Chromosome numbers and meiotic analysis in the pre-breeding of <Emphasis Type="BoldItalic">Brachiaria decumbens</Emphasis> (Poaceae)

A total of 44 accessions of Brachiaria decumbens were analysed for chromosome count and meiotic behaviour in order to identify potential progenitors for crosses. Among them, 15 accessions presented 2n = 18; 27 accessions, 2n = 36; and 2 accessions, 2n = 45 chromosomes. Among the diploid accessions, the rate of meiotic abnormalities was low, ranging from 0.82% to 7.93%. In the 27 tetraploid accessions, the rate of meiotic abnormalities ranged from 18.41% to 65.83%. The most common meiotic abnormalities were related to irregular chromosome segregation, but chromosome stickiness and abnormal cytokinesis were observed in low frequency. All abnormalities can compromise pollen viability by generating unbalanced gametes. Based on the chromosome number and meiotic stability, the present study indicates the apomictic tetraploid accessions that can act as male genitor to produce interspecific hybrids with B. ruziziensis or intraspecific hybrids with recently artificially tetraploidized accessions.     

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 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.     

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.     

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.     

Multiple spindles and cellularization during microsporogenesis in an artificially induced tetraploid accession of Brachiaria ruziziensis (Gramineae)

The genus Brachiaria is characterized by a majority of polyploid accessions--mainly tetraploid--and apomictic reproduction. Sexuality is found among diploids. To overcome incompatibility barriers, accessions with the same ploidy level are necessarily used in hybridization. Thus, sexual diploid accessions were tetraploidized to be used as female genitors. This paper reports microsporogenesis in an artificially induced tetraploid accession of Brachiaria ruziziensis. Chromosome pairing at diakinesis ranged from univalents to tetravalents, with predominance of bivalents. Irregular chromosome segregation was frequent in both meiotic divisions. During the first division, multiple spindles showing different arrangements were recorded. The spindle position determined the plane of first cytokinesis and the number of chromosomes determined the size of the cell. Meiotic products were characterized by polyads with spores of different sizes. Pollen sterility was estimated at 61.38%. The limitations of using this accession in the breeding program are discussed.     

Unusual cytological patterns of microsporogenesis in Brachiaria decumbens: abnormalities in spindle and defective cytokinesis causing precocious cellularization

Cytogenetic studies carried out in the tetraploid accession BRA001068 of Brachiaria decumbens, also known as cv. Basilisk, revealed an unusual pattern of microsporogenesis. The spindle in metaphase I and anaphase I became heavily stained with propionic carmine. In telophase I, the interzonal microtubules continued to be intensely stained, and during the phragmoplast formation the fibers were pushed to the cell wall, persisting until prophase II, even after cytokinesis. Due to its tetraploid condition, the accession presented many cells with precocious chromosome migration to the poles in metaphase I and laggards in anaphase I that gave rise to micronuclei in telophase I. While in other polyploid accessions of Brachiaria micronuclei remained in this condition until the second cytokinesis, the micronuclei in this accession organized their own spindle in the second division. In several microsporocytes, the micronuclei with their minispindle were divided further into microcytes by additional cytokinesis. Some curious planes of cytokinesis were found in some cells, with partitioning of cytoplasm into cells of irregular shape. The result consisted of a high frequency of abnormal products of meiosis. Quadrivalents were observed in diakinesis at low frequency, which suggests a segmental allotetraploid and the inability of both genomes to co-ordinate their activities, leading to multiple spindle and precocious cellularization. In spite of abnormal meiotic products reducing pollen fertility, seed production was normal. Enough normal pollen was available to fertilize the central-cell nucleus of the embryo sac and produce normal endosperm in this pseudogamous aposporous apomictic accession.     

A differential phenotypic expression of a divergent spindle mutation in interspecific Brachiaria hybrids

Several mutations are known to alter the normal progression of meiosis and can be correlated with defects in microtubule distribution. The dv mutation affects the spindle organization and chromosomes do not converge into focused poles. Two Brachiaria hybrids presented the phenotypic expressions of dv mutation but exhibited many more details in the second division. Bivalents were distantly positioned and spread over a large metaphase plate and failed to converge into focused poles. Depending on the distance of chromosomes at the poles, telophase I nuclei were elongated or the chromosomes were grouped into various micronuclei of different sizes in each cell. The first cytokinesis occurred. However, when there were micronuclei, a second cytokinesis immediately took place dividing the prophase II meiocytes into three or four cells. In each meiocyte, meiosis progressed to the second division. Slightly elongated nuclei or micronuclei were recorded in telophase II. After a third cytokinesis, hexads or octads were formed. Pollen grains of different sizes were generated. One of these hybrids presented a higher frequency of abnormal cells than when previously analyzed. The fate of these hybrids as genitors or as candidates for cultivars in the Brachiaria breeding program is discussed.     

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.     

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.     

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.     

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.     

Meiotic abnormalities as expression of nuclear-cytoplasmic incompatibility in crosses of <Emphasis Type="Italic">Pisum sativum</Emphasis> subspecies

Meiosis in anthers and mitosis in somatic cells were studied in reciprocal F₁ hybrids of the accession VIR320, which belonged to wild Pisum sativum ssp. elatius (Bieb.) Schmal., and the laboratory line Sprint-1. When VIR320 was used as a maternal form, the hybrids displayed nuclear-cytoplasmic conflict, which caused chlorophyll defects and meiotic abnormalities. One or two chromosomes lagged in the equatorial region during chromosome segregation to the poles, distorting cytokinesis and yielding abnormal microspores. Chlorophyll defects were not observed, and meiotic abnormalities were far less frequent in reciprocal hybrids and in the case of an abnormal paternal inheritance of plastids from Sprint-1. Mitosis lacked overt abnormalities in all of the hybrids.     

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.     

Microsporogenesis in inbred line of popcorn (Zea mays L.)

Endogamy places genes for several characteristics in homozygosis, which include those related to meiosis causing abnormalities that may impair gamete viability. An original population (S0) of popcorn (CMS-43) produced by Embrapa Maize and Sorghum was self-pollinated for seven years, generating inbred lines (S1 to S7). Conventional studies of microsporogenesis revealed that meiotic abnormalities did not increase with endogamy. Univalent chromosomes, irregular chromosome segregation, abnormal cell shape, partial asynapsis, cell fusion, absence of cytokinesis, abnormal spindle orientation, and chromosome stickiness were recorded in low frequency in meiocytes. Since the frequency of abnormalities was low, mainly in S7, inbred lines from CMS-43 have a high potential for hybridization.     

Synaptonemal complex analysis of hybrid cattle. II. Bos indicus x Bos taurus F1 and backcross hybrids

The levels of meiotic chromosome pairing abnormalities observed in six Australian F1 Bos indicus x Bos taurus cattle crosses (mean = 23%) were significantly higher than those of the full-blood breeds (9%). The abnormal configurations in the F1 hybrids included partial pairing failure, multivalents, interlocks, and inversion pairing. Abnormal configurations were also present, but at lower frequency, in backcross hybrid bulls. The main types of abnormal configurations and the levels of XY-autosomal associations and autosomal asynapsis observed were unlikely to cause significant fertility problems in the hybrids.     

Cytomixis and meiotic abnormalities during microsporogenesis are responsible for male sterility and chromosome variations in Houttuynia cordata

Houttuynia cordata (Saururaceae) is a leaf vegetable and a medicinal herb througout much of Asia. Cytomixis and meiotic abnormalities during microsporogenesis were found in two populations of H. cordata with different ploidy levels (2n = 38, 96). Cytomixis occurred in pollen mother cells during meiosis at high frequencies and with variable degrees of chromatin/chromosome transfer. Meiotic abnormalities, such as chromosome laggards, asymmetric segregation and polyads, also prevailed in pollen mother cells at metaphase of the first division and later stages. They were caused by cytomixis and resulted in very low pollen viability and male sterility. Pollen mother cells from the population with 2n = 38 showed only simultaneous cytokinesis, but most pollen mother cells from the population with 2n = 96 showed successive cytokinesis; a minority underwent simultaneous cytokinesis. Cytomixis and irregular meiotic divisions appear to be the origin of the intraspecific polyploidy in this species, which has large variations in chromosome numbers.     

Cytogenetics of Festulolium (Festuca x Lolium hybrids)

Grasses are the most important and widely cultivated crops. Among them, ryegrasses (Lolium spp.) and fescues (Festuca spp.) provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Species from the two genera display complementary agronomic characteristics and are often grown in mixtures. Breeding efforts to combine desired features in single entities culminated with the production of Festuca x Lolium hybrids. The so called Festuloliums enjoy a considerable commercial success with numerous cultivars registered all over the world. They are also very intriguing from a strictly cytogenetic point of view as the parental chromosomes recombine freely in hybrids. Until a decade ago this phenomenon was only known in general quantitative terms. The introduction of molecular cytogenetic tools such as FISH and GISH permitted detailed studies of intergeneric chromosome recombination and karyotyping of Festulolium cultivars. These tools were also invaluable in revealing the origin of polyploid fescues, and facilitated the development of chromosome substitution and introgression lines and physical mapping of traits of interest. Further progress in this area will require the development of a larger set of cytogenetic markers and high-resolution cytogenetic maps. It is expected that the Lolium-Festuca complex will continue providing opportunities for breeding superior grass cultivars and the complex will remain an attractive platform for fundamental research of the early steps of hybrid speciation and interaction of parental genomes, as well as the processes of chromosome pairing, elimination and recombination.