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.     

Enriching Ploidy Level Diversity: the Role of Apomictic and Sexual Biotypes of Hieracium subgen. Pilosella (Asteraceae) that Coexist in Polyploid Populations

The capacity to generate variation in ploidy and reproductive mode was compared in facultatively apomictic versus sexual maternal plants that coexist in two model populations. The population structure was studied in polyploid hybrid swarms comprised of Hieracium pilosella (usually sexual, less commonly apomictic), H. bauhini (apomictic), and their hybrids (sexual, apomictic, or sterile). Relationships among established biotypes were proposed on the basis of their DNA ploidy level/chromosome number, reproductive mode and morphology. Isozyme phenotypes and chloroplast DNA haplotypes were assayed in the population that was richer in hybrids. The reproductive origin of seed progeny was identified in both sexual and apomictic mothers, using alternative methods: the karyological, morphological and reproductive characters of the cultivated progeny were compared with those of respective mothers, or flow cytometric seed screening was used. In both populations, the progeny of sexual mothers mainly retained a rather narrow range of ploidy level/chromosome number, while the progeny of facultatively apomictic mothers was more variable. The high-polyploid hybrids, which had arisen from the fertilization of unreduced egg cells of apomicts, mainly produced aberrant non-maternal progeny (either sexually and/or via haploid parthenogenesis). Apparently, such versatile reproduction resulted in genomic instability of the recently formed high-polyploid hybrids. While the progeny produced by both true apomictic and sexual mothers mostly maintained the maternal reproductive mode, the progeny of those ‘versatile’ mothers was mainly sexual. Herein, we argue that polyploid facultative apomicts can considerably increase population diversity.     

A further study of microsatellite on apomixis in cassava

Cassava is the main staple crop in the tropics. It is vegetatively propagated by stem cuttings that maintain superior genotypes, but favors disease accumulation. In this report we present the results of the screening of the progeny and the second generation of the clone UnB 307 for apomixis using microsatellites. A total of 29 plants were screened, representing the maternal plant, its first and second generations, that were left to open pollination. About 20% of the offspring were rated as genetically identical plants. This result confirms the facultative apomictic nature of cassava, with high environment effect.     

Spore germination and isozyme patterns in the apomictic fern Cyrtotnium falcatum

Progeny from a plant of the apomictic species Cyrtomium falcatum were examined by protein electrophoresis. These progeny were produced from spores originating in sporangia with differing spore numbers. Analysis of zymograms indicates that variant banding patterns were exhibited in 16.1% of the progeny sampled. These anomalous phenotypes suggest that some apomictically reproducing plants can produce genetically variable progeny.     

Molecular analysis of apomixis in cassava

Cassava is the main staple for more than 800 million people in the tropics. It is propagated vegetatively by stem cuttings, which maintains superior genotypes but favors disease accumulation and spread. In this report, we present the results of the screening of the progeny and the second generation of the clone UnB 307 for apomixes using microsatellites. A total of 29 plants were screened, representing the maternal plant, its first and second generations that were left to open pollination. About 20% of the offspring were rated as genetically identical plants. This result confirms the facultative apomictic nature of cassava, with high environmental effect.     

Genetic diversity in sexual diploid and apomictic tetraploid populations of Paspalum notatum situated in sympatry or allopatry

Paspalum notatum is a subtropical grass widely distributed in the temperate areas of America. Diploids are sexual while polyploids give rise to clonal seeds through aposporous apomixis. RAPD markers were used to analyze the genetic structure of three natural populations: i) diploids reproducing sexually (R2X); ii) sympatric apomictic tetraploids collected in the vicinity of the diploids (R4X); iii) allopatric apomictic tetraploids growing in isolation (C4X). The apomictic reproduction rate was evaluated by the use of molecular markers in progeny tests, while chromosome-counting allowed the verification of ploidy levels. Data revealed that the R4X group presented a variation considerably higher than that observed for C4X. Jaccards coefficients were used to produce a cluster diagram using the UPGMA method. All but one tetraploid genotypes grouped together and were associated to diploid genotype A21. The possibility of occasional generation of novel tetraploid clones from the interaction between tetraploid and diploid individuals is discussed.     

The Detection,Rate and Manifestation of Residual Sexuality in Apomictic Populations of Pilosella (Asteraceae,Lactuceae)

The effect of maternal, facultatively apomictic plants on population diversity was evaluated in seven hybridizing polyploid Pilosella populations, where apomictic (P. bauhini or P. aurantiaca) and sexual (P. officinarum) biotypes coexist. The ploidy level, reproductive system, morphology, clonal structure and chloroplast DNA haplotypes were used to characterize these plants and their hybrids. The reproductive origins of the progeny were assessed through either a flow cytometric seed screen and/or a comparison between the ploidy level of progeny embryos/seedlings and the maternal ploidy level. The cultivated progeny derived from residual sexuality in maternal apomicts were also identified based on their morphology and reproductive behaviour. The progeny different from their maternal parents (0.6?92.3 % of progeny embryos and 0?100 % of progeny seedlings) originated either sexually or via haploid parthenogenesis. Comparing the facultatively apomictic and sexual mothers, the progeny arrays generated in the field showed that apomictic mothers produce progeny that is more variable in ploidy level. This effect was demonstrated at both the embryonic and seedling stages of progeny development. Residual sexuality in apomicts was also effective in experimental crosses, generating progeny similar to spontaneous hybrids in the field. The 2n + n hybrids produced from an apomictic and a sexual parent displayed similar reproductive behaviour, producing polyhaploid, sexual and apomictic progeny in variable ratios. Repeated hybridizations between parental species and/or multi-step crosses can result in hybrid swarms rich in cytotypes and morphotypes. The variation recorded in these populations suggests prevailing introgressive hybridization towards the sexual species P. officinarum.     

Quantification of progeny classes in two facultatively apomictic accessions of Hieracium

Hieracium is an established model system for studying the cytological and genetic basis of gametophytic apomixis. In common with most known apomicts, the formation of 'maternal seed' is not exclusive in Hieracium, as apomixis operates in conjunction with a low level of sexuality. When this occurs the form of apomixis is described as 'facultative'. The formation of maternal seed in these plants is characterised by the avoidance of meiosis followed by the parthenogenetic development of an unreduced egg cell. In some ovules, however, meiosis does proceed, and sometimes the fertilisation of an egg cell presages embryogenesis. As a result, this mechanism of facultative apomixis leads to the formation of several different types of progeny, each representing a unique combination of meiosis/apomeiosis and fertilisation/parthenogenesis. Furthermore, fertilisation may involve either self or non-self pollen, leading to the recognition of six progeny classes from each individual plant. To facilitate an understanding of these processes we have developed a method for identifying individuals from different progeny classes based on the inheritance of introduced heterologous marker genes. This technique permits the screening of many thousands of seedlings at germination, and the consequent isolation of individuals associated with rare classes. Progeny profiles were determined for two apomictic accessions of Hieracium. Both were found to develop approximately 2.5% of their seed from meiotically derived eggs under the experimental conditions used and to have a rate of hybridity of approximately 2%. Evidence was also found for the action of a self-incompatibility mechanism operating in these plants despite the autonomous nature of apomixis in Hieracinum. As a demonstration of the utility of this approach, a study was conducted of polyembryony in one accession. The results indicate that there was a 7 fold greater likelihood that a meiotically derived seedling would arise in a polyembryonic seed than in a single-embryo seed. This indicates that facultative apomixis in Hieracium not only results from the simultaneous occurrence of sexual and asexual seed formation in the same capitulum as previously demonstrated, but most often as parallel processes within the same ovule.     

兼性无融合生殖龙须草SSR引物开发及杂交后代的检测

以来自中国5个省份12个居群的龙须草为材料,通过锚定PCR开发复合SSR引物、数据库检索和近缘物种SSR引物的引用等3种方法开发龙须草的SSR引物.结果显示:在锚定PCR开发复合SSR位点所设计的33对引物中筛选到有效扩增引物13对,表现多态性的引物有6对;利用基因特异序列法得到多态性引物3个;所用18条同源物种引物中有83%的SSR引物在供试的12个龙须草居群中都有清晰的SSR条带,其中56%的引物表现出多态性.3种方法在龙须草12个居群中共筛选得到了19对多态性引物,多态性比率约为73%.利用筛选得到的SSR多态性引物对来自2个居群的杂交F1代进行检测,鉴别出只有母本带型的无融合生殖后代和具有双亲带型的杂交后代,在子代中还出现偏父本带型、亲本缺失带型和变异带型等,证明所开发的SSR引物可以揭示龙须草生殖方式的复杂性,适用于龙须草遗传分析及亲缘关系鉴定.     

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.     

Ploidy level selection during germination and early stages of seedling growth in the progeny of allohexaploid facultative apomict,Hieracium rubrum (Asteraceae)

Selection within progeny of a facultative apomict,Hieracium rubrum, was studied using flow cytometry of embryos in seeds (a modified method of Flow Cytometric Seed Screen) and seedlings. Flow cytometric screening of particular progeny classes was based on distinct ploidy categories, reflecting the way of their origin. The results of both estimations of progeny composition significantly differed, which makes direct comparison of proportions detected in seeds or seedling stage impossible. The results suggest that progeny originating from reduced egg cells have higher mortality during germination and the early establishment phase than that from unreduced egg cells. Within the progeny of emasculated plants, the proportion of polyhaploid progeny decreased in favor of apomictically derived plants. Within the progeny of plants pollinated byH. pilosella, the proportion of polyhaploid progeny decreased significantly in favor of apomictically derived plants and 2n + n hybrids. It is argued that at least a proportion of the sexually derived progeny of this facultatively apomictic maternal parent has a lower survival rate than apomictically derived progeny.     

Somatic chimerism, genetic inheritance, and mapping of the fleshless berry (flb) mutation in grapevine (Vitis vinifera L.).

The fleshless berry (flb) mutation of grapevine (Vitis vinifera L. 'Ugni Blanc') impairs the differentiation and division of inner mesocarp cells responsible for flesh in grapevine berries. In order to study the inheritance of the mutation and to map the flb locus, 5 segregating populations were created. Progeny plants were classified as mutant or wild type by scoring for the presence of an ovary phenotype associated with the Flb- phenotype at anthesis. Phenotypic segregation revealed the involvement of a single dominant allele that was heterozygous in the original mutant. Through bulk segregant analysis, microsatellite (simple sequence repeat (SSR)) markers linked to the mutation were identified, and the flb locus was assigned to linkage group 18. The locus position was then refined by analyzing individual progeny and the segregation of SSR markers in the target region with the closest marker 5.6 cM distant from the flb locus. All progeny with the Flb- ovary phenotype differed from the original fleshless berry mutant in that no berries formed after anthesis. Our data suggest that the original mutant plant was a chimera with the mutated allele present in only 1 cell layer (L2 layer) of the ovary and berry.     

Paternity and ploidy segregation of progenies derived from tetraploid Malus xiaojinensis

Apomixis is usually subdivided into parthenogenesis, apogamy, and apospory on the basis of different mechanisms of development and cytogenetic effects. Although most plants and animals undergo sexual reproduction to multiply, it is important to understand the role of apomixis in plant reproduction because of its useful applications in the breeding and propagation of some crops. Tetraploid Malus xiaojinensis Cheng et Jiang is a typical facultative apomictic plant species. Paternity and ploidy analyses of M. xiaojinensis seedlings were performed with flow cytometry in conjunction with microsatellite and single nucleotide polymorphism markers. The proportion of apomictic derived progeny of M. xiaojinensis × Malus baccata was 54.05, 53.96, and 43.55 % from 2008 to 2010, respectively. The progeny of M. xiaojinensis × M. baccata comprised hybrids of 2x, 3x, 4x, or 5x ploidy, whereas apomictic offspring were 2x, 3x, or 4x. Among the apomictic derived progeny, the ploidy of restituted apomictic progeny was 2x, 3x, or 4x, whereas that of nonrestituted apomictic progeny was 4x only. The proportions of the different types of apomictic derived progeny differed among the years 2008 to 2010. The ploidy of progeny from emasculated flowers also segregated into 2x, 3x, and 4x individuals. These results indicate that both parthenogenesis and unreduced meiotic diplospory play important roles in apomictic reproduction in M. xiaojinensis.     

Expression of lorelei-like genes in aposporous and sexual Paspalum notatum plants

Gametophytic apomictic plants form non-reduced embryo sacs that generate clonal embryos by parthenogenesis, in the absence of both meiosis and egg-cell fertilization. Here we report the sequence and expression analysis of a lorelei-like Paspalum notatum gene, n20gap-1, which encodes a GPI-anchored protein previously associated with apomixis in this species. Phylogeny trees showed that n20gap-1 was evolutionary related to the Arabidopsis thaliana lorelei genes At4g26466 and At5g56170. The lorelei At4g26466 disruption was shown to be detrimental to sperm cell release in arabidopsis. RFLP (Restriction Fragment Length Polymorphism) analysis revealed the occurrence of several homologous sequences in the Paspalum notatum genome, exhibiting polymorphisms genetically linked to apomixis. Real-time PCR showed that lorelei-family genes present a minor activity peak at pre-meiosis and a major one at anthesis. The apomictic genotype analyzed showed a significantly increased activity at pre-meiosis, post-meiosis and anthesis with respect to a sexual genotype. In situ hybridization assays revealed expression in integuments, nucellus and the egg-cell apparatus. Several n20gap-1 alleles differing mainly at the 3' UTR sequence were identified. Allele-specific real-time PCR experiments showed that allele 28 was significantly induced in reproductive tissues of the apomictic genotype with respect to the sexual genotype at anthesis. Our results indicate that P. notatum lorelei-like genes are differentially expressed in representative sexual (Q4188) and apomictic (Q4117) genotypes, and might play a role in the final stages of the apomixis developmental cascade. However, the association of n20gap-1 expression with the trait should be confirmed in significant number of sexual and apomictic genotypes.     

EVIDENCE OF PARTIAL APOMIXIS IN ANTENNARIA MEDIA (ASTERACEAE: INULEAE) DETECTED BY THE SEGREGATION OF GENETIC MARKERS

The interplant variation in sexual and asexual reproduction in an Oregon population of the alpine perennial Antennaria media was investigated. Four polymorphic loci were assayed by enzyme electrophoresis of the progeny of 72 families from two subpopulations of A. media. The population was divided into two spatially distinct subpopulations. A multilocus model, incorporating a mixture of apomixis and random outcrossing, was used to estimate the mating system of pistillate plants both on the population and individual levels with statistical significance of the estimates based on bootstrap methods. The population contained a mixture of sexual individuals, partial apomicts, and obligate apomicts. The first subpopulation contained individuals that were partially apomictic and presumably produced both reduced and unreduced embryo sacs. There was a conspicuous difference in the breeding system composition between the two subpopulations. The first subpopulation had a “female” biased gender ratio and contained mostly obligate apomicts, some partial apomicts, and some outcrossing amphimicts. The second subpopulation, which had a nearly balanced gender ratio, contained mostly amphimicts, some obligate apomicts, but no facultative apomicts. This is the first study to document partial apomixis in individual plants by the use of genetic markers.     

Use of the SSLP-based method for detection of rare apomictic events in a sexual AtSERK1 transgenic Arabidopsis population

Here we present a screening method to evaluate the potential of genes to transfer aspects of apomixis into sexual crop plants. Based on the assumption that an apomictic progeny is an exact genetic replica of the mother plant we employed a set of single sequence length polymorphism (SSLP) markers to identify individuals displaying heterozygosity fixation in segregating sexual populations as an indication of rare apomictic events. Here we present the results of such a study using the Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (AtSERK1) gene expressed under the control of the AtLTP1 promoter in sexual Arabidopsis plants. In one of the three tested F2 transgenic populations expressing the AtLTP1::AtSERK1 construct we observed two plants with heterozygosity maintenance for the full set of SSLP markers indicating a possible clonal inheritance. However, as their offspring revealed a close to binomial segregation for a number of heterozygous loci, it was concluded that these two putative apomictic plants either lost their clonal ability in the next generation or resulted from incidental recombination events displaying the genotype of the parent. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Apomixis in Coix aquatica Roxb

When plants of Coix aquatica Roxb. were grown in isolation orbagged, with removal of staminate spikelets several producedone or two seeds, and one plant formed several seeds. Thesewere presumably formed through apomixis, of the autogamous type.Apomixis occurs side by side with sexual reproduction, and istherefore facultative. The fact that one of the plants grownunder the same conditions had higher apomictic seed set thanothers, and both its apomictic and selfed progeny also showedhigher apomictic seed formation suggests that these have greaterapomictic potentialities than others. Genetic analysis of apomixissuggests that it is recessive to sexuality, and is probablygoverned by a number of genes. A few triploids tested did notshow any apomictic seed set indicating that polyploidy per semay not be responsible for initiation of apomixis. Except thatit is a diploid, C. aquatica seems to fulfil the criteria forapomixis, yet it reproduces predominantly by sexual means.     

The chromosome segment related to apomixis in Paspalum simplex is homoeologous to the telomeric region of the long arm of rice chromosome 12

Apomixis is a form of asexual reproduction that in plants leads to the production of seed progeny that are exact copies of the mother individual. A mapping population generated by backcrossing a sexual with an apomictic genotype of Paspalum simplex, both at the tetraploid level, was used to find markers co-segregating with apomixis. Genetic analysis showed that apomixis is under the control of a single dominant allele assuming a random chromatid assortment. Five rice markers, mapped in the telomeric region of the long arm of rice chromosome 12, showed tight linkage with apomixis. Genetic and molecular data strongly indicate that the potentiality to express apomixis in P. simplex is given by a relatively large chromosome segment that is inherited as a single genetic unit.