Meiotic analyses show adaptations to maintenance of fertility in X1Y1X2Y2X3Y3X4Y4X5Y5 system of amazon frog Leptodactylus pentadactylus (Laurenti, 1768)

Heterozygous chromosomal rearrangements can result in failures during the meiotic cycle and the apoptosis of germline, making carrier individuals infertile. The Amazon frog Leptodactylus pentadactylus has a meiotic multivalent, composed of 12 sex chromosomes. The mechanisms by which this multi-chromosome system maintains fertility in males of this species remain undetermined. In this study we investigated the meiotic behavior of this multivalent to understand how synapse, recombination and epigenetic modifications contribute to maintaining fertility and chromosomal sexual determination in this species. Our sample had 2n = 22, with a ring formed by ten chromosomes in meiosis, indicating a new system of sex determination for this species (X1Y1X2Y2X3Y3X4Y4X5Y5). Synapsis occurs in the homologous terminal portion of the chromosomes, while part of the heterologous interstitial regions performed synaptic adjustment. The multivalent center remains asynaptic until the end of pachytene, with interlocks, gaps and rich-chromatin in histone H2A phosphorylation at serine 139 (γH2AX), suggesting transcriptional silence. In late pachytene, paired regions show repair of double strand-breaks (DSBs) with RAD51 homolog 1 (Rad51). These findings suggest that Rad51 persistence creates positive feedback at the pachytene checkpoint, allowing meiosis I to progress normally. Additionally, histone H3 trimethylation at lysine 27 in the pericentromeric heterochromatin of this anuran can suppress recombination in this region, preventing failed chromosomal segregation. Taken together, these results indicate that these meiotic adaptations are required for maintenance of fertility in L. pentadactylus.

Monocentric chromosomes in Juncus (Juncaceae) and implications for the chromosome evolution of the family

Holocentric chromosomes are rare among angiosperms, but have been suggested to be shared by all or most of the species of Cyperaceae and Juncaceae. However, no clear demonstration of the centromere type in Juncus, the largest genus of Juncaceae, has so far been published. Thus, we conducted a detailed chromosomal investigation of four Juncus spp. aiming to identify their centromere type. Mitotic chromosomes were analysed using the fluorochromes CMA and DAPI, fluorescent in situ hybridization (FISH) with rDNA probes and immunodetection of histones H3 phosphorylated at serine 10 (H3-S10ph) and H2A phosphorylated at threonine 133 (H2A-T133ph). DAPI-stained chromosomes of all species displayed typical primary constrictions, which were not related to AT-poor CMA+ heterochromatin or rDNA sites (usually negatively stained with DAPI). Immunodetection with H3-S10ph and H2A-T133ph revealed hyperphosphorylation of pericentromeric and centromeric regions, respectively, in a restricted area, as observed in monocentric chromosomes. Meiotic analyses in J. microcephalus showed no indication of inverted meiosis, commonly found in plants with holocentric chromosomes. Since the species investigated here belong to four different sections of Juncus and all of them display typical monocentric chromosomes, it seems that this kind of centromere is common in the genus and may represent the standard centromere organization for Juncus. If Juncus has monocentric chromosomes, there is no reason to hypothesize that other genera of Juncaceae for which centromeres have not been carefully investigated have holocentric chromosomes.

Cytogenetic Characterization of Brown Howler Monkeys, Alouatta guariba clamitans (Atelidae, Platyrrhini): Meiotic Confirmation of an X1X1X2X2X3X3/X1X2X3Y1Y2 Sex Chromosome System

For brown howler monkeys (Alouatta guariba clamitans), diploid chromosome numbers varying from 2n = 45 to 2n = 52, with XX/XY, X1X1X2X2/X1X2Y, and X1X1X2X2X3X3/X1X2X3Y1Y2 sex chromosome systems have been described by mitotic studies but still await confirmation by meiotic analyses. We analyzed 3 male individuals sampled in the wild (in the municipality of Santa Maria, RS, Brazil) as well as 1 male and 1 female individual in captivity at the São Braz breeding center. Peripheral blood samples and testicular biopsies were taken. We found different diploid numbers for both sexes in somatic cells, 2n = 45,X1X2X3Y1Y2 in males and 2n = 46,X1X1X2X2X3X3 in females, with 4 metacentric (9-12), 7 submetacentric (1-6, 8), and 9 acrocentric autosomal chromosome pairs (13-20, 22). X1 and X2 were submetacentric chromosomes, while X3, Y1, and Y2 were acrocentric ones. Spermatocyte microspreads were examined for synaptonemal complexes. Pachytene spermatocyte analysis was done to verify the chromosome number and morphologies observed in mitotic karyotypes. Immunodetection was performed using anti-SMC3 and anti-CREST antibodies. The presence of a sex chromosome pentavalent X1X2X3Y1Y2 in the males was confirmed by C-banding in metaphase I and by immunodetection in prophase I by the clear identification of 5 centromeres. The G-banded karyotype corresponded to that previously described for A. g. clamitans in the south of Brazil (Curitiba, Parana State, and Blumenau, Santa Catarina State) and for the Misiones Province, Argentina.

Meiotic and mitotic behaviour of B chromosomes of ryegrass

Mitotic and meiotic analyses using conventional and fluorescent stains were employed in plants (accession ETBAZ 055 - origin: Capão do Leão, Rio Grande do Sul State, Brazil) from the Germplasm Active Bank of Ryegrass (Banco Ativo de Germoplasma de Azevém) of Embrapa, for observing the behaviour of B chromosomes. In several meiotic stages, there were observed up to two B chromosomes, which have presented an unstable behaviour regarding their precocious ascension to metaphase I or delays during anaphase I. At the end of the process, the Bs showed predominantly segregation for the nuclei under formation in order to guarantee their propagation. Concerning the cells that comprise the anther tapetum and the root meristems, the B chromosomes have presented a more stable behaviour.

Chromosome 5D instability in cell lines of Triticum tauschii and morphological variation in regenerated plants

Immature embryos of Triticum tauschii cultured on L2 medium with 30 g∙L−1maltose gave rise to compact and highly structured callus that readily regenerated shoots when transferred to MS medium supplemented with zeatin and indole-3-acetic acid. Two cell suspensions were initiated from the callus induced on L2 medium. An analysis of chromosome number in these cell suspensions after 3 months of culture showed that 95 and 75% of the cells had a normal complement. After 5 months, in both lines the majority of cells had chromosome complements of 2n = 13 and many of these aneuploid cells possessed a dicentric chromosome. C-banding indicated that the dicentric was often formed from chromosomes 2D and 5D. Fifty-six plants regenerated from callus were grown to maturity. Variation was observed in tiller number, flowering time, and seed-set; in two cases, the variation in early flowering was shown to be heritable. All the regenerants analysed had normal chromosome counts (2n = 2x = 14) and meiotic analyses of 35 plants revealed no obvious structural rearrangements.Key words: immature embryo culture, regeneration, chromosome, cell suspension, Triticum tauschii.

Comparison of crossover frequencies in barley (Hordeum vulgare) and H. vulgare × H. bulbosum hybrids using a paracentric inversion

Crosses between different parental ploidy combinations of barley (Hordeum vulgare L.) and H. bulbosum L. have been carried out principally to obtain hybrids with high allosyndetic chromosome pairing. Fertility has been observed in tetraploid and triploid hybrids, but there has been little evidence of gene introgression from H. bulbosum into H. vulgare in their progeny. To investigate whether crossing-over takes place between homoeologous chromosomes of H. vulgare and H. bulbosum, diploid hybrids were obtained from crosses between a barley mutant (wst3) homozygous for a paracentric inversion on chromosome 3 and one H. bulbosum genotype. Meiotic analyses at metaphase I and anaphase I and II were carried out on several viable hybrids and compared with control diploid hybrids without the inversion and H. vulgare plants heterozygous for the inversion. Aberrations (bridges and (or) fragments) in H. vulgare × H. bulbosum pollen mother cells at anaphase I were significantly less frequent than in the barley hétérozygote. It is concluded that reduced crossing-over between the parental chromosomes in hybrids contributes to the lack of success in obtaining genetic recombinations between the two species.Key words: Hordeum vulgare, Hordeum bulbosum, interspecific hybrid, paracentric inversion, crossing-over.