Seed Paternity Analysis Using SSR Markers to Assess Successful Pollen Donors in Mixed Olive Orchards

The olive tree (Olea europaea L.) is a wind-pollinated crop that exhibits an extreme alternate bearing habit. To improve fruit set, several methods have been used to determine the most successful compatible combinations of cultivars. In this study, priority is given to seed paternity analysis based on simple sequence repeats (SSRs), microsatellite markers used for the identification of potential pollen donors of cultivar ‘Oblica’ in a mixed olive orchard during two consecutive years. Seven microsatellite primers were successfully used to examine the paternity of olive embryos from ‘Oblica’ mother trees. Embryos were considered as a product of self-fertilization if only maternal alleles were present, but not a single case of self-fertilization was found among all the embryos analyzed. Two dominant pollen donors were not the closest nor the cultivars with the highest number of trees in the orchard, suggesting that cross-compatibility may have a key role in determining pollen donor success. In our earlier studies, pollen tube growth and fertilization success correlated with fruit set when controlled crosses between cultivars were performed; however, some discrepancy might appear compared to paternity analyses when mother trees have a free choice among different pollen sources from cultivars growing in their surroundings.

Pollen Competition and Paternal Contribution during Artificially Controlled Pollination of Black Locust (Robinia pseudoacacia L.) without Castration

(1) Background: Considering the serious damage caused by castration and the extremely high outcrossing rate in nature, we hypothesized that artificial controlled pollination of black locust without castration could be conducted for hybridization breeding. (2) Methods: The study conducted controlled pollination on 20 mating combinations of black locust without castration using a single or mixed male parent. Offspring of different developmental stages and the leaves of parents were collected to extract DNA and perform paternity analysis using SSR molecular markers. (3) Results: The contribution rate of each male parent differed according to developmental stage after pollination using different pollens mixed in equal proportions. There were significant correlations between the genetic similarity between each male parent and female parent and contribution rate of each male parent at three different developmental stages after pollination. (4) Conclusions: The composition of offspring pollen donors showed no bias toward selfing or outcrossing when artificially pollinated without castration. Hybrid breeding of black locust by artificially controlled pollination without castration may not be feasible, given that our manual method resulted in a large number of abortive and abnormal offspring. Introduction of honeybees in a limited space to conduct controlled pollination of black locust for hybrid breeding may be feasible.

Depicting the mating system and patterns of contemporary pollen flow in trees of the genus Anadenanthera (Fabaceae)

Anadenanthera (Fabaceae) is endemic to the Neotropics and consists of two tree species: A. colubrina (Vell.) Brenan and A. peregrina (L.) Speg. This study examined the mating system and contemporary gene flow of A. colubrina (Acol) and A. peregrina (Aper) in a highly fragmented area of the Atlantic Forest to provide valuable information that informs conservation strategies. Reproductive adults from forest remnants [nA. colubrina = 30 (2.7 ha), nA. peregrina = 55 (4.0 ha)] and progeny-arrays (nA. colubrina = 322, nA. peregrina = 300) were genotyped for seven nuclear microsatellite markers. Mating system analyses revealed that A. colubrina is a mixed mating species (tm = 0.619) while A. peregrina is a predominantly outcrossing species (tm = 0.905). For both Anadenanthera species, high indices of biparental inbreeding were observed (Acol = 0.159, Aper = 0.216), resulting in low effective pollination neighborhood sizes. Categorical paternity analysis revealed different scales of pollen dispersal distance: the majority of crossings occurring locally (i.e., between nearby trees within the same population), with moderate pollen dispersal coming from outside the forest fragments boundaries (Acolmp = 30%, Apermp = 35%). Nevertheless, pollen immigration from trees outside the populations for both species suggests that the populations are not reproductively isolated. This study highlights the importance of evaluating both mating system and contemporary gene flow for a better understanding of the biology of Anadenanthera species. This information should be considered to ensure the effective conservation and management practices of these plant species.

Spatial asymmetry of the paternity success in nests of a fish with alternative reproductive tactics

Guard-sneaker tactics are widespread among fish, where territorial males defend a nest and provide parental care while sneakers try to steal fertilizations. Territorials and sneakers adopt diverse pre- and post-mating strategies, adjusting their ejaculate investment and/or behavioural responses to the presence of competitors. The relative distance of competitors from the spawning female plays a major role in influencing male mating strategies and the resulting paternity share. However, territorial male quality and sneaking intensity do not fully account for the variability in the relative siring success occurring among species. An often neglected factor potentially affecting sneakers proximity to females is the nest structure. We conducted a field experiment using the black goby, whose nests show two openings of different size. We found that territorial males defend more and sneaking pressure is higher at the front, larger access of the nest than at the back, smaller one. Moreover, microsatellite paternity analysis shows that territorials sire more offspring at the back of their nest. Such a predictable spatial distribution of the paternity share suggests that nest structure might work as an indirect cue of male relative siring success, potentially influencing the territorial male investment in parental care and/or the female egg deposition strategy.

Reintroduced Native Populus nigra in Restored Floodplain Reduces Spread of Exotic Poplar Species

Exotic Populus taxa pose a threat to the success of riparian forest restoration in floodplain areas. We evaluated the impact of exotic Populus taxa on softwood riparian forest development along the river Common Meuse after introducing native Populus nigra and after the re-establishment of the natural river dynamics. We sampled 154 poplar seedlings that spontaneously colonized restored habitat and assessed their taxonomy based on diagnostic chloroplast and nuclear microsatellite markers. Furthermore, by using a paternity analysis on 72 seedlings resulting from six open pollinated P. nigra females, we investigated natural hybridization between frequently planted cultivated poplars and native P. nigra. The majority of the poplar seedlings from the gravel banks analyzed where identified as P. nigra; only 2% of the sampled seedlings exhibited genes of exotic poplar species. Similarly, the majority of the seedlings from the open pollinated progenies were identified as P. nigra. For three seedlings (4%), paternity was assigned to a cultivar of P. × canadensis. Almost two decades after reintroducing P. nigra, the constitution of the seed and pollen pools changed in the study area in favor of reproduction of the native species and at the expense of the exotic poplar species. This study indicates that, although significant gene flow form exotic poplars is observed in European floodplains, restoration programs of the native P. nigra can vigorously outcompete the exotic gene flows and strongly reduce the impact of exotic Populus taxa on the softwood riparian forest development.

Polyandry and paternity affect disease resistance in eusocial wasps

Polyandry (multiple mating by females) is a central challenge for understanding the evolution of eusociality. Several hypotheses have been proposed to explain its observed benefits in eusocial Hymenoptera, one of which, the parasite–pathogen hypothesis (PPH), posits that high genotypic variance among workers for disease resistance prevents catastrophic colony collapse. We tested the PPH in the polyandrous wasp Vespula shidai. We infected isolated workers with the entomopathogenic fungus Beauveria bassiana and quantified their survival in the laboratory. Additionally, we conducted a paternity analysis of the workers using nine microsatellite loci to investigate the relationship between survival and the matriline and patriline membership of the workers. As predicted by the PPH, nestmate workers of different patrilines showed differential resistance to B. bassiana. We also demonstrated variation in virulence among strains of B. bassiana. Our results are the first to directly support the PPH in eusocial wasps and suggest that similar evolutionary pressures drove the convergent origin and maintenance of polyandry in ants, bees, and wasps.

Reintroduced native Populus nigra in restored floodplain reduces spread of exotic poplar species

1.AbstractExotic Populus taxa pose a threat to the success of riparian forest restoration in floodplain areas. We evaluated the impact of exotic Populus taxa on softwood riparian forest development along the river Common Meuse after introducing native Populus nigra and after the re-establishment of the natural river dynamics. We sampled 154 poplar seedlings that spontaneously colonised restored habitat and assessed their taxonomy based on diagnostic chloroplast and nuclear microsatellite markers. Furthermore, by using a paternity analysis on 72 seedlings resulting from six open pollinated P. nigra females, we investigated natural hybridization between frequently planted cultivated poplars and native P. nigra. The majority of the poplar seedlings from the gravel banks analyzed where identified as P. nigra; only 2% of the sampled seedlings exhibited genes of exotic poplar species. Similarly, the majority of the seedlings from the open pollinated progenies were identified as P. nigra. For three seedlings (4%), paternity was assigned to a cultivar of P. x canadensis. Almost two decades after reintroducing P. nigra, the constitution of the seed and pollen pools changed in the study area in favour of reproduction of the native species and at the expense of the exotic poplar species. This study indicates that, although significant gene flow form exotic poplars is observed in European floodplains, restoration programmes of the native P. nigra can vigorously outcompete the exotic gene flows and strongly reduce the impact of exotic Populus taxa on the softwood riparian forest development.

Selfing and correlated paternity in relation to pollen management in western red cedar seed orchards

We used microsatellite genetic markers to evaluate the mating system of western red cedar (Thuja plicata Donn ex D. Don) under various seed orchard pollen management schemes. We primarily examined whether supplemental mass pollination (SMP) can reduce the observed selfing rates. Pollen blowing and “hooding” were also examined in smaller tests. Only SMP was consistently effective in reducing the selfing rate, from 30% to 20%. The correlation of paternity was quite high (60%–90%) in two of three orchards, and in these two orchards the application of SMP reduced this correlation by about 10% as well. The correlation of paternity is the fraction of full-sibling vs. half-sibling progeny, and unbiased estimates can be obtained with few loci, even single loci, in contrast to other types of paternity analysis. We also find the microsatellite amplicon sizes should be pooled into “bins” of 2–4 nucleotides, owing to unintended errors of assay; otherwise the estimates are biased. This new feature of mating system estimation was incorporated into the computer program MLTR.

Compatibility between “Arbequina” and “Souri” Olive Cultivars May Increase Souri Fruit Set

The “Souri” olive cultivar, which is autochthonous to the eastern Mediterranean region, has been the major olive variety cultivated traditionally under rain-fed conditions in northern Israel. The aim of this study was to determine the optimal pollen donor for the olive cultivar Souri in order to maximize Souri fruit set. Artificial cross pollination of Souri flowers with several local varieties has identified the “Nabali” as the most efficient pollinizer of the Souri. However, further experiments using artificial cross pollination conducted with cultivars not common to this region have revealed the “Arbequina” as a more efficient pollinizer of the Souri cultivar than the Nabali. Based on a preliminary paternity analysis, the Nabali was identified as the dominant pollinizer of Souri trees in traditional olive orchards in the north of Israel. However, in a multi-variety orchard, molecular paternity analysis has shown Arbequina to be the most frequent pollinizer. We then tested, during two consecutive years, whether the presence of a pollen-producing Arbequina tree adjoining Souri trees in the field will increase their fruit set. We found that Souri fruit set was 8.36% when pollinized by an Arbequina tree in close proximity to them, significantly higher than the fruit set of 5.6% for Souri trees without the nearby Arbequina cultivar. On the basis of these trials, we expect that the yield of Souri orchards will improve if Arbequina trees are planted.