Clonal Orchard Research Articles

Clone variation of seed traits, germination and seedling growth in Dalbergia sissoo Roxb. clonal seed orchard

A clonal seed orchard (CSO) of Dalbergia sissoo Roxb. at Hoshiarpur, India consisting of 20 clones originating from different agro-climatic conditions of four northern states (Uttar Pradesh, Rajasthan, Haryana and Uttarakhand) was the source of seeds for variability studies. There was lot of variation in seed size, seed weight, germination percent, germination value and growth rate in nursery of different clones over the years. Seed length, seed width and seed weight were positively correlated to each other but seed size had no effect on germination percent and germination value under laboratory conditions. However, seed weight was found positively correlated with germination percent in nursery with the seed lot of 2008 collection. The genetic parameters for seed traits and seedling growth also showed a wide range of variations in the orchard clones. Heritability values were found to be over 50 percent for seed weight and seed length. However, only seed weight showed high heritability value coupled with more genetic gain across the years, which indicate the presence of good amount of heritable additive component in seed weight. There was no consistency in the seed characters, germination and seedling growth parameters studied across the two years. Effect of clones was dominant and accounted for variation in seed size, seed weight, seed germination and growth parameters. Seed size or seed weight should not be used as criteria for grading of bulked seed lots of different clones, as it can narrow down genetic diversity by rejecting small seeds. The impact of these genetic differences in handling of seed lots during bulking and grading for mass propagation of nursery planting stock of D. sissoo is also discussed.

Genes expressed in Taiwania reproductive organs include two male-cone protein 1 precursor homologs

Taiwania (Taiwania cryptomerioides Hayata), a conifer native to Taiwan, is an economically important tree species due to its high resistance to insect infection and high wood utilization properties; however, when it was grown in clonal orchards, no filled seeds or fertile pollen were obtained. To address this reproductive dilemma, a suppressive subtractive hybridization (SSH) library was constructed from male cones, female cones and leaves. Reverse transcription-polymerase chain reaction (RT-PCR) analysis confirmed that two bark storage proteins, a beta-phellandrene synthase, an endochitinase A2 precursor and two male-cone protein 1 precursors were specifically expressed in the reproductive organs of Taiwania. Comparisons of these two male-cone protein 1 precursors with those of Pinus radiata suggest that these genes may have different functions during different stages of male cone development and that different specific genes may be obtained from male cones collected at different stages of development.

Finalizing the selection of parents for the Sitka spruce (Picea sitchensis (Bong.) Carr) breeding population in Britain using Mixed Model Analysis

The Best Linear Unbiased Predictor method was used to derive breeding values of over 1000 Sitka spruce (Picea sitchensis (Bong.) Carr) plus trees based on data from 69 half-sibling progeny tests. Roughly half the progeny was derived from open pollination of the tree standing in the forest. The balance followed controlled pollination of female flowers on grafted copies of plus trees growing in clonal achives with a polymix (PO) of selected pollens. It was necessary to adjust for the selected nature of the PO pollens during analysis. The breeding objective was to maximize gain for stem diameter and stem form while avoiding a fall in wood density. The study investigated numerous economic weightings attached to the three selection traits and used a multi-trait selection index method to rank parents. Sub-populations of the best 120 parents required to complete the breeding population, and the best 40 parents required for new tested clonal orchards were reselected and tested against the breeding objective. The strong inverse relationship between stem diameter and wood density remains the main challenge to meeting the breeding objective. The study demonstrates, however, that provided sufficient plus trees are initially selected, it is possible to meet the breeding objective despite this difficult inter-trait relationship.

Genetic identification of clones and the genetic structure of seed crops in a Pinus brutia seed orchard

Megagametophyte tissue in seeds from a Pinus bruia Ten. seed orchard was analyzed for 9 enzyme systems (aconitase, alcohol dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate-transaminase, malate dehydrogenase, menadione reductase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, and shikimate dehydrogenase) encoded by 14 loci using starch gel electrophoresis. Twenty-eight clones and 3 ramets per clone were sampled from the orchard for analysis. Isozyme patterns of the clones clearly indicate that each clone had a different genotype when overall loci were taken into consideration. In 5 of the 28 clones, 1 of the 3 ramets did not match the other 2 ramets at 2 or more loci. This indicates that about 6% of the studied ramets in the seed orchard were mislabeled during and/or before being planted in the orchard. The Adh2-1 allele was unique to clone no.14; therefore, such an allele could be used to determine the extent to which a specific clone contributes to the seed crop. Of the 14 loci studied, 7 (50%) were polymorphic (0.95 criterion). Overall, 28 alleles were observed in the orchard trees and seed crops. Three of the 28 alleles (allele 2 at Aco, allele 2 at Got1, and allele 3 at Pgi2) were not observed in the orchard clones, but were observed only in the orchard seed crops, which indicates that these alleles originated from outside pollen sources. Important allelic differences were noted between the gene pool of the orchard clones and the gene pool of the orchard seed crops, most probably due to the presence of these alien alleles in the orchard crops. We also observed that there was 14.3%, 4.2%, and 13.5% deficiency of heterozygotes at Mnr2, Pgd3, and Sdh1 loci, respectively, in the orchard seed crops. Deficiency of heterozygosity could be the result of the Wahlund effect, positive assortative mating, and/or selection for homozygotes and mating among relatives, either alone or in combination.

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.

Mating patterns in an indoor miniature Cryptomeria japonica seed orchard as revealed by microsatellite markers

To evaluate the practical application of an indoor seed orchard, we compared the seed production, final germination rates and mating patterns of potted Cryptomeria japonica D. Don clones in two miniature seed orchards: one in a glasshouse and the other outdoors. There was no statistically significant difference in total seed production between the orchards (P = 0.275, ANOVA). However, the final germination rate of seeds produced in the indoor orchard was significantly lower than that of seeds produced in the outdoor orchard (P < 0.01, Wilcoxon test). The average self-fertilization rate was higher in the indoor orchard (27.2%) than in the outdoor orchard (5.6%), and this parameter was strongly associated with the reduced final germination rates of the seeds harvested in the glasshouse. Pollen contamination was considerably lower in the indoor seed orchard (48.9% in the outdoor seed orchard and 4.4% in the indoor seed orchard). χ2 tests indicated that the paternal contributions of each constituent clone differed significantly in both orchards (P < 0.001 in both cases). The strong variations in paternal contributions among indoor orchard clones was significantly correlated with their respective pollen production capacities (Spearman ρ = 0.693, P < 0.001). To reduce the high self-fertilization rates, we recommend the use of orchard clones with similar pollen production capacity in indoor seed orchards.

The development of population structure of Cryphonectria parasitica on European chestnut (Castanea sativa Mill.) in the Experimental Castanetarium Horné Lefantovce, observed over a 12-year study period

: We studied occurrence of chestnut blight disease in the Experimental Castanetarium Horné Lefantovce, SW Slovakia. The study ran in years 2006–2007 on a set consisting of 889 chestnut trees growing in the clonal orchard on Biological Plot 105. From this number, 857 trees were found healthy without disease symptoms. The chestnut blight disease was identified on 32 of them. The infected trees were examined for presence of pycnidia and perithecia of the causal agent. In all positive cases, the observed morphological characters indicated virulency of the obtained isolates. No hypovirulent isolate was detected on the evaluated experimental plot. In total, six vegetative compatibility (vc) types were specified in the sample consisting of 31 isolates. Our vc types corresponded to the European vc types EU 2, EU 12, EU 13, EU 14, EU 17, EU 19. Two vc types – EU 2 and EU 19 – were dominant. Vc type EU 19 accounted 35.5% and EU 2 32.2% of isolates. The vc type EU 19, which was the most frequent one in the evaluated site, was detected in Slovakia for the first time.

The importance of being the first pollen in the strobili of Scots pine

Timing of pollen arrival may affect the level of seeds fertilized by pollen from outside seed orchards, especially in seed orchards of Scots pine ( Pinus sylvestris L.) established in southern Finland with stock from northern populations. We performed sequential pollination experiments with Scots pine pollen collected from seed orchard clones originating from southern and northern Finland and recipient strobili in clones originating from southern Finland. When southern pollen was introduced first, seeds were sired equally by northern and southern genotypes. When northern pollen was introduced first, northern genotypes were found in 75% of the resulting seeds. When both pollen types were introduced at the same time, the resulting seed crop was split almost equally between northern and southern genotypes. These results do not unconditionally support the idea that the first pollen grain in the pollen chamber always fertilizes the ovum; instead, they suggest a more complex way of competition between pollen grains.

Variation in Clone Fertility and its Effect on the Gene Diversity of Seeds From a Seed Orchard of Chamaecyparis obtusa in Korea

Abstract Male and female strobilus production was assessed annually over a four-year period for a clonal seed orchard of hinoki (Chamaecyparis obtusa Endl.) in Korea. Clonal fertility and fertility variation, expressed by both sibling coefficient and coefficient of variation in strobilus production among 50 orchard clones, were reported. Fertility varied among clones and among years producing four-year averages per ramet of 510.2 and 1050.6 for female and male strobili, respectively. The correlation between female and male strobilus production was positive in each of the four years studied and, with the exception of one year, statistically significant. The clonal status number (Ns), a measure of gene diversity, was calculated based on the observed clonal fertility variation and varied from 28.0 (N = 50) in the poorest flowering year (2002) to 46.7 in the best year (2005). On average (pooled), the relative status number was 95% of the census number (N). Variation in female fertility was generally higher than that for male fertility, and this variation was reflected in the status numbers of female and male parents. The pooled Ns estimated from all four years was higher than that for any single year, implying that gene diversity would increase when seeds collected from different years are pooled. Sexual asymmetry calculations showed that clonal contributions would be balanced between genders.

Deterministic Simulation of Gains for Seedling and Cloned Main and Elite Breeding Populations of Pinus radiata and Implications for Strategy

Abstract Genetic gains in breast-height diameter were estimated using deterministic simulation. Simulations of gain from one generation of selection were undertaken in large Main and small Elite breeding populations for a range of heritabilities, with varying numbers of parents, families, seedlings/family, clones/family and ramets/ clone to aid revision of the New Zealand Pinus radiata breeding strategy. Cloned versus seedling populations of equal numbers of plants were simulated, derived from open pollination, polycrossing, and pair crossing. Balanced within-family selection was used for 200, 400 and 800-parent Main breeding populations and among- and within-family selection for 25-parent Elite populations of 25 up to 100 full-sib families. Predicted gains from within-family selection in the Main population were highest from cloned polycross families at all heritabilities and lowest for seedling fullsib families. Gains from cloned populations were higher than seedling equivalents at heritabilities ≤0.5, and their advantage in gain was greatest at lower heritabilities. Elite populations of 25 parents showed similar trends but intensive among- and within-family selection resulted in much higher gains than from the Main, highest from the cloned options. The increase in gain with increased number of families diminished with more than 2-3 times as many families as parents. A new strategy was proposed for P. radiata, based on the simulation results, involving an expanded Main breeding population of open-pollinated (OP) seedling families, together with pair-cross family seedlots already available, supported by parentage reconstruction using DNA markers. Forwards selection in small cloned Elite populations was proposed as the main source of seed orchard clones.

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.

Selection of Mediterranean stone pine clones for cone production

Though stone pine is currently a genuine forest tree of Mediterranean ecosystems, its use as grafted orchards crop for its edible pine nuts is potentially feasible. Therefore, genetic improvement of the species has been undertaken by several Spanish forest administrations, establishing in the Nineties experimental clonal orchards by grafting scions from plus trees, selected for their outstanding cone yield in pine forests of the main Spanish provenance regions. This paper analyses the coming into production of four clonal trials located at the National Forest Breeding Centre «Puerta de Hierro», Madrid. The four different-aged orchards showed a strong, synchronized masting habit. Estimations are given for the degree of genetic determination of cone production and for clonal improvement values. Due to the presence of strong size-yield correlations and spatial autocorrelations, genetic parameters were estimated by an individual tree cone-yield model adjusted for environmental tree size differences and for correlations between nearest neighbours (NN). The degree of genetic determination H2, estimated in less than 20% without adjustments, reached 30-38% adjusting for environmental size variation and 45-73% after spatial adjustment, with parallel increments in clonal improvement values and expected genetic gain. The results allow rogueing the four clone banks, progressing in the domestication of the stone pine as nut crop.

Major chemotypes and antioxidative activity of the leaf essential oils of Cinnamomum osmophloeum Kaneh. from a clonal orchard

Essential oils of 92 cutting clones from a clonal orchard of Cinnamomum osmophloeum Kaneh. were obtained by hydrodistillation and characterised by gas chromatography–mass spectrometry. Our results showed that the yields of essential oils ranged between 0.09% and 2.65% (vol/fresh wt). The constituents of essential oils varied among samples. The major chemotypes classified in the individual cutting clones were cinnamaldehyde (50 plants, representing 50–95% of the total volatiles), linalool (1 plant, 73.3%), β-cubebene (2 plants, 59.4% and 78.7%), and cinnamyl acetate (1 plant, 61.8%). The antioxidant activities of the four chemotypes were determined using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The antioxidant activities of the essential oil decreased in the order of cinnamyl acetate > cinnamaldehyde > β-cubebene > linalool. Indigenous cinnamon oil extract showed a good free radical-scavenging capacity at all concentrations studied, except at 2 μg/ml. The scavenging activity increased with increasing concentration of the extract. The capability of the four essential oil chemotypes to reduce the stable radical, DPPH, to DPPH-H was assayed by a decrease in the IC 50 values of 10.4 (cinnamyl acetate type) to 29.7 (linalool type) μg/ml. These results suggest that the leaf essential oil of C. osmophloeum possesses chemical compounds with antioxidant activity which can be used as natural preservatives in food and/or by the pharmaceutical industry. Trees in this plantation which can be used for further propagation for the production of chemotypes of interest were identified.

Mating System and Pollen Contamination in a Pinus brutia Seed Orchard

Mating system parameters and pollen contamination were estimated in an 11-year-old Pinus brutia Ten. clonal seed orchard, located in Antalya, Turkey, with the aid of isozymes. Isozyme analysis was performed on both maternal (megagametophyte) and embryo tissues of seeds collected from the seed orchard and the surrounding natural stand. Fourteen loci encoding nine enzyme systems were assayed. Based on a multilocus estimator, the proportion of viable seeds originating from outcrossing (tˆ m ) in the seed orchard was found to be 0.947; the remainder (0.053) was due to selfing. We estimated that 85.7% of the orchard seed resulted from pollen contamination by trees in surrounding stands. This may be related to the young age and subsequent low pollen production of the orchard. Only 9% [i.e., 1.0−0.857 (contamination)−0.053 (selfing) = 0.090] of the orchard offspring was due to cross-pollination among clones within the orchard. Due to the high level of pollen contamination, it is estimated that genetic gain in this seed crop is less than 57% of that expected if all offspring resulted from random mating among orchard clones (i.e., no contamination).

Reproductive Success of Pollen Derived From Selected and Non-Selected Sources and its Impact on the Performance of Crops in a Nematode-Resistant Japanese Black Pine Seed Orchard

Abstract The reproductive success of pollen derived from selected and non-selected sources and its impact on the performance of orchard crops were evaluated, using five pairs of microsatellite markers, in a Japanese black pine (Pinus thunbergii Parl.) clonal seed orchard consisting of 16 nematode-resistant clones. The paternity of each open-pollinated seed was determined by comparing the genotypes of seeds from six clones (24 trees) with genotypes of the 16 orchard clones and two trees (N1, N2) representing other genotypes that had been inadvertently included in the orchard. Out of 384 seeds examined, the paternity of 316 seeds (82.3%) was assigned to the clones within the seed orchard. On average, the male reproductive success of orchard clones varied from 0.0% to 10.5%, and was significantly related to the male-flowering fecundity of each clone. It was not related to the synchrony of flowering phenology between mates. The expected proportions of seeds produced by clonal trees as a result of pollination by orchard clones, and by contaminating pollen originating from internal and external sources were estimated at 86.8%, 3.3% and 9.9%, respectively. Nematode-resistant seedlings of Japanese black pine were produced from surviving 2-yr seedlings that had previously been inoculated with pinewood nematode (Bursaphelenchus xylophilus). Without pollen contamination, the survival rate of seedlings produced by mating between resistant clones is expected to be 62.4%. However, in this orchard the figure was reduced to 57.5%, due to pollen contamination from both internal and external sources.

Lodgepole Pine and White Spruce Germination: Effects of Stratification and Simulated Aging

Abstract The effects of seed pre-treatment (stratification/prechilling) and simulated aging on germination parameters (germination capacity, speed and value and peak value) were evaluated for several seedlots originating from seed orchard clones of lodgepole pine (Pinus contorta var. latifolia DOUGL. ex LOUD.) and white spruce (Picea glauca (MOENCH) VOSS). Region of origin and stratification had little effect on white spruce, while stratification enhanced germination speed and completeness of lodgepole pine. Broad-sense heritability for germination parameters ranged from 70 to 97% (unstratified) and from 81 to 96% (stratified) for pine, and from 95 to 97% (unstratified) and from 93 to 97% (stratified) for spruce. Simulated aging (short-term storage at high temperature and relative humidity, approximating the physiological effects of long-term storage) resulted in rapid deterioration of white spruce, with very little germination after six days of aging. Lodgepole pine germination increased during the first several aging treatments relative to the control, but germination capacity decreased following twelve days of aging, and was very low after 18 days. White spruce was nondormant and responded primarily to moisture conditions, whereas pine was strongly influenced by maternal effects. These results can be incorporated for more efficacious nursery production practices for commercial reforestation seedling production as well as ex-situ gene conservation strategies.

Fertility Variation, Genetic Relatedness, and Their Impacts on Gene Diversity of Seeds From a Seed Orchard of Pinus thunbergii

Abstract Clonal differences in the number of male and female strobili were determined for five consecutive years in a clonal seed orchard of Pinus thunbergii in Korea. The effects of relatedness and clonal differences in reproductive development on gene diversity of seed (in terms of accumulated relatedness by status number) were estimated. While clonal differences were found, fertility variation was not large through all studied years. The orchard clones were divided into different regions and locations based on the geographical distribution and distance of natural stands that plus trees were selected from. Assuming that there was no relatedness among regions, locations and clones, the status number (Ns) was varied from 47.6 to 55.5 for five successive years. On average (pooling), Ns was 92% of census number (N). Assumed relatedness among regions, locations and/or clones decreased the status number. Effect of parental selection on relatedness and orchard management was also 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.

Consistency of resistance to attack by the green spruce aphid (Elatobium abietinum Walker) in different ontogenetic stages of Sitka spruce

Abstract 1 The susceptibility of different genotypes of 29‐year‐old Sitka spruce to damage by the green spruce aphid, Elatobium abietinum, was investigated in a progeny trial where aphid damage on individual trees had previously been assessed twice in an earlier stage of ontogenetic development. The progeny trial comprised 14 open‐pollinated families originating from a clonal seed orchard that had been established using mature spruce trees selected for aphid resistance.2 Previous investigations had demonstrated that resistance was inherited by the offspring, and that differences in resistance between progenies of the individual orchard clones were highly significant.3 Susceptibility to aphid attack was recorded as the percentage loss of previous year's needles. Differences in susceptibility recorded between the juvenile trees were found to persist after the trees had developed into the closed‐canopy, sexually reproducing stage. Needle loss of the families was significantly less than that of the reference population of Sitka spruce.4 Hybrids between Sitka spruce and white spruce were defoliated more heavily than pure Sitka spruce, and the difference increased with age.5 Family heritability of resistance was estimated as 0.60 compared to 0.73 when the trees were assessed in the juvenile stage. The genetic correlation based on family means between damage in the juvenile and sexually reproducing stand was high (0.83), indicating a high consistency of resistance to the aphid over years and ontogenetic stages.6 A skewed distribution of defoliation indicated that major genes are involved in the expression of resistance, and that the genetics behind resistance has a nonadditive component.

Flowering and Airborne Pollen Occurrence in a Pinus sylvestris Seed Orchard Consisting of Northern Clones

To explain the high background pollination rate detected in earlier studies, this study investigated the flowering phenology of a Scots pine seed orchard with respect to airborne pollen occurrence during 1992 and 1993. The receptive period of the female strobili and the shedding period of the male strobili were observed visually. Airborne pollen was monitored with Rotorod traps located inside and outside the seed orchard. There was no time gap in the onset of flowering between the earliest female and male strobili in 1992. Female flowering began 3 days earlier than male flowering in 1993, and the earliest strobili were probably pollinated by pollen transported long distance from more southerly areas. The almost simultaneous peak in pollen occurrence inside the seed orchard and in surrounding pine stands indicates that the seed orchard clones are not temporally isolated from sources of contamination. However, pollen concentrations inside the seed orchard were higher than those in the surrounding stands. Therefore, flowering phenology cannot sufficiently explain the observed pollen contamination rates in this seed orchard.