Apomictic Species Research Articles

Optimization of protein extraction and proteomic studies in Cenchrus polystachion (L.) Schult

Apomicts have been studied at their genetic levels, but there are no any direct evidence of its mechanism. In order to understand the mechanism involved, a close relative of Pennisetum, Cenchrus polystachion, an apomictic species was explored for more insights into protein expression in reproductive structures. Optimization of protein extraction was studied with the leaf tissue and optimized protocol was extrapolated to other five tissues. The phenol-based protein extraction emerged as the best method for plant leaf tissue providing a better protein yield, separation of bands, removal of non-protein components like polyphenolic compounds and nucleic acids. The proteome analysis of leaf, stigma, immature ovary, seed, anther sac and pollen tissues of Cenchrus polystachion were carried out identifying a total of 135407 proteins against the Poaceae database from UNIPROT/TrEMBL. The target candidate proteins found in all the tissues were identified and mainly comprised of Actin Protein, PIP, Starch Synthase, ATP Synthase, Glutathione S Transferase, Dehydroascorbate reductase, Ascorbate peroxidase and heat shock proteins. Visualization and descriptive statistics conveyed all the necessary information to understand the differential expression of proteins in Cenchrus polystachion. This study forms a base to understand the role of tissue specific expressed proteins in an apomictic plant.

An unexpected new diploid Hieracium from Europe: Integrative taxonomic approach with a phylogeny of diploid Hieracium taxa

AbstractHieracium s.str. (Asteraceae) is one of the largest angiosperm genera notorious for its taxonomic complexity caused by widespread interspecific hybridization. This process is tightly coupled with polyploidization and apomixis – asexual reproduction by seeds, which has ensured the persistence of otherwise sterile interspecific hybrids. As a result, apomictic polyploid taxa dominate the taxonomic diversity of the genus whilst sexual diploid species are extremely rare and mostly confined to southern Europe. As diploid taxa are assumed to be the parents of apomictic lineages, the discovery of any new diploid species is important for understanding evolutionary processes and diversity patterns in the genus. Here, we describe a new diploid species, Hieracium vranceae, a narrow endemic to the Munţii Vrancei (Vrancea Mountains, Eastern Carpathians, Romania). This taxon with a distinctive morphology and a strong affinity to relic rocky habitats was first collected in 2013. This means that, even in Europe, some regions, like the Eastern Carpathians, are botanically underexplored and might still preserve an undiscovered diversity of plants. Phylogenetic analyses with multiple molecular markers (low‐copy nuclear genes gsh1 and sqs, nuclear ribosomal ETS, four cpDNA loci) applied to all diploid Hieracium species support specific rank for H. vranceae. In contrast, molecular data suggest conspecifity of two pairs of species, H. lucidum/H. cophanense from Sicily and H. naegelianum/H. renatae from the Balkans, which are moreover only weakly differentiated morphologically. Molecular (cpDNA) and cytogenetic (GISH, FISH) analyses furthermore revealed that H. vranceae has been involved as a maternal parent in the origin of an allotriploid apomictic species, H. telekianum, to which H. vranceae contributed one haplome. The other putative parents of H. telekianum are the diploid narrow endemic H. pojoritense and probably H. sparsum s.l. Our data thus stress the importance of interspecific hybridization for an evolutionary shift from sexuality to apomixis in Hieracium. Our findings furthermore highlight the significance of that part of the Carpathians as one of the most important evolutionary/refugial centres of Hieracium and suggest dynamic species ranges at a regional scale allowing physical contact of taxa whose distributional ranges are nowadays completely allopatric.

Genetic polymorphism in Chondrilla (Asteraceae) in southern European Russia and the nature of Chondrilla juncea L

We studied genetic diversity in 54 populations of nine sexual and apomictic species of the genus Chondrilla (C. acantholepis, C. ambigua, C. brevirostris, C. canescens, C. graminea, C. juncea, C. laticoronata, C. latifolia and C. pauciflora) in SE European Russia and neighboring territories of NW Kazakhstan. We analysed the trnT–trnF region of plastid DNA and the internal transcribed spacer of ribosomal DNA (ITS1–5.8S–ITS2) using statistical parsimony, maximum likelihood and neighbor net methods. Two major evolutionary lineages, roughly corresponding to the two subgenera traditionally recognized in the region, were revealed. Within the first evolutionary lineage (subgenus Brachyrhynchus), the sexual diploid C. ambigua and its putatively hybrid apomictic derivatives C. brevirostris, C. laticoronata and C. pauciflora could be recognized. Their identity was also confirmed by analyses of ISSR markers. The second evolutionary lineage (subgenus Chondrilla) comprises C. juncea, C. acantholepis, C. canescens, C. graminea and C. latifolia in European Russia, but analyses of morphological variability and the genealogy of plastid and nuclear markers favor their treatment as the single facultatively apomictic species C. juncea. The results demonstrate that an apomictic mode of reproduction does not necessarily result in the formation of genetically separated microspecies.

Plant apomixis is rare in Himalayan high-alpine flora

Gametophytic apomixis is a way of asexual plant reproduction by seeds. It should be advantageous under stressful high altitude or latitude environment where short growing seasons, low temperatures, low pollinator activity or unstable weather may hamper sexual reproduction. However, this hypothesis remains largely untested. Here, we assess the reproductive mode in 257 species belonging to 45 families from the world’s broadest alpine belt (2800–6150 m) in NW Himalayas using flow cytometric seed screen. We found only 12 apomictic species, including several members of Poaceae (Festuca, Poa and Stipa), Rosaceae (Potentilla) and Ranunculaceae (Halerpestes, Ranunculus), which are families typical for high apomict frequency. However, several apomictic species were newly discovered, including the first known apomictic species from the family Biebersteiniaceae (Biebersteinia odora), and first apomicts from the genera Stipa (Stipa splendens) and Halerpestes (Halerpestes lancifolia). Apomicts showed no preference for higher elevations, even in these extreme Himalayan alpine habitats. Additional trait-based analyses revealed that apomicts differed from sexuals in comprising more rhizomatous graminoids and forbs, higher soil moisture demands, sharing the syndrome of dominant species with broad geographical and elevation ranges typical for the late-successional habitats. Apomicts differ from non-apomicts in greater ability of clonal propagation and preference for wetter, more productive habitats.

In vitro morphogenetic responses from obligatory apomictic Taraxacum belorussicum Val. N. Tikhom seedlings explants

Taraxacum belorussicum Val. N. Tikhom, a poorly known and obligatory apomictic species, is an attractive plant material for studying the embryological, genetic and molecular mechanisms of apomixis. This work aims to obtain an efficient protocol for Taraxacum belorussicum regeneration. Four types of explants (cotyledons, hypocotyls, meristems and roots) that were taken from 2-weeks-old seedlings were used for in vitro cultures, and a fast and efficient protocol of T. belorussicum regeneration was obtained. Various ½ MS-based media containing IAA (5.71 µM), TDZ (4.54 µM) and PSK (100 nM) were chosen to assess the morphogenetic abilities of selected T. belorussicum explants. Studies on the role of PSK were done in three independent experiments, where the most significant factors were always light and darkness. All explants produced callus by the third day of culture and adventitious shoots after 7 days, although in an asynchronous indirect manner, and with different intensities for all explant types. The most preferred medium culture for hypocotyl, cotyledon and meristem explants was ½ MS + TDZ, and ½ MS + IAA + TDZ + PSK for roots which were the only explant sensitive to PSK. A short darkness pretreatment (8 days) in PSK medium was found suitable to enhance organogenesis. Secondary organogenesis was observed for regenerated plants on meristem explants from the ½ MS + IAA + TDZ + PSK medium. A weak somatic embryogenesis was observed for hypocotyl and cotyledon explants from ½ MS + IAA + TDZ and ½ MS + IAA + TDZ + PSK media. Histological and scanning electron microscope images (SEM) of T. belorussicum confirmed indirect organogenesis and somatic embryogenesis. Plant material treated with aniline blue solution revealed the presence of callose in the cell walls of cotyledon and hypocotyl explants. The presence of extracellular matrix (ECM) and heterogenic structure of callus was also verified by scanning electron microscopy and light microscopy, confirming the high morphogenetic ability of T. belorussicum.

A High-Density Linkage Map of the Forage Grass Eragrostis curvula and Localization of the Diplospory Locus.

Eragrostis curvula (Schrad.) Nees (weeping lovegrass) is an apomictic species native to Southern Africa that is used as forage grass in semiarid regions of Argentina. Apomixis is a mechanism for clonal propagation through seeds that involves the avoidance of meiosis to generate an unreduced embryo sac (apomeiosis), parthenogenesis, and viable endosperm formation in a fertilization-dependent or -independent manner. Here, we constructed the first saturated linkage map of tetraploid E. curvula using both traditional (AFLP and SSR) and high-throughput molecular markers (GBS-SNP) and identified the locus controlling diplospory. We also identified putative regulatory regions affecting the expressivity of this trait and syntenic relationships with genomes of other grass species. We obtained a tetraploid mapping population from a cross between a full sexual genotype (OTA-S) with a facultative apomictic individual of cv. Don Walter. Phenotypic characterization of F1 hybrids by cytoembryological analysis yielded a 1:1 ratio of apomictic vs. sexual plants (34:27, X2 = 0.37), which agrees with the model of inheritance of a single dominant genetic factor. The final number of markers was 1,114 for OTA-S and 2,019 for Don Walter. These markers were distributed into 40 linkage groups per parental genotype, which is consistent with the number of E. curvula chromosomes (containing 2 to 123 markers per linkage group). The total length of the OTA-S map was 1,335 cM, with an average marker density of 1.22 cM per marker. The Don Walter map was 1,976.2 cM, with an average marker density of 0.98 cM/marker. The locus responsible for diplospory was mapped on Don Walter linkage group 3, with other 65 markers. QTL analyses of the expressivity of diplospory in the F1 hybrids revealed the presence of two main QTLs, located 3.27 and 15 cM from the diplospory locus. Both QTLs explained 28.6% of phenotypic variation. Syntenic analysis allowed us to establish the groups of homologs/homeologs for each linkage map. The genetic linkage map reported in this study, the first such map for E. curvula, is the most saturated map for the genus Eragrostis and one of the most saturated maps for a polyploid forage grass species.

Extending the range of Rubus ser. Micantes (Rosaceae) southward: Rubus vallis-cembrae, a unique new species from Italy

Rubus ser. Micantes Sudre (Rosaceae) includes apomictic polyploid species of hybrid origin, which occur in north-western and central Europe, with rare outposts south-east of the Alps. The first regionally distributed representative of the series occurring in Italy, Rubus vallis-cembrae Prosser & Kiraly, is described here as a morphologically unique, isolated tetraploid species. The new species is morphologically compared with related taxa of the series. Furthermore, comprehensive iconography and data on distribution and ecology are presented.Citation: Prosser F. & Kiraly G. 2019: Extending the range of Rubus ser. Micantes (Rosaceae) southward: Rubus vallis-cembrae, a unique new species from Italy. – Willdenowia 49: 167–175. doi: https://doi.org/10.3372/wi.49.49205Version of record first published online on 27 June 2019 ahead of inclusion in August 2019 issue.

Ecological, genetic, and reproductive features of Cenchrus species indicate evolutionary superiority of apomixis under environmental stresses

Many Cenchrus species are resilient to harsh environmental conditions and reproduce through apomixis (asexual seed production), and the resulting progeny is genetically identical to the mother plant. Although the apomictic mode of reproduction restricts the efforts of plant breeders to pyramiding desirable traits, it facilitates the fixation of genetic combinations over multiple generations. Eight Cenchrus species are found in India of which only four apomictic species are grown in sown pastures. We assessed the variability in the morphological, behavioral, ecological, reproductive, genetic and molecular features among the different Cenchrus species, and envisaged a relationship between apomixis and stress tolerance for survival and species propagation under unfavorable ecological conditions. Our findings revealed considerable variability among the Cenchrus species. Molecular diversity analysis grouped the Cenchrus species into two distinct clusters. The apomictic species exhibited a lower diversity than the sexual species. Reproductive pathway diversity (BII and BIII seed formation) among the apomictic Cenchrus species indicated segregation of the components of apomixis, namely parthenogenesis, apomeiosis and functional endosperm formation. For the first time we report partitioning of the components of apomixis in C. glaucus. The earlier reported correlation between apomixis and polyploidy was not observed in the genus Cenchrus. Apomictic Cenchrus species are usually perennial, highly productive and invasive, and adapted to yearly fluctuations in the weather. Sexual Cenchrus species are usually annual, relatively less productive and do not survive during winter. Therefore, we concluded that apomixis facilitates the expansion of species by enabling reproduction even under stressful conditions. The information on genetic and reproductive diversity among the Cenchrus species presented in this study might be useful for interspecific hybridization among these species, as well as to decipher the molecular basis of apomixis in plants.

Transference of natural diversity from the apomictic germplasm of Paspalum notatum to a sexual synthetic population

AbstractGenetic improvement in apomictic forage species has been restricted because of the absence of genetic variability in sexual germplasm with the same ploidy level. Following a new breeding scheme, a sexual synthetic tetraploid population (SSTP) of Paspalum notatum has been generated. The objectives of this work were: (a) to evaluate the genetic variability in SSTP by means of molecular markers, morphologic and agronomic traits, and seed fertility and quality traits and (b) to assess the transference of genetic variability from the apomictic germplasm to the sexual one. Molecular markers revealed a twofold higher level of variability in the SSTP in comparison with the sexual germplasm utilised for its generation, and similar levels with the apomictic ones; moreover, markers showed that most of the variability was inherited from the apomictic germplasm. Morphologic and agronomic traits and seed fertility and quality traits showed high levels of variation in the three groups of genotypes indicating that the new breeding scheme was effective in transferring variability from the apomictic germplasm to the SSTP. This new population will be useful in breeding of P. notatum, and the breeding scheme used for its generation may be used in other apomictic species.

The morphometrics of autopolyploidy: insignificant differentiation among sexual-apomictic cytotypes.

Polyploidization of the plant genome affects the phenotype of individuals including their morphology, i.e. size and form. In autopolyploids, we expect mainly nucleotypic effects, from a number of monoploid genomes (i.e. chromosome sets) or genome size, seen from an increase in size or dimension of the polyploids compared with the diploids (or lower ploids). To identify nucleotypic effects, confounding effects of hybridity (observed in allopolyploids), postpolyploidization processes or environmental effects need to be considered. We morphometrically analysed five ploidy cytotypes of the sexual–apomictic species Potentilla puberula cultivated ex situ under the same experimental conditions. Sexuals are mainly tetraploid, while higher ploidy (penta- to octoploidy) is typically associated with the expression of apomixis. The cytotypes likely arose via autopolyploidization although historic involvement of another species in the origin of apomicts cannot be fully ruled out, suggested by a slight molecular differentiation among reproductive modes. We (i) revisited molecular differentiation using amplified fragment length polymorphisms and performed a morphometric analysis to test (ii) if cytotypes are morphologically differentiated from each other and (iii) if the size of individuals is related to their ploidy. Weak molecular differentiation of sexual versus apomictic individuals was confirmed. Cytotypes and reproductive modes were also morphologically poorly differentiated from each other, i.e. apomicts largely resampled the variation of the sexuals and did not exhibit a unique morphology. Overall size of individuals increased moderately but significantly with ploidy (ca. 14 % in the comparison of octo- with tetraploids). The results support an autopolyploid origin of the P. puberula apomicts and suggest a nucleotypic effect on overall plant size. We discuss taxonomic consequences of the results in the context of data on reproductive relationships among cytotypes and their ecological preferences and evolutionary origin, and conclude that cytotypes are best treated as intraspecific variants within a single species.

The Boechera Genus as a Resource for Apomixis Research.

The genera Boechera (A. Löve et D. Löve) and Arabidopsis, the latter containing the model plant Arabidopsis thaliana, belong to the same clade within the Brassicaceae family. Boechera is the only among the more than 370 genera in the Brassicaceae where apomixis is well documented. Apomixis refers to the asexual reproduction through seed, and a better understanding of the underlying mechanisms has great potential for applications in agriculture. The Boechera genus currently includes 110 species (of which 38 are reported to be triploid and thus apomictic), which are distributed mostly in the North America. The apomictic lineages of Boechera occur at both the diploid and triploid level and show signs of a hybridogenic origin, resulting in a modification of their chromosome structure, as reflected by alloploidy, aneuploidy, substitutions of homeologous chromosomes, and the presence of aberrant chromosomes. In this review, we discuss the advantages of the Boechera genus to study apomixis, consider its modes of reproduction as well as the inheritance and possible mechanisms controlling apomixis. We also consider population genetic aspects and a possible role of hybridization at the origin of apomixis in Boechera. The molecular tools available to study Boechera, such as transformation techniques, laser capture microdissection, analysis of transcriptomes etc. are also discussed. We survey available genome assemblies of Boechera spp. and point out the challenges to assemble the highly heterozygous genomes of apomictic species. Due to these challenges, we argue for the application of an alternative reference-free method for the comparative analysis of such genomes, provide an overview of genomic sequencing data in the genus Boechera suitable for such analysis, and provide examples of its application.

The Occurrence of Seedlessness in Higher Plants; Insights on Roles and Mechanisms of Parthenocarpy.

Parthenocarpy in a broad sense includes those processes that allow the production of seedless fruits. Such fruits are favorable to growers, because they are set independently of successful pollination, and to processors and consumers, because they are easier to deal with and to eat. Seedless fruits however represent a biological paradox because they do not contribute to offspring production. In this work, the occurrence of parthenocarpy in Angiosperms was investigated by conducting a bibliographic survey. We distinguished monospermic (single seeded) from plurispermic (multiseeded) species and wild from cultivated taxa. Out of 96 seedless taxa, 66% belonged to plurispermic species. Of these, cultivated species were represented six times higher than wild species, suggesting a selective pressure for parthenocarpy during domestication and breeding. In monospermic taxa, wild and cultivated species were similarly represented. The occurrence of parthenocarpy in wild species suggests that seedlessness may have an adaptive role. In monospermic species, seedless fruits are proposed to reduce seed predation through deceptive mechanisms. In plurispermic fruit species, parthenocarpy may exert an adaptive advantage under suboptimal pollination regimes, when too few embryos are formed to support fruit growth. In this situation, parthenocarpy offers the opportunity to accomplish the production and dispersal of few seeds, thus representing a selective advantage. Approximately 20 sources of seedlessness have been described in tomato. Excluding the EMS induced mutation parthenocarpic fruit (pat), the parthenocarpic phenotype always emerged in biparental populations derived from wide crosses between cultivated tomato and wild relatives. Following a theory postulated for apomictic species, we argument that wide hybridization could also be the force driving parthenocarpy, following the disruption of synchrony in time and space of reproductive developmental events, from sporogenesis to fruit development. The high occurrence of polyploidy among parthenocarpic species supported this suggestion. Other commonalities between apomixis and parthenocarpy emerged from genetic and molecular studies of the two phenomena. Such insights may improve the understanding of the mechanisms underlying these two reproductive variants of great importance to modern breeding.

Cytogenetic relationships, polyploid origin and taxonomic issues in Paspalum species: inter- and intraspecific hybrids between a sexual synthetic autotetraploid and five wild apomictic tetraploid species.

Paspalum is a noteworthy grass genus due to the forage quality of most species, with approximately 330 species, and the high proportion of those that reproduce via apomixis. Harnessing apomictic reproduction and widening knowledge about the cytogenetic relationships among species are essential tools for plant breeding. We conducted cytogenetic analyses of inter- and intraspecific hybridisations involving a sexual, colchicine-induced autotetraploid plant of P. plicatulum Michx. and five indigenous apomictic tetraploid (2n=40) species: P. compressifolium Swallen, P. lenticulare Kunth, two accessions of P. nicorae Parodi, P. rojasii Hack. and two accessions of P.plicatulum. Fertility of the hybrids was investigated and their reproductive system was analysed considering the relative embryo:endosperm DNA content from flow cytometry. Morphological, nomenclatural and taxonomic issues were also analysed. Cytogenetic analysis suggested that all indigenous tetraploid accessions of five apomictic species are autotetraploid or segmental allotetraploid. If segmental allotetraploids, they probably originated through autoploidy followed by diploidisation processes. Autosyndetic male chromosome pairing observed in all hybrid families supported this assertion. Allosyndetic chromosome associations were also observed in all hybrid families. In the hybrids, the proportion of male parent chromosomes involved in allosyndesis per pollen mother cell varied from 5.5% to 35.0% and the maximum was between 25% and 60%. The apomictic condition of the indigenous male parents segregated in the hybrids. These results confirm a strong association between autoploidy and apomixis in Paspalum, and the existence of cytogenetic relationships between different species of the Plicatula group. Allosyndetic chromosome pairing and seed fertility of the hybrids suggest the feasibility of gene transfer among species.

Multiple independent origins of intermediate species between Sorbus aucuparia and S. hybrida (Rosaceae) in the Baltic region

Populations intermediate between the diploid and sexual Sorbus aucuparia and the tetraploid and facultative apomictic Sorbus hybrida are scattered in coastal regions of southern Scandinavia. Our aims were to investigate whether these populations were of local and independent origins, whether they were morphologically and molecularly distinct from each other and whether they could give rise to constant offspring by apomixis. Six intermediate populations from the Baltic Sea basin were studied for variation at nuclear and plastid microsatellite loci, for morphological differentiation patterns, and for ploidy level by flow cytometry. Seeds were sown to check for constancy with the mother plants. Three populations from Gotland and possibly the small population from Uppland were in each case fixed for a unique combination of genetic markers, had restricted variation in leaf shape and were morphologically divergent from each other. Flow cytometry analysis showed that the four genetically monomorphic populations were consistently triploid. Cultivation showed that offspring from trees within two of the monomorphic populations had leaf shapes closely similar to the mother individuals. In contrast, a population from Gotland and a population from Åland were variable at molecular marker loci and were variable in leaf shape. The three monomorphic Gotland populations and possibly also the Uppland population propagate asexually by apomixis, and they have independent origins. The two variable populations are apparently composed of hybrids that have been formed repeatedly and independently between individuals of the two parental species growing at the same sites. One of the Gotland apomicts has already been described as Sorbus teodori. Sorbus faohraei Hedrén & J.Levin and Sorbus atrata Hedrén & J.Levin are described as new apomictic species from Gotland.

Mendelian segregation of leaf phenotypes in experimental F2hybrids elucidates origin of morphological diversity of the apomicticRanunculus auricomuscomplex

AbstractApomictic plant complexes exhibit a stunning morphological diversity. TheRanunculus auricomuscomplex with more than 800 morphologically recognized asexual species and just 4 known sexual species is a model group to study origins of high morphological diversity in asexually reproducing lineages. During the Pleistocene, the sexuals of theR. auricomuscomplex diverged in Central Europe into two morphological groups of contrasting basal leaf phenotypes, the undivided forms represented by diploidR. carpaticolaand the deeply divided forms represented by diploidR. notabilis. An experimental interspecific crossing ofR. carpaticola×R. notabilisrevealed an extensive phenotypic diversity of the sexually formed F2hybrid generation. We hypothesize that apomictic species of the EuropeanR. auricomuscomplex might have established from hybrid derivatives ofcarpaticola‐andnotabilis‐like sexual ancestors. Here we investigate segregation of F2basal leaf phenotypes from experimentalR. carpaticola×R. notabiliscrosses. We tested the hypothesis whether F2hybrid leaf phenotypes segregate in a Mendelian manner and whether any segregant phenotypes matchR. variabilis, an apomictic species and natural hybrid derivative ofR. notabilis. Using landmark‐based geometric morphometrics of basal leaves, we recognized two major morphological clusters within theR. carpaticola×R. notabilisF2hybrid population. Thecarpaticola‐like morphological cluster included undivided or less‐divided leaf phenotypes without lateral sinuses and thenotabilis‐like morphological cluster contained deeply divided leaf phenotypes with 1–2 lateral sinuses. The overall ratio of the both morphological clusters within the F2hybrid population was approximately 1 : 1, suggesting Mendelian inheritance and partial dominance of the allele for the divided leaf phenotype. The morphological cluster of divided leaf phenotypes included 11% of F2individuals with a higher similarity to the apomictic speciesR. variabilisthan to the parental sexuals. Thevariabilis‐like F2segregant phenotypes often resulted from F1 crosses ofcarpaticola‐like mother plants withnotabilis‐like pollen donors; such crossing lines reached the highest morphological disparity. Equivalent hybridizations in nature followed by Mendelian segregation of phenotypes, which became later on fixed via the shift to apomictic reproduction, might have established the extant diversity of apomictic complexes. Microsatellite genotyping with six SSR loci revealed no correlation of genetic and phenotypic variation. We conclude that former “morpho‐groups” or “main species” based on phenotypic similarity only are unsuitable units for taxonomic classification.

Establishment of Apomixis in Diploid F2 Hybrids and Inheritance of Apospory From F1 to F2 Hybrids of the Ranunculus auricomus Complex.

Hybridization and polyploidization play important roles in plant evolution but it is still not fully clarified how these evolutionary forces contribute to the establishment of apomicts. Apomixis, the asexual reproduction via seed formation, comprises several essential alterations in development compared to the sexual pathway. Furthermore, most natural apomicts were found to be polyploids and/or hybrids. The Ranunculus auricomus complex comprises diploid sexual and polyploid apomictic species and represents an excellent model system to gain knowledge on origin and evolution of apomixis in natural plant populations. In this study, the second generation of synthetically produced homoploid (2x) and heteroploid (3x) hybrids derived from sexual R. auricomus species was analyzed for aposporous initial cell formation by DIC microscopy. Complete manifestation of apomixis was determined by measuring single mature seeds by flow cytometric seed screen. Microscopic analysis of the female gametophyte formation indicated spontaneous occurrence of aposporous initial cells and several developmental irregularities. The frequency of apospory was found to depend on dosage effects since a significant increase in apospory was observed, when both F1 parents, rather than just one, were aposporous. Other than in the F1 generation, diploid Ranunculus F2 hybrids formed BIII seeds and fully apomictic seeds. The results indicate that hybridization rather than polyploidization seems to be the functional activator of apomictic reproduction in the synthetic Ranunculus hybrids. In turn, at least two hybrid generations are required to establish apomictic seed formation.

Asexual Female Gametogenesis Involves Contact with a Sexually-Fated Megaspore in Apomictic Hieracium.

Apomixis results in asexual seed formation where progeny are identical to the maternal plant. In ovules of apomictic species of the Hieracium subgenus Pilosella, meiosis of the megaspore mother cell generates four megaspores. Aposporous initial (AI) cells form during meiosis in most ovules. The sexual pathway terminates during functional megaspore (FM) differentiation, when an enlarged AI undergoes mitosis to form an aposporous female gametophyte. Then, the mitotically programmed FM dies along with the three other megaspores by unknown mechanisms. Transcriptomes of laser-dissected AIs, ovule cells, and ovaries from apomicts and AI-deficient mutants were analyzed to understand the pathways involved. The steps leading to AI mitosis and sexual pathway termination were determined using antibodies against arabinogalactan protein epitopes found to mark both sexual and aposporous female gametophyte lineages at inception. At most, four AIs differentiated near developing megaspores. The first expanding AI cell to contact the FM formed a functional AI that underwent mitosis soon after megaspore degeneration. Transcriptome analyses indicated that the enlarged, laser-captured AIs were arrested in the S/G2 phase of the cell cycle and were metabolically active. Further comparisons with AI-deficient mutants showed that AIs were enriched in transcripts encoding homologs of genes involved in, and potentially antagonistic to, known FM specification pathways. We propose that AI and FM cell contact provides cues required for AI mitosis and megaspore degeneration. Specific candidates to further interrogate AI-FM interactions were identified here and include Hieracium arabinogalactan protein family genes.

Assembly of the Boechera retrofracta Genome and Evolutionary Analysis of Apomixis-Associated Genes.

Closely related to the model plant Arabidopsis thaliana, the genus Boechera is known to contain both sexual and apomictic species or accessions. Boechera retrofracta is a diploid sexually reproducing species and is thought to be an ancestral parent species of apomictic species. Here we report the de novo assembly of the B. retrofracta genome using short Illumina and Roche reads from 1 paired-end and 3 mate pair libraries. The distribution of 23-mers from the paired end library has indicated a low level of heterozygosity and the presence of detectable duplications and triplications. The genome size was estimated to be equal 227 Mb. N50 of the assembled scaffolds was 2.3 Mb. Using a hybrid approach that combines homology-based and de novo methods 27,048 protein-coding genes were predicted. Also repeats, transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were annotated. Finally, genes of B. retrofracta and 6 other Brassicaceae species were used for phylogenetic tree reconstruction. In addition, we explored the histidine exonuclease APOLLO locus, related to apomixis in Boechera, and proposed model of its evolution through the series of duplications. An assembled genome of B. retrofracta will help in the challenging assembly of the highly heterozygous genomes of hybrid apomictic species.

Rubus prissanicus (Rosaceae), a new bramble species from North West Poland

Rubus prissanicus, a new regional apomictic species, is described from north-western Poland. It is similar to R. lindleianus and R. langei, however it differs from the former species by broader leaflets and less numerous prickles on the inflorescence axis, and from the latter by unequally serrate leaflet margin, and from both these species by different types of trichomes on abaxial leaflet surface. Unlike other Polish species of R. ser. Rhamnifolii, in which the indumentum of abaxial leaf surface consists mainly of branched hairs, simple trichomes are the main component of indumentum of R. prissanicus leaves. The new species has been observed mainly on fertile soils, in broadleaf forest habitats, usually in fairly sunny places, along forest margins, by forest roads, in clearings, and in wayside thickets.

Discovery and Identification of Meloidogyne Species Using COI DNA Barcoding.

DNA barcoding with a new cytochrome oxidase c subunit 1 primer set generated a 721 to 724 bp fragment used for the identification of 322 Meloidogyne specimens, including 205 new sequences combined with 117 from GenBank. A maximum likelihood analysis grouped the specimens into 19 well-supported clades and four single-specimen lineages. The "major" tropical apomictic species ( Meloidogyne arenaria , Meloidogyne incognita , Meloidogyne javanica ) were not discriminated by this barcode although some closely related species such as Meloidogyne konaensis were characterized by fixed diagnostic nucleotides. Species that were collected from multiple localities and strongly characterized as discrete lineages or species include Meloidogyne enterolobii , Meloidogyne partityla , Meloidogyne hapla , Meloidogyne graminicola , Meloidogyne naasi , Meloidogyne chitwoodi , and Meloidogyne fallax . Seven unnamed groups illustrate the limitations of DNA barcoding without the benefit of a well-populated reference library. The addition of these DNA sequences to GenBank and the Barcode of Life Database (BOLD) should stimulate and facilitate root-knot nematode identification and provide a first step in new species discovery.