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Revista Brasileira de Zootecnia

versão On-line ISSN 1806-9290

R. Bras. Zootec. vol.46 no.3 Viçosa mar. 2017 

Forage Crops

Determination of the mode of reproduction of bahiagrass hybrids using cytoembryological analysis and molecular markers

Roberto Luis Weiler1  * 

Miguel Dall’Agnol1 

Carine Simioni1 

Karine Cristina Krycki2 

Nair Dahmer3 

Divanilde Guerra4 

1Universidade Federal do Rio Grande do Sul, Faculdade de Agronomia, Departamento de Plantas Forrageiras e Agrometeorologia, Porto Alegre, RS, Brazil.

2Universidade Federal do Rio Grande do Sul, Programa de Pós-graduação em Zootecnia, Porto Alegre, RS, Brazil.

3Sociedade Educacional Três de Maio, Três de Maio, RS, Brazil.

4Universidade Estadual do Rio Grande do Sul, Três Passos, RS, Brazil.


The aim of this study was to determine the mode of reproduction of a hybrid progeny derived from intraspecific crosses of Paspalum notatum through cytoembryological analysis and use of RAPD (random amplification of polymorphic DNA) molecular markers. Cytoembryological analysis allowed identification of the mode of reproduction of 28 plants that were selected after agronomic productivity evaluations. Of these, 19 had embryo sac morphology compatible with an apomictic mode of reproduction and nine had embryo sac morphology compatible with a sexual mode of reproduction. Meanwhile, molecular marker analysis for 194 individuals showed 54 sexual and 140 apomictic plants; of the 28 plants analyzed by the two methods, ten results (35.7%) were in disagreement. In this paper, through cytoembryological analyses, a ratio of 1:2.1 of sexual to apomictic plants was found. The BCU 243 marker showed a stable pattern of amplification, but some results differed with cytoembryological analyses, demonstrating that these analyses are more reliable when determining the mode of sexual reproduction for the plants of P. notatum. Apomictic plants characterized in this work can be tested in the field to check their agronomic value and registration as plant varieties, while the sexual plants can be used as potential parents in future crosses.

Key words: apomixis; molecular analysis; Paspalum notatum; plant breeding; RAPD


Animal production based on native pastures figures prominently in the state of Rio Grande do Sul, where it represents the dietary basis for this livestock. The Pampa biome has a great diversity of plant taxa, with about 400 species of grasses and 150 species of legumes (Boldrini, 1997).

Among the grasses natifve to Brazil, the genus Paspalum L. is representative of various herbaceous ecosystems and is responsible for producing a large portion of the available forage; thus it is an important tool used for the production of beef cattle (Valls, 1980). In the genus Paspalum, there is a close correlation between ploidy level and mode of reproduction, in which diploidy is correlated with sexual reproduction and outcrossing and polyploidy is correlated with apomixis (Adamowski et al., 2005).

The native germplasm of Paspalum notatum Flüggé is tetraploid and shows characteristics of apomictic lineages. In the ecotypes of P. notatum that have an apomictic mode of reproduction, female meiosis does not occur or is not functional. In “bahiagrass”, apomixis is aposporic and pseudogamous, in which embryo sac arises by mitotic division of a somatic cell of the embryo sac and endosperm develops by the union of a male nucleus of the pollen grain with the polar nuclei (Acuña et al., 2009).

In crosses between apomictic and sexual plants, there is a wide segregation between plants of the two modes of reproduction. The most reliable way to assess whether an individual plant is apomictic or sexual is through cytoembryological analysis of the sexual embryo sacs and apomictic embryo sacs. Another way to evaluate the mode of reproduction is through the use of molecular markers, which requires a marker linked or at least very close to the gene region that controls the apomixis. In P. notatum, two RAPD (random amplification of polymorphic DNA) molecular markers were selected, completely linked to the expression of apospory: BCU 243 and BCU 259 (Martínez et al., 2003). The use of molecular markers allows rapid determination of the mode of reproduction at the seedling stage, enabling a screening of plants that will be taken to the field.

The objective of this study was to determine the mode of reproduction of a hybrid progeny derived from intraspecific crosses of P. notatum through cytoembryological analysis and through the use of RAPD molecular markers.

Material and Methods

In collaboration with IBONE (Instituto de Botânica del Nordeste), located in Corrientes, Argentina, three genotypes artificially polyploidizated by colchicine were obtained, called Q4188, Q4205 (Quarin et al. 2003), and C44X (Quarin et al., 2001). These three genotypes are sexual tetraploids and were used as female parents in artificial crosses with elite 100% apomictic tetraploid genotypes native from Rio Grande do Sul (ecotypes André da Rocha and Bagual), used as the male parents. The crosses were performed using the methodology described by Burton (1948) to obtain an F1 hybrid progeny. Crossing schemes allowed six combinations of parents that gave rise to the families Q4188 × André da Rocha (Progeny A), Q4188 × Bagual (Progeny B), Q4205 × André da Rocha (Progeny C), Q4205 × Bagual (Progeny D), C44X × André da Rocha (Progeny E), and C44X × Bagual (Progeny F).

The determination of the mode of reproduction through cytoembryological analysis was performed on 28 hybrids, selected from 196 evaluated for their dry mass productivity. To confirm their reproductive mode, cytoembryological analyses were also performed in all the parents involved in the crosses and the cultivar Pensacola of P. notatum, used as a control in all agronomic evaluations performed in hybrid progeny.

For the cytoembryological analysis, the inflorescences in anthesis (when the embryo sac is fully developed) were collected and the dissected flowers were fixed in FAA (95% ethanol, 40 mL; distilled water, 14 mL; 40% formalin, 3 mL; and Glacial acetic acid, 3 mL) for 24 h at room temperature. After this period, they were stored in 70% ethanol and kept refrigerated until the extraction of ovaries that were dissected from the flowers. The samples containing the ovaries underwent a clarifying process consisting of a series of dehydrations in alcohol with methyl salicylate, following the protocol proposed by Young et al. (1979) and modified by Acuña et al. (2007).

Clarified ovaries were stored in a solution of methyl salicylate (100%) until the interferential contrast microscopy analyses. For this, they were arranged on a slide and kept moist with methyl salicylate. For each hybrid plant, a minimum of 30 ovaries were analyzed to determine the mode of reproduction.

Single embryo sacs containing the egg apparatus, the binucleated central cell, and a mass of antipodals at the chalazal end were classified as sexual. In contrast, multiple or single embryo sacs with the egg apparatus, the central cell, and no antipodals were classified as apomictic. Plants producing ovules with either sexual or apomictic embryo sacs were classified as facultative (Acuña et al., 2007).

Of the hybrid plants resulting from crosses made, 194 (including the 28 individuals studied by cytoembryological analysis) were evaluated for their mode of reproduction using RAPD molecular markers. Extraction of DNA was performed according to the methodology described by Ferreira and Grattapaglia (1998), with the modification of making a microextraction of about 1 cm2 of a young leaf, ignoring the midrib. The DNA samples were quantified by electrophoresis (100 V for 1 h) in 1% agarose gel, stained with ethidium bromide for visualization of bands, and compared with standards of known concentration (50, 100, 200, and 500 ng DNA).

The polymerase chain reactions (PCR) were performed in a final volume of 13 µL containing 1.5 units Taq polymerase, 10 mmol L–1 dNTP mix (dATP, dTTP, dCTP, dGTP), 2.5 µL 10x buffer (10 mmol L–1 Tris - HCl (pH 8.3), 50 mmol L–1 MgCl, 30 pg of each primer, 20 ng of genomic DNA, and autoclaved water and amplified in MJ thermocycler. Visualization was done on 1.8% agarose gel prepared with TAE buffer (0.04 M Tris acetate, 1 mM EDTA) at 100 volts for 3 h in horizontal wells.

The analysis of the gels was made by comparison of banding patterns between hybrids and the banding pattern of the parents (female and male), repeating the analysis of gel bands that were doubtful or not very clear.

Results and Discussion

Cytoembryological analyses allowed for the identification of the reproductive mode of 28 hybrid plants (Table 1) that were selected after agronomic evaluations of productivity (Weiler, 2013). The mode of reproduction of all 28 plants was successfully determined; 19 showed the morphology of the embryo sac compatible with apomictic reproduction (Figure 1) and nine had morphology of the embryo sac compatible with sexual reproduction (Figure 2). Two hybrid plants, A16 and B26, demonstrated facultative apomictic characteristics.

Table 1 Mode of reproduction of 28 selected hybrid plants obtained from intraspecific crosses of Paspalum notatum, determined by cytoembryological analyses 

Genotypes Sexual ovaries Apomictic ovaries Atrophied ovaries Sterile ovaries Abnormal ovaries Total ovaries Mode of reproduction
D3 - 24 - - 18 42 Apomictic
C17 - 22 - - 18 40 Apomictic
D16 30 - - - 8 38 Sexual
B17 7 1 - 1 97 106 Apomictic
C2 1 21 - 2 14 38 Apomictic
B43 - 41 3 - 1 45 Apomictic
C24 17 - 3 - 14 34 Sexual
D25 23 - - - 8 31 Sexual
C18 24 - - 1 16 41 Sexual
F15 - 40 - - - 40 Apomictic
F29 2 26 - - 10 38 Apomictic
B26 26 16 - - 14 56 Apomictic facultative
B37 - 31 - - 7 38 Apomictic
F24 - 29 - - 10 39 Apomictic
D17 28 - 4 - 24 56 Sexual
C32 4 - - - 26 30 Sexual
C9 2 3 - - 28 33 Apomictic
C6 - 21 - - 9 30 Apomictic
C15 - 29 - 1 1 31 Apomictic
B29 2 - - - 28 30 Apomictic
D23 23 - - - 17 40 Sexual
B35 26 - - 7 12 45 Apomictic
A20 2 34 1 - 3 40 Apomictic
C22 - 24 - 1 6 31 Apomictic
A16 12 19 - - 33 64 Apomictic facultative
C23 - 27 2 - 10 39 Apomictic
B2 4 1 - - 26 31 Sexual
B28 24 - - - 16 40 Sexual
Bagual - 28 - 1 9 38 Apomictic
André da Rocha - 22 1 1 14 38 Apomictic
Pensacola 19 - 2 3 12 36 Sexual

Arrows indicate multiple sacs.

Scale bar: 10 µm.

Figure 1 Morphology of the embryo sac in the apomictic hybrid B43. 

Arrow indicates the antipodes.

Scale bar: 10 µm.

Figure 2 Morphological features of the embryo sac of the sexual hybrid plant B37. 

During the aposporic development, the megaspore mother cell can degenerate before or after the initial differentiation of apospory or suffer meiosis and form a reduced embryo sac. Thus, the sexual process can co-exist with unreduced embryo sacs, becoming initially aposporic in one embryo sac (Karasawa, 2009) and aposporic apomictic plants may also exhibit some sexual reproduction at different frequencies and are, therefore, characterized as facultative apomictic plants. In these cases, a number of factors, such as seasonal fluctuations associated with photoperiod during development of the inflorescence and responses to day length, light intensity, temperature, and type and level of soil fertility, cause changes in the frequency of incidences of sexual and apomictic embryos (Koltunow, 1993).

The genetic control of apomixis in tetraploid P. notatum is by a single dominant locus and with Mendelian segregation, but in controlling the expression of apomixis, there is probably a pleiotropic lethal effect of the dominant locus or partial lethality factors linked to the aposporic gene, possibly causing segregation distortion in favor of plants with the sexual mode of reproduction (Martínez et al., 2001).

Valle and Savidan (1996) reported that the occurrence of two types of sacs in the same embryonic egg in apomictic plants of Brachiaria is considered indicative of facultative apomixis. Quarin et al. (2001) also mentioned that an artificially induced tetraploid population may have apomictic, sexual, or facultative mode of reproduction, indicating that apomictic genes exist in diploid plants and that expression of these genes is repressed in the diploid level.

In Brachiaria, it was found that the sexual embryo sac present in facultative apomictic plants can be fertilized, form a zygote, and generate a plant sexually, thus characterizing facultative apomixis that, by definition, is the occurrence of viable seeds derived from fertilization of a sexual embryo sac in a apomictic plant (Nogler, 1984; Koltunow, 1993). In this genus, the frequency of embryo sacs with reduced apospory can vary from 0 to 50% (Valle et al., 1994).

A high number of abnormal ovaries were found in almost all plants (Table 1), ratio of 1:2.4 (abnormal:total ovaries), possibly because the ovaries were not in the best period of anthesis when inflorescences were collected for analysis.

In this paper, 28 plants were selected for the cytoembryological analysis, because these plants had the largest dry mass production; a ratio of 1:2.1 was found (sexual:apomictic), which did not differ (P>0.05) from the expected ratio of 1:3 (sexual:apomictic). The genetic aposporic segregation was previously explained by a genetic model of simple Mendelian inheritance with dominance of apospory over sexual reproduction, but with a strong distortion of segregation (Martínez et al., 2001). Contrary to that found in this study, Fortes et al. (2004) mentioned that this distortion had been found responsible for values of 4.3:1 (sexual:apomictic), differing from the expected 1:1. Martínez et al. (2001) found values of 2.8:1 (sexual:apomictic), while the work of Stein et al. (2004) found 6.5:1 (sexual:apomictic). All these studies were conducted with Paspalum notatum genotypes.

The analyses with molecular markers showed that only the primer BCU 243 had a stable pattern of band amplification in ten different reactions with the female parents (Q4188, Q4205 and C44X), the male parents (Bagual and André da Rocha), and the cultivar Pensacola. In sexual plants, a band of 700 bp was found and for apomictic plants, two bands were found, one at 700 bp and the other at 750 bp (Figure 3). Primer BCU 259 showed no stable pattern of differentiation of apomictic and sexual plants when parents were tested. The work of Martínez et al. (2003) showed that the BCU 243 primer amplified a fragment specific to apomictic parents in bulk analysis.

Indicated bands are 700 and 750 bp.

Figure 3 Agarose gel at 1.8% with the sexual female parents Q4188, Q4205, and C44X (columns 1, 2, and 3), apomictic male parents André da Rocha and Bagual (columns 4 and 5), Pensacola (column 6), hybrids A24, A25, A26, A28, A29 (columns 7, 8, 9, 11, and 12), and column 10, a fail amplification. 

The ratio of 1:2.6 (sexual plants:apomictic) was found; however, in studies conducted at IBONE, evaluating 44 hybrids, Martínez et al. (2001) found a ratio of 4.5:1 (sexual:apomictic). This distorted segregation is possibly related to the lethal effect of the dominant pleiotropic locus or partial lethality factors linked to the aposporic gene.

The findings of the molecular marker analysis in this study were somewhat similar to the expected, with 54 sexual plants to 140 apomictic plants (Table 2), meaning a ratio similar to the expected of 1:2.6 (sexual:apomictic).

Table 2 Mode of reproduction of intraspecific hybrid progeny of Paspalum notatum, determined by BCU 243 RAPD molecular marker 

Hybrid Mode of reproduction Hybrid Mode of reproduction Hybrid Mode of reproduction Hybrid Mode of reproduction
A 2 Sexual B22 Apomictic C28 Sexual E15 Apomictic
A 7 Sexual B23 Apomictic C29 Apomictic E16 Sexual
A 8 Apomictic B25 Apomictic C30 Apomictic E17 Apomictic
A10 Apomictic B26 Apomictic C31 Apomictic E18 Apomictic
A11 Apomictic B27 Sexual C32 Apomictic E19 Apomictic
A12 Apomictic B28 Apomictic C33 Apomictic E20 Sexual
A13 Apomictic B29 Apomictic C34 Apomictic E21 Apomictic
A14 Apomictic B30 Apomictic C35 Apomictic E22 Apomictic
A15 Apomictic B31 Sexual C36 Apomictic E23 Sexual
A16 Sexual B32 Apomictic D1 Apomictic E24 Apomictic
A17 Apomictic B33 Apomictic D2 Apomictic F1 Sexual
A18 Apomictic B34 Apomictic D3 Apomictic F2 Apomictic
A20 Apomictic B35 Apomictic D4 Sexual F3 Apomictic
A21 Apomictic B36 Apomictic D5 Apomictic F4 Apomictic
A22 Sexual B37 Sexual D6 Apomictic F5 Apomictic
A23 Sexual B38 Apomictic D7 Apomictic F6 Sexual
A24 Apomictic B39 Sexual D8 Apomictic F7 Sexual
A25 Apomictic B40 Apomictic D9 Apomictic F8 Apomictic
A26 Apomictic B41 Apomictic D10 Apomictic F9 Apomictic
A27 Apomictic B42 Apomictic D11 Apomictic F10 Apomictic
A28 Apomictic B43 Apomictic D12 Sexual F11 Sexual
A29 Apomictic B44 Apomictic D13 Sexual F12 Apomictic
A31 Sexual B52 Sexual D14 Sexual F13 Sexual
A32 Apomictic C1 Apomictic D15 Apomictic F14 Apomictic
A33 Apomictic C2 Apomictic D16 Apomictic F15 Apomictic
A35 Apomictic C3 Apomictic D17 Apomictic F16 Apomictic
A36 Sexual C4 Apomictic D18 Sexual F17 Apomictic
A37 Apomictic C5 Apomictic D19 Apomictic F18 Apomictic
A38 Sexual C6 Apomictic D20 Sexual F19 Apomictic
B1 Apomictic C7 Apomictic D21 Apomictic F20 Apomictic
B2 Sexual C8 Apomictic D22 Sexual F21 Sexual
B3 Sexual C9 Apomictic D23 Apomictic F22 Apomictic
B4 Sexual 10 Sexual D24 Apomictic F23 Apomictic
B5 Apomictic C11 Sexual D25 Apomictic F24 Apomictic
B6 Sexual C12 Apomictic D26 Apomictic F25 Apomictic
B7 Apomictic C13 Apomictic D27 Apomictic F26 Apomictic
B8 Sexual C14 Sexual E1 Apomictic F27 Apomictic
B 9 Sexual C15 Apomictic E2 Sexual F28 Apomictic
B10 Apomictic C16 Sexual E3 Apomictic F29 Apomictic
B11 Sexual C17 Apomictic E 4 Apomictic F30 Apomictic
B12 Apomictic C18 Sexual E 5 Apomictic F31 Apomictic
B13 Sexual C19 Apomictic E 6 Apomictic F32 Apomictic
B14 Apomictic C20 Apomictic E 7 Apomictic F33 Apomictic
B15 Apomictic C21 Apomictic E 8 Apomictic Bagual Apomictic
B16 Sexual C22 Apomictic E 9 Sexual André da Rocha Apomictic
B17 Sexual C23 Apomictic E10 Sexual Q4188 Sexual
B18 Sexual C24 Apomictic E11 Sexual Q4205 Sexual
B19 Apomictic C25 Apomictic E12 Apomictic C44X Sexual
B20 Sexual C26 Sexual E13 Sexual Pensacola Sexual
B21 Apomictic C27 Apomictic E14 Sexual

In progeny A, of the 29 hybrids obtained, 21 were considered apomictic and eight were sexual. In progeny B, of 44 hybrids, 27 were apomictic and 17 were sexual. In progeny C, of 37 hybrids, 30 were apomictic and seven were sexual. In progeny D, of 27 hybrids, 20 were apomictic and seven were sexual. In progeny E, of 24 hybrids, 15 were apomictic and nine were sexual. In progeny F, of 33 hybrids, 27 were apomictic and six were sexual (Table 2). In all progenies, fewer sexual hybrids were found in comparison with apomictic, ranging from a ratio of 1:1.4 (sexual:apomictic) in family E to a ratio of 1:4.5 (sexual:apomictic) in family F.

The cytoembryological analysis is made with direct visualization of the ovaries and the determination of the mode of reproduction occurs through the structures observed. Many ovaries of different flowers and inflorescences are measured, resulting in higher reliability.

Of the 28 plants analyzed by the two methods, ten results (35.7%) were in disagreement. They were plants A16, B17, B28, B37, C24, C32, D16, D17, D23, and D25. Possibly, the variance of the results can be attributed to the marker used; even if this marker worked in the IBONE studies, it did not show satisfactory repeatability of the results. According to Meunier and Grimont (1993), the low reproducibility of RAPD could be explained by the low touchdown of primers (~30 °C). Thus, the annealing was done at a higher touchdown temperature (35 °C), which gave higher stringency conditions for good day-to-day reproducibility. However, the data shown by Fortes et al. (2004), after analyzing 44 hybrids using RAPD markers, showed similar results to the cytoembryological analysis, finding more apomictic than sexual plants.

Rebozzio et al. (2012), using RAPD markers BCU 243 and BCU 259 along with eleven AFLP markers, showed that these markers, previously connected to the aposporic region in the apomictic plant Q4117, were also conserved in other apomictic accessions, although the marker of the BCU 243 primer in particular presented a band clearly linked to the aposporic region. According to the same authors, in an attempt to obtain SCAR (sequence-characterized amplified regions) markers, which would be more specific in the identification of apomictic plants, it was only possible to develop a SCAR marker from an AFLP (amplified fragment length polymorphism) marker (Espinoza et al., 2006).

Molecular markers can be a useful tool in distinguishing sexual and apomictic plants by accelerating the process of identification and selection of apomictic plants. However, results must be validated for each laboratory by comparison with embryological data. As a result, the repetition of the molecular analyses, including other markers, should be performed.

For all the parents involved in the crosses and the cultivar Pensacola of P. notatum, the mode of reproduction was confirmed by both analyses.

In a breeding program, it is essential to determine the mode of reproduction in segregating progenies obtained by crosses between sexual and apomictic genotypes, allowing the formation of groups of plants separated by the mode of reproduction. Thus, apomictic plants can be used directly as cultivars if they are of agronomic interest, while sexual plants can be inserted as female parents in programs of continued crosses.

The data obtained in this work represent a major breakthrough for the breeding program. This makes it possible to directly use selected plants by their agronomic performance, subjecting apomictic plants to new field trials to prove their agronomic value and also allow the registration and protection of plant varieties. Besides, sexual plants could be used for future crosses, making them potential parents within this program.


The predominant mode of reproduction is the apomictic in a ratio of 1:2.1 of sexual to apomictic plants.

It is possible to determine the mode of reproduction through cytoembryological and RAPD molecular marker analyses.


The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support.


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Received: March 18, 2016; Accepted: October 27, 2016

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