Chromosome numbers and microsporogenesis in Brachiaria brizantha (Gramineae)

The genus Brachiaria, native to the African tropical savannas, has achieved significance as a pasture grass in many tropical and subtropical countries, including Brazil. Many species and accessions are polyploid and apomictic, which complicates the improvement of breeding stocks through hybridization. In support of breeding programs, cytogenetic characterization, including chromosome counts and evaluation of the meiotic behavior in the accessions of the Brachiaria has been undertaken at the Embrapa Beef Cattle Center. In this study, 22 accessions of B. brizantha were analyzed of which one was found to be diploid (2n = 2x = 18), 18 were tetraploid (2n = 4x = 36) and three were hexaploid (2n = 6x = 54). The meiotic chromosome behavior was slightly irregular in the diploid and in some tetraploid accessions, and highly irregular in most tetra- and hexaploid accessions. Meiotic abnormalities were those common to polyploidy, i.e., multivalent chromosome association at diakinesis and irregular chromosome segregation leading to micronuclei formation in the tetrad stage. Low frequencies of multivalent chromosome associations among polyploids suggest that they may be segmental allopolyploids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Analysis of meiotic behavior in selecting potential genitors among diploid and artificially induced tetraploid accessions of Brachiaria ruziziensis (Poaceae)

Sexuality is correlated with diploidy and apomixis with polyploidy in the Brachiaria genus. Brachiaria ruziziensis is a key species in Brachiaria breeding due to its obligate sexuality and intrinsic agronomic qualities. Interspecific crosses in the genus became feasible only when a few diploid accessions of B. ruziziensis were artificially tetraploidized and remained sexual. Hybridization has been done since, using natural tetraploid apomictic accessions of B. brizantha or B. decumbens as pollen donors. Twenty two accessions of B. ruziziensis from the Embrapa Beef Cattle germplasm collection (Campo Grande, MS, Brazil) were cytologically analyzed: 16 are natural diploids (2n = 2x = 18) and six are artificially induced tetraploids (2n = 4x = 36). The meiotic behavior in the 16 diploid accessions varied. The mean of meiotic abnormalities per accession ranged from zero to 24.46%. Meiotic behavior in the induced tetraploid accessions also varied with the mean of meiotic abnormalities ranging from 5.20% to 54.71%. The most common abnormalities observed in both the diploid and the tetraploid accessions, were those related to irregular chromosome segregation. In one tetraploid accession, with a high frequency of those, other irregularities involving chromosome orientation at metaphase plate and chromosome convergence to the poles, a meiotic mutation known as divergent spindle, were recorded. Meiotic behavior should be considered in selecting potential genitors for breeding.     

Abnormal pollen development of bread wheat-<Emphasis Type="Italic">Leymus mollis</Emphasis> partial amphiploid

Summary Octoploid Tritileymus was the partial amphiploid obtained from Triticum aestivum × Leymus mollis, its chromosome constitution was 14 Leymus chromosomes and 42 wheat chromosomes. In the pollen development of Tritileymus, some abnormalities were observed, such as the absence of tetrad formation, cytomixis of microspores, irregular shape of some microspores and multi-germination pores, symmetrical first mitosis, chromosomes or chromatids randomly dispersed in the cytoplasm of microspore.     

Meiotic behavior in Brachiaria humidicola (Poaceae) hybrids

In the genus Brachiaria, genetic variation can be exploited directly from germplasm collections or released using sexual reproduction in normally apomictic polyploids. The discovery of a natural sexual polyploid accession H031 of Brachiaria humidicola collected in Africa, opened new opportunities to exploit the genetic variation in this species. This accession was crossed with an apomictic cultivar BRS Tupi with the same chromosome number (2n = 36) and 361 F₁ hybrids were obtained. Following visual selection for leafiness, vigor, growth habit, and the mode of reproduction, 50 hybrids were selected for further agronomic evaluation. The parents and 45 of the 50 selected hybrids were evaluated for the regularity of meiosis. In the female parent (H031), meiosis was somewhat irregular, with 16.3% of abnormal tetrads, whereas the male (cv. BRS Tupi) meiosis was very regular, with only 3.1% of abnormal tetrads. Among hybrids (sexual and apomictic), the percentage of abnormal tetrads ranged from 15.8 to 98.3%. The abnormalities included irregular chromosome segregation, chromosome stickiness and the absence of cytokinesis. Considering that apomixis in the genus Brachiaria is pseudogamic, and that meiotic aberrations can compromise pollen viability, the results of this study present another parameter to aid selection for more stable microsporogenesis. Apomictic derivatives with stable meiosis are candidates for new cultivars whereas sexual hybrids can be retained in breeding for another round of recombination.     

A New Intergeneric Hybrid between Triticum aestivum L. and Agropyron fragile (Roth) Candargy: Variation in A. fragile for Suppression of the Wheat Ph-Locus Activity

New intergeneric hybrids were obtained between Triticum aestivum L. cv. Tukuho’ (2n = 6x = 42, AABBDD) and Agropyron fragile (Roth) Candargy PGR 8097 (2n = 4x = 28, PPPP) at a frequency of 1.06 %, through the use of direct embryo culture and in ovulo embryo culture. Such hybrids could be used to transfer barley yellow dwarf virus (BYDV) resistance and winterhardiness into bread wheat. The somatic chromosome number in all the hybrid plants was 2n = 5x = 35, as expected. Considerable variation in chromosome pairing was observed among the different hybrid plants. Average meiotic chromosome configuration at metaphase I was 17.29 Is + 6.57 rod Us + 1.97 ring Us + 0.18 III + 0.03 IV + 0.002 VI. The high level of chromosome pairing in some F₁ hybrids was attributed to Ph-suppressor gene(s) present in A. fragile. The hybrids could not be backcrossed to wheat, but amphiploid seeds have been obtained by colchicine treatment.     

Cytology and reproductive behavior of diploid, tetraploid and hexaploid germplasm accessions of a wild forage grass: Paspalum compressifolium

Summary Fourteen germplasm accessions of Paspalum compressifolium native from southern Brazil were cytologically and embryologically analysed. The study revealed that one accession was diploid (2n=20), twelve were tetraploid (2n=40) and one was hexaploid (2n=60). This is the first report of diploid and hexaploid cytotypes for this species. Studies on microsporogenesis, megasporogenesis, and embryo sac development indicated that the diploid cytotype had regular meiotic behavior and reproduces sexually. Tetraploid cytotype usually had an important proportion of chromosomes that associated as quadrivalents during meiosis and reproduced by mean of aposporous apomixis. The hexaploid cytotype showed irregular meiotic behavior with about one third of the chromosomes associated as multivalents and reproduced by aposporous apomixis. Thus, P. compressifolium could be an agamic complex. Breeding in this complex is possible due to the presence of diploid sexuals which can be treated by colchicine and pollinated by apomicts.     

Occurrence of 2n and jumbo pollen in the Brazilian ginseng (Pfaffia glomerata and P. tuberosa)

An analysis of microsporogenesis was carried out in four populations of Pfaffia tuberosa (2n=4x=68) and in ten populations of P. glomerata(2n=2x=34). Monads, dyads and triads were reported at high frequency in all populations of P. tuberosa. In P. glomerata, on the other hand, only four populations presented monads, and at low frequency. Dyads and triads were observed in both species, albeit at a higher frequency in P. tuberosa populations. Certain abnormalities of spindles in the second division, such as sequential and tripolar spindles, led to triad formation by the rejoining of two chromosome sets. The precise causes of monad and dyad formation have not been found during meiosis, although evidence exists that dyads originated by omission of second meiosis and monads by omission of both meiotic divisions. This revised version was published online in July 2006 with corrections to the Cover Date.     

N-banded karyotype of Aegilops ovata and chromosomal constitution of its amphiploid with Triticum aestivum

The N-banded karyotype of an Aegilops ovata (2n = 4x = 28) accession was produced, and the chromosomal constitution of its partial amphiploid with bread wheat Triticum aestivum‘Chinese Spring’ was established. All A. ovata chromosomes showed specific N-banding patterns and can easily be distinguished from each other. As a result of irregular meiotic behaviour of the chromosomes in the amphiploid, some structural rearrangements have probably occurred in most of the A. ovata chromosomes. The wheat chromosomes remained unmodified in their morphology and the N-banding patterns.     

2n Gamete formation in the genus Brachiaria (Poaceae: Paniceae)

Microsporogenesis of several Brachiaria species of the Brazilian collection at Embrapa Beef Cattle has been analyzed in detail. This paper reports abnormal cytokinesis in three accessions of three different species (Brachiaria humidicola, 2n = 4x = 36, Brachiaria decumbens, 2n = 4x = 36, and Brachiaria dura, 2n = 6x = 54). Chromosomes paired in bi-, tri-, and quadrivalents in these accessions, whereas chromosome segregation at meiosis I was characterized by exclusion of laggards as micronuclei. In a high number of meiocytes, the first sign of cytokinesis appeared only in metaphase II and did not divide the meiocyte into a dyad. Total absence of cytokinesis was also detected among meiocytes in the second division. Since in both cases the two metaphase plates were very close, they favored the rejoining of chromosome sets after anaphase II and formed a restitutional nucleus in telophase II. Second cytokinesis occurred after telophase II in most meiocytes. Monads, dyads, and triads with n or 2n nuclei were observed among meiotic products. The 2n gametes observed correspond to the first division restitution (FDR). The number of affected cells in each accession was variable, but the number of microspores with restitutional nucleus, including those scored in tetrads and the released ones, did not exceed 9%. Although polyploidy is common in the genus Brachiaria, its origin is still unclear. Current results suggest that 2n gametes may have contributed to the evolutionary history of the genus.     

Meiotic behavior in interspecific hybrids between Brachiaria ruziziensis and Brachiaria brizantha (Poaceae)

The meiotic behavior of two half-sib interspecific tetraploid (2n = 4x = 36) promising hybrids, a sexual and an apomictic one, from crosses B. ruziziensis and B. brizantha, was evaluated. Although chromosome paired predominantly as bivalents, a few tri- and quadrivalents were recorded. Results suggest that B. brizantha and B. ruziziensis are closely related and genetic recombination is expected in hybrids. Introgression of specific target genes from B. ruziziensis into B. brizantha and vice-versa may be foreseen. However, abnormalities such as irregular chromosome segregation, chromosome stickiness and abnormal cytokinesis reported in these hybrids affect pollen fertility. More than 65% of pollen grains are sterile. Since the distinctive cytological feature of these hybrids is abnormal cytokinesis, this fact suggests that both parental genomes are unable to coordinate their activities with regard to this cytological phenomenon. Deployment of such hybrids in the process of developing varieties is discussed.     

Identification and cytological analysis of 2n-pollen producers in Lotus tennis Wald. et Kit.

Two meiotic mutants of L. tennis (2n = 2x = 12) producing unreduced pollen are described. When crossed to male sterile L. corniculatus (2n = 4x = 24) plants, all progeny plants were morphologically similar to L. corniculatus, had 2n = 24 chromosomes, and in the cross, were fully compatible with L. corniculatus, indicating that the male parent plants were 2n-pollen producers. One of them also had ‘giant’ pollen grains. In metaphase II of both genotypes, there were parallel and tripolar spindles leading to dyad and triad formation, the latter being found most frequently. Since both the above-mentioned mechanisms result in first-division restitution-type microspores, the genotypes examined could be useful in breeding Lotus.     

Ploidy races in Actinidia chinensis

Summary Ploidy levels were examined in 26 accessions of Actinidia chinensis: 20 accessions were diploid (2n=2x=58) and 6 accessions were tetraploid (2n=4x=116). There was no evidence of variation in ploidy level within an accession. Our results are consistent with tetraploid A. chinensis coming from a restricted part of China.Interploid crosses within A. chinensis produced only low numbers of seedlings which were mainly triploid. Crossing hexaploid A. deliciosa with pollen of tetraploid A. chinensis produced a large family of plants and those checked were pentaploid.Counts on 83 genotypes of different ploidy levels (2x, 3x, 4x, and 5x) confirmed that the basic chromosome number in Actinidia is 29.     

A comparative study of the cytological and reproductive behaviour of wheat and triticale subjected to two temperature regimes

Summary A comparison was made of the meiotic stability and reproductive behaviour ofTriticale hexaploide cv. Rosner andT. aestivum cv. Pitic in response to two temperature regimes (15° and 30°C) imposed continuously from premeiosis to maturity. Mean frequencies of MI open bivalents, univalents, and lagging chromosomes at AI were significantly higher in Rosner than in Pitic. Such irregularity would appear to arise from the asynchronous disjunction of chromosomes between Metaphase I and Anaphase I, resulting in meiotic disturbances and a high frequency of abnormal microspores. Despite such abnormalities, floret fertility was equivalent to that observed in Pitic, and the proportion of aneuploids recovered in the progeny, although greater than expected in wheat, was much lower than would be predicted. It is suggested that there is a strong certation effect in the transmission of aneuploid microspores and that the irregularities observed during microsporogenesis do not apply directly to megasporogenesis.No appreciable effect of temperature on meiotic chromosome behaviour during microsporogenesis was observed in either wheat or Triticale. In Triticale, high temperatures (30°C) substantially reduced spikelet fertility, and in contrast, low temperature (15°C) increased the frequency of aneuploids.Contribution No 232, Department of Plant Science, University of Manitoba, Winnipeg 19, Manitoba, Canada     

Further cytological characteristics of a male-sterile mutant in soybean affecting cytokinesis and microspore development

A spontaneous male‐sterile mutation affecting microspore development has been found in BR97‐13509H line of the official Brazilian soybean breeding programme. Slightly abnormal and irregular chromosome segregation occurred in both meiotic divisions, albeit insufficient to explain total pollen sterility. The main cause of male sterility was the absence of cytokinesis after telophase II. Instead of the typical tetrads of microspores, four‐nucleate coenocytic microspores were formed. Coenocytic microspores resulting from the absence of cytokinesis have also been described in ms1 and ms4 male‐sterile soybean mutants, although with certain differential characteristics. In the BR97‐13509H mutant line, after interphase the four nuclei were rejoined in one or two metaphase plates and progressed through the first pollen mitosis. Pollen then underwent a progressive process of degeneration characterized by continuous cytoplasmic shrinkage. The second pollen mitosis failed to occur, and pollen sterility became total.     

Stabilization of new types of diploids (2n=22, 24) through selfing of aneuploids (2n=21, 22) derived from crossing of sesquidiploids (2n=29, AAC) and Brassica campestris (2n=20 AA)

Summary Aneuploids with 2n=21 and 2n=22 derived from crossing of sesquidiploids (2n=29, AAC) and Brassica campestris (2n=20, AA) were selfed successively in order to follow the changes in chromosome number of the progenies for three consecutive generations. Progenies with 2n=22, 23 and 24 obtained after selfing of S₀ generation and the succeeding S₁, S₂ and S₃ generations were analyzed in terms of pollen stainability, % seed set as well as cytogenetically based on meiotic behaviour with the aim of determining the possibility of addition of one or more alien chromosomes into n=10 species which may lead to differentiation of single or plural disomic addition lines. The generation of aneuploids with 2n=21 progressed in such a way that most plants seem to revert to the 2n=20 chromosome number of B. campestris after selfing. From 2n=22 aneuploids, however, the succeeding progenies showed high frequency of plants with two additional chromosomes which accounted for 50.6% and 52.9% of total S₃ progenies via 2n=22 and 2n=24 S₂ generations, respectively. The meiotic behaviour of these progenies indicated evidence for a rule governing the frequency distribution of chromosome number among these addition lines and high possibility to breed such disomic plants with 2n=22. A method of selecting stable aneuploids was suggested in addition to the possible role of pollination biology at various processes of such breeding program.     

Cytogenetic analyses of sugarcane relatives (Andropogoneae: Saccharinae)

Summary Meiosis was studied in 31 wild Saccharum relatives, including Erianthus (8 clones), Miscanthus (5 clones), Narenga prophyrocoma (1 clone), S. robustum (3 clones), and S. spontaneum (14 clones). Chromosome number for 18 clones confirmed published counts or was typical of the particular species. Chromosome number for seven clones (Djatiroto 2n=58, Molokai 5099 2n=80, SES 84/58 2n=58, SES 114 2n=64, SES 260 2n=64, Taiwan 100 2n=112, and US 57-11-2 2n=60) differed from published counts (2n=112, 86-100, 64, 60, 60, 96, and 30, respectively). Counts were obtained for the first time for six clones (Local escape 2n=96, Nepal 2n=72, NG 77-77 2n=108–112, NG 77-199 2n=166, US 57-60-2 2n=20, and US 68-1-4 2n=38). Bivalent chromosome pairing predominated in all clones. Meiotic irregularity (numeric aberrations, univalents, multivalents, and telophase II micronuclei) tended to be associated with taxonomic grouping and level of polyploidy. Clones in Erianthus, Miscanthus, and Narenga were apparent euploids (2n=20–60) and tended to have fewer meiotic irregularities than Saccharum clones. Differences in level of meiotic stability among taxonomic groups may reflect error in chromosome association and synapsis associated with high chromosome number.     

An ms2 male-sterile, female-fertile soybean sharing phenotypic expression with other ms mutants

Genetic and cytological studies of microsporogenesis and microgametogenesis were conducted with a natural male‐sterile, female‐fertile soybean mutant (BR97‐17971) found in an F₄ breeding line of the Brazilian soybean breeding programme. Allele tests with ms Type Collection from USDA/ARS showed that the gene was allelic to ms2. Conventional analysis of microsporogenesis revealed a differential meiotic behaviour from the normal one reported in ms2 male‐sterile plants. Sterile plants have different levels of meiotic abnormalities related to chromosome segregation. Despite segregational abnormalities in the first and second divisions leading to micronuclei formation, the main meiotic cause of pollen sterility was absent or the defective cytokinesis following telophase II. Absent or defective cytokinesis has never been reported in ms2 mutants, but it was reported in the ms1 and ms4 mutants. After telophase II, one to four nucleate microspores underwent degeneration. Cross‐sections of sterile plant anthers showed that, at telophase II, the tapetal cells with large vacuoles degenerated. Despite callose deposition around meiocytes, sterile plants did not form tetrads. They degenerated and collapsed after callose dissolution, forming an amorphous mass. Anthers from male‐sterile plants were shrivelled.     

Production of 27-, 28-, and 56-chromosome apomictic hybrid derivatives between pearl millet (2n=14) and Pennisetum squamulatum (2n=54)

Summary An interspecific hybridization program designed to transfer gene(s) controlling apomixis from Pennisetum squamulatum Fresen. (2n=6x=54) to induced tetraploid (2n=4x=28) cultivated pearl millet, Pennisetum americanum (L.) Leeke resulted in four offtype plants, two with 27 chromosomes and two with 28 chromosomes. These plants were found among 217 spaced plants established from open-pollinated seed of an apomictic 21-chromosome polyhaploid (2n=21) plant derived from an apomictic interspecific hybrid (2n=41) between tetraploid pearl millet and Pennisetum squamulatum. It appeared that a 21- (or 20-) chromosome unreduced egg from the apomictic polyhaploid united with a 7-chromosome pearl millet (2n=2x=14) gamete to produce a 28- (or 27-) chromosome offspring. Meiotic chromosome behavior was irregular averaging from 3.60 to 4.05 bivalents per microsporocyte in the 27- and 28-chromosome hybrids. The 27- or 28-chromosome hybrids, like the 21-chromosome female parent, shed no pollen, but set from 1.8 to 28 seed per panicle when allowed to outcross with pearl millet. Progeny of the 28-chromosome hybrids were uniform and identical to their respective female parents, indicating that apomixis had been effectively transferred through the egg. In addition, a 56-chromosome plant resulting from chromosome doubling of a 28-chromosome hybrid was identified. Pollen was 68 per cent stainable and the plant averaged 2.3 selfed seeds per panicle. Chromosomes of the 56-chromosome plant paired as bivalents (x=10.67) or associated in multivalents. Three to nine chromosomes remained unpaired at metaphase I. Multiple four-nucleate embryo sacs indicated the 56-chromosome hybrid was an obligate apomict. The production of 27-, 28-, and 56-chromosome hybrid derivatives were the results of interspecific hybridization, haploidization, fertilization of unreduced apomictic eggs, and spontaneous chromosome doubling. These mechanisms resulted in new unique genome combinations between x=7 and x=9 Pennisetum species.     

Meiotic instability of common wheat strains derived from Triticum timopheevi Zhuk. crosses

Summary Meiotic instability was studied in four strains of common wheat derived from crosses involving a Triticum timopheevi derivative, C.I. 13093, and the common wheat varieties Cheyenne and Minturki. A wide range in the percentage of normal cells at six stages of meiosis, MI, AI, dyad, MII, AII, and quartet, was found. The date of sporocyte sampling influenced meiotic irregularities at six stages of meiosis. Florets from the same spikes differed in the percentage of normal cells at all the meiotic stages except MII. Plants within strains differed significantly at the MI and quartet stages only. Except at AI stage, spikes collected on the same day did not differ significantly. Apparently, environment during premeiotic phase determined the extent and the pattern of meiotic instability in spikes of these strains.Chromosome counts were taken on 213 of the highly aberrant PMC at MI. Among these, 44 different chromosome numbers ranging from 4 to 76 per cell were recorded. Cells with deviating numbers from 22 to 28 were the most frequent (30.52 percent). Chromosome numbers 12, 17, 21, 22, 24, 26 and 28 occurred in 43.9 percent of the aberrant PMC, and the number of cells with these chromosome numbers ranged from 9 to 15.The metaphase 1 chromosome pairing in deviating PMC was not related to the number of chromosomes in these PMC. Apparently, deviating PMC had a random assortment of chromosomes. In aberrant PMC and others with apparently normal chromosome numbers (but irregular meiosis) homeologous chromosomes may pair. Functional gametes from such cells may perpetuate meiotic instability that had persisted in these three advanced generation common wheat strains of hybrid origin.     

Meiotic behavior as a selection tool in the breeding of <Emphasis Type="Italic">Brachiaria humidicola</Emphasis> (Poaceae)

Brachiaria humidicola is a tropical grass that grows in seasonally swampy grasslands in Africa. In Brazil, two apomictic cultivars (2n = 54) of this species are widely used as pastures in poorly drained soils. The recent discovery of a sexual polyploid accession (2n = 36) in the germplasm collection at the Embrapa Beef Cattle Research Center allowed intraspecific hybridization with the objective of broadening the genetic variability and selection of superior genotypes in this species. Hybridization, however, depends on accessions with the same ploidy level. Cytological analyses of 55 accessions revealed that 19 apomictic accessions also presented 2n = 36 chromosomes. Chromosome pairing in hexavalent association at diakinesis and metaphase I suggested that the basic chromosome number for this species is x = 6. Cytological analysis revealed abnormalities in variable frequencies in the meiosis of these hexaploid (2n = 6x = 36) accessions. The most common were those related to irregular chromosome segregation which led to unbalanced gamete formation, but chromosome stickiness was also recorded. These results clearly demonstrate the value of cytogenetics in the choice of genitors and for superior hybrids to be obtained in the breeding of this species. For that both the ploidy level and the frequency of abnormalities need to be considered, besides other favorable agronomic characteristics.