Meiotic behavior of Brachiaria decumbens hybrids.

Brachiaria decumbens is a forage grass of inestimable value for livestock in Brazil due to its production of good quality forage even when planted on acid and poor soils, although it is susceptible to pasture spittlebugs. Only one cultivar, cv. Basilisk, has been used as the pollen donor in crosses with Brachiaria ruziziensis since 1988 at Embrapa Gado de Corte Research Center. Breeding within the species only became possible from 2009 when sexual accessions were successfully tetraploidized using colchicine. Three sexual genotypes were obtained and hybridization within B. decumbens was finally achieved. Here, we evaluated microspore tetrads using conventional cytology and found meiotic indexes above 78% for all three female genitors (cD24-2, cD24-27, cD24-45), but a low meiotic index (<22%) in the natural apomictic genitor D62 (cv. Basilisk) and in 49 hybrids. Analysis of the relationship between abnormal tetrad frequency and non-viable pollen grains yielded a highly significant Pearson correlation coefficient. The t-test proved significant for the progeny of cD24-45 x D62, with lower abnormalities and pollen sterility when compared to the other two progenies resulting from cD24-2 and cD24-27 crossed to D62, but these two did not differ. Apomictic hybrids such as S036 and X030 with low pollen sterility have the potential for use in cultivar development, whereas the sexual hybrids T012, X072, and X078 might be of use as female genitors in polycross blocks if they display good agronomic traits.

Meiotic behavior and chromosome number of Urochloa adspersa (Trin.) R. D. Webster from the Brazilian Chaco.

This is the first report of meiotic division in Uro-chloa adspersa (Trin.) collected from the Brazilian Chaco. Meiotic analyses were performed on three specimens of U. adspersa named G10, G15, and G16. Inflorescences were collected and fixed in a mixture of ethanol and acetic acid (3:1, v/v) for 24 h and then stored in 70% alcohol. Diakinesis revealed different chromosome numbers and ploidy levels. All three plants were polyploids: G10 and G15 exhibited 2n = 6x = 54 chromosomes (arranged in 27 bivalents), while G16 exhibited 2n = 4x = 36 chromosomes (18 bivalents). Meiotic behavior was mainly normal in the hexaploid G15 and the tetraploid G16 (5.3 and 6.2% of the cells were abnormal, respective-ly), revealing only a few meiotic abnormalities that are common to polyploids, i.e., those related to irregular chromosome segregation. G10 exhibited other meiotic abnormalities during meiosis II, such as chromosome stickiness, irregular spindle orientation, and irregular cytokinesis, which led to the formation of a few triads, resulting in 16.9% of the cells being abnormal. The origin of these abnormalities is discussed, and we suggest that the genes that control meiotic steps may be present in the Urochloa gene pool.

Chromosome stickiness impairs meiosis and influences reproductive success in Panicum maximum (Poaceae) hybrid plants.

Chromosome stickiness has been studied in several species of higher plants and is characterized by sticky clumps of chromatin resulting in sterility. Chromosome stickiness was recorded in Panicum maximum hybrid plants that were cultivated in the field. In the meiocytes affected, chromosomes clumped into amorphous masses that did not orient themselves on the equatorial plate, and anaphase I disjunction failed to occur. After a normal cytokinesis, the masses of chromatin were divided between both daughter cells. Metaphase and anaphase of the second division also did not occur, and after the second cytokinesis, polyads were formed. This abnormality arose spontaneously. Abnormalities that cause male sterility are an important tool for obtaining hybrid seeds in plant breeding. This is the first report of an abnormality affecting pollen viability in P. maximum. This finding can open a new opportunity in the breeding program of this species that is devoted to hybridization where manual cross-pollination is difficult and time consuming.

Complete asynapsis resulting in 2n pollen formation in Paspalum jesuiticum Parodi (Poaceae).

Cytological investigation revealed complete asynapsis during microsporogenesis in 2 wild accessions of Paspalum jesuiticum collected in distinct Brazilian regions. Both accessions were hexaploid (2n = 6x = 60) and 60 univalents could be counted at diakinesis. In this phase, the majority of meiocytes exhibited univalents with both chromatids. After alignment at the metaphase plate, the chromatids segregated to the poles. Only 1 meiotic division (equational) occurred, and after cytokinesis, a dyad with 2n microspores was formed. The genetic constitution of the 2n gametes was equivalent to that of first division restitution (FDR). Since recombination did not occur, 100% transmission of parental heterozygosity could be expected from the FDR 2n gametes. The meiotic behavior of both accessions suggested that they resulted from a recent natural hybridization event. The potential use of the 2n gametes in Paspalum breeding programs has been discussed.

Microsporogenesis in Paspalum conspersum Schrad. (Virgata group) with different ploidy levels.

Knowledge about the cytology and reproductive behavior of a species is indispensable for hybridization programs. This is especially true for species belonging to the genus Paspalum, among which apomixis and a wide range of ploidy levels are frequently found. Paspalum conspersum Schrad. is a robust and warm-season perennial bunchgrass native to South America. Previous studies have indicated that both tetraploid and hexaploid races exist in this species; however, only information related to tetraploids has been applied to another taxon. In this study, a cytological investigation in two Brazilian accessions collected in different regions revealed tetraploidy in the accession BRA-012823 (2n = 4x = 40), with chromosome pairing in bivalents and normal meiosis and tetrad formation, and pentaploidy (2n = 5x = 50) in the accession BRA-022748, which presented total asynapsis. In this latter accession, 50 univalents could be scored at diakinesis. After alignment at the metaphase plate, sister chromatids segregated to the poles. Only one meiotic division (equational) occurred, and after cytokinesis, 100% of the dyads that formed had 2n microspores. The meiotic behavior during microsporogenesis, which showed 10 delayed univalents to reach the metaphase plate, suggests that this accession is a recent natural hybrid constituted by a parental genome with 40 chromosomes and another with 10 chromosomes. The potential usage of these accessions in Paspalum breeding has been discussed.

Stigma receptivity, mode of reproduction, and mating system in Mesosetum chaseae Luces (Poaceae), a native grass of the Brazilian Pantanal.

Mesosetum chaseae Luces, known regionally as "grama-do-cerrado", is abundant in the Pantanal region in Brazil and contributes significantly to livestock and environmental conservation. This species is under basic studies at Embrapa Pantanal (Nhecolândia subregion, Pantanal, Corumbá, MS, Brazil). In this study, we present data about stigma receptivity, mode of reproduction, and mating system for 10 accessions collected in Nhecolândia subregion (Pantanal). Stigma receptivity was optimal, producing innumerous oxygen bubbles upon testing with hydrogen peroxidase. Clarified ovaries analyzed under interference microscopy showed an embryo sac of the Polygonum type, typical of sexual species. The mating system, tested in protected flowers, indicated allogamy. These data are important for subsidizing future breeding programs for this species.

Chromosome number, microsporogenesis, microgametogenesis, and pollen viability in the Brazilian native grass Mesosetum chaseae (Poaceae).

The genus Mesosetum is a primarily South American genus with 42 species. Mesosetum chaseae, regionally known as 'grama-do-cerrado', is abundant in the Pantanal Matogrossense (Brazil); it is a valuable resource for livestock and for environmental conservation. We collected specimens from the Nhecolandia sub-region of the Brazilian Pantanal, located in Corumbá, Mato Grosso do Sul, Brazil. We examined chromosome number, ploidy level, meiotic behavior, microgametogenesis, and pollen viability of 10 accessions. All the accessions were diploid, derived from x = 8, presenting 2n = 2x = 16 chromosomes. Chromosomes paired as bivalents showing, predominantly, two terminal chiasmata. Interstitial chiasmata were rare. Meiosis was quite normal producing only a few abnormal tetrads in some accessions. Microgametogenesis, after two mitotic divisions, produced three-celled pollen grains. Pollen viability was variable among plant and accessions and was not correlated with meiotic abnormalities.

Microsporogenesis in Brachiaria brizantha (Poaceae) as a selection tool for breeding.

The genus Brachiaria comprises more than 100 species and is the single most important genus of forage grass in the tropics. Brachiaria brizantha, widely used in Brazilian pastures for beef and dairy production, is native to tropical Africa. As a subsidy to the breeding program underway in Brazil, cytological studies were employed to determine the chromosome number and to evaluate microsporogenesis in 46 accessions of this species available at Embrapa Beef Cattle (Brazil). Thirty-four accessions presented 2n = 36; seven had 2n = 45, and five had 2n = 54 chromosomes. Based on the higher level of chromosome association observed in diakinesis, in tetra-, penta-, and hexavalents, respectively, it was concluded that they are derived from x = 9; consequently, these accessions are tetra- (2n = 4x = 36), penta- (2n = 5x = 45), and hexaploids (2n = 6x = 54). The most common meiotic abnormalities were irregular chromosome segregation due to polyploidy. Chromosome stickiness, abnormal cytokinesis, non-congressed bivalents in metaphase I and chromosomes in metaphase II, and chromosome elimination were recorded at varying frequencies in several accessions. The mean percentage of meiotic abnormalities ranged from 0.36 to 95.76%. All the abnormalities had the potential to affect pollen viability by generating unbalanced gametes. Among the accessions, only the tetraploid ones with less than 40% of abnormalities are suitable as pollen donors in intra- and interspecific crosses. Currently, accessions with a high level of ploidy (5 and 6n) cannot be used as male genitors in crosses because of the lack of sexual female genitors with the same levels of ploidy.

ms17: a meiotic mutation causing partial male sterility in a corn silage hybrid.

Cytological analysis under light microscopy of the single hybrid P30R50 of silage corn revealed an abnormal pattern of microsporogenesis that affected the meiotic products. Meiosis progressed normally until diakinesis, but before migration to the metaphase plate, bivalents underwent total desynapsis and 20 univalent chromosomes were scattered in the cytoplasm. At this stage, meiocytes also exhibited a number of chromatin-like fragments scattered throughout the cell. Metaphase I was completely abnormal in the affected cells, and univalent chromosomes and fragments were distributed among several curved spindles. Anaphase I did not occur, and each chromosome or group of chromosomes originated a micronucleus. After this phase, an irregular cytokinesis occurred, and secondary meiocytes with several micronuclei were observed. Metaphase II and anaphase II also did not occur, and after the second cytokinesis, the genomes were fractionated into polyads, generating several unbalanced microspores, with various-sized nuclei. About 35% of the tetrads were abnormal in the hybrid. This spontaneous mutation had been previously reported in a USA maize line called ms17 and was found to cause male sterility.

Meiotic behavior and pollen viability in Moringa oleifera (Moringaceae) cultivated in southern Brazil.

Although originally from India, Moringa oleifera is now cultivated throughout most of the tropics, including Brazil. Despite its multipurpose value for food and traditional medicine, little is known about the meiotic behavior and pollen viability of M. oleifera. We evaluated microsporogenesis and pollen viability in eleven plants grown in southern Brazil (Maringá, Paraná). Bud flowers were collected in different stages of development. All plants that we analyzed presented 2n = 28 chromosomes, as previously reported for this species. Chromosomes paired as bivalents. Meiotic abnormalities were rare and metaphase I was the most affected phase. Pollen viability was superior to 88%. Tripolar spindles in metaphase II, leading to the formation of unreduced gametes, were recorded in some plants at a low frequency.

Meiotic behavior in nonaploid accessions of Brachiaria humidicola (Poaceae) and implications for breeding.

Brachiaria humidicola is a grass adapted to seasonally swampy grasslands in Africa; two cultivars, 'common' and Llanero, are widely used in Brazilian pastures. New cultivars are in great demand in order to diversify current production systems to achieve improved quality and yield. Cytological analyses of 55 accessions of this species available from the Embrapa Beef Cattle germplasm collection revealed that 27 are apomictic and have 2n = 54 chromosomes. Chromosome pairing as bi- to nonavalent associations at diakinesis indicated a basic chromosome number in this species of x = 6, as found in other closely related Brachiaria species. Thus, these 27 accessions are nonaploid (2n = 9x = 54). Abnormalities were found in the meiosis of these accessions, at variable frequencies. The most common abnormalities were those related to irregular chromosome segregation, which led to unbalanced gamete formation; but chromosome stickiness, cell fusion, and absence of cytokinesis were also recorded. Although some accessions have a low frequency of meiotic abnormalities, ensuring potentially good pollen viability, these cannot be used in hybridization due to a lack of sexual accessions with the same ploidy level.

Genome elimination during microsporogenesis in two pentaploid accessions of Brachiaria decumbens (Poaceae).

Polyploidy is a prominent and significant force in plant evolution, taking place since ancient times and continuing until today. Recent cytogenetic studies in the genus Brachiaria using germplasm collected from wild African savannas in the 1980s revealed that most species and accessions within species are polyploid. Diploid, tetraploid, and pentaploid accessions have been found. We found asynchronous meiosis during microsporogenesis, followed by genome elimination, in two pentaploid (2n = 5x = 45) accessions (D53 and D71) of a hardy, invasive pasture grass, introduced from Africa to Brazil, Brachiaria decumbens. In these accessions, chromosomes paired as 18 bivalents and nine univalents during diakinesis, suggesting that these accessions resulted from a recent event of natural hybridization. The lack of chromosome associations in the genomes suggests that these accessions resulted from hybridization between two genotypes that are not closely related, with low genome affinity and with different meiotic rhythms. This supposition is reinforced by the meiotic behavior of the nine univalents, which were always laggard in relation to the other chromosomes and eliminated as micronuclei in microspores. The behavior of these accessions, which have an odd level of ploidy and confirmed genome elimination, supports the general assumption that a polyploid accession can undergo a new event of polyploidization by natural hybridization (neopolyploidyzation). This evidence for natural hybridization in Brachiaria shows that this is a wild genus in an ongoing evolutionary process.

Genetic diversity and population structure analysis of the tropical pasture grass Brachiaria humidicola based on microsatellites, cytogenetics, morphological traits, and geographical origin.

Brachiaria humidicola (Rendle) Schweick. is a warm-season grass commonly used as forage in the tropics. Accessions of this species were collected in eastern Africa and massively introduced into South America in the 1980s. Several of these accessions form a germplasm collection at the Brazilian Agricultural Research Corporation. However, apomixis, ploidy, and limited knowledge of the genetic basis of this germplasm collection have constrained breeding activities. The objectives of this work were to identify genetic variability in the Brazilian B. humidicola germplasm collection using microsatellite markers and to compare the results with information on the following: (1) collection sites of the accessions; (2) reproductive mode and ploidy levels; and (3) genetic diversity revealed by morphological traits. The evaluated germplasm population is highly structured into four major groups. The sole sexual accession did not group with any of the clusters. Genetic dissimilarities did not correlate with either geographic distances or genetic distances inferred from morphological descriptors. Additionally, the genetic structure identified in this collection did not correspond to differences in ploidy level. Alleles exclusive to either sexual or apomictic accessions were identified, suggesting that further evaluation of the association of these loci with apospory should be carried out.

Meiotic behavior as a selection tool in silage corn breeding.

In breeding programs, commercial hybrids are frequently used as a source of inbred lines to obtain new hybrids. Considering that maize production is dependent on viable gametes, the selection of populations to obtain inbred lines with high meiotic stability could contribute to the formation of new silage corn hybrids adapted to specific region. We evaluated the meiotic stability of five commercial hybrids of silage corn used in southern Brazil with conventional squashing methods. All of them showed meiotic abnormalities. Some abnormalities, such as abnormal chromosome segregation and absence of cytokinesis, occurred in all the genotypes, while others, including cytomixis and abnormal spindle orientation, were found only in some genotypes. The hybrid SG6010 had the lowest mean frequency of abnormal cells (21.27%); the highest frequency was found in the hybrid P30K64 (44.43%). However, the frequency of abnormal meiotic products was much lower in most genotypes, ranging from 7.63% in the hybrid CD304 to 43.86% in Garra. Taking into account the percentage of abnormal meiotic products and, hence, meiotic stability, only the hybrids CD304, P30K64, SG6010, and P30F53 are recommended to be retained in the breeding program to obtain inbred lines to create new hybrids.

Meiotic behavior of a nonaploid accession endorses x = 6 for Brachiaria humidicola (Poaceae).

Brachiaria humidicola (Poaceae), originally from Africa, is an economically important pasture plant in tropical South America. An accession of B. humidicola (H038) collected from the wild African savanna (Mbeya, Tanzania) showed irregular microsporogenesis. This meiotic behavior was consistent with an allopolyploid origin. Multivalent chromosome association at diakinesis gave tri- to octavalents, associated with two nucleoli in some cells. Six non-congregated univalents in metaphase I and anaphase I, along with previous lines of evidence for x = 6 in B. humidicola, confirm H038 as a nonaploid accession, 2n = 9x = 54. Asynchrony in the genome during microsporogenesis also corroborated this assumption. Its putative origin could be a cross between two related species with different rhythms in meiosis. The meiotic behavior of this accession reinforces the hypothesis of the existence of a new basic chromosome number (x = 6) for Brachiaria. The use of this accession in the breeding of this important forage grass for the tropics is discussed.

Origin of a polyploid accession of Brachiaria humidicola (Poaceae: Panicoideae: Paniceae).

Brachiaria humidicola, a species adapted to poorly drained and infertile acid soils, is widely used throughout the tropics. Cytological characterization of 54 accessions of B. humidicola for breeding purposes revealed 2n = 36, 42, and 54 chromosomes. One accession (H030), with 2n = 42 chromosomes, showed a different meiotic behavior. In most accessions from the genus Brachiaria previously studied, the basic chromosome number is x = 9, but the putative basic number in H030 appears to be x = 6. Since six univalent chromosomes were found in diakinesis and metaphase I, and these behaved as laggards in anaphase I, it was hypothesized that both genitors were derived from x = 6, and that this accession is a heptaploid 2n = 7x = 42. The parental genomes did not have the same meiotic behavior, particularly during anaphase, when one genome consisting of six univalents remained as laggards and underwent sister-chromatid segregation. At telophase, 18 segregated chromosomes were found at each pole. The laggard genome did not reach the poles at telophase I or II in time to be included in the nucleus and was eliminated as micronuclei.

Mitotic behavior in root tips of Brachiaria genotypes with meiotic chromosome elimination during microsporogenesis.

Three accessions of Brachiaria brizantha, three of B. humidicola, and two interspecific hybrids between B. ruziziensis and B. brizantha were analyzed with regard to their mitotic behavior in root tips. All these genotypes revealed chromosome elimination or lack of chromosome affinity in previous analyses of microsporogenesis. Analyses of root tips showed a normal mitotic division in all accessions and hybrids, reinforcing the notion that the genetic control of meiosis is totally independent of that of mitosis. The implications of these findings for the Brachiaria breeding program are discussed.

Evaluation of microsporogenesis in an interspecific Brachiaria hybrid (Poaceae) collected in distinct years.

Microsporogenesis in an interspecific Brachiaria hybrid, grown in the field under natural environmental conditions in Brazilian savannas, was analyzed in three distinct years of collection. Several types of meiotic abnormalities were recorded during those three years, but varied in type and frequency depending on the year. The average temperature and rainfall 15 days before collection was unusually high in those years. The percentage of abnormal meiocytes recorded was 62% in 2001, 73% in 2004, and 77% in 2005. The abnormalities observed during microsporogenesis compromised pollen viability by generating unbalanced gametes or affecting nucleolus organization. The environmental conditions under which the hybrid was growing could have affected the genetic control of meiosis. More detailed studies, under controlled conditions, are necessary to better understand the effects of environmental factors on Brachiaria microsporogenesis hybrids.

Evaluation of microsporogenesis in an interspecific Brachiaria hybrid (Poaceae) collected in distinct years

Microsporogenesis in an interspecific Brachiaria hybrid, grown in the field under natural environmental conditions in Brazilian savannas, was analyzed in three distinct years of collection. Several types of meiotic abnormalities were recorded during those three years, but varied in type and frequency depending on the year. The average temperature and rainfall 15 days before collection was unusually high in those years. The percentage of abnormal meiocytes recorded was 62% in 2001, 73% in 2004, and 77% in 2005. The abnormalities observed during microsporogenesis compromised pollen viability by generating unbalanced gametes or affecting nucleolus organization. The environmental conditions under which the hybrid was growing could have affected the genetic control of meiosis. More detailed studies, under controlled conditions, are necessary to better understand the effects of environmental factors on Brachiaria microsporogenesis hybrids.