Camilo L. Quarin

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 fingerprinting for determining the mode of reproduction in Paspalum notatum, a subtropical apomictic forage grass

Abstract Paspalum is an important genus of the family Gramineae that includes several valuable forage grasses. Many of the species are polyploid and either obligate or facultative apomicts. Cyto-embryological observations of several tetraploid genotypes of P. notatum were performed to determine their mode of reproduction. Afterwards, selfed progenies of the genotypes F131, Q3664 and Q4117 were analysed using RFLP and RAPD genetic fingerprints to identify maternal and non-maternal (aberrant) plants, and to establish the degree of apomictic reproduction. Five maize clones and six primers were used for detecting genetic deviations from the maternal profile. Maize clones umc379, umc384 and umc318 and primers OPG10 and OPI4 were the most informative for discriminating between maternal and aberrant individuals within the progenies of F131 and Q3664. The combined results of three RFLP clones or 4–6 RAPD primers were necessary to ascertain the mode of reproduction in plants F131 and Q3664. The results obtained with the RFLP and RAPD markers were in agreement with the cyto-embryological studies in ascertaining the mode and degree of apomictic reproduction. Plant F131 showed a completely sexual reproductive behaviour, Q3664 an elevated expression of sexuality, while Q4117 was highly apomictic. A fingerprint analysis of an outcrossing population, aimed at the identification of hybrid plants, was also performed. Maize clones um318 and umc379 and primers OPC2 and OPC9 were used. The presence of specific bands belonging to the male parent permitted a rapid and easy detection of hybrids. The methodology described here can be applied both for the characterisation of P. notatum populations and to identify hybrid progenies in Paspalum breeding programs.

Effect of pollen source and pollen ploidy on endosperm formation and seed set in pseudogamous apomictic Paspalum notatum

Abstract It is generally accepted that most angiosperms require an accurate balance between maternal and paternal genome contribution for endosperm development. The endosperm balance number (EBN) hypothesis postulates that each species has an effective number which must be in a 2:1 maternal to paternal ratio for normal endosperm development and seed formation. The aim of this work was to investigate the effect of different sources and ploidy levels of pollen donors on endosperm formation and seed production of aposporous tetraploid (2n=4×=40) Paspalum notatum. Hand-emasculated spikelets of an apomictic 4× plant were dusted with pollen of 2×, 4×, 5×, 6× and 8× races of the same species; 3× and 4× races of a phylogenetically closely related species, P. cromyorrhizon; and 2× and 4× races of P. simplex, a species of a different subgenus. Experiments including self-pollination as well as emasculation without pollination were conducted for controls. Results indicated that apomictic 4×P. notatum is a pseudogamous species with effective fertilization of the two unreduced (2n) polar nuclei by a reduced (n) sperm. Endosperm development and seed production occurred independently of the species or the ploidy level of the pollen donor. However, seed germination rates were significantly lower than in the self-pollinated control when the pollen donor was 3×P. cromyorrhizon or 2× and 4×P. simplex. Aposporous embryo sacs in Paspalum contribute to endosperm formation with two unreduced (2n) polar nuclei, while the male contribution is the same as in sexual plants (n). Since sexual Paspalum plants fit the EBN hypothesis, the EBN insensitivity observed in apomictic plants might be a prerequisite for the spread of pseudogamous apomixis. The EBN insensitivity could have arisen as an imprinting consequence of a high maternal genome contribution.

Tetraploid races of Paspalum notatum show polysomic inheritance and preferential chromosome pairing around the apospory-controlling locus

The objective of this work was to determine the type of inheritance (disomic/polysomic) in tetraploid (2n=4x=40) Paspalum notatum and investigate the transmission pattern of the chromosome region associated with apospory. An F1 family segregating for the reproductive mode (aposporous vs non-aposporous) was generated by crossing a tetraploid sexual plant as female parent with an apomictic individual as pollen donor. Pollen mother cells from both parental plants were examined to ascertain chromosome-pairing behavior at meiosis. The high rate of quadrivalent chromosome associations indicated an autotetraploid origin of the species, although bivalent pairing and occasional univalents were detected. The observation of a lagging bivalent, a bridge of chromatin, or two aligned laggards in the aposporous parent suggested a chromosome inversion in this strain. Segregation ratios of AFLP markers and the proportion of linkages in repulsion versus coupling phase denoted tetrasomic inheritance, but markers displaying disomic ratios were also observed. Preferential chromosome pairing (disomic inheritance) in the chromosome segment related to apospory was detected. The possible relationship between a chromosome rearrangement and the inheritance of apospory is discussed.

Evolutionary implications of meiotic chromosome behavior, reproductive biology, and hybridization in 6x and 9x cytotypes of Andropogon gerardii (Poaceae).

Andropogon gerardii, big bluestem, has 60 and 90 chromosome cytotypes. Meiosis in the hexaploid was shown to be regular, although some secondary associations of bivalents form. Meiosis in the enneaploid (2n = 9z = 90) is irregular, leading to most gametes having unbalanced chromosome complements. Both cytotypes show considerable self-incompatibility. Cytotypes crossed freely, forming a variety of fertile euploids and aneuploids. Indistinguishable exomorphology, intermixing in natural populations, and compatibility suggest that A. gerardii is best understood as a cytotypically complex single species.

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.

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

Abstract.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. Jaccard’s 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.

Cytology of Intra- and Interspecific Hybrids between two Cytotypes of Paspalum notatum and P. cromyorrhizon

Intra- and interspecific hybrids were made between diploid and tetraploid cytotypes of Paspalum notatum Flugge and P. cromyorrhizon Trin, to study their phylogenetic relationships. Two P. notatum (2n = 4x = 40) x P. notatum var. saurae Parodi (2n = 2x = 20) hybrids had 2n = 3x = 30. At metaphase I of meiosis, the mean chromosome associations were 1.93 I, 1.93 II, and 8.07 III, confirming that tetraploid P. notatum is an autotetraploid. Because P. notatum var. saurae has the genome formula NN, P. notatum can be given the formula NNNN. Two hybrids were recovered from the P. cromyorrhizon (2n = 2x = 20) x P. cromyorrhizon (2n = 4x = 40) cross: one had 30, and the other 40, chromosomes. Meiosis was irregular in both hybrids. The mean meiotic chromosome associations for the triploid were 4.81 I, 4.73 II, 4.92 III, 0.1 IV, 0.03 V, and 0.07 VI, and for the tetraploid, 0.84 I, 10.56 II, 0.21 III, 4.15 IV, 0.11 VI, and 0.03 VIII. Of seven P. notatum var. saurae x P. cromyorrhizon (2n = 2x = 20) hybrids, six had 20 chromosomes and a mean chromosome association of 9.04 I, 5.42 II, and 0.04 III, and one had 40 chromosomes and a mean association of 1.2 I, 6.33 II, 0.4 III, 6.13 IV, and 0.07 VI. The frequency of bivalents in the 20-chromosome hybrids and tetravalents in the 40-chromosome hybrid indicates that the genomes of these two species are partially homologous and that both species have forms of the N genome. Two hybrids from the P. notatum x P. cromyorrhizon (2n = 2x = 20) cross had 2n = 3x = 30. The mean pairing behavior was 9.38 I, 9.09 II, 0.78 III, and 0.02 IV, and some cells had as many as 5 III. These findings support the traditional taxonomic treatment that P. notatum and P. cromyorrhizon are separate species.

Genetic and embryological evidences of apomixis at the diploid level in Paspalum rufum support recurrent auto-polyploidization in the species

Gametophytic apomixis is an asexual mode of reproduction by seeds. This trait is present in several plant families and is strongly associated with polyploidy. Paspalum rufum is a forage grass with sexual self-incompatible diploids (2n = 2x = 20) and aposporous-apomictic pseudogamous tetraploids (2n = 4x = 40). In previous work embryological observations of the diploid genotype Q3754 showed 8.8–26.8% of the ovaries having one meiotic plus an aposporous-like embryo sac, suggesting some capability for apomictic reproduction. The objective of this work was to characterize progenies derived from Q3754 to determine if aposporous sacs were functional and generated progenies via apomixis at the diploid level. Re-examination of Q3754 ovaries showed that 12.5% of them contained one sexual plus an aposporous sac confirming previous results. Progeny tests were carried out on two experimental families (H1 and S1) employing heterozygous RAPD marker loci. Family H1 was obtained crossing Q3754 with a natural diploid genotype (Q3861) and S1 derived from the induced self-pollination of Q3754. Genetic analysis of H1 showed that all individuals derived from sexual reproduction. However, 5 out of 95 plants from S1 showed the same heterozygous state as the mother plant for 14 RAPD loci suggesting a clonal origin. Further experiments, designed to test the functionality of aposporous sacs by flow cytometric analyses, were carried out on a third family (M1) obtained by crossing Q3754 with the tetraploid plant Q3785. Histograms of 20 M1 plants showed 15 diploids (75%), 4 triploids (20%) and 1 tetraploid (5%). Triploids and the tetraploid may have originated from functional aposporous embryo sacs. Likewise, the reconstruction of the developmental route of 40 individual seeds demonstrated that 11 of them (27.5%) derived from fertilized aposporic sacs. The results presented in this work indicate that gametophytic apomixis is effectively expressed at the diploid level in Paspalum rufum and could be the foundation of a recurrent auto-polyploidization process in the species.

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.