Evolution in the Cycles of Life.

The life cycles of eukaryotes alternate between haploid and diploid phases, which are initiated by meiosis and gamete fusion, respectively. In both ascomycete and basidiomycete fungi and chlorophyte algae, the haploid-to-diploid transition is regulated by a pair of paralogous homeodomain protein encoding genes. That a common genetic program controls the haploid-to-diploid transition in phylogenetically disparate eukaryotic lineages suggests this may be the ancestral function for homeodomain proteins. Multicellularity has evolved independently in many eukaryotic lineages in either one or both phases of the life cycle. Organisms, such as land plants, exhibiting a life cycle whereby multicellular bodies develop in both the haploid and diploid phases are often referred to as possessing an alternation of generations. We review recent progress on understanding the genetic basis for the land plant alternation of generations and highlight the roles that homeodomain-encoding genes may have played in the evolution of complex multicellularity in this lineage.

The novel expression of clonality following whole-genome multiplication compensates for reduced fertility in natural autopolyploids.

Exploring the fitness consequences of whole-genome multiplication (WGM) is essential for understanding the establishment of autopolyploids in diploid parental populations, but suitable model systems are rare. We examined the impact of WGM on reproductive traits in three major cytotypes (2x, 3x, 4x) of Pilosella rhodopea, a species with recurrent formation of neo-autopolyploids in mixed-ploidy populations. We found that diploids had normal female sporogenesis and gametogenesis, high fertility, and produced predominantly euploid seed progeny. By contrast, autopolyploids had highly disturbed developmental programs that resulted in significantly lower seed set and a high frequency of aneuploid progeny. All cytotypes, but particularly triploids, produced gametes of varying ploidy, including unreduced ones, that participated in frequent intercytotype mating. Noteworthy, the reduced investment in sexual reproduction in autopolyploids was compensated by increased production of axillary rosettes and the novel expression of two clonal traits: adventitious rosettes on roots (root-sprouting), and aposporous initial cells in ovules which, however, do not result in functional apomixis. The combination of increased vegetative clonal growth in autopolyploids and frequent intercytotype mating are key mechanisms involved in the formation and maintenance of the largest diploid-autopolyploid primary contact zone ever recorded in angiosperms.

An exonuclease V homologue is expressed predominantly during early megasporogenesis in apomictic Brachiaria brizantha.

MAIN CONCLUSION: An exonuclease V homologue from apomictic Brachiaria brizantha is expressed and localized in nucellar cells at key moments when these cells differentiate to give rise to unreduced gametophytes. Brachiaria is a genus of forage grasses with economical and agricultural importance to Brazil. Brachiaria reproduces by aposporic apomixis, in which unreduced embryo sacs, derived from nucellar cells, other than the megaspore mother cell (MMC), are formed. The unreduced embryo sacs produce an embryo without fertilization resulting in clones of the mother plant. Comparative gene expression analysis in ovaries of sexual and apomictic Brachiaria spp. revealed a sequence from B. brizantha that showed a distinct pattern of expression in ovaries of sexual and apomictic plants. In this work, we describe a gene named BbrizExoV with strong identity to exonuclease V (Exo V) genes from other grasses. Sequence analysis in signal prediction tools showed that BbrizExoV might have dual localization, depending on the translation point. A longer form to the nucleus and a shorter form which would be directed to the chloroplast. This is also the case for monocot sequences analyzed from other species. The long form of BbrizExoV protein localizes to the nucleus of onion epidermal cells. Analysis of ExoV proteins from dicot species, with exception of Arabidopsis thaliana ExoVL protein, showed only one localization. Using a template-based AlphaFold 2 modelling approach the structure of BbrizExoV in complex with metal and ssDNA was predicted based on the holo structure of the human counterpart. Features predicted to define ssDNA binding but a lack of sequence specificity are shared between the human enzyme and BbrizExoV. Expression analyses indicated the precise site and timing of transcript accumulation during ovule development, which coincides with the differentiation of nucelar cells to form the typical aposporic four-celled unreduced gametophyte. A putative function for this protein is proposed based on its homology and expression pattern.

TRIMETHYLGUANOSINE SYNTHASE1 mutations decanalize female germline development in Arabidopsis.

Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in Paspalum grasses. Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of TGS1 in female reproduction using reporter lines and loss-of-function mutants in Arabidopsis thaliana. pAtTGS1:AtTGS1 reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous-like lineage. Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants.

Identification of IPT9 in Brachiaria brizantha (syn. Urochloa brizantha) and expression analyses during ovule development in sexual and apomictic plants.

BACKGROUND: In Brachiaria sexual reproduction, during ovule development, a nucellar cell differentiates into a megaspore mother cell (MMC) that, through meiosis and mitosis, gives rise to a reduced embryo sac. In aposporic apomictic Brachiaria, next to the MMC, other nucellar cells differentiate into aposporic initials that enter mitosis directly forming an unreduced embryo sac. The IPT (isopentenyltransferase) family comprises key genes in the cytokinin (CK) pathway which are expressed in Arabidopsis during ovule development. BbrizIPT9, a B. brizantha (syn. Urochloa brizantha) IPT9 gene, highly similar to genes of other Poaceae plants, also shows similarity with Arabidopsis IPT9, AtIPT9. In this work, we aimed to investigate association of BbrizIPT9 with ovule development in sexual and apomictic plants. METHODS AND RESULTS: RT-qPCR showed higher BbrizIPT9 expression in the ovaries of sexual than in the apomictic B. brizantha. Results of in-situ hybridization showed strong signal of BbrizIPT9 in the MMC of both plants, at the onset of megasporogenesis. By analyzing AtIPT9 knockdown mutants, we verified enlarged nucellar cell, next to the MMC, in a percentage significantly higher than in the wild type, suggesting that knockout of AtIPT9 gene triggered the differentiation of extra MMC-like cells. CONCLUSIONS: Our results indicate that AtIPT9 might be involved in the proper differentiation of a single MMC during ovule development. The expression of a BbrizIPT9, localized in male and female sporocytes, and lower in apomicts than in sexuals, and effect of IPT9 knockout in Arabidopsis, suggest involvement of IPT9 in early ovule development.

Evolution of Transcriptomes in Early-Generation Hybrids of the Apomictic Ranunculus auricomus Complex (Ranunculaceae).

Hybridisation in plants may cause a shift from sexual to asexual seed formation (apomixis). Indeed, natural apomictic plants are usually hybrids, but it is still unclear how hybridisation could trigger the shift to apomixis. The genome evolution of older apomictic lineages is influenced by diverse processes such as polyploidy, mutation accumulation, and allelic sequence divergence. To disentangle the effects of hybridisation from these other factors, we analysed the transcriptomes of flowering buds from artificially produced, diploid F2 hybrids of the Ranunculus auricomus complex. The hybrids exhibited unreduced embryo sac formation (apospory) as one important component of apomixis, whereas their parental species were sexual. We revealed 2915 annotated single-copy genes that were mostly under purifying selection according to dN/dS ratios. However, pairwise comparisons revealed, after rigorous filtering, 79 genes under diversifying selection between hybrids and parents, whereby gene annotation assigned ten of them to reproductive processes. Four genes belong to the meiosis-sporogenesis phase (ASY1, APC1, MSP1, and XRI1) and represent, according to literature records, candidate genes for apospory. We conclude that hybridisation could combine novel (or existing) mutations in key developmental genes in certain hybrid lineages, and establish (together with altered gene expression profiles, as observed in other studies) a heritable regulatory mechanism for aposporous development.

A study of the heterochronic sense/antisense RNA representation in florets of sexual and apomictic Paspalum notatum.

BACKGROUND: Apomixis, an asexual mode of plant reproduction, is a genetically heritable trait evolutionarily related to sexuality, which enables the fixation of heterozygous genetic combinations through the development of maternal seeds. Recently, reference floral transcriptomes were generated from sexual and apomictic biotypes of Paspalum notatum, one of the most well-known plant models for the study of apomixis. However, the transcriptome dynamics, the occurrence of apomixis vs. sexual expression heterochronicity across consecutive developmental steps and the orientation of transcription (sense/antisense) remain unexplored. RESULTS: We produced 24 Illumina TruSeq®/ Hiseq 1500 sense/antisense floral transcriptome libraries covering four developmental stages (premeiosis, meiosis, postmeiosis, and anthesis) in biological triplicates, from an obligate apomictic and a full sexual genotype. De novo assemblies with Trinity yielded 103,699 and 100,114 transcripts for the apomictic and sexual samples respectively. A global comparative analysis involving reads from all developmental stages revealed 19,352 differentially expressed sense transcripts, of which 13,205 (68%) and 6147 (32%) were up- and down-regulated in apomictic samples with respect to the sexual ones. Interestingly, 100 differentially expressed antisense transcripts were detected, 55 (55%) of them up- and 45 (45%) down-regulated in apomictic libraries. A stage-by-stage comparative analysis showed a higher number of differentially expressed candidates due to heterochronicity discrimination: the highest number of differential sense transcripts was detected at premeiosis (23,651), followed by meiosis (22,830), postmeiosis (19,100), and anthesis (17,962), while the highest number of differential antisense transcripts were detected at anthesis (495), followed by postmeiosis (164), meiosis (120) and premeiosis (115). Members of the AP2, ARF, MYB and WRKY transcription factor families, as well as the auxin, jasmonate and cytokinin plant hormone families appeared broadly deregulated. Moreover, the chronological expression profile of several well-characterized apomixis controllers was examined in detail. CONCLUSIONS: This work provides a quantitative sense/antisense gene expression catalogue covering several subsequent reproductive developmental stages from premeiosis to anthesis for apomictic and sexual P. notatum, with potential to reveal heterochronic expression between reproductive types and discover sense/antisense mediated regulation. We detected a contrasting transcriptional and hormonal control in apomixis and sexuality as well as specific sense/antisense modulation occurring at the onset of parthenogenesis.

Manifestation of apomictic potentials in the line AS-3 of Sorghum bicolor (L.) Moench.

MAIN CONCLUSION: AS-3 line of Sorghum bicolor possesses functional components of apomixis-apospory, parthenogenesis and autonomous endospermogenesis. The data obtained indicate efficiency of selection for apomixis components in diploid species of cultivated crops. Apomixis (seed formation without fertilization) is one of most attractive phenomena in plant biology. In this paper, we provide the results of long-term selection for apomixis components in the progeny of grain sorghum (Sorghum bicolor (L.) Moench) hybrid plants with male sterility mutation. Selection was carried out for a high frequency of aposporous embryo sacs (ESs), autonomous pro-embryos, and the presence of maternal-type plants in test crosses with the line Volzhskoe-4v (V4v) homozygous for the Rs1 genes determining the red color of the leaves and stem of the hybrids. As a result of using this approach, the line, AS-3, was created, in which the frequency of ovaries with parthenogenetic embryos reached 42-45%. The autonomous development of embryos and endosperm was observed in the panicles of each of the 10 cytologically studied plants of this line. The frequency of parthenogenesis positively correlated with the high average daily air temperature during the first five out of 10 days preceding the onset of flowering (r = 0.75; P > 0.01). Genotyping of the plants from the progeny of hand-emasculated panicles of AS-3 pollinated with V4v performed using co-dominant SSR markers revealed that the F1 hybrids carrying the Rs1 gene (chromosome 6) possessed both paternal and maternal alleles of Sb1-10 (chromosome 4) and Xtxp320 (chromosome 10) markers, while in the maternal-type plants (rs1rs1), only the maternal alleles of these markers were present. In the endosperm of the kernels from which the maternal-type seedlings were obtained, only the maternal alleles were present, while in the endosperm of the kernels that produced hybrid seedlings, both the paternal and maternal alleles were observed. The data obtained indicate the presence of functional components of apomixis (apospory, parthenogenesis, autonomous endospermogenesis) in the grain sorghum line AS-3, and the efficiency of selection for apomixis in functionally diploid species of cultivated crops.

Small RNA-seq reveals novel regulatory components for apomixis in Paspalum notatum.

BACKGROUND: Apomixis is considered an evolutionary deviation of the sexual reproductive pathway leading to the generation of clonal maternal progenies by seeds. Recent evidence from model and non-model species suggested that this trait could be modulated by epigenetic mechanisms involving small RNAs (sRNAs). Here we profiled floral sRNAs originated from apomictic and sexual Paspalum notatum genotypes in order to identify molecular pathways under epigenetic control that might be involved in the transition from sexuality to agamospermy. RESULTS: The mining of genes participating in sRNA-directed pathways from floral Paspalum transcriptomic resources showed these routes are functional during reproductive development, with several members differentially expressed in apomictic and sexual plants. Triplicate floral sRNA libraries derived from apomictic and a sexual genotypes were characterized by using high-throughput sequencing technology. EdgeR was apply to compare the number of sRNA reads between sexual and apomictic libraries that map over all Paspalum floral transcripts. A total of 1525 transcripts showed differential sRNA representation, including genes related to meiosis, plant hormone signaling, biomolecules transport, transcription control and cell cycle. Survey for miRNA precursors on transcriptome and genome references allowed the discovery of 124 entities, including 40 conserved and 8 novel ones. Fifty-six clusters were differentially represented in apomictic and sexual plants. All differentially expressed miRNAs were up-regulated in apomictic libraries but miR2275, which showed different family members with opposed representation. Examination of predicted miRNAs targets detected 374 potential candidates. Considering sRNA, miRNAs and target surveys together, 14 genes previously described as related with auxin metabolism, transport and signaling were detected, including AMINO ACID/AUXIN PERMEASE 15, IAA-AMIDO SYNTHETASE GH3-8, IAA30, miR160, miR167, miR164, miR319, ARF2, ARF8, ARF10, ARF12, AFB2, PROLIFERATING CELL FACTOR 6 and NITRATE TRANSPORTER 1.1. CONCLUSIONS: This work provides a comprehensive survey of the sRNA differential representation in flowers of sexual and apomictic Paspalum notatum plants. An integration of the small RNA profiling data presented here and previous transcriptomic information suggests that sRNA-mediated regulation of auxin pathways is pivotal in promoting apomixis. These results will underlie future functional characterization of the molecular components mediating the switch from sexuality to apomixis.

Translocation of a parthenogenesis gene candidate to an alternate carrier chromosome in apomictic Brachiaria humidicola.

BACKGROUND: The apomictic reproductive mode of Brachiaria (syn. Urochloa) forage species allows breeders to faithfully propagate heterozygous genotypes through seed over multiple generations. In Brachiaria, reproductive mode segregates as single dominant locus, the apospory-specific genomic region (ASGR). The AGSR has been mapped to an area of reduced recombination on Brachiaria decumbens chromosome 5. A primer pair designed within ASGR-BABY BOOM-like (BBML), the candidate gene for the parthenogenesis component of apomixis in Pennisetum squamulatum, was diagnostic for reproductive mode in the closely related species B. ruziziensis, B. brizantha, and B. decumbens. In this study, we used a mapping population of the distantly related commercial species B. humidicola to map the ASGR and test for conservation of ASGR-BBML sequences across Brachiaria species. RESULTS: Dense genetic maps were constructed for the maternal and paternal genomes of a hexaploid (2n = 6x = 36) B. humidicola F1 mapping population (n = 102) using genotyping-by-sequencing, simple sequence repeat, amplified fragment length polymorphism, and transcriptome derived single nucleotide polymorphism markers. Comparative genomics with Setaria italica provided confirmation for x = 6 as the base chromosome number of B. humidicola. High resolution molecular karyotyping indicated that the six homologous chromosomes of the sexual female parent paired at random, whereas preferential pairing of subgenomes was observed in the apomictic male parent. Furthermore, evidence for compensated aneuploidy was found in the apomictic parent, with only five homologous linkage groups identified for chromosome 5 and seven homologous linkage groups of chromosome 6. The ASGR mapped to B. humidicola chromosome 1, a region syntenic with chromosomes 1 and 7 of S. italica. The ASGR-BBML specific PCR product cosegregated with the ASGR in the F1 mapping population, despite its location on a different carrier chromosome than B. decumbens. CONCLUSIONS: The first dense molecular maps of B. humidicola provide strong support for cytogenetic evidence indicating a base chromosome number of six in this species. Furthermore, these results show conservation of the ASGR across the Paniceae in different chromosomal backgrounds and support postulation of the ASGR-BBML as candidate genes for the parthenogenesis component of apomixis.

The steps from sexual reproduction to apomixis.

MAIN CONCLUSION: In this paper, an interaction model of apomixis-related genes was constructed to analyze the emergence of apomictic types. It is speculated that apomixis technology will be first implemented in gramineous plants. Apomixis (asexual seed formation) is a phenomenon in which a plant bypasses the most fundamental aspects of sexual reproduction-meiosis and fertilization-to form a viable seed. Plants can form seeds without fertilization, and the seed genotype is consistent with the female parent. The development of apomictic technology would be revolutionary for agriculture and for food production as it would reduce costs and breeding times and also avoid many complications typical of sexual reproduction (e.g. incompatibility barriers) and of vegetative propagation (e.g. viral transfer). The application of apomictic reproductive technology has the potential to revolutionize crop breeding. This article reviews recent advances in apomixis in cytology and molecular biology. The general idea of identifying apomixis was proposed and the process of the emergence of non-fusion types was discussed. To better understand the apomixis mechanism, an apomixis regulatory model was established. At the same time, the realization of apomixis technology is proposed, which provides reference for the research and application of apomixis.

Heterochronic reproductive developmental processes between diploid and tetraploid cytotypes of Paspalum rufum.

BACKGROUND AND AIMS: Apomixis is an asexual reproductive mode via seeds that generate maternal clonal progenies. Although apomixis in grasses is mainly expressed at the polyploid level, some natural diploid genotypes of Paspalum rufum produce aposporous embryo sacs in relatively high proportions and are even able to complete apomixis under specific conditions. However, despite the potential for apomixis, sexuality prevails in diploids, and apomixis expression is repressed for an as yet undetermind reason. Apomixis is thought to derive from a deregulation of one or a few components of the sexual pathway that could be triggered by polyploidy and/or hybridization. The objectives of this work were to characterize and compare the reproductive development and the timing of apospory initial (AI) emergence between diploid genotypes with potential for apomixis and facultative apomictic tetraploid cytotypes of P. rufum. METHODS: Reproductive characterization was performed by cytoembryological observations of cleared ovaries and anthers during all reproductive development steps and by quantitative evaluation of the ovule growth parameters. KEY RESULTS: Cytoembryological observations showed that in diploids, both female and male reproductive development is equally synchronized, but in tetraploids, megasporogenesis and early megagametogenesis are delayed with respect to microsporogenesis and early microgametogenesis. This delay was also seen when ovary growth was taken as a reference parameter. The analysis of the onset of AIs revealed that they emerge during different developmental periods depending on the ploidy level. In diploids, the AIs appeared along with the tetrad (or triad) of female meiocytes, but in tetraploids they appeared earlier, at the time of the megaspore mother cell. In both cytotypes, AIs can be seen even during megagametogenesis. CONCLUSIONS: Overall observations reveal that female sexual reproductive development is delayed in tetraploids as compared with diploid genotypes, mainly at meiosis. In tetraploids, AIs appear at earlier sexual developmental stages than in diploids, and they accumulate up to the end of megasporogenesis. The longer extension of megasporogenesis in tetraploids could favour AI emergence and also apomixis success.

Structure, target-specificity and expression of PN_LNC_N13, a long non-coding RNA differentially expressed in apomictic and sexual Paspalum notatum.

KEY MESSAGE: ncRNA PN_LNC_N13 shows contrasting expression in reproductive organs of sexual and apomictic Paspalum notatum genotypes. Apomictic plants set genetically maternal seeds whose embryos derive by parthenogenesis from unreduced egg cells, giving rise to clonal offspring. Several Paspalum notatum apomixis related genes were identified in prior work by comparative transcriptome analyses. Here, one of these candidates (namely N13) was characterized. N13 belongs to a Paspalum gene family including 30-60 members, of which at least eight are expressed at moderate levels in florets. The sequences of these genes show no functional ORFs, but include segments of different protein coding genes. Particularly, N13 shows partial identity to maize gene BT068773 (RESPONSE REGULATOR 6). Secondary structure predictions as well as mature miRNA and target cleavage detection suggested that N13 is not a miRNA precursor. Moreover, N13 family members produce abundant 24-nucleotide small RNAs along extensive parts of their sequences. Surveys in the GREENC and CANTATA databases indicated similarity with plant long non-coding RNAs (lncRNAs) involved in splicing regulation; consequently, N13 was renamed as PN_LNC_N13. The Paspalum BT068773 predicted ortholog (N13TAR) originates floral transcript variants shorter than the canonical maize isoform and with possible structural differences between the apomictic and sexual types. PN_LNC_N13 is expressed only in apomictic plants and displays quantitative representation variation across reproductive developmental stages. However, PN_LNC_N13-like homologs and/or its derived sRNAs showed overall a higher representation in ovules of sexual plants at late premeiosis. Our results suggest the existence of a whole family of N13-like lncRNAs possibly involved in splicing regulation, with some members characterized by differential activity across reproductive types.

A reference genetic linkage map of apomictic Hieracium species based on expressed markers derived from developing ovule transcripts.

BACKGROUND AND AIMS: Apomixis in plants generates clonal progeny with a maternal genotype through asexual seed formation. Hieracium subgenus Pilosella (Asteraceae) contains polyploid, highly heterozygous apomictic and sexual species. Within apomictic Hieracium, dominant genetic loci independently regulate the qualitative developmental components of apomixis. In H. praealtum, LOSS OF APOMEIOSIS (LOA) enables formation of embryo sacs without meiosis and LOSS OF PARTHENOGENESIS (LOP) enables fertilization-independent seed formation. A locus required for fertilization-independent endosperm formation (AutE) has been identified in H. piloselloides. Additional quantitative loci appear to influence the penetrance of the qualitative loci, although the controlling genes remain unknown. This study aimed to develop the first genetic linkage maps for sexual and apomictic Hieracium species using simple sequence repeat (SSR) markers derived from expressed transcripts within the developing ovaries. METHODS: RNA from microdissected Hieracium ovule cell types and ovaries was sequenced and SSRs were identified. Two different F1 mapping populations were created to overcome difficulties associated with genome complexity and asexual reproduction. SSR markers were analysed within each mapping population to generate draft linkage maps for apomictic and sexual Hieracium species. KEY RESULTS: A collection of 14 684 Hieracium expressed SSR markers were developed and linkage maps were constructed for Hieracium species using a subset of the SSR markers. Both the LOA and LOP loci were successfully assigned to linkage groups; however, AutE could not be mapped using the current populations. Comparisons with lettuce (Lactuca sativa) revealed partial macrosynteny between the two Asteraceae species. CONCLUSIONS: A collection of SSR markers and draft linkage maps were developed for two apomictic and one sexual Hieracium species. These maps will support cloning of controlling genes at LOA and LOP loci in Hieracium and should also assist with identification of quantitative loci that affect the expressivity of apomixis. Future work will focus on mapping AutE using alternative populations.

The LOSS OF APOMEIOSIS (LOA) locus in Hieracium praealtum can function independently of the associated large-scale repetitive chromosomal structure.

Apomixis or asexual seed formation in Hieracium praealtum (Asteraceae) is controlled by two independent dominant loci. One of these, the LOSS OF APOMEIOSIS (LOA) locus, controls apomixis initiation, mitotic embryo sac formation (apospory) and suppression of the sexual pathway. The LOA locus is found near the end of a hemizygous chromosome surrounded by extensive repeats extending along the chromosome arm. Similar apomixis-carrying chromosome structures have been found in some apomictic grasses, suggesting that the extensive repetitive sequences may be functionally relevant to apomixis. Fluorescence in situ hybridization (FISH) was used to examine chromosomes of apomeiosis deletion mutants and rare recombinants in the critical LOA region arising from a cross between sexual Hieracium pilosella and apomictic H. praealtum. The combined analyses of aposporous and nonaposporous recombinant progeny and chromosomal karyotypes were used to determine that the functional LOA locus can be genetically separated from the very extensive repeat regions found on the LOA-carrying chromosome. The large-scale repetitive sequences associated with the LOA locus in H. praealtum are not essential for apospory or suppression of sexual megasporogenesis (female meiosis).

Alternative Evolutionary Pathways in Paspalum Involving Allotetraploidy, Sexuality, and Varied Mating Systems.

The genetic systems of Paspalum species have not been extensively studied. We analyzed the ploidy, reproductive mode, mating system, and fertility of four Paspalum species-Paspalum durifolium, Paspalum ionanthum, Paspalum regnellii, and Paspalum urvillei. An analysis of 378 individuals from 20 populations of northeastern Argentina was conducted. All populations of the four Paspalum species were pure tetraploid and had a sexual and stable reproductive mode. However, some populations of P. durifolium and P. ionanthum showed low levels of apospory. Populations of P. durifolium and P. ionanthum had low seed sets under self-pollination but were fertile under open pollination, showing that self-incompatibility likely caused self-sterility. In contrast, populations of P. regnellii or P. urvillei showed no evidence of apospory, and seed sets in both self- and open pollination conditions were high, suggesting that they are self-compatible due to the absence of pollen-pistil molecular incompatibility mechanisms. The evolutionary origin of the four Paspalum species could explain these differences. This study supplies valuable insights into the genetic systems of Paspalum species, which could have implications for their conservation and management.

Spotting the Targets of the Apospory Controller TGS1 in Paspalum notatum.

Sexuality and apomixis are interconnected plant reproductive routes possibly behaving as polyphenic traits under the influence of the environment. In the subtropical grass Paspalum notatum, one of the controllers of apospory, a main component of gametophytic apomixis reproduction, is TRIMETHYLGUANOSINE SYNTHASE 1 (TGS1), a multifunctional gene previously associated with RNA cleavage regulation (including mRNA splicing as well as rRNA and miRNA processing), transcriptional modulation and the establishment of heterochromatin. In particular, the downregulation of TGS1 induces a sexuality decline and the emergence of aposporous-like embryo sacs. The present work was aimed at identifying TGS1 target RNAs expressed during reproductive development of Paspalum notatum. First, we mined available RNA databases originated from spikelets of sexual and apomictic plants, which naturally display a contrasting TGS1 representation, to identify differentially expressed mRNA splice variants and miRNAs. Then, the role of TGS1 in the generation of these particular molecules was investigated in antisense tgs1 sexual lines. We found that CHLOROPHYLL A-B BINDING PROTEIN 1B-21 (LHC Ib-21, a component of the chloroplast light harvesting complex), QUI-GON JINN (QGJ, encoding a MAP3K previously associated with apomixis) and miR2275 (a meiotic 24-nt phasi-RNAs producer) are directly or indirectly targeted by TGS1. Our results point to a coordinated control exercised by signal transduction and siRNA machineries to induce the transition from sexuality to apomixis.

Variation of Residual Sexuality Rates along Reproductive Development in Apomictic Tetraploids of Paspalum.

Most apomictic plants are facultative, maintaining the ability to reproduce sexually at different frequencies depending on the taxa, ploidy, and reproductive stage. In this context, Paspalum species are good model systems for studies evaluating the varying levels of apomixis expression. We aimed to identify, in apomictic tetraploid Paspalum species, the degree of apomixis and residual sexuality in three stages of reproductive development, and if their expression varies along them in order to predict their realized impact on the genetic diversity of future generations. Three main stages in the reproductive development (i.e., ovule, seed, and progeny) were studied in tetraploids from populations of P. cromyorhizon and P. maculosum. Mature ovules were studied using cytoembryological analysis, seeds by flow cytometry, and progeny tests with molecular markers. The expression of sexuality and apomixis was compared in each stage. We observed a decline in expression of sexual reproduction through the consecutive stages, jointly with an increase of apomixis expression. Both species showed at least one tetraploid plant capable of producing progeny by sexual means. These small rates of sexually originated progeny prove the ability of apomictic plants to produce low levels of genetic variation through rare events of sexuality. This study also demonstrates the importance of analyzing different reproductive stages in order to get a whole picture of the reproductive outcomes in plant evolution.

Environmental and Genetic Factors Affecting Apospory Expressivity in Diploid Paspalum rufum.

In angiosperms, gametophytic apomixis (clonal reproduction through seeds) is strongly associated with polyploidy and hybridization. The trait is facultative and its expressivity is highly variable between genotypes. Here, we used an F1 progeny derived from diploid apomictic (aposporic) genotypes of Paspalum rufum and two F2 families, derived from F1 hybrids with different apospory expressivity (%AES), to analyze the influence of the environment and the transgenerational transmission of the trait. In addition, AFLP markers were developed in the F1 population to identify genomic regions associated with the %AES. Cytoembryological analyses showed that the %AES was significantly influenced by different environments, but remained stable across the years. F1 and F2 progenies showed a wide range of %AES variation, but most hybrids were not significantly different from the parental genotypes. Maternal and paternal genetic linkage maps were built covering the ten expected linkage groups (LG). A single-marker analysis detected at least one region of 5.7 cM on LG3 that was significantly associated with apospory expressivity. Our results underline the importance of environmental influence in modulating apospory expressivity and identified a genomic region associated with apospory expressivity at the diploid level.

High-Resolution Linkage Map With Allele Dosage Allows the Identification of Regions Governing Complex Traits and Apospory in Guinea Grass (Megathyrsus maximus).

Forage grasses are mainly used in animal feed to fatten cattle and dairy herds, and guinea grass (Megathyrsus maximus) is considered one of the most productive of the tropical forage crops that reproduce by seeds. Due to the recent process of domestication, this species has several genomic complexities, such as autotetraploidy and aposporous apomixis. Consequently, approaches that relate phenotypic and genotypic data are incipient. In this context, we built a linkage map with allele dosage and generated novel information of the genetic architecture of traits that are important for the breeding of M. maximus. From a full-sib progeny, a linkage map containing 858 single nucleotide polymorphism (SNP) markers with allele dosage information expected for an autotetraploid was obtained. The high genetic variability of the progeny allowed us to map 10 quantitative trait loci (QTLs) related to agronomic traits, such as regrowth capacity and total dry matter, and 36 QTLs related to nutritional quality, which were distributed among all homology groups (HGs). Various overlapping regions associated with the quantitative traits suggested QTL hotspots. In addition, we were able to map one locus that controls apospory (apo-locus) in HG II. A total of 55 different gene families involved in cellular metabolism and plant growth were identified from markers adjacent to the QTLs and APOSPORY locus using the Panicum virgatum genome as a reference in comparisons with the genomes of Arabidopsis thaliana and Oryza sativa. Our results provide a better understanding of the genetic basis of reproduction by apomixis and traits important for breeding programs that considerably influence animal productivity as well as the quality of meat and milk.