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.

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.

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.

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 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.

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.

Abnormal cytokinesis in microsporogenesis of Brachiaria humidicola (Poaceae: Paniceae).

Microsporogenesis was evaluated in the Brachiaria humidicola collection of the Embrapa Beef Cattle Center, represented by 60 accessions. One accession (H121) presented an abnormal pattern of cytokinesis that had never been reported in this genus. Among 900 meiocytes analyzed in the first division, 10.7% underwent precocious and multiple cytokinesis in metaphase I, fractionating the genome and the cytoplasm into two or more parts. The expected cytokinesis after telophase I did not occur. The abnormal meiocytes from the first division entered the second division but the second cytokinesis after telophase II was also abnormal. Among the 857 meiocytes analyzed in the second division, 10.9% presented abnormal, incomplete or total absence of cytokinesis. Dyads and binucleated microspores were recorded among the meiotic products. The use of this accession in the Embrapa breeding program is compromised.

Evidence of programmed cell death during microsporogenesis in an interspecific Brachiaria (Poaceae: Panicoideae: Paniceae) hybrid.

Morphological changes have been investigated during plant programmed cell death (PCD) in the last few years due to the new interest in a possible apoptotic-like phenomenon existing in plants. Although PCD has been reported in several tissues and specialized cells in plants, there have been few reports of its occurrence during microsporogenesis. The present study reports a typical process of PCD during meiosis in an interspecific Brachiaria hybrid leading to male sterility. In this hybrid, some inflorescences initiated meiosis but it was arrested in zygotene/pachytene. From this stage, meiocytes underwent a severe alteration in shape showing substantial membrane blebbing; the cytoplasm became denser at the periphery; the cell nucleus entered a progressive stage of chromatin disintegration, and then the nucleolus disintegrated, and the cytoplasm condensed and shrunk. The oldest flowers of the raceme showed only the callose wall in the anthers showing obvious signs of complete sterility.

Microsporogenesis in sexual Brachiaria hybrids (Poaceae).

Three sexual interspecific hybrids of Brachiaria (HBGC076, HBGC009, and HBGC014) resulting from crosses between B. ruziziensis (female genitor) and B. decumbens and B. brizantha (male genitors) produced by Embrapa Beef Cattle in the 1980s were cytologically analyzed by conventional methods for meiotic studies. The cytogenetic analysis showed the occurrence of common meiotic abnormalities among them. The most frequent abnormalities were those related to irregular chromosome segregation due to polyploidy. Other abnormalities, such as chromosome stickiness, absence of cytokinesis, irregular cytokinesis, abnormal spindle orientation, and abnormal nucleolus disintegration, were found in the three hybrids, while, chromosome disintegration was detected only in HBGC014. All the abnormalities, except for abnormal nucleolus disintegration, can cause unbalanced gamete formation, leading to pollen sterility. Multivalent chromosome association at diakinesis revealed genome affinity between the two parental species in the hybrids, suggesting some possibility for gene introgression. Presently, the Brachiaria breeding program has the objective of releasing, primarily, apomictic hybrids as new cultivars since they do not segregate but preserve the genetic makeup indefinitely. Besides, they result in homogeneous pastures which are easier to manage. The sexual hybrids, however, are paramount in the breeding program: they work as 'bridges' to introgress traits of interest into the apomictic genotypes. The cytogenetic analyses of these three hybrids substantiate their maintenance in the breeding program due to low frequency of meiotic abnormalities, complemented by interesting agronomic traits. They may be used in crosses to generate new cultivars in the future.

Evidence of programmed cell death during microsporogenesis in an interspecific Brachiaria (Poaceae: Panicoideae: Paniceae) hybrid

Morphological changes have been investigated during plant programmed cell death (PCD) in the last few years due to the new interest in a possible apoptotic-like phenomenon existing in plants. Although PCD has been reported in several tissues and specialized cells in plants, there have been few reports of its occurrence during microsporogenesis. The present study reports a typical process of PCD during meiosis in an interspecific Brachiaria hybrid leading to male sterility. In this hybrid, some inflorescences initiated meiosis but it was arrested in zygotene/pachytene. From this stage, meiocytes underwent a severe alteration in shape showing substantial membrane blebbing; the cytoplasm became denser at the periphery; the cell nucleus entered a progressive stage of chromatin disintegration, and then the nucleolus disintegrated, and the cytoplasm condensed and shrunk. The oldest flowers of the raceme showed only the callose wall in the anthers showing obvious signs of complete sterility.

Microsporogenesis in sexual Brachiaria hybrids (Poaceae)

Three sexual interspecific hybrids of Brachiaria (HBGC076, HBGC009, and HBGC014) resulting from crosses between B. ruziziensis (female genitor) and B. decumbens and B. brizantha (male genitors) produced by Embrapa Beef Cattle in the 1980s were cytologically analyzed by conventional methods for meiotic studies. The cytogenetic analysis showed the occurrence of common meiotic abnormalities among them. The most frequent abnormalities were those related to irregular chromosome segregation due to polyploidy. Other abnormalities, such as chromosome stickiness, absence of cytokinesis, irregular cytokinesis, abnormal spindle orientation, and abnormal nucleolus disintegration, were found in the three hybrids, while, chromosome disintegration was detected only in HBGC014. All the abnormalities, except for abnormal nucleolus disintegration, can cause unbalanced gamete formation, leading to pollen sterility. Multivalent chromosome association at diakinesis revealed genome affinity between the two parental species in the hybrids, suggesting some possibility for gene introgression. Presently, the Brachiaria breeding program has the objective of releasing, primarily, apomictic hybrids as new cultivars since they do not segregate but preserve the genetic makeup indefinitely. Besides, they result in homogeneous pastures which are easier to manage. The sexual hybrids, however, are paramount in the breeding program: they work as ‘bridges’ to introgress traits of interest into the apomictic genotypes. The cytogenetic analyses of these three hybrids substantiate their maintenance in the breeding program due to low frequency of meiotic abnormalities, complemented by interesting agronomic traits. They may be used in crosses to generate new cultivars in the future.

Abnormal cytokinesis in microsporogenesis of Brachiaria humidicola (Poaceae: Paniceae)

Microsporogenesis was evaluated in the Brachiaria humidicola collection of the Embrapa Beef Cattle Center, represented by 60 accessions. One accession (H121) presented an abnormal pattern of cytokinesis that had never been reported in this genus. Among 900 meiocytes analyzed in the first division, 10.7% underwent precocious and multiple cytokinesis in metaphase I, fractionating the genome and the cytoplasm into two or more parts. The expected cytokinesis after telophase I did not occur. The abnormal meiocytes from the first division entered the second division but the second cytokinesis after telophase II was also abnormal. Among the 857 meiocytes analyzed in the second division, 10.9% presented abnormal, incomplete or total absence of cytokinesis. Dyads and binucleated microspores were recorded among the meiotic products. The use of this accession in the Embrapa breeding program is compromised.

Evidence of allopolyploidy in Brachiaria brizantha (Poaceae: Paniceae) through chromosome arrangement at metaphase plate during microsporogenesis.

In the hexaploid (2n = 6x = 54) accession B176 of Brachiaria brizantha, one cytological characteristic differentiated it from the other accessions previously analyzed with the same ploidy level. Nearly 40% of meiocytes displayed the chromosome set arranged at two metaphase plates at the poles of the cell, close to the membrane. In these cells, both metaphase plates were arranged in an angle to form a typical tripolar spindle. Therefore, cells did not show normal chromosome segregation at anaphase I. Only nine univalent chromosomes migrated from each plate to the opposite pole with the remainder staying immobile on the plate. As a result of such spindle orientation and chromosome behavior, trinucleate telophases I were recorded. After telophase, cytokinesis eliminated the small nuclei into a microcyte. The second division proceeded normally, with the presence of microcytes in all phases. The origin of such an abnormality was explained on the hexaploid level of the accession which could have resulted by chromosome doubling of a triploid derived from species that did not display the same behavior for spindle organization. The high percentage of meiotic abnormalities recorded in this accession compromises fertility and renders it inadequate for the breeding program.