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.     

Physical mapping of rDNA genes corroborates allopolyploid origin in apomictic Brachiaria brizantha

Brachiaria (Trin.) Griseb belongs to the family Poaceae, and within the genus, apomixis or sexuality is present in different accessions of the same species. The majority of Brachiaria species are polyploid and apomictic, making strategies for crop improvement by breeding very intricate. In spite of the high frequency of apomictic polyploids, the relationship of polyploidy and hybridization with apomixis in Brachiaria is still unclear. Further analysis requires detailed knowledge regarding the genomic composition of the polyploids. The present work introduces the use of fluorescent in situ hybridization (FISH) into cytogenetic analysis of Brachiaria. Physical mapping of heterologous rDNA sequences, associated with conventional karyotyping of the B. brizantha diploid sexual (BRA 002747) and the tetraploid apomictic (BRA000591) accessions, provided evidence of the latter being of allopolyploid origin. Based on our results and on previous knowledge on apomixis in B. brizantha, we suggest that the origin of apomixis was probably a consequence of hybridization.     

Meiotic behaviour in three interspecific three-way hybrids between Brachiaria ruziziensis and B. brizantha (Poaceae: Paniceae)

The meiotic behaviour of three three-way interspecific promising hybrids (H17, H27, and H34) was evaluated. These hybrids resulted from the crosses between B. ruziziensis X B. brizantha and crossed to another B. brizantha. Two half-sib hybrids (H27 and H34) presented an aneuploid chromosome number (2n = 4x = 33), whereas hybrid H17 was a tetraploid (2n = 4x = 36), as expected. Chromosome paired predominantly as multivalents suggesting that genetic recombination and introgression of specific target genes from B. brizantha into B. ruziziensis can be expected. Arrangement of parental genomes in distinct metaphase plates was observed in H27 and H34, which have different male genitors. Hybrids H17 and H34 have the same male genitor, but did not display this abnormality. In H17, abnormalities were more frequent from anaphase II, when many laggard chromosomes appeared, suggesting that each genome presented a different genetic control for meiotic phase timing. Despite the phylogenetic proximity among these two species, these three hybrids presented a high frequency of meiotic abnormalities, mainly those related to irregular chromosome segregation typical of polyploids, H34, 69.1%; H27, 56.1% and H17, 44.9%. From the accumulated results obtained through cytological studies in Brachiaria hybrids, it is evident that cytogenetical analysis is of prime importance in determining which genotypes can continue in the process of cultivar development and which can be successfully used in the breeding. Hybrids with high frequency of meiotic abnormalities can seriously compromise seed production, a key trait in assuring adoption of a new apomictic cultivar of Brachiaria for pasture formation.     

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.     

Karyotype analysis in Brachiaria (Poaceae) species

This is the first karyotype characterization of Brachiaria species. Twelve accessions belonging to five species were analysed. The basic chromosome number was x = 9 and 7, the same reported for the tribe Paniceae. Variations in the chromosome number were observed in B. decumbens (2n = 18; 36) and B. humidicola (2n = 36; 42; 54). Chromosome numbers of 2n = 18 in B. ruziziensis and 2n = 36 in B. brizantha and B. jubata were recorded. Inter- and intraspecific karyotype differentiation of the accessions analysed was facilitated by variations in karyotypic symmetry. The karyotypes were generally considered symmetrical, with a tendency to asymmetry in the direction of the polyploids. It is suggested that addition, deletions and mainly polyploidy have been the most direct causes involved in the chromosome evolution of this genus.     

Anther development in Brachiaria brizantha (syn. Urochloa brizantha) and perspective for microspore in vitro culture

Protoplasma - Brachiaria, a genus from the Poaceae family, is largely cultivated as forage in Brazil. Among the most cultivated varieties of Brachiaria spp., B. brizantha cv. Marandu (syn. Urochloa...     

Sexual reproduction development in apomictic Eulaliopsis binata (Poaceae)

Apomixis is a particular mode of reproduction that allows progeny formation without meiosis and fertilization. Eulaliopsis binata, a tetraploid apomictic species, is widely used for making paper, rope and mats. There is great potential for fixation of heterosis in E. binata due to autonomous endosperm formation in this species. Although most of its embryo sac originates from nucellus cells, termed apospory, we observed sexual reproduction initiation in 86.8 to 96.8% of the ovules, evidenced by callose deposition on the walls of cells undergoing megasporogenesis. However, only 2-3% mature polygonum-type sexual embryo sacs were confirmed by embryological investigation. Callose was not detected on aposporous initial cell walls. The aposporous initial cells differentiated during pre- and post-meiosis of the megaspore mother cell, while the sexual embryo sac degenerated at the megaspore stage. DNA content ratio of embryo and endosperm in some individuals was 2C:3C, based on flow cytometry screening of seed, similar to that of normal sexual seed. These results confirm that apomictic E. binata has conserved sexual reproduction to a certain degree, which may contribute to maintaining genetic diversity. The finding of sexual reproduction in apomictic E. binata could be useful for research on genetic mechanism of apomixis in E. binata.     

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.     

Dosage effects in the endosperm of diplosporous apomictic Tripsacum (Poaceae)

 Imprinting in the endosperm of angiosperms, a phenomena by which expression of alleles differs depending on whether they originate from the male or female parent, has been shown to explain most failure of interploidy or interspecific crosses in plants. Because of imprinting, seeds develop normally only if a specific dosage is represented in the endosperm, with the relative contributions of genomes in the ratio of two maternal doses to one paternal dose (2m:1p). In Tripsacum, a wild relative of maize, all polyploids reproduce through the diplosporous type of apomixis. Diplospory results from meiotic failure in megasporocytes that develop into eight-nucleate unreduced female gametophytes. The male gametophytes remain unaffected. Flow cytometry was used to determine ploidy levels in the endosperm of both apomictic and sexual Tripsacum accessions. In both cases, fertilization appeared to involve only one sperm nucleus. Therefore, endosperm of apomictic Tripsacum develops normally even though the ratio of genomic contributions deviates from the normal 2m:1p ratio. Ratios of 2:1, 4:1, 4:2, 8:1 and 8:2 were observed, depending on both the ploidy level of the parents and the mode of reproduction. Thus, specific dosage effects are seemingly not required for endosperm development in Tripsacum. These findings suggest that evolution of diplosporous apomixis might have been restricted to species with few or no imprinting requirements, and the findings have strong implications regarding the transfer of apomixis to sexually reproducing crops. Received: 17 February 1997 / Revision accepted: 7 July 1997     

Molecular and morphological phylogenetic analysis of Brachiaria and Urochloa (Poaceae)

The taxonomic relationships of Brachiaria and Urochloa have been questioned based on previous morphological studies. In this paper, we reconsider the phylogenetic relationships of these genera using 22 species of Brachiaria and Urochloa and six species of Paniceae as out-groups. The ITS1, 5.8S, and ITS2 region (internal transcribed spacer) of nuclear ribosomal DNA and eight morphological characters of the inflorescence were compiled into a data matrix. The cladistic analyses suggest that Urochloa-Brachiaria as a complex is paraphyletic with Eriochloa and Melinis. Species of all these genera share molecular synapomorphies and belong to the same monophyletic groups. The results confirm the continuous gradation between those genera previously found in several morphological studies. Therefore, the following eight new combinations are made: Urochloa bovonei (Chiov.) A.M. Torres & C.M. Morton, Urochloa dura (Stapf) A.M. Torres & C.M. Morton, Urochloa dura var. dura (Stapf) A.M. Torres & C.M. Morton, Urochloa dura var. pilosa (J.G. Anderson) A.M. Torres & C.M. Morton, Urochloa lachnantha (Hochst.) A.M. Torres & C.M. Morton, Urochloa leersioides (Hochst.) A.M. Torres & C.M. Morton, Urochloa nigropedata (Munro ex Ficalho & Hiern) A.M. Torres & C.M. Morton, and Urochloa subulifolia (Mez) A.M. Torres & C.M. Morton.     

Desynapsis and precocious cytokinesis in Brachiaria humidicola (Poaceae) compromise meiotic division

The forage grass species Brachiaria humidicola is native to African savannas. Owing to its good adaptation to poorly drained and infertile acid soils, it has achieved wide utilization for pastures in Brazilian farms. Among the 55 accessions of B. humidicola analysed from the Embrapa Beef Cattle collection, one (H022), presented desynapsis and an abnormal pattern of cytokinesis in the first meiotic division. Among 28 inflorescences analysed in this accession, 12 were affected by the anomaly. In affected meiocytes, the first cytokinesis occurred in metaphase I and was generally perpendicular to a wide-metaphase plate, dividing the genome into two parts with an equal or unequal number of chromosomes. The normal cytokinesis after telophase I did not occur, and the meiocytes entered metaphase II, progressing to the end of meiosis with the occurrence of the second cytokinesis. As the first cytokinesis occurred precociously, whereas the second was normal, tetrads were formed but with unbalanced chromosome numbers in microspores. Abnormal cytokinesis occurred only in those meiocytes that underwent desynapsis after diakinesis. The implications of this abnormality in the Brachiaria breeding programme are discussed.     

Genetic Diversity and Population Structure of the Brachiaria brizantha Germplasm

Brachiaria brizantha (Hochst. ex A. Rich.) Stapf. (syn. Urochloa brizantha (Hochst. ex A. Rich.) R.D. Webster) is a species used primarily as forage in tropical America and Southeast Asia. B. brizantha has been extensively researched since the 1980s with the initiation of the Tropical Forages Breeding Program conducted by the Brazilian Agricultural Research Corporation (Empresa Brasileira de Pesquisa Agropecuária; EMBRAPA), holding one of the largest germplasm collections in the world. This work has identified 15 new microsatellite markers for this species, which have been used in addition to five previously reported markers, to estimate the genetic similarities among 172 accessions and six cultivars of this species. Similarity index values ranged from 0.40 to 1.00. Two duplications were found in the germplasm. A Bayesian analysis performed using the STRUCTURE 2.3.3 program revealed the presence of three clusters with different allelic pools. This analysis is valuable for the performance of crosses to explore heterosis; however, the mode of reproduction of the accessions and ploidy barriers must be observed for effective exploration. A grouping analysis using the neighbor-joining method was consistent with the STRUCTURE analysis, and a combination approach suggested that this germplasm collection does not exhibit considerable genetic variability despite the presence of three distinct allelic pools. The lack of correlation between the genetic and geographic distances is also discussed.     

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.     

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.     

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.     

Silicon distribution and accumulation in shoot tissue of the tropical forage grass Brachiaria brizantha

Silicon (Si) accumulation in organs and cells is one of the most prominent characteristics of plants of the family Poaceae. Many species from this family are used as forage plants for animal feeding. The present study investigates in Brachiaria brizantha (Hochst. ex A. Rich.) Stapf. cv. Marandu: (1) the dry matter production and Si content in shoot due to soil Si fertilizations; (2) the Si distribution among shoot parts; and (3) the silica deposition and localization in leaves. Plants of B. brizantha cv. Marandu were grown under contrasting Si supplies in soil and nutrient solution. Silica deposition and distribution in grass leaf blades were observed using light microscope and scanning electron microscope equipped with an energy dispersive X-ray spectrometer (SEM-EDXS). Silicon concentration in the B. brizantha shoot increased according to the Si supply. Silicon in grass leaves decreased following the order: mature leaf blades > recently expanded leaf blades > non-expanded leaf blades. Silicon accumulates mainly on the upper (adaxial) epidermis of the grass leaf blades and, especially, on the bulliform cells. The Si distribution on adaxial leaf blade surface is non uniform and reflects a silica deposition exclusively on the cell wall of bulliform cells.     

Microsporogenesis in Brachiaria dictyoneura (Fig. & De Not.) Stapf (Poaceae: Paniceae)

Microsporogenesis was analyzed in five accessions of Brachiaria dictyoneura presenting x = 6 as the basic chromosome number. All accessions were tetraploid (2n = 4x = 24) with chromosome pairing in bi-, tri-, and quadrivalents. The recorded meiotic abnormalities were those typical of polyploids, including precocious chromosome migration to the poles, laggard chromosomes, and micronucleus formation. The frequency of these abnormalities, however, was lower than those reported for other polyploid accessions previously analyzed for other Brachiaria species. Cell fusion and absence of cytokinesis were also recorded in some accessions, leading to restitutional nucleus formation in some cells. Genetically unbalanced microspores, binucleate, and 2n microspores were found among normal meiotic products as results from these abnormalities. The limitation in using these accessions as pollen donor in interspecific crosses with sexual species with x = 7 or x = 9 in breeding programs is discussed.     

Abnormal meiosis in tetraploid genotypes ofBrachiaria brizantha (Poaceae) induced by colchicine: its implications for breeding

Meiotic behavior was analyzed in 6 progenies from 3 artificially induced tetraploid (2n = 4x = 36) sexual genotypes (C31, C41, and C48) of the normally apomicticBrachiaria brizantha (Hochst. ex A. Rich.) Stapf., syn.Urochloa brizantha (Hochst. ex A. Rich.) R. Webster. These are key plants to allow intraspecific hybridization of this important forage species, widely used for pastures in the tropics. The percentage of abnormal cells among the plants ranged from 39.8% to 63.2%. In the single plant derived from C48, only the common meiotic abnormalities typical of polyploids were observed, while in plants derived from C31 and C41, a distinct behavior was found. In the majority of cells of those plants, the chromosomes remained scattered in the cytoplasm in the first division, without forming a metaphase plate. This abnormality blocked chromosome movements at anaphase I. Several micronuclei of various sizes were formed and, after the occurrence of an irregular first cytokinesis, the meiocytes progressed normally to the second division, generating polyads with unbalanced microspores. Pollen viability was not correlated with meiotic abnormalities. The importance of these findings to theBrachiaria breeding program is discussed. The sexual progeny of C48 seems most suitable as female parents to be used in intra-and interspecific hybridization.     

Importance of the nitrogen source in the grass species Brachiaria brizantha responses to sulfur limitation

Background and aims

Plant responses to S supply are highly dependent on N nutrition. We investigated the effect of S status on metabolic, nutritional, and production variables in Brachiaria brizantha treated with different N forms. Additionally, ¹⁵N and ³⁴S root influx were determined in plants under short- and long-term S deprivation.

Methods

Plants were submitted to soil fertilization treatments consisted of combinations of N forms [without N, ammonium (NH₄ ⁺), nitrate (NO₃ ^?) or NH₄ ⁺+NO₃ ^?] at S rates (0, 15, 30, or 45 mg dm^?3). N and S influx capacity was determined in hydroponically-grown plants.

Results

Shoot production due to S supply increased 53, 145 and 196 % with NH₄ ⁺, NH₄ ⁺+NO₃ ^? and NO₃ ^? treatments, respectively. No or low S impaired protein synthesis and led to high accumulation of N-NO₃ ^? and asparagine in NO₃ ^?-fed plants, both alone and with NH₄ ⁺. Proline accumulation was observed in NH₄ ⁺-fed plants. Short- and long-term S deprivation did not promote considerable changes in ¹⁵N influx. ³⁴S absorption decreased depending on the N form provided: NH₄ ⁺+NO₃ ^? > only NH₄ ⁺ > only NO₃ ^? > low N.

Conclusions

Including both NH₄ ⁺ and NO₃ ^? forms in fertilizer increases N and S intake potential and thereby enhances plant growth, nutritional value and production.     

Isolation and characterization of microsatellite markers for Brachiaria brizantha (Hochst. ex A. Rich.) Stap

The first set of nuclear simple sequence repeat (SSR) loci for Brachiaria brizantha (Hochst. ex A. Rich.) Stap is described. A microsatellite-enriched library was constructed and 19 loci were characterized. About 13 SSR loci were found to be polymorphic and across-taxa amplification tests showed that six of them can be transferred to four other species of Brachiaria. This new SSR resource will be a powerful tool for population genetic studies of B. brizantha, for interspecific genetic studies within the genus Brachiaria, for mapping and for marker assisted selection in breeding.