Comparison of genetic diversity of the invasive weed Rubus alceifolius poir. (Rosaceae) in its native range and in areas of introduction, using amplified fragment length polymorphism (AFLP) markers

Theory predicts that colonization of new areas will be associated with population bottlenecks that reduce within-population genetic diversity and increase genetic differentiation among populations. This should be especially true for weedy plant species, which are often characterized by self-compatible breeding systems and vegetative propagation. To test this prediction, and to evaluate alternative scenarios for the history of introduction, the genetic diversity of Rubus alceifolius was studied with amplified fragment length polymorphism (AFLP) markers in its native range in southeast Asia and in several areas where this plant has been introduced and is now a serious weed (Indian Ocean islands, Australia). In its native range, R. alceifolius showed great genetic variability within populations and among geographically close populations (populations sampled ranging from northern Vietnam to Java). In Madagascar, genetic variability was somewhat lower than in its native range, but still considerable. Each population sampled in the other Indian Ocean islands (Mayotte, La Réunion, Mauritius) was characterized by a single different genotype of R. alceifolius for the markers studied, and closely related to individuals from Madagascar. Queensland populations also included only a single genotype, identical to that found in Mauritius. These results suggest that R. alceifolius was first introduced into Madagascar, perhaps on multiple occasions, and that Madagascan individuals were the immediate source of plants that colonized other areas of introduction. Successive nested founder events appear to have resulted in cumulative reduction in genetic diversity. Possible explanations for the monoclonality of R. alceifolius in many areas of introduction are discussed.     

Identification of the male parent of superior half-sib <Emphasis Type="Italic">Populus tomentosa</Emphasis> individuals based on SSR markers

When hundreds of cross-pollinated trees planted in The National Gene Bank of Populus tomentosa blossom and bear fruit, in theory, half-sib progeny should originate from hundreds of combinations. Paternity testing was performed on the superior half-sib progeny with outstanding performance using microsatellite markers, a process that can be used to rapidly screen for superior cross combinations and significantly improve the efficiency and effectiveness of genetic improvements. Based on the comprehensive evaluation of the eight main growth traits of 2292 plants of 30 half-sib families, 77 plants consisting of 43 triploid and 34 diploid plants were selected as superior. We used 14 pairs of highly polymorphic SSR primers to identify the male parents of the 77 superior plants. Our results identified 29 male trees as the pollen parents of 59 superior plants from 49 cross combinations. The probabilities of 2-14, 3-48-1, 1903, 2-1, 4234, 5099, and 4114 as male parents were significantly higher than those of other males, suggesting that these male parents have potential for applications in breeding strategies of P. tomentosa. Thus, this study used SSR molecular markers to identify the male parents of superior individuals in a P. tomentosa population, laying a foundation for the application of maternal efficiency and construction of optimal hybrid parent groups.     

Pollen flow in Eucalyptus grandis determined by paternity analysis using microsatellite markers

Gene flow by pollen dispersal from forestry plantations containing introduced species, provenances or selected elite breeding material may impact on local native forest by changing the genetic diversity, introducing new genes or gene combinations, or causing the extinction of rare genotypes in adjacent native forest areas. Patterns of pollen flow can be used to assess the risk of genetic pollution of native forest areas from nearby plantations. Pollen flow in an artificial population of Eucalyptus grandis was estimated using molecular markers and paternity analysis. Microsatellite genotyping was used to identify pollen parents of progeny arrays from six mother trees. Of 329 progeny analysed, 178 (54%) were assigned to pollen parents within the population. Pollen parents located within the population were between 0–192 m from the respective mother trees, with an average pollination distance of 57.96 m. Pollination of mother trees was outcrossed, not by nearest neighbours, and displayed a preference for inter-provenance matings within the population. Progeny that could not be assigned pollen parents within the population (46%) were assumed to have resulted from pollen immigration from external sources. These pollen flow parameters provide useful information about the dynamics of pollen movement within E. grandis populations and may be used in risk assessment of gene flow from plantations to adjacent areas of native forest.     

Evidence of sexuality in European Rubus (Rosaceae) species based on AFLP and allozyme analysis

Reproduction of polyploid Rubus species is described as facultatively apomictic. Pollination is needed for seed set, but most seedlings are produced asexually by pseudogamy. Although sexual processes may occur, clonal diversity can be extremely low. We performed a pollination experiment to investigate the breeding system and used allozyme and AFLP markers to analyze genetic variation among and within seed families in R. armeniacus and R. bifrons. Pollination either with self or outcross pollen was necessary to trigger seed set. Outbreeding marginally increased the number and quality of seeds compared with selfing. The enzyme PGI revealed some genetic variation within seed families. Seven other enzyme systems were monomorphic. The more detailed AFLP analyses with five primer pairs detected the same rate of genetic variation (14-17% of seedlings were genetically distinct) and confirmed the allozyme results for the same individuals. No genetic variation was found between the seed families from within a species collected in widely separated populations, but clear species-specific differences were observed. The results support the view that polyploid Rubus species are pseudogamous apomicts with low genetic diversity among and within seed families. However, sexual reproduction occasionally occurs and contributes to the maintenance of genetic variation within natural populations.     

No evolutionary shift in the mating system of north American Ambrosia artemisiifolia (Asteraceae) following its introduction to China

The mating system plays a key role during the process of plant invasion. Contemporary evolution of uniparental reproduction (selfing or asexuality) can relieve the challenges of mate limitation in colonizing populations by providing reproductive assurance. Here we examined aspects of the genetics of colonization in Ambrosia artemisiifolia, a North American native that is invasive in China. This species has been found to possess a strong self-incompatibility system and have high outcrossing rates in North America and we examined whether there has been an evolutionary shift towards the dependence on selfing in the introduced range. Specifically, we estimated outcrossing rates in one native and five invasive populations and compared levels of genetic diversity between North America and China. Based on six microsatellite loci we found that, like the native North American population, all five Chinese populations possessed a completely outcrossing mating system. The estimates of paternity correlations were low, ranging from 0.028-0.122, which suggests that populations possessed ~8-36 pollen donor parents contributing to each maternal plant in the invasive populations. High levels of genetic diversity for both native and invasive populations were found with the unbiased estimate of gene diversity ranging from 0.262-0.289 for both geographic ranges based on AFLP markers. Our results demonstrate that there has been no evolutionary shift from outcrossing to selfing during A. artemisiifolia's invasion of China. Furthermore, high levels of genetic variation in North America and China indicate that there has been no erosion of genetic variance due to a bottleneck during the introduction process. We suggest that the successful invasion of A. artemisiifolia into Asia was facilitated by repeated introductions from multiple source populations in the native range creating a diverse gene pool within Chinese populations.     

Lowland Switchgrass Plants in Populations Set Completely Outcrossed Seeds Under Field Conditions as Assessed with SSR Markers

Switchgrass has long been characterized as an outcrossing species. However, mating behavior of plants in populations grown in the field allowing open pollination has not been documented. Accordingly, the objectives of this study were to determine the fertilization mode (i.e., selfing vs. outcrossing) of two self-compatible plants and to assess the mating behavior variability of lowland switchgrass genotypes in populations under field conditions. In Experiment I, two self-compatible genotypes ‘NL94 LYE 16?×?13’ and ‘SL93 7?×?15’ along with two populations were planted with two replications on the OSU Agronomy Research Farm, Stillwater, OK. Sixty-four progeny derived from half-sib seeds of each genotype per replication per year were genotyped with 4 to 20 simple sequence repeat (SSR) markers. In both 2010 and 2011, all progeny plants of the two parents were completely outcrossed exhibiting 100 % self-incompatibility. In Experiment II, two genetically narrow-based (NL94 C2-3 and SL93 C2-3), each having five parents, and two broad-based (NL94 C3 and SL93 C3), each comprising 26 parents, switchgrass populations with three replications were field established at the OSU Cimarron Valley Research Station, Perkins, OK. The DNA samples were isolated from 1700 open-pollinated progeny of 62 seed parents in 2010 and 773 progeny of 42 parents in 2011. Among all the progeny genotyped with eight to 16 SSR markers, only one was identified as a selfed progeny, indicating very little variability in outcrossing behavior under the field conditions. The identification of specific genotypes like NL94 LYE 16?×?13 and SL93 7?×?15 which are self-incompatible in the open field but self-compatible under the controlled conditions potentially enables efficient production of F1 hybrid seed in switchgrass.     

Asexual reproduction in a close relative of Arabidopsis: a genetic investigation of apomixis in Boechera (Brassicaceae)

Understanding apomixis (asexual reproduction through seeds) is of great interest to both plant breeders and evolutionary biologists. The genus Boechera is an excellent system for studying apomixis because of its close relationship to Arabidopsis, the occurrence of apomixis at the diploid level, and its potentially simple inheritance by transmission of a heterochromatic (Het) chromosome. Diploid sexual Boechera stricta and diploid apomictic Boechera divaricarpa (carrying a Het chromosome) were crossed. Flow cytometry, karyotype analysis, genomic in situ hybridization, pollen staining and seed-production measurements were used to analyse the parents and resulting F1, F2 and selected F3 and test-cross (TC) generations. The F1 plant was a low-fertility triploid that produced a swarm of aneuploid and polyploid F2 progeny. Two of the F2 plants were fertile near-tetraploids, and analysis of their F3 and TC progeny revealed that they were sexual and genomically stabilized. The apomictic phenotype was not transmitted by genetic crossing as a single dominant locus on the Het chromosome, suggesting a complex genetic control of apomixis that has implications for future genetic and evolutionary analyses in this group.     

Genetic diversity and reproductive biology in ecotypes of the facultative apomict Hypericum perforatum L

Apomixis is a mode of asexual reproduction through seed. Progeny produced by apomixis are clonal replicas of a mother plant. The essential feature of apomixis is that embryo sacs and embryos are produced in ovules without meiotic reduction or egg cell fertilisation. Thus, apomixis fixes successful gene combinations and propagates high fitness genotypes across generations. A more profound knowledge of the mechanisms that regulate reproductive events in plants would contribute fundamentally to understanding the evolution and genetic control of apomixis. Molecular markers were used to determine levels of genetic variation within and relationship among ecotypes of the facultative apomict Hypericum perforatum L. (2n = 4x = 32). All ecotypes were polyclonal, being not dominated by a single genotype, and characterised by different levels of differentiation among multilocus genotypes. Flow cytometric analysis of seeds indicated that all ecotypes were facultatively apomictic, with varying degrees of apomixis and sexuality. Seeds set by haploid parthenogenesis and/or by fertilisation of aposporic egg cells were detected in most populations. The occurrence of both dihaploids and hexaploids indicates that apospory and parthenogenesis may be developmentally uncoupled and supports two distinct genetic factors controlling apospory and parthenogenesis in this species. Cyto-embryological analysis showed that meiotic and aposporic processes do initiate within the same ovule: the aposporic initial often appeared evident at the time of megaspore mother cell differentiation. Our observations suggest that the egg cell exists in an active metabolic state before pollination, and that its parthenogenetic activation leading to embryo formation may occur before fertilisation and endosperm initiation.     

AFLP markers closely linked to a major gene essential for nucellar embryony (apomixis<Emphasis Type="Italic">)</Emphasis> in <Emphasis Type="Italic">Citrus maxima</Emphasis> × <Emphasis Type="Italic">Poncirus trifoliata</Emphasis>

Some citrus varieties express a form of apomixis termed nucellar embryony in which the adventive embryos develop from nucellus tissue surrounding the embryo sac. This trait results in many seeds containing multiple embryos (polyembryony). Inheritance of the frequency of polyembryony was studied in 88 progeny from a cross of Citrus maxima (monoembryonic) × Poncirus trifoliata (polyembryonic). The frequency of polyembryonic seed produced by each progeny was determined by scoring 100–500 seeds for the number of seedlings to emerge from each seed. Two groups of eight individuals from each extreme of the population were chosen for bulked segregant analysis with amplified fragment length polymorphism markers amplified with 256 primer combinations. Candidate markers identified in the bulks as linked to the trait were tested on the 32 individuals used to create the bulks and then on the remaining plants in the population. Five candidate markers tightly linked to polyembryony in P. trifoliata were identified. Specific marker alleles were present in nearly all progeny that produced polyembryonic seed, and alternate alleles were present in nearly all progeny that produced only monoembryonic seed. The region defined by these markers very likely contains a gene that is essential for the production of polyembryonic seeds by apomixis, but also shows segregation distortion. The proportion of polyembryonic seeds varied widely among the hybrid progeny, probably due to other genes. Scoring 119 progeny of a P. trifoliata selfed population for the closely linked markers and the proportion of polyembryonic seeds confirmed close linkage between these markers and polyembryony.     

濒危植物焕镛木的兼性无融合生殖

2001～2002年连续两年在广西环江木论和广西罗城大黄泥的2个焕镛木(Woonyoungia septentrionalis(Dandy)Law)自然种群中,对单性异株的濒危植物焕镛木进行繁育系统测定,对即将开花的雌花花蕾分别进行套袋、套网、人工授粉处理,并用自然授粉雌花作对照,其座果率和结实率统计结果表明:自然授粉、人工授粉、套袋和套网处理的花均能结实,但它们的座果率和结实率存在较大的差异.在两个种群中,人工授粉和自然授粉的总结实率(PERS)均比套袋和套网处理的高,其中人工授粉的最高,套网处理的最低.由此可见,焕镛木既能通过有性生殖方式结实,又能通过无融合生殖方式结实,而且这两种生殖方式获得的种子均能萌发成幼苗,由此断定,焕镛木的繁育系统为兼性无融合生殖.这是首次报道木兰科植物存在无融合生殖现象.     

Patterns of pollen flow in a dense population of the insect-pollinated canopy tree species Castanopsis sieboldii

Insect pollinations of tree species with high-density populations have rarely been studied. Since the density of adults can affect effective pollen dispersal, short-distance pollination, even by insects, may frequently occur in high-density populations. To test this prediction, we investigated pollination patterns in a high-density population of the insect-pollinated canopy tree species Castanopsis sieboldii by paternity analysis using genotypes at 8 microsatellite loci of 145 adult trees and 439 seeds from 11 seed parents in a 4-ha plot. We then explored their genetic effects on the population by calculating other population genetics parameters. Although C. sieboldii has high potential for long-distance dispersal of pollen (as indicated by a fat-tailed dispersal kernel), the cumulative pollination at the local scale was spatially limited and strongly dependent on the distance between parents due to the high density of adults. Genetic diversity estimates for pollen pools accepted by each seed parent converged on a maximum as the effective number of pollen parents increased. The genetic diversity of pollen pool bulked over all the seed parents from inside the plot did not differ from that of the total pollen pools. Therefore, although pollen flow from distant pollen parents may help to maintain the genetic diversity of offspring, pollen parents neighboring seed parents may be the main contributors to the genetic diversity of the offspring at the seed stage.     

濒危植物焕镛木的兼性无融合生殖

2001—2002年连续两年在广西环江木论和广西罗城大黄泥的2个焕镛木(Woonyoungia septentrionalis (Dandy) Law)自然种群中,对单性异株的濒危植物焕镛木进行繁育系统测定,对即将开花的雌花花蕾分别进行套袋、套网、人工授粉处理,并用自然授粉雌花作对照,其座果率和结实率统计结果表明：自然授粉、人工授粉、套袋和套网处理的花均能结实,但它们的座果率和结实率存在较大的差异。在两个种群中,人工授粉和自然授粉的总结实率(PERS)均比套袋和套网处理的高,其中人工授粉的最高,套网处理的最低。由此可见,焕镛木既能通过有性生殖方式结实,又能通过无融合生殖方式结实,而且这两种生殖方式获得的种子均能萌发成幼苗,由此断定,焕镛木的繁育系统为兼性无融合生殖。这是首次报道木兰科植物存在无融合生殖现象。     

Population estimators or progeny tests: what is the best method to assess null allele frequencies at SSR loci?

Nuclear SSRs are notorious for having relatively high frequencies of null alleles, i.e. alleles that fail to amplify and are thus recessive and undetected in heterozygotes. In this paper, we compare two kinds of approaches for estimating null allele frequencies at seven nuclear microsatellite markers in three French Fagus sylvatica populations: (1) maximum likelihood methods that compare observed and expected homozygote frequencies in the population under the assumption of Hardy-Weinberg equilibrium and (2) direct null allele frequency estimates from progeny where parent genotypes are known. We show that null allele frequencies are high in F. sylvatica (7.0% on average with the population method, 5.1% with the progeny method), and that estimates are consistent between the two approaches, especially when the number of sampled maternal half-sib progeny arrays is large. With null allele frequencies ranging between 5% and 8% on average across loci, population genetic parameters such as genetic differentiation (F _ST) may be mostly unbiased. However, using markers with such average prevalence of null alleles (up to 15% for some loci) can be seriously misleading in fine scale population studies and parentage analysis.     

Isolation and characterization of microsatellites in Rattus rattus

We isolated and characterized 10 microsatellite loci in the black rat Rattus rattus (Muridae, Rodentia), a widespread invasive species largely known to cause serious problems in agriculture and human health. Polymorphism was studied in two populations, one from Madagascar and one from Senegal. It ranged from three to 12 alleles in Madagascar, and from two to five alleles in Senegal. Together with the loci previously adapted from Rattus norvegicus, this set of markers should allow the conduct of thorough studies on the genetic structure of natural populations of R. rattus.     

DNA fingerprinting—A useful tool in the taxonomy of apomictic plant groups

DNA fingerprinting has proved useful for the analysis of genotypic distribution and estimation of genetic relatedness in apomictic plant groups likeRubus, Amelanchier, Hieracium andTaraxacum. Speciation through interspectific hybridization has been demonstrated in one case involving apomicticRubus species. The application of molecular markers to progeny derived from experimental cross-pollinations has yielded information on levels of apomixis as well as on its inheritance and regulation in e.g.Rubus, Poa, Pennisetum andTripsacum.     

Microsatellite analysis of seed dispersal and parentage of saplings in bur oak, Quercus macrocarpa

Microsatellite analysis was used to examine parentage and spatial distributions of 62 adult bur oaks Quercus macrocarpa, and 100 saplings in a single stand. Using genotypes scored by PCR products at four microsatellite loci, we determined that 94 saplings matched at least one parent in the stand. Saplings often occur as dense clusters of half-sibs around the presumed maternal parent, and only four adults were seed parents to a large proportion of the saplings sampled. A stump apparently was the seed parent of the largest cluster of half-sibs, which occupied a sizeable light gap opened up by the death of their maternal tree. Approximately half of the saplings appeared to have grown from seeds that had not been removed after falling from the tree, and half from seeds that were dispersed beyond the crown of their maternal parent. Long-distance seed dispersal may be more common than has been previously reported. Extremely high levels of long-distance pollination were indicated, and pollen donors within the stand were generally distributed randomly around maternal trees. More than half of the saplings had paternal parents outside of the stand. This study demonstrates the utility of microsatellite analysis for studying mating systems, seed dispersal and seedling establishment in natural plant populations.     

Using genetic markers to directly estimate male selection gradients

We present an analysis of Raphanus raphanistrum and simulations illustrating the utility of directly estimating male phenotypic selection gradients using genetic markers. The method offers a much more refined characterization of selection than attempting to assign paternity to individual progeny. Our analysis of R. raphanistrum reveals selection on remarkably fine features of floral morphology, including anther exsertion, that were opaque to previous approaches. The new results also undermine a previous conclusion that selection on wild radish floral morphology acts primarily through female fitness. Simulation results show that selection gradients on the order of beta = 0.1-0.2 can be readily detected with allozyme markers in moderate-sized (< 200 paternal individuals) populations. Highly polymorphic (e.g., microsatellite) markers will likely detect fine scale selection (beta < 0.1) in larger populations (> or = 400 individuals). Increased progeny sample size, by sampling either additional maternal families or more progeny per maternal parent, partly compensates for low exclusion probability. Increasing the number of possible fathers without changing progeny sample size decreases the ability to detect selection, especially at lower exclusion probabilities. Sampling only some male genotypes reduces the power to detect selection and biases (underestimates) the magnitude of the selection gradient estimate.     

A model for linkage analysis with apomixis

Apomixis, or asexual reproduction through seeds, occurs in over 400 species of angiosperms. Although apomixis can favorably perpetuate desired genotypes through successive seed generation, it may also bring about some difficulty for linkage analysis and quantitative trait locus mapping. In this article, we explore the issue of how apomixis affects the precision and power of linkage analysis with molecular markers. We derive a statistical model for estimating the linkage between different markers when some progeny are derived from apomixis. The model was constructed within the maximum likelihood framework and implemented with the EM algorithm. A series of procedures are formulated to test the linkage of markers, the rate of apomixis, and the degree of genetic interference during meiosis. The model was examined and validated through simulation studies. The model will provide a tool for linkage mapping and evolutionary studies for plant species that undergo apomixis.     

Determining genetic origins of aberrant progeny from facultative apomictic Kentucky bluegrass using a combination of flow cytometry and silver-stained RAPD markers

Seeded plants that reproduce through facultative apomixis produce two types of progeny: (1) apomictic progeny genetically identical to the maternal genotype, and (2) aberrant progeny genetically different from the maternal genotype. Aberrant progeny have at least nine different genetic origins depending on gametic ploidy level and whether fertilization was self, cross, or absent. Multiple genetic origins of aberrant progeny complicate the results of basic and applied genetic studies. Determining the genetic origin of progeny plants using traditional techniques, such as cytology, embryology, and segregational studies, is technically difficult in Kentucky bluegrass. We have found that two relatively new techniques, flow cytometry and silver-stained RAPD (ssRAPD) markers, are powerful tools for rapidly determining the genetic origins of aberrant Kentucky bluegrass progeny. Our application of these techniques demonstrate that (1) flow cytometry accurately distinguishes progeny ploidy levels, and (2) ssRAPD markers distinguish progeny resulting from cross-fertilization. Therefore, a combination of flow cytometry and ss-RAPD data would be useful for most genetic studies of aberrant individuals. Moreover, ssRAPD s were found to be of value for measuring the loss of genetic markers from polyhaploids and quantifying the inheritance of parental genomes in polydiploid B_n (n+n) and polytriploid B_III (2n+n) hybrids. Quantifying shared ss-RAPD markers may also be useful for determining genetic relatedness between varieties and germplasm sources.