Preferential exclusion of hybrids in mixed pollinations between oilseed rape (Brassica napus) and weedy B. campestris (Brassicaceae)

In most experimental hybridizations between oilseed rape (Brassica napus) and weedy B. campestris, either intra- or interspecific pollen has been applied to individual flowers. Under field conditions, however, stigmas will often receive a mixture of the two types of pollen, thereby allowing for competition between male gametophytes and/or seeds within pods. To test whether competition influences the success of hybridization, pollen from the two species was mixed in different proportions and applied to stigmas of both species. The resulting seeds were scored for paternity by isozyme and randomly amplified polymorphic DNA analysis. Using data on the proportion of fully developed seeds and the proportion of these seeds that were hybrids, a statistical model was constructed to estimate the fitness of conspecific and heterospecific pollen and the survival of conspecific and heterospecific zygotes to seeds. B. campestris pollen in B. napus styles had a significantly lower fitness than the conspecific pollen, whereas no difference between pollen types was found in B. campestris styles. Hybrid zygotes survived to significantly lower proportions than conspecific zygotes in both species, with the lowest survival of hybrid zygotes in B. napus pods. This is in contrast to the higher survival of hybrid seeds in B. napus than in B. campestris pods when pollinations are made with pure pollen. Altogether, the likelihood of a foreign pollen grain producing a seed was much lower on B. napus than on B. campestris. In addition, pods on B. napus developed to a lower extent the more heterospecific pollen was in the mix, whereas this had no effect on B. campestris.     

Primary pollinator exclusion has divergent consequences for pollen dispersal and mating in different populations of a bird‐pollinated tree

Pollination by nectarivorous birds is predicted to result in different patterns of pollen dispersal and plant mating compared to pollination by insects. We tested the prediction that paternal genetic diversity, outcrossing rate and realized pollen dispersal will be reduced when the primary pollinator group is excluded from bird‐pollinated plants. Pollinator exclusion experiments in conjunction with paternity analysis of progeny were applied to Eucalyptus caesia Benth. (Myrtaceae), a predominantly honeyeater‐pollinated tree that is visited by native insects and the introduced Apis mellifera (Apidae). Microsatellite genotyping at 14 loci of all adult E. caesia at two populations (n = 580 and 315), followed by paternity analysis of 705 progeny, revealed contrasting results between populations. Honeyeater exclusion did not significantly impact pollen dispersal or plant mating at Mount Caroline. In contrast, at the Chiddarcooping site, the exclusion of honeyeaters led to lower outcrossing rates, a threefold reduction in the average number of sires per fruit, a decrease in intermediate‐distance mating and an increase in near‐neighbour mating. The results from Chiddarcooping suggest that bird pollination may increase paternal genetic diversity, potentially leading to higher fitness of progeny and favouring the evolution of this strategy. However, further experimentation involving additional trees and study sites is required to test this hypothesis. Alternatively, insects may be effective pollinators in some populations of bird‐adapted plants, but ineffective in others.     

Mating system parameters at hierarchical levels of fruits, individuals and populations in the Brazilian insect-pollinated tropical tree, Tabebuia roseo-alba (Bignoniaceae)

For many tree species, mating system analyses have indicated potential variations in the selfing rate and paternity correlation among fruits within individuals, among individuals within populations, among populations, and from one flowering event to another. In this study, we used eight microsatellite markers to investigate mating systems at two hierarchical levels (fruits within individuals and individuals within populations) for the insect pollinated Neotropical tree Tabebuia roseo-alba. We found that T. roseo-alba has a mixed mating system with predominantly outcrossed mating. The outcrossing rates at the population level were similar across two T. roseo-alba populations; however, the rates varied considerably among individuals within populations. The correlated paternity results at different hierarchical levels showed that there is a high probability of shared paternal parentage when comparing seeds within fruits and among fruits within plants and full-sibs occur in much higher proportion within fruits than among fruits. Significant levels of fixation index were found in both populations and biparental inbreeding is believed to be the main cause of the observed inbreeding. The number of pollen donors contributing to mating was low. Furthermore, open-pollinated seeds varied according to relatedness, including half-sibs, full-sibs, self-sibs and self-half-sibs. In both populations, the effective population size within a family (seed-tree and its offspring) was lower than expected for panmictic populations. Thus, seeds for ex situ conservation genetics, progeny tests and reforestation must be collected from a large number of seed-trees to guarantee an adequate effective population in the sample.     

Outcrossing between an agroforestry plantation and remnant native populations of Eucalyptus loxophleba

Gene dispersal among populations of a species is an important force influencing their genetic structure. Dispersal may also occur between taxa that would normally be isolated when nonendemic, domesticated or transgenic species are planted within the natural range of interfertile taxa. Such a mosaic of populations is typical of many agricultural landscapes, and investigations are needed to assess the risks of genetic contamination of the endemic populations but a combination of approaches may be necessary because of the limitations of research in this landscape. This study used microsatellite markers and a range of analyses (mating system, paternity exclusion, Bayesian assignment) to examine gene dispersal between remnants of the endemic Eucalyptus loxophleba ssp. supralaevis and a plantation of a nonendemic subspecies. Our results indicate that remnant populations are connected by significant dispersal to pollen sources up to 1.94 km away including the plantation. The combined analyses showed that the pollen pool and outcrossing rates of individuals within remnants varied significantly probably because of asynchronous flowering and that the likelihood of paternity was not correlated with spatial proximity. More than half of all progeny had male parents from outside their stand with the largest proportions estimated to come from the plantation by exclusion (42.4%) or Bayesian analyses (18.8–76%). Fragmentation may not be associated with decreased gene dispersal between populations of tree species, natural or planted, so that the distances required to buffer endemic trees in fragmented rural landscapes are likely to be large.     

Heritable paternal cytoplasmic organelles in alfalfa sperm cells: ultrastructural reconstruction and quantitative cytology.

Sperm cells within pollen grains and pollen tubes of alfalfa (Medicago sativa L.) were observed at the ultrastructural level, and their plastid DNA was detected by DAPI (4,6-diamidino-2-phenylindole) staining. One sperm pair within the pollen grain and three sperm pairs within pollen tubes were reconstructed in three-dimensions from serial ultrathin sections. The two sperm cells are linked by cytoplasmic bridges in both pollen grains and tubes, and the vegetative nucleus is closely associated with the sperm cells within the pollen tube. The number of plastids and plastid nucleoids (DNA aggregates) in the sperm cell pair, collectively, is not significantly different from that in the generative cell; however, over 60% of the sperm cell plastids contain no DNA detectable with DAPI. The mean number of mitochondria in sperm cells is reduced from that in the generative cell (from 54 to 17), which suggests that paternal mitochondrial inheritance probably does not occur in the genotype investigated. Sperm cells of a pair may vary in their shape within the pollen grain and tube, but the number of plastids and mitochondria is not significantly different between the sperm cells. Therefore, heterospermy is not a factor determining cytoplasmic inheritance patterns in this species.     

Real-time patterns of pollen flow in the wild-service tree, Sorbus torminalis (Rosaceae). III. Mating patterns and the ecological maternal neighborhood

Understanding the role of mother plants as pollen recipients in shaping mating patterns is essential for understanding the evolution of populations and in particular to predict the consequence of habitat fragmentation. Here, we investigated variation in mating patterns due to maternal phenotypic traits, phenological variance, and landscape features in Sorbus torminalis, a hermaphroditic, insect-pollinated and low-density, European temperate forest tree. The diversity and composition of pollen clouds received by maternal trees in S. torminalis were mainly determined by their conspecific neighborhood: isolated individuals sample more diversity through more even paternal contributions, low relatedness among paternal genes, and high rates of long-distance pollen dispersal within their progenies. Maternal phenotypic traits related to pollinator attractiveness also had an effect, but only when competition was strong: in this case, larger mother trees with more flowers sampled more diversity. The floral architecture of S. torminalis, with multiple-seeded fruit, strongly shaped mating patterns, with higher levels of correlated paternity among seeds belonging to the same fruit (30% full sibs) than among seeds belonging to different fruits (14% full sibs). Finally, flowering phenology affected the distribution of diversity among maternal pollen clouds, but the earliest and latest mother trees did not receive less diversity of pollen than the others.     

High rates of extra‐pair paternity in a socially monogamous beetle with biparental care

1. Nest construction and paternity assurance are predicted to favour biparental care in insects. The horned passalus (Odontotaenius disjunctus) is a socially monogamous beetle with biparental care that breeds in decaying logs. The genetic mating system of the horned passalus was investigated to determine if paternity assurance is likely to drive the evolution or maintenance of paternal care in this system. Parental time budgets were also examined to better understand the types and frequencies of behaviours performed by parents. 2. Genotyping‐by‐sequencing revealed high levels of extra‐pair paternity, with 54.8% of offspring sired by extra‐pair males and 70% of nests containing extra‐pair young. 3. More heterozygous social males were cuckolded less than more homozygous social males. Extra‐pair mating, however, seems unlikely to increase offspring genetic diversity as extra‐pair offspring were not more heterozygous than within‐pair offspring, and average brood heterozygosity did not increase with higher rates of extra‐pair paternity. 4. Behavioural observations demonstrated that parents spent on average 46.5% of their time processing the decaying wood resource for larval offspring. Because resource processing is a by‐product of feeding and provides shareable benefits for all larvae in the brood, this form of paternal care could be favoured despite low paternity.     

Patterns of pollen dispersal in a small population of Pinus sylvestris L. revealed by total-exclusion paternity analysis

Patterns of pollen dispersal were investigated in a small, isolated, relict population of Pinus sylvestris L., consisting of 36 trees. A total-exclusion battery comprising four chloroplast and two nuclear microsatellites (theoretical paternity exclusion probability EP=0.996) was used to assign paternity to 813 seeds, collected from 34 trees in the stand. Long-distance pollen immigration accounted for 4.3% of observed matings. Self-fertilization rate was very high (0.25), compared with typical values in more widespread populations of the species. The average effective pollen dispersal distance within the stand was 48 m (or 83 m excluding selfs). Half of effective pollen was dispersed within 11 m, and 7% beyond 200 m. A strong correlation was found between the distance to the closest tree and the mean mating-distance calculated for single-tree progenies. The effective pollen dispersal distribution showed a leptokurtic shape, with a large and significant departure from that expected under uniform dispersal. A maximum-likelihood procedure was used to fit an individual pollen dispersal distance probability density function (dispersal kernel). The estimated kernel indicated fairly leptokurtic dispersal (shape parameter b=0.67), with an average pollen dispersal distance of 135 m, and 50% of pollen dispersed beyond 30 m. A marked directionality pattern of pollen dispersal was found, mainly caused by the uneven distribution of trees, coupled with restricted dispersal and unequal male success. Overall, results show that the number and distribution of potential pollen donors in small populations may strongly influence the patterns of effective pollen dispersal.     

POLLEN DISPERSAL DYNAMICS IN AN ALPINE WILDFLOWER,POLEMONIUM VISCOSUM

The alpine wildflower, Polemonium viscosum, depends on insect visitors for effective pollination. Here, I examine experimentally the effects of pollinator visitation on pollen removal, pollen dispersal success, paternity, and gene flow. Bumble bee pollinators visited donor individuals homozygous for marker alleles at an isozyme (GOT-2) encoding locus and then were presented with arrays of recipient plants lacking the marker alleles. Four aspects of male fitness were estimated for each donor: the number of pollen grains dispersed to flowers of the first recipient visited, the number of offspring sired on that recipient, the proportion of offspring sired in the full array, and the proportion of mates in the array bearing seeds of the donor. Pollen removal was strongly influenced by the number of bee visits to donor flowers. The amount of pollen removed in turn significantly affected the number of pollen grains reaching flowers of the first recipient. However, because seed production decelerates with stigma pollen load, the relationship between pollen export and paternal success at this proximate scale showed diminishing returns. The probability of reaching mates within the array also increased with pollen export. These findings show that floral characters enhancing pollinator visitation rate in P. viscosum have positive effects on paternity and gene flow.     

Hierarchical patterns of paternity within crowns of Albizia julibrissin (Fabaceae)

The floral architecture and phenology of the tree species Albizia julibrissin (Fabaceae) offer the potential for flowers within inflorescences to share common pollen donors. Patterns of paternity within individual tree crowns may differ among isolated individuals and those in populations due to differences in pollinator foraging behavior. To determine how genetic diversity is partitioned within individual seed pools and whether these patterns differ among isolated and population trees, we obtained all fruits from three inflorescences from four clusters from three isolated trees and from three population trees in Athens, Georgia. We assayed 14 polymorphic allozymes to genotype all progeny within singly sired fruits to determine the multilocus genotype of each fruit's pollen donor. Inflorescences had multiple pollen donors, but simulation analyses revealed that redundancy of pollen donors tended to be more likely within inflorescences than randomly across the crown. Analysis of genetic and genotypic diversity indicated that individual maternal trees received pollen from many donors in uneven frequencies. Results suggest that isolated trees receive pollen from slightly fewer pollen donors and experience more within-plant pollinator movement than trees in populations. However, isolated trees receive qualitatively similar pollen from many sources, suggesting that these trees are not effectively isolated and that pollen moves long distances in this species.     

Pollen load size, reproductive success, and progeny kinship of naturally pollinated flowers of the tropical dry forest tree Pachira quinata (Bombacaceae)

Several studies have demonstrated, using controlled pollinations, that the number and identity of pollen grains deposited onto a flower's stigma affect the reproductive success of plants. However, few studies have shown this relationship under conditions of natural pollination. Using the tropical dry forest tree Pachira quinata, we evaluated the relationship between the number of microgametophytes per pistil and the number of sires with respect to the production of fruits and seeds in a natural population of Pachira quinata. Our study demonstrates that fruit and seed production are directly related to the number of microgametophytes per pistil in natural populations of P. quinata. Only 6% of the marked flowers developed into mature fruits and 10% of the marked flowers initiated fruits but later aborted them. A mean of 23 pollen grains were required to produce a seed. Flowers with >400 pollen grains on the stigma always developed into mature fruits, whereas flowers that received <200 grains never matured fruits. Half of the pollen grains transferred to a flower stigma germinated and developed pollen tubes to the base of the style. The number of pollen grains on a stigma explained 34% of the variation in seed number per fruit, and the number of seeds produced per fruit is positively correlated with the size of the seeds. The population of P. quinata studied is predominantly outcrossing, and seeds within fruits are sired by one or a few donors. The total seed crop within trees was sired by three to five donors. Our study examined the implications of the variation in size of microgametophyte loads per pistil with respect to the breeding system and the paternity of progeny under natural conditions. The competitive ability of pollen and pollen tube attrition are important factors regulating fruit production in P. quinata.     

Under how wide a set of conditions will nonrandom mating occur in Raphanus sativus (Brassicaceae)?

Studies of the weedy annual Raphanus sativus have demonstrated that nonrandom mating, a prerequisite for sexual selection, can occur in greenhouse plants. To determine whether this nonrandom mating pattern can occur under a wide range of conditions, including conditions that might occur in the field, we considered variation in both maternal condition and pollen load size. Maternal condition was varied by altering the watering regime. Pollen load size was varied from approximately 26 to 343 pollen grains per stigma. At the smallest pollen load size, patterns of seed paternity were altered in two of the three pollen donor pairs; seed paternity became more equal among donors. For one of three pollen donor pairs, seed paternity was more divergent among donors on stressed maternal plants. Finally, for one pollen donor pair, rank order of pollen donor performance changed from the medium to the small pollen loads on stressed vs. control maternal plants. Thus, some field conditions may alter patterns of nonrandom mating in wild radish.     

Multiple paternity in common bean (Phaseolus vulgaris L., Fabaceae)

We report on two field experiments that were conducted in 1991 and 1992 at the South Coast Extension and Research Center, Irvine, CA, to study the incidence of multiple paternity in the common bean (Phaseolus vulgaris L.). Hypocotyl color and shikimate dehydrogenase (Skdh) isozymes were used as genetic markers. The white-seeded cultivar ‘Ferry Morse 53’ (FM 53) was used as the female parent. This cultivar is homozygous recessive (pp) for hypocotyl color. The pollen source parents were three homozygous dominant (PP) purple-hypocotyled, black-seeded cultivars. Three cultivars, ‘ICA Pijao,’ G4459, and the maternal parent FM 53, are of Mesoamerican origin and homozygous for the fast (F) allele at the Skdh locus. The other cultivar, Black Valentine, is of Andean origin and is homozygous for the slow (S) allele at the Skdh locus. Overall, 6 125 pods were obtained from 57 and 111 plants harvested individually in 1991 and 1992, respectively. All progeny, 28938 seeds, were scored for hypocotyl color at the seedling stage. The purple-hypocotyled seedlings were genotyped for the Skdh locus to identify their pollen parents. Multiple paternity was identified in all the pods with hybrid seeds (i.e., those of intercultivar crosses) at 5.8% and 8.1% in 1991 and 1992, respectively. All multiply sired pods produced both nonhybrid and hybrid seeds. As many as three successful fathers per pod were identified, but the number of markers limited measuring higher levels of multiple paternity. Most multiply sired pods (≈70%) were filled by nonhybrid seeds plus a single hybrid seed. Ovule position effect within multiply sired pods was inferred from the nonrandom distribution of hybrid seeds within a pod. On average, hybrid seeds occurred more frequently in ovules in position 7 (most basal) and in position 1 (most stylar) than in ovules in the middle positions of the pod.     

Pollen flow in the wildservice tree, Sorbus torminalis (L.) Crantz. I. Evaluating the paternity analysis procedure in continuous populations

The joint development of polymorphic molecular markers and paternity analysis methods provides new approaches to investigate ongoing patterns of pollen flow in natural plant populations. However, paternity studies are hindered by false paternity assignment and the nondetection of true fathers. To gauge the risk of these two types of errors, we performed a simulation study to investigate the impact on paternity analysis of: (i) the assumed values for the size of the breeding male population (NBMP), and (ii) the rate of scoring error in genotype assessment. Our simulations were based on microsatellite data obtained from a natural population of the entomophilous wild service tree, Sorbus torminalis (L.) Crantz. We show that an accurate estimate of NBMP is required to minimize both types of errors, and we assess the reliability of a technique used to estimate NBMP based on parent-offspring genetic data. We then show that scoring errors in genotype assessment only slightly affect the assessment of paternity relationships, and conclude that it is generally better to neglect the scoring error rate in paternity analyses within a nonisolated population.     

Mating system and pollen gene flow in Mediterranean maritime pine

Mating systems define the mode of gene transmission across generations, helping to determine the amount and distribution of genetic variation within and among populations of plant species. A hierarchical analysis of Mediterranean maritime pine mating system (61 mother trees from 24 plots, clustered in three populations) was used to identify factors affecting mating patterns and to fit pollen dispersal kernels. Levels of ovule and seed abortion, multi- and single-locus outcrossing rates and correlated paternity were estimated from progeny arrays and correlated with ecological stand variables and biometric tree measures. Pollen dispersal kernels were fitted using TwoGener and KinDist indirect methods and simulations were carried out to identify relevant factors affecting correlated paternity. Maritime pine showed high outcrossing rates (t(m) and t(s) approximately 0.96) and relatively low levels of correlated paternity [an r(p) of 0.018 (Ritland's estimate) or 0.048 (Hardy's estimate)], although higher than in other anemophilous tree species. Mating system parameters had high variation at the single-tree level (99-100%) but no stand or population effect was detected. At the single-tree level, outcrossing rates were correlated with tree (diameter and height) and crown size. In addition, correlated paternity showed a significant negative correlation with tree height, height to crown base and height to the largest crown width, probably reflecting the importance of the trees' 'ecological neighborhoods'. Indirectly estimated pollen dispersal kernels were very leptokurtic (exponential-power distributions with beta<0.5), with mean dispersal distances from 78.4 to 174.4 m. Fitted dispersal kernels will be useful in building explicit simulation models that include dispersal functions, and which will contribute to current conservation and management programs for maritime pine. Nevertheless, the numerical simulations showed that restricted dispersal, male fertility and phenological overlap could only partially explain the observed levels of correlated paternity; so other factors may also be relevant for the management of this valuable forest tree species.     

Effects of pollen load size and composition on pollen donor performance in wild radish, Raphanus sativus (Brassicaceae)

A critical concern in the debate over the importance of sexual selection in plants is whether the nonrandom mating demonstrable in greenhouse crosses can occur in the field. Field populations likely experience smaller and more variable pollen load sizes than those that have been used in many greenhouse experiments. Therefore, we performed a greenhouse experiment in which we varied both pollen load size and composition in wild radish, Raphanus sativus, and examined the paternity of seeds. We used five maternal plants and four pairs of pollen donors. We were able to produce pollen loads of 40, 118, and 258 grains per stigma. The smallest of the pollen loads was scant enough to result in a slight, but significant reduction in seed number per fruit. While variation in pollen load composition significantly affected the proportions of seeds fathered by different donors, variation in pollen load size did not. The relative performance of different donors was constant across pollen load sizes, suggesting that, for this species, differential performance of pollen donors can occur at pollen load sizes that are likely to occur in field populations.     

Comparison of pollen gene flow among four European beech (Fagus sylvatica L.) populations characterized by different management regimes

The study of the dispersal capability of a species can provide essential information for the management and conservation of its genetic variability. Comparison of gene flow rates among populations characterized by different management and evolutionary histories allows one to decipher the role of factors such as isolation and tree density on gene movements. We used two paternity analysis approaches and different strategies to handle the possible presence of genotyping errors to obtain robust estimates of pollen flow in four European beech (Fagus sylvatica L.) populations from Austria and France. In each country one of the two plots is located in an unmanaged forest; the other plots are managed with a shelterwood system and inside a colonization area (in Austria and France, respectively). The two paternity analysis approaches provided almost identical estimates of gene flow. In general, we found high pollen immigration (~75% of pollen from outside), with the exception of the plot from a highly isolated forest remnant (~50%). In the two unmanaged plots, the average within-population pollen dispersal distances (from 80 to 184 m) were higher than previously estimated for beech. From the comparison between the Austrian managed and unmanaged plots, that are only 500 m apart, we found no evidence that either gene flow or reproductive success distributions were significantly altered by forest management. The investigated phenotypic traits (crown area, height, diameter and flowering phenology) were not significantly related with male reproductive success. Shelterwood seems to have an effect on the distribution of within-population pollen dispersal distances. In the managed plot, pollen dispersal distances were shorter, possibly because adult tree density is three-fold (163 versus 57 trees per hectare) with respect to the unmanaged one.     

Variation in the breeding behavior of the dry forest tree Enterolobium cyclocarpum (guanacaste) in Costa Rica

We studied the breeding systems of four populations of Enterolobium cyclocarpum (guanacaste, earpod tree) in Costa Rica. Multilocus estimates of the outcrossing rate indicate that E. cyclocarpum is a predominant outcrossing species (t(m) ranged between 0.881 and 0.901) and biparental inbreeding is low (range between 0.058 and 0.079). Overall, our analyses showed significant differences in the gene frequencies between pollen and ovules and significant differences in pollen gene frequencies between the four populations. We also found significant differences in the pollen gene frequencies calculated for single trees in the same population. Outcrossing rates and pollen gene frequencies varied in two consecutive years in two populations of E. cyclocarpum. The correlated mating model revealed that there are differences in the correlation of paternity between populations and years. These findings indicate that there is variation in the average number of trees that father the seed crop of each tree and/or that some fathers are overrepresented in the seed crop of each tree. The implication of these findings for the development of strategies for conservation and management of this species are discussed.     

Phenological match drives pollen‐mediated gene flow in a temporally dimorphic tree

• Variation in flowering phenology is common in natural populations, and is expected to be, together with inter‐mate distance, an important driver of effective pollen dispersal. In populations composed of plants with temporally separated sexual phases (i.e. dichogamous or heterodichogamous populations), pollen‐mediated gene flow is assumed to reflect phenological overlap between complementary sexual phases. In this study, we conducted paternity analyses to test this hypothesis in the temporally dimorphic tree Acer opalus.
• We performed spatially explicit analyses based on categorical and fractional paternity assignment, and included tree size, pair‐wise genetic relatedness and morph type as additional predictors. Because differences between morphs in flowering phenology may also influence pollination distances, we modelled separate pollen dispersal kernels for the two morphs.
• Extended phenological overlap between male and female phases (mainly associated with inter‐morph crosses) resulted in higher siring success after accounting for the effects of genetic relatedness, morph type and tree size, while reduced phenological overlap (mainly associated with intra‐morph crosses) resulted in longer pollination distances achieved. Siring success also increased in larger trees.
• Mating patterns could not be predicted by phenology alone. However, as heterogeneity in flowering phenology was the single morph‐specific predictor of siring success, it is expected to be key in maintaining the temporal dimorphism in A. opalus, by promoting not only a prevalent pattern of inter‐morph mating, but also long‐distance pollination resulting from intra‐morph mating events.

     

Leaf-rust resistance in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.). 2. Genetic analysis and mapping of resistance genes <Emphasis Type="Italic">Pr3</Emphasis>, <Emphasis Type="Italic">Pr4</Emphasis>, and <Emphasis Type="Italic">Pr5</Emphasis>

The conventional way to drive modifications in old forest tree seed orchards is to establish progeny trials involving each parent tree and then evaluate its contribution to the performance of the progeny by estimating its general and specific combining ability (GCA and SCA). In this work, we successfully applied an alternative parent selection tactic based on paternity testing of superior offspring derived from a hybrid seed orchard established with a single Eucalyptus grandis seed parents and six E. urophylla pollen parents. A battery of 14 microsatellite markers was used to carry out parentage tests of 256 progeny individuals including two independent samples of selected trees and one control unselected sample, all derived from 6-year-old forest stands in eastern Brazil. Paternity determination was carried out for all progeny individuals by a sequential paternity exclusion procedure. Exclusion was declared only when the obligatory paternal allele in the progeny tree was not present in the alleged parent tree for at least four independent markers to avoid false exclusions due to mutation or null alleles. After maternity checks to identify seed mixtures and selfed individuals, the paternity tests revealed that approximately 29% of the offspring was sired by pollen parents outside the orchard. No selfed progeny were found in the selected samples. Three pollen parents were found to have sired essentially all of the offspring in the samples of selected and non-selected progeny individuals. One of these three parents sired significantly more selected progeny than unselected ones (P0.0002 in a Fisher exact test). Based on these results, low-reproductive-successful parents were culled from the orchard, and management procedures were adopted to minimize external pollen contamination. A significant difference (P<0.01) in mean annual increment was observed between forest stands produced with seed from the orchard before and after selection of parents and revitalization of the orchard. An average realized gain of 24.3% in volume growth was obtained from the selection of parents as measured in forest stands at age 2–4 years. The marker-assisted tree-breeding tactic presented herein efficiently identified top parents in a seed orchard and resulted in an improved seed variety. It should be applicable for rapidly improving the output quality of seed orchards, especially when an emergency demand for improved seed is faced by the breeder.Communicated by D.B. Neale