Pollination type modulates residual sexuality in apomictic Paspalum rufumABSTRACT
Paspalum rufum Nees is a subtropical species that presents diploid individuals that primarily reproduce sexually and through allogamy due to self-incompatibility, as well as tetraploid races that are self-fertile and reproduce via apomixis. Embryological studies have shown that tetraploid races can occasionally produce offspring through sexual reproduction, making them facultative apomicts. This study aimed to analyze the impact of pollen source on the rate of apomixis in facultative apomictic accessions of P. rufum. Six progenies were obtained through self-pollination and cross-pollination from three facultative apomictic tetraploid (4×) genotypes of P. rufum: Q3756, Q3785, and Q4185. The progeny origin, whether maternal or non-maternal, was determined using molecular markers. Our findings indicate that the pollen source significantly influenced the progeny origin across three P. rufum accessions. In instances of self-pollination by a facultative apomictic plant, most of its descendants originate from aposporous embryo sacs (AES) and are genetically identical to the mother plant, with the percentage of non-maternal plants ranging from 1 % to 6 %. However, when the same genotype undergoes cross-pollination, the percentage of non-maternal offspring can rise to 19 % in the progeny derived from the Q4185 × Q3785 cross. This residual sexuality and the manipulation of pollination conditions could be effective strategies for generating genetic variability in apomictic species for which fully sexual counterparts are not available.
Keywords:
reproduction mode; progeny test; apomixis expressivity; maternal plant; non-maternal plant
Introduction
Paspalum L. is a substantial genus within the Gramineae family, comprising ∼310 species that are primarily native to tropical and warm-temperate regions in America (Soreng et al., 2022). In this genus, apomixis – a form of asexual reproduction through seeds (Nogler, 1984) – and polyploidy are the most prevalent reproductive strategies. Obligate apomicts in Paspalum exclusively produce aposporous sacs, resulting in progenies that are maternal clones. In contrast, facultative apomicts generate both meiotic and aposporous sacs, producing progeny that includes both maternal and aberrant (non-maternal) individuals (Quarin et al., 1998; Espinoza et al., 2002). Notably, despite its genetic basis, reproduction in some facultative apomicts can be influenced by environmental factors. Conditions such as temperature, light, water stress, pollination, and chemical exposure may alter reproductive behavior (Asker and Jerling, 1992).
Recent studies have reported that sexual reproduction is enhanced at lower temperatures in facultative apomictic plants of Paspalum intermedium Munro (Karunarathne et al., 2020). Additionally, both average temperature and latitude influence the expressivity of apospory in diploid and tetraploid genotypes of Paspalum rufum Ness (Soliman et al., 2021a). Studies conducted on apomictic grasses such as Dichanthium aristatum (Poir.) C.E. Hubb. (Knox, 1967), Paspalum cromyorrhizon Trin. (Quarin, 1986), Ranunculus auricomus L. (Klatt et al., 2016), and Cenchrus ciliaris L. (Sánchez et al., 2023) have revealed that light regimes significantly influence the development rate of aposporous embryo sacs (AES) in plants grown in both field and controlled environmental settings. Moreover, the timing of pollination can affect apomixis rates. In facultative apomicts like Paspalum notatum Flüggé (Martínez et al., 1994) and Pennisetum ciliare L. (Burson et al., 2002), changes in pollination timing have been shown to increase the frequency of BII (n + n) and BIII (2n + n) hybrids, suggesting a potential epigenetic influence. This approach may facilitate the generation of novel apomictic hybrids when sexual germplasm is non-existent.
P. rufum has been used as a model organism to study apomixis (Delgado et al., 2014; Soliman et al., 2021a, b). This species comprises sexual diploid cytotypes (2n = 2x = 20) and obligate or facultative apomicts tetraploid cytotypes (2n = 4x = 40) (Norrmann et al., 1989; Quarin et al., 1998). This study aimed to assess the extent to which pollen source and pollination method affect apomixis rates in facultative apomictic genotypes of P. rufum.
Materials and Methods
Study site and plant material
The study was conducted in the experimental field of the Faculty of Agricultural Sciences at the National University of the Northeast in Corrientes, Argentina. The site is located at coordinates 27°27’50" S, 58°50’22" W, altitude 58 m. Three tetraploid accessions of P. rufum were used: Q3756 (2n = 4x = 40), collected in Uruguay; Q3785 (2n = 4x = 40), collected in Paso de los Libres, Corrientes, Argentina; and Q4185 (2n = 4x = 40), an artificial BIII hybrid (2n + n) developed by crossing a diploid plant (Q3754) with Q3756 (Quarin et al., 1998). Progenies from these accessions were produced through self- and cross-pollinations using the techniques described by Quarin and Norrmann (1990). Self-pollinations were conducted by bagging young inflorescences before anthesis and keeping them covered until maturity. For cross-pollinations, the target inflorescence was isolated in a glassine bag throughout the procedure to prevent pollen contamination from unintended foreign genotypes in bloom within the living Paspalum collection. Pollen of the selected male parent was collected, the glassine bag was removed from the mother plant's inflorescence, and the pollen was applied to the emerged stigmata. The inflorescence was then resealed in the bag. This process was repeated over three to five days until all spikelets of the inflorescences had completed blooming. Following self- and cross-pollination, the inflorescences were kept in bags for approximately one month. Upon reaching maturity, the inflorescences were harvested, threshed, and the spikelets were counted, with empty ones separated from those containing caryopses using a seed blower. Seed set was calculated by dividing the number of florets containing caryopses by the total number of pollinated florets, then multiplying by 100. These progenies were subsequently analyzed using random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers to distinguish between maternal and non-maternal individuals.
Cytoembryological studies
The mode of reproduction was assessed by observing the development of embryo sacs. Spikelets at anthesis were fixed for 24 h in an FAA solution (70 % ethanol, glacial acetic acid, and 37 % formaldehyde in a ratio of 9 × 10−1: 5 × 10−2: 5 × 10−2) and subsequently preserved in 70 % ethanol at 4 °C. Ovaries were selected from different clusters of inflorescences collected from the same plant, then dissected and dehydrated using a tertiary butyl alcohol series. The samples were embedded in paraffin, sectioned at 12 µm, and stained with a series of safranin-fast green before being examined under a light microscope. Ploidy level in plants classified as non-maternal was determined by counting the chromosome numbers in root-tip cells. Young roots were harvested, treated in a saturated solution of α-bromonaphthalene for 2 h, hydrolyzed in 1 N HCl at 60 °C for 10 min, stained with 1 % basic fuchsine, and then squashed with a drop of 2 % aceto-orcein on slides for observation under a phase contrast microscope.
Molecular studies and statistical analysis
Genomic DNA was extracted from the young leaves of both the parental plants and their progeny, following the protocol described by Ortiz et al. (1997). For the RAPD analysis, primers from the University of British Columbia in Canada [set 5 (BC401 to BC444) and set 8 (BC701 to BC758)] were used. The reactions were carried out according to the procedures outlined by Espinoza et al. (2002). Briefly, screening was conducted using decamer primers to identify polymorphisms between the parental samples. Subsequently, the most informative primers were employed to analyze the progeny, ensuring that all individuals inherited bands specific to the male parent. Additionally, RFLPs were generated as described by Ortiz et al. (1997), using anchored probes from maize (UMC – University of Missouri – Columbia) and rice (RZ – Cornell University).
Statistical analyses
A chi-square (χ2) test was used to compare the observed numbers of non-maternal and maternal individuals under self- and cross-pollination conditions across three genotypes of P. rufum.
Results
Cytoembryological analysis of the reproductive behavior of parental plants
The mode of reproduction of accessions Q3756 and Q4185 was previously determined by cytoembryological methods by Norrmann et al. (1989) and Quarin et al. (1998). However, in this study, we re-analyzed these two genotypes and, for the first time, examined accession Q3785, which had not been characterized before. Ovules were fixed concurrently with the crosses or self-pollinations conducted. Most ovules examined showed different structural types: 1) Ovules with a single meiotic embryo sac (MES), characterized by the presence of an egg cell and two synergids (egg apparatus) at the micropylar end, a large binucleate central cell, and a cluster of antipodal cells at the chalazal end; 2) Ovules bearing one to more AES, which featured an egg apparatus and a binucleate central cell, but lacked antipodal cells; 3) Ovules containing both meiotic and AES. The findings from the cytoembryological analysis are presented in Table 1. All accessions were classified as facultative apomicts, as they exhibited both MES and AES. For ovules that exhibited MES alone or a combination of MES and AES, the potential for progeny production by sexual means ranged from 57.5 % to 65.8 % in these genotypes (Table 1).
Genesis of progeny families
A facultative apomictic plant can generate both maternal and non-maternal progeny. Maternal progeny originates from the parthenogenetic egg cell of an AES (2n + 0), whereas non-maternal progeny can originate from MES, either through the fertilization of a reduced egg cell to create a BII hybrid (n + n) or from fertilization of an unreduced egg cell in an AES, resulting in a BIII hybrid (2n + n). These two types of hybrids (BII and BIII) are often referred to as "aberrant" or "off-type" plants (Delgado et al., 2014). Progeny families were obtained from the three accessions (Q3756, Q4185, and Q3785) through both self- and cross-pollination. Seed set in these three accessions ranged from 49.2 % to 68.4 % during cross-pollinations and from 16 % to 62 % during self-pollinations (Table 2). A total of 24 to 30 individuals from each progeny family were selected to assess their genetic origin (Table 2).
Seed set and origin of different progenies obtained through self- and cross-pollination of three tetraploid accessions of Paspalum rufum Nees.
Screening and selection of informative markers
Progeny analysis aims to identify deviations from the maternal genotype as indicators of a sexual reproduction event. In this context, RAPD and RFLP markers were employed to detect non-maternal offspring by examining individual DNA fingerprints that differ from the maternal profile due to marker segregation. The detection of specific bands inherited from the male parent enabled the rapid and straightforward identification of non-maternal individuals (BII or BIII hybrids) in progeny from cross-pollination. A screening was conducted on the accessions used as parents to generate the progeny families and select the most suitable RAPD primers. In total, 102 oligonucleotides (BC401 to BC444 and BC701 to BC758) were tested to identify polymorphisms among these accessions. Twelve primers (11.7 %) failed to produce amplification. Among the remaining 90 primers, 72 (80 %) generated monomorphic markers, while 18 (20 %) displayed polymorphisms among the analyzed accessions. Thirteen primers (BC406, BC414, BC415, BC425, BC428, BC429, BC721, BC730, BC731, BC734, BC755, BC757, BC758) were selected for further analysis of the different progenies, based on the polymorphism level detected between the parents of each cross. These primers produced clear and well-defined bands during electrophoresis. In progenies obtained from crosses, male-specific bands were used to identify individuals resulting from sexual reproduction (n + n or 2n + n). In progenies obtained from selfing, non-maternal individuals were identified by the absence of bands found in the female parent. RFLP screenings were performed using 26 probes (RZ-56, RZ-69, RZ-104, RZ-125, RZ-166, RZ-206, RZ-224, RZ-244, RZ-245, RZ-273, RZ-413, RZ-455, RZ-574, RZ-590, RZ-630, RZ-753, UMC-16, UMC-55, UMC-65, UMC-80, UMC-104, UMC-127, UMC-128, UMC-147, UMC-374, UMC-475). Only six of the parental accessions (23 %) showed polymorphisms among the samples analyzed (RZ-69, RZ-166, RZ-206, RZ-224, RZ-590, UMC-127). Of the remaining probes, seven (27 %) either did not hybridize or showed smearing, while 13 (50 %) were monomorphic. Given the limited number of bands detected per probe (from 1 to 3), only progenies derived from the male parent Q3785 were analyzed. This approach was used because Q3785 produced unique bands, which enabled the identification of non-maternal plants within the progeny.
Q3756 progeny analysis
Thirty individuals obtained through selfing Q3756 were analyzed using five RAPD primers (BC414, BC415, BC730, BC734, and BC755), yielding 20 markers. The analysis of the banding patterns in the progeny revealed non-maternal profiles in two plants when primers BC730 and BC734 were used, due to segregation of a maternal marker. The remaining 28 plants exhibited the same amplification pattern as the maternal line, Q3756. Notably, primers BC414, BC415, and BC755 consistently displayed the same banding pattern as the mother plant. This result indicates that 6.6 % of this progeny originated through sexual reproduction, while 93.4 % resulted from apomixis. Additionally, a progeny was produced by crossing Q3756 with Q3785 to investigate reproductive behavior under this pollination condition. Twenty-four individuals were analyzed using the same five primers: BC406, BC415, BC428, BC721, and BC731, which produced five male-specific bands. These primers facilitated the identification of four non-maternal individuals based on the inheritance of the specific paternal bands, while the remaining 20 were confirmed to be of maternal origin. The DNA of this progeny was also analyzed using RFLP with probes RZ-69, RZ-166, and RZ-224. These probes identified two plants as non-maternal, which had also been identified as non-maternal using RAPD markers. Altogether, the results of both RAPD and RFLP analyses showed that 16.6 % of this progeny was produced sexually, while 83.4 % originated from parthenogenesis of an unreduced egg cell (Table 2). The differences observed between self- and cross-pollinations were statistically not significant (χ2 = 3.35; p > 0.05). Furthermore, all non-maternal plants were found to be tetraploid, suggesting that these individuals originated from the fertilization of reduced egg cells from a MES by a reduced male gamete (BII hybrids).
Q3785 progeny analysis
Twenty-seven individuals were generated through selfing Q3785 and subsequently genetically profiled using RAPD with primers BC414, BC415, BC730, BC755, and BC757, yielding 22 markers. The analysis of the amplification products revealed that 26 individuals exhibited a banding pattern identical to that of the female parent, indicating that 96.3 % of the plants were produced through apomixis. The remaining individual exhibited genotypic differences at the BC414, BC415, and BC755 primers due to marker segregation and was therefore classified as non-maternal (3.7 %). All progeny exhibited an identical banding pattern consistent with the maternal line when analyzed with probes RZ-66 and RZ-224. The accession Q3785 was also used as the female parent in a cross-pollination with Q3756. From this cross, 30 individuals were obtained and analyzed using RAPD markers with primers BC428, BC429, BC731, BC755, and BC758. Three plants were classified as hybrids due to the inheritance of a specific paternal marker, while the remaining 27 exhibited a banding pattern identical to that of the female parent, indicating that 10 % of this progeny originated from hybridization, whereas 90 % arose through parthenogenesis (Table 2). Notably, the differences in the apomixis rates observed between the two types of pollination (selfing and cross-pollination) were not statistically significant (χ2 = 3.34; p > 0.05). Chromosome counting in non-maternal plants indicated that these individuals were tetraploid (BII hybrids).
Q4185 progeny analysis
RAPD analysis using primers BC414, BC415, BC428, BC730, BC755, and BC757 was performed on 30 individuals obtained from selfing Q4185, yielding 23 markers. Six plants (20 %) were classified as non-maternal because they deviated from the maternal banding pattern, indicating their hybrid origin (Figure 1A). In contrast, the remaining 24 plants (80 %) displayed a banding pattern identical to that of the mother plant, confirming their maternal origin. A second progeny was obtained by crossing Q4185 with Q3785. Thirty (30) individuals were analyzed using RAPD with primers BC406, BC415, BC428, BC721, and BC731, and RFLP with probes RZ-69, RZ-166, and RZ-244. Primers BC721 and BC731 detected 12 non-maternal plants, while BC419 detected 9, and both BC406 and BC425 detected 2 each. The identification of specific bands originating from the male parent enabled the identification of four non-maternal plants using the RZ-224 probe (Figure 1B), while RZ-69 and RZ-166 each identified two hybrid plants. The analysis of amplification products and hybridization patterns of the probes allowed the classification of 19 plants (63.3 %) as non-maternal (indicating hybrid origin) and 11 plants (36.7 %) as maternal (indicating apomictic origin) (Table 2). These results showed that the Q4185 accession exhibits a differential capacity to generate progeny through sexual reproduction or apomixis, depending on the type of pollination (self- or cross-pollination), with highly significant differences observed (χ2 = 35.21; p < 0.05). Additionally, ploidy level was determined for all non-maternal plants, which were consistently tetraploid (BII hybrids).
Genetic fingerprints of Paspalum rufum Nees and its progeny. A) Polymerase Chain Reaction amplification profiles obtained with primer BC414 from self-pollinated progeny of Q4185. B) DNA hybridization patterns using probe RZ-224 in progeny derived from a cross between Q4185 (female parent) and Q3785 (male parent). *Indicate individuals of non-maternal origin.
Discussion
The examination of female meiotic division (megasporogenesis) and the subsequent embryo sac development (megagametogenesis) represents a common and effective approach for determining the mode of reproduction in higher plants. Notably, cytoembryological analyses have been widely employed to identify the apomictic reproductive mode in many species of the genus Paspalum (Burson and Bennett, 1970; Norrmann et al., 1989; Quarin et al., 1998). In most apomictic Paspalum species, apospory is the predominant mechanism. Mature AES can readily be distinguished from their meiotic counterparts due to differences in structure and cellular organization (Ortiz et al., 2013). However, apomixis and sexual reproduction are not mutually exclusive; thus, meiotic and AES may coexist within the same ovule, with both capable of generating progeny from different origins (Espinoza et al., 2002; Reutemann et al., 2022). In facultative aposporous species, meiotic and apomictic reproductive pathways develop simultaneously, often competing to ultimately produce a seed. Along these pathways, intense competition can occur between apomictic and sexual reproduction and ultimately influence the development of an individual into an adult. Analyses of various genotypes of Paspalum malacophyllum Trin. suggest that intense competition, specific (epi)genetic deregulation that triggers apomixis and alters the normal meiotic progression, as well as the effects of inbreeding depression, are key factors supporting the establishment of clonality (Hojsgaard et al., 2013). Our cytoembryological studies confirm that these three P. rufum accessions reproduce via facultative apomixis and have the potential to generate multiple progeny types:
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maternal descendants by parthenogenesis of an unreduced egg cell (2n + 0);
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non-maternal descendants when parthenogenesis of the unreduced egg cell fails (2n + n);
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non-maternal descendants from MES containing reduced egg cells (n + n).
Furthermore, RAPD and RFLP fingerprinting have demonstrated their efficacy as screening tools for identifying non-maternal or off-type progeny, as well as for evaluating reproductive mode in apomictic species. The use of molecular markers enables the estimation of the actual proportion of sexual reproduction in progeny tests of P. rufum accessions, and our findings are consistent with previous studies on other apomictic species (Rebozzio et al., 2011; Hojsgaard et al., 2013; Reutemann et al., 2022). Our study indicates that, at least in some cases, the pollen source can influence progeny origin in facultative apomicts like P. rufum. In cases of self-pollination by a facultative apomictic plant, most descendants are derived from AES and are genetically identical to the mother plant. However, when the same genotype undergoes cross-pollination, the number of non-maternal individuals increases. This pattern was observed across all progenies analyzed, although it was statistically significant only in the Q4185 accession.
Recent studies have reported changes in the incidence of apomixis within certain facultative apomictic grass species. In the short-day species D. aristatum, belonging to the tribe Andropogoneae, experimental findings have indicated that apomictic expression is associated with the photoperiod during inflorescence development (Knox and Heslop-Harrison, 1963). A strong correlation was identified between day length and the incidence of apomixis, with higher apomictic expression observed in northern Australia, where photoperiods were shorter than 14 h during inflorescence development. Conversely, significantly lower expression was recorded in southern regions characterized by photoperiods exceeding 14 h (Knox, 1967). These findings align with observations by Quarin (1986) regarding the facultative apomict Paspalum cromyorryzon Trin., where the maximum potential for apomictic reproduction coincided with the flowering peak, while sexual reproduction increased as conditions favorable for flowering diminished toward the end of the season. Similar results were observed in facultative apomictic genotypes of P. notatum, where apomictic expression varied with the flowering season, peaking in summer while decreasing during autumn and spring (Rios et al., 2013). In buffelgrass (P. ciliare) accessions, a facultative apomictic species, the frequency of MES decreased when tillers developed during the hottest months of the year, regardless of photoperiod. These findings suggest that environmental factors, beyond photoperiod, influence the sexual expression in facultative apomicts (Hussey et al., 1991). Similarly, variations in the frequencies of MES and AES were reported in three accessions of Brachiaria decumbens Stapf, analyzed in early summer versus late autumn, although these differences were not statistically significant (Naumova et al., 1999). Additionally, notable differences were observed between sexual expression at flowering (assessed by cytological techniques) and the final level of sexual reproduction (determined by polymorphic fingerprinting in the progeny) within facultative apomictic plants of P. notatum. The proportion of non-maternal plants in the progeny of two facultative apomictic accessions was significantly lower than the initially predicted based on the potential sexual reproduction assessments conducted at flowering in the same genotypes. These results suggest a marked advantage in seed development from AES over those from meiotic sacs (Rebozzio et al., 2011).
The presence of residual sexuality, along with the ability to manipulate pollination conditions, could serve as a valuable tool for generating variability in apomictic species where 100 % sexual counterparts are unavailable. Genotypes derived from crossing events may gradually accumulate desirable traits, with significant implications for both fundamental research and the genetic improvement of apomictic Paspalum species. Thus, maintaining a residual sexual pathway provides an opportunity for genetic diversification and evolution via sexual recombination (Chapman et al., 2003; Hörandl and Hojsgaard, 2012).
Declaration of use of AI technologies
Data availability statement
The authors confirm that the data supporting the findings of this study are available within the article.
Acknowledgments
The authors would like to thank the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Universidad Nacional del Nordeste (UNNE). This research was founded by the CONICET – pluriannual research project (PIP) 11220220100189; Secretaría General de Ciencia y Técnica, Universidad Nacional del Nordeste (UNNE) research project (PI) 2021 A 003; Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), scientific and technological research projects (PICT/2019) - No. 01675. We also thank Dr. Silvina C. Pessino for her kind review of the manuscript for English style and grammar.
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