水平最高,8号家系则最低。(4)比较发现,天然居群子代的遗传多样性显著或极显著地高于孤立木子代。(5)F统计量和分子方差分析(AMOVA)均表明,红豆树优树子代群体的遗传变异主要存在于家系内,家系间的遗传分化相对较小。(6)相关性分析发现,子代遗传多样性参数与种子性状、子代年高生长量呈显著正相关(r=0.378~0.527)。[结论]较大的红豆树天然居群在维持其子代较高遗传多样性中发挥了重要作用,子代遗传多样性显著影响苗木生长,这为红豆树遗传保育和优良家系选择提供了理论依据。