Eric J. Martínez

A rise of ploidy level induces the expression of apomixis in Paspalum notatum

Abstract The common races of bahiagrass, Paspalum notatum, are tetraploid (2n=4x=40) and reproduce by aposporous apomixis. Paspalum notatum var. saurae is the corresponding diploid (2n=2x=20) sexual race that outbreeds due to self-incompatibility. Chromosome doubling was induced by colchicine treatments in three individual plants from a natural diploid population. Embryological studies demonstrated that one of the induced autotetraploid plants reproduced sexually. The other two autotetraploids were facultative apomicts. These results indicate that an unexpressed gene(s) for apomixis exists at the diploid level. The expression of the trait is ploidy-dependent. The ploidy dependency may act either on the locus controlling apomixis through some transcription factors or via a secondary locus which requires a higher allele dosage to affect the expression of the main locus.

Genetic characterization of apospory in tetraploid Paspalum notatum based on the identification of linked molecular markers

Tetraploid Paspalum notatum (bahiagrass) is a valuable forage grass with aposporous apomictic reproduction. In a previous study, we showed that apospory in bahiagrass is under the control of a single dominant gene with a distorted segregation ratio. The objective of this work was to identify molecular markers linked to apospory in tetraploid P. notatum and establish a preliminary syntenic relationship with the genomic region associated with apospory in P. simplex. A F1 population of 290 individuals, segregating for apospory, was generated after crossing a completely sexual plant (Q4188) with a natural aposporous apomictic plant (Q4117). The whole progeny was classified as sexual or aposporous by embryo sacs analysis. A bulked segregant analysis was carried out to identify molecular markers co-segregating with apospory. Four hundred RAPD primers, 30 AFLP primers combinations and 85 RFLP clones were screened using DNA from both parental genotypes and aposporous and sexual bulks. Linkage analysis was performed with cytological and genetic information from the complete progeny. Cytoembryological analysis showed 219 sexual and 71 aposporous F1 individuals. Seven different molecular markers (2 RAPD, 4 AFLP and 1 RFLP) were found to be completely linked to apospory. The RFLP probe C1069, mapping to the telomeric region of the long arm of rice chromosome 12, was one of the molecular markers completely linked to apospory in P. notatum. This marker had been previously associated with apospory in P. simplex. A preliminary map of the chromosome region carrying the apospory locus was constructed.

Harnessing apomictic reproduction in grasses: what we have learned from Paspalum

BACKGROUND Apomixis is an alternative route of plant reproduction that produces individuals genetically identical to the mother plant through seeds. Apomixis is desirable in agriculture, because it guarantees the perpetuation of superior genotypes (i.e. heterotic hybrid seeds) by self-seeding without loss of hybrid vigour. The Paspalum genus, an archetypal model system for mining apomixis gene(s), is composed of about 370 species that have extremely diverse reproductive systems, including self-incompatibility, self-fertility, full sexual reproduction, and facultative or obligate apomixis. Barriers to interspecific hybridization are relaxed in this genus, allowing the production of new hybrids from many different parental combinations. Paspalum is also tolerant to various parental genome contributions to the endosperm, allowing analyses of how sexually reproducing crop species might escape from dosage effects in the endosperm. SCOPE In this article, the available literature characterizing apomixis in Paspalum spp. and its use in breeding is critically reviewed. In particular, a comparison is made across species of the structure and function of the genomic region controlling apomixis in order to identify a common core region shared by all apomictic Paspalum species and where apomixis genes are likely to be localized. Candidate genes are discussed, either as possible genetic determinants (including homologs to signal transduction and RNA methylation genes) or as downstream factors (such as cell-to-cell signalling and auxin response genes) depending, respectively, on their co-segregation with apomixis or less. Strategies to validate the role of candidate genes in apomictic process are also discussed, with special emphasis on plant transformation in natural apomictic species.

Competition between meiotic and apomictic pathways during ovule and seed development results in clonality

Meiotic and apomictic reproductive pathways develop simultaneously in facultative aposporous species, and compete to form a seed as a final goal. This developmental competition was evaluated in tetraploid genotypes of Paspalum malacophyllum in order to understand the low level of sexuality in facultative apomictic populations. Cyto-embryology on ovules, flow cytometry on seeds and progeny tests by DNA fingerprinting were used to measure the relative incidence of each meiotic or apomictic pathway along four different stages of the plants life cycle, namely the beginning and end of gametogenesis, seed formation and adult offspring. A high variation in the frequencies of sexual and apomictic pathways occurred at the first two stages. A trend of radical decline in realized sexuality was then observed. Sexual and apomictic seeds were produced, but the efficiency of the sexual pathway dropped drastically, and exclusively clonal offspring remained. Both reproductive pathways are unstable at the beginning of development, and only the apomictic one remains functional. Key factors reducing sexuality are the faster growth and parthenogenetic development in the aposporous pathway, and an (epi)genetically negative background related to the extensive gene de-regulation pattern responsible for apomixis. The effects of inbreeding depression during post-fertilization development may further decrease the frequency of effective sexuality.

Sequence characterization, in silico mapping and cytosine methylation analysis of markers linked to apospory in Paspalum notatum

In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum. The objective of this work was to sequence these markers, obtain their flanking regions by chromosome walking and perform an in silico mapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen molecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2–4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize. Two loci associated with apomixis locus were tested in methylation-sensitive RFLP experiments using genomic DNA extracted from leaves. Although both target sequences were methylated no methylation polymorphisms associated with the mode of reproduction were detected.

Sexual diploid and apomictic tetraploid races in Thrasya petrosa (Gramineae)

Thrasya petrosa (Trin.) Chase is the most widespread species of a grass genus indigenous to the New World. Genetic systems in diploid (2n = 2x = 20) and tetraploid (2n = 4x = 40) races of T. petrosa were investigated. The diploid race exhibited embryological development typical of sexual reproduction, but failed to produce seed because of self-incompatibility, whereas the tetraploid showed embryological pathways characteristic of facultative apomixis. Consequently, some ovules showed a normal meiotic embryo sac, others had one to several aposporous sacs, and, finally, some ovules had one or more aposporous sacs beside the meiotic one. A uniform progeny test assisted by molecular markers confirmed that the main reproductive mode for the tetraploid race was apomixis, despite some sexual reproductive structures observed by cytoembryological analyses. The chromosome pairing patterns at meiosis suggested that autoploidy was the genetic origin of the tetraploid races of T. petrosa. In addition, the close relationship between Thrasya Kunth and Paspalum L. previously supported by phylogenetic analyses is further sustained by the particular genetic system shared by the two genera. The system involves co-specific sexual self-incompatible diploids and apomictic, pseudogamous and self-compatible polyploids.

Apospory followed by sterility in a hypotriploid hybrid (2x X 4x) of Paspalum

Abstract – A self-incompatible diploid plant (2n = 2x = 20) of Paspalum limbatum grown in a field nursery surrounded by tetraploid accessions of several Paspalum species produced a hypotriploid descendant (2n-1 = 3x = 29). Molecular fingerprinting by RAPD markers indicated that an apomictic 4x accession of P. guenoarum was the pollen parent. Meiotic chromosome pairing in the hypotriploid hybrid averaged 16.3 univalents (range 9-29), 5.4 bivalents (0-9), and 0.7 trivalents (0-3). Since P. guenoarum has an autopolyploid or segmental allopolyploid origin, the bivalent associations in the hypotriploid hybrid could be ascribed to an autosyndetic pairing between the two chromosome sets contributed by the male parent. The trivalent chromosome associations suggested some degree of homology between the chromosomes of P. limbatum and P. guenoarum. Embryological analysis of the hypotriploid hybrid showed complete abortion of the megaspore mother cell before or during the first meiotic division. One to several aposporous embryo sacs developed from somatic nucellar cells in over 97% of the ovules analysed. The plant was 100% male sterile and seed sterility reached 99.9%, despite hand-pollination with several pollen sources. Thus, apospory was fully inherited from the apomictic 4x male parent, but the trait did not prevent seed sterility.

Unravelling the ambiguous reproductive biology of Paspalum malacophyllum: a decades old story clarified

In a recent manuscript published by our group we analyzed the reproductive biology of the grass Paspalum malacophyllum by using traditional embryological techniques combined with current cytological and molecular methods. Our findings confirmed apparent contradictions regarding the reproductive behavior of P. malacophyllum from six independently published reports over the past six decades. Herein we summarize the main findings, conclusions, and validations of all previous studies, highlighting the need for multiple approaches to characterize reproductive systems when using apomictic plants in a breeding program.