CHROMOSOME-STUDIES IN HYPOPTOPOMATINAE (PISCES, SILURIFORMES, LORICARIIDAE) .2. ZZ ZW SEX-CHROMOSOME SYSTEM, B-CHROMOSOMES, AND CONSTITUTIVE HETEROCHROMATIN DIFFERENTIATION IN MICROLEPIDOGASTER-LEUCOFRENATUS

Cytogenetic analysis of two local populations of microlepidogaster leucofrenatus showed a basic diploid chromosome number (2N) of 54 in both populations. Some fishes were found to have a 2N = 55 or 56 chromosomes due to the presence of one or two large heterochromatic B chromosomes. Specimens of M. leucofrenatus from the Poco Grande stream had 24 metacentrics, 24 submetacentrics, four subtelocentrics, and one submetacentric homomorphic pair in males and one submetacentric/subtelocentric heteromorphic pair in females, whereas individuals of this species from the Marumbi River had 22 metacentrics, 24 submetacentrics, four subtelocentrics, two acrocentrics, and one submetacentric/subtelocentric heteromorphic pair in females. The occurrence of the heteromorphic pair in the females was due to the presence of an extra C-banded segment on the W chromosome. Ag-NORs in both populations were located interstitially on the short arm of the largest metacentric pair. The Poco Grande population had less constitutive heterochromatin than did the Marumbi River population. The speciation process in this fish species is discussed on the basis of heterochromatin distribution.

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 (20 = 36; 42; 54). Chromosome numbers of 20 = 18 in B. ruziziensis and 20 = 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.

SYNAPTONEMAL COMPLEX-ANALYSIS OF THE SEX BIVALENT IN GOATS

Synaptonemal complex (SC) analysis of XY pairing in the goat (Capra hircus; 2n = 60) was investigated by electron microscopy for the first time in this species. Synapsis of the X and Y chromosomes begins during the mid-late zygotene stage as the autosomes complete their pairing. Only a small portion of the total length of the Y is paired with the X chromosome at this time. By the early pachytene, almost 90% of the Y is paired with the X. All the observed stages of the sex bivalent pairing showed the structural difference between the differential and pairing regions. In the pairing region, a synaptonemal complex is formed, while in the differential region the chromosome axes remain free.

A natural triploid in Trichomycterus davisi (Siluriformes, Trichomycteridae): mitotic and meiotic characterization by chromosome banding and synaptonemal complex analyses

Within a total of 50 analyzed specimens a male individual of Trichomycterus davisi has been recorded with 81 chromosomes including 60 metacentric, 18 submetacentric and three subtelocentric chromosomes. When compared with diploid individuals (2n = 54) and the morphological standard of chromosomes, this male is a triploid with 3 = 81 chromosomes. Since staining with silver nitrate indicates three active nucleolar organizer regions (NORs), the three NOR- bearing chromosomes in this individual are genetically active. Analysis of the synaptonemal complex (SC) by electronic microscopy shows that there is an incomplete pairing of the third set of chromosomes in the triploid individual.

Use of primers derived from a new sequence of the bovine Y chromosome for sexing Bos taurus and Bos indicus embryos

A bovine male-specific marker was identified in our laboratory through random amplified polymorphic DNA (RAPD) analysis. This fragment of 3216 bp was cloned, sequenced and mapped by fluorescent in situ hybridization (FISH) on the taurine Yq. Primers derived from this sequence were initially screened by polymerase chain reaction (PCR) for their ability to detect Y-specific segments in zebu and taurine genomic DNA. Two of these primers amplified a 655 bp Y-specific sequence present in taurine and zebu male genomic DNA. These primers were then used for detecting the 655 bp male sequence in DNA from 173 zebu and 30 taurine embryos, which had been previously sexed using primers for the sequence BC 1.2. The results revealed an accuracy of 100%. (C) 2002 Elsevier B.V. All rights reserved.

CYTOGENETIC ANALYSIS OF THE BRAZILIAN BLIND CATFISH PIMELODELLA-KRONEI AND ITS PRESUMED ANCESTOR, P-TRANSITORIA

Karyotypes of the blind catfish Pimelodella kronei from three different caves in the southwestern part of the State of São Paulo, Brazil, and of its presumed ancestor Pimelodella transitoria were examined. Chromosome number and formula, as well as C-banding pattern and NOR location were found to be the same in both species, supporting the hypothesis of a close relationship and even conspecificity of P. kronei and P. transitoria.

CYTOGENETICS OF 4 BRAZILIAN HYLA SPECIES (AMPHIBIA-ANURA) AND DESCRIPTION OF A CASE WITH A SUPERNUMERARY CHROMOSOME

The chromosomes of Hyla fuscovaria, H. hayii and II. prasina, with 2n=24, and of Hyla sp. (aff. circumdata), a new species, with 2n=24 and 2n=25, were studied.The karyotypes with 2n=24 in the four species were very similar, with almost no differences in the size and morphology of the chromosomes. The numerical variability found in Hyla sp. (aff. circumdata) is due to the occurrence of a supernumerary chromosome in some specimens. NOR data obtained for the first time in the four species and C banding analysis of H. prasina indicate that such types of banding may be useful to differentiate species with very similar karyotypes, contributing to the understanding of chromosome evolution and the establishment of phylogenetic relationships among Brazilian Hyla species.

SYNAPTONEMAL COMPLEX-ANALYSIS IN GOATS CARRYING THE 5/15 ROBERTSONIAN TRANSLOCATION

Synaptonemal complexes were analysed by electron microscopy in 2 bucks heterozygous for the 5/15 Robertsonian translocation. The cis configuration (free homologous 5 and 15 chromosomes on the same side of the 5/15 translocated chromosome) was found in all 50 cells examined. This feature is considered a prerequisite for the development of balanced gametes. No association between the sex bivalent and trivalent was observed.

RFLP analysis of genetic variation in species of section Arachis, genus Arachis (Leguminosae)

Four A-genome species of the genus Arachis ( A. cardenasii, A. correntina, A. duranensis, A. kempff-mercadoi), three B genomes species ( A. batizocoi, A. ipaensis and A. magna), the AABB allotetraploid A. hypogaea (cultivated peanut) and introgression lines resulting from a cross between A. hypogaea and A. cardenasii were analyzed by RFLP. The A genome species (cytologically characterized by the presence of a small chromosome pair 'A') were closely similar to each other and shared a large number of restriction fragments. In contrast, the B genome species differed more from one another and shared few fragments. The results of this study indicate that the absence of the small chromosome pair is not a good criterion for grouping species of section Arachis as B genome species, since their genome might be quite distinct from the B genome of A. hypogaea. The lowest genetic variation was detected within accessions of A. duranensis (17 accessions), followed by A. batizocoi (4 accessions) and A. cardenasii (9 plants of accession GKP 10017). The high level of genetic variation found in A. cardenasii might indicate that not all accessions of wild species of Arachis are autogamous, as reported for A. hypogaea.

CYTOGENETIC STUDIES ON THE SUBFAMILY HYPOPTOPOMATINAE (PISCES, SILURIFORMES, LORICARIIDAE) .3. ANALYSIS OF 7 SPECIES

Cytogenetic studies carried out on nine species belonging to five genera of the subfamily Hypoptopomatinae showed that this group has a relatively constant diploid number, 2n = 54, with only one species having 2n = 72 chromosomes. Nevertheless, the karyotypic formulae, NOR position and C-band pattern are very different among species and sometimes among local populations, with species having undifferentiated sex chromosomes and species having the XX/XY or the ZZ/ZW mechanisms. The population structure of species belonging to the subfamily Hypoptopomatinae suggests that many chromosome rearrangements have been fixed in the different species and populations due to their geographic isolation and these karyotypic differences may be very important today for species definition.

Chromosomal analysis of the leptodactylids Pleurodema diplolistris and Physalaemus nattereri (Amphibia, Anura)

Detailed characterizations of the karyotypes of the Brazilian leptodactylid frogs Pleurodema diplolistris, the only species of Pleurodema not studied cytogenetically so far, and Physalaemus nattereri, a species in the Ph. biligonigerus group, are presented. Both karyotypes had 2n = 22 and their chromosomes had a very similar morphology, except for pair 11, which was metacentric in Pl. diplolistris and telocentric in Ph. nattereri. The localization of nucleolar organizer regions (NORs) and heterochromatic bands allowed the differentiation of chromosomes that were morphologically indistinguishable between these species, such as pairs 1, 3 and 10, which showed interstitial C-bands in Ph. nattereri, and pair 8, that had an NOR and an adjacent C-band in Pl. diplolistris. Pair 8 also has NOR-bearing chromosomes in many other Pleurodema species. However, in these species, the NOR is located proximal to the centromere on the short arm, while in Pl. diplolistris it occurred distally on the long arm, a condition that may be considered a derived state. In Ph. nattereri, the NOR occurred on chromosome I 1 and differed from the other species of the Ph. biligonigerus group. In contrast, C-banding revealed a heterochromatic block near the centromere on the short arm of pair 3, a characteristic common to all members of this group of Physalaemus.

SYNAPTONEMAL COMPLEX-ANALYSIS IN SPERMATOCYTES OF TILAPIA, OREOCHROMIS-NILOTICUS (PISCES, CICHLIDAE)

Some adaptations of the synaptonemal complex (SC) whole-mounting technique first used in plants permitted its application to meiotic studies in tilapia, Oreochromis niloticus. Direct observation of the chromosome pairing process and bivalent structure during the meiotic prophase of this fish species by light and electron microscopy permitted the analysis of SCs in autosomes and the possible identification of sex chromosomes. The analysis of SCs in spermatocytes of 0. niloticus revealed that all 22 bivalent chromosomes completely paired, except for the occurrence of a size heteromorphism in the terminal region of the largest bivalent associated with the presence of an incompletely paired segment during the synapsis process, which may be the cytological visualization of an XX/XY sex chromosome system in this species.

CYTOGENETIC ANALYSIS OF THE PAMPAS DEER, OZOTOCEROS-BEZOARTICUS (MAMMALIA, CERVIDAE)

The chromosome constitution of five males and three females of the Pampas deer (Ozotoceros bezoarticus) coming mainly from the region of Corumba-MS, was studied. The diploid number of the species was reconfirmed as 68 chromosomes with Fundamental Number (FN) = 74. The X chromosome was the largest and the Y the smallest in the genome. Constitutive heterochromatin demonstrated by C banding was present in the centromeric region of all chromosomes, except in pair number two, which had none, and in chromosome X which had a stained region in the telomere on the long arm, Chromosomes pairs 3 and 4 bore Ag-NORs. The banding patterns differed from those of previous reports for this species. This may be due to subspecific differences.

A LINE2 repetitive DNA sequence from the cichlid fish, Oreochromis niloticus: sequence analysis and chromosomal distribution

We report the cloning and characterization of a long interspersed nucleotide element (LINE) fi-om a cichlid fish, Oreochromis niloticus, and show the distribution of this element, called CiLINE2 for cichlid LINE2, in the chromosomes of this species. The identification of an open reading frame in CiLINE2 with amino acid sequence similarity to reverse transcriptases encoded by LINE-like elements in Caenorhabditis elegans, Platemys spixii, Schistosoma mansoni, Gallus gallus (CRI), Drosophila melanogaster (I factor), and Homo sapiens (LINE2), as well as the structure of the element, suggest it is a member of this family of non-long terminal repeat-containing retrotransposons. Search of a DNA sequence database identified sequences similar to CiLINE2 in four other fish species (Haplotaxodon microlepis, Oreochromis mossambicus, Pseudotropheus zebra, and Fugu rubripes). Southern blot hybridization experiments revealed the presence of sequences similar to CiLINE2 in all Tilapiini species analyzed from the genera Oreochromis, Tilapia, and Sarotherodon, and gave an estimated copy number of about 5500 for the haploid genome of O. niloticus. Fluorescent in situ hybridization showed that CiLINE2 sequences were organized in small clusters dispersed over all chromosomes of O. niloticus, with a higher concentration near chromosome ends. Furthermore the long arm of chromosome 1 was strikingly enriched with this sequence. The distribution of LINE2-related elements might underlie the difference in chromosome banding patterns observed between cold-blooded vertebrates and mammals.

Cytogenetical and cytotaxonomical analysis of some Brazilian species of Eleocharis (Cyperaceae)

Karyotype analysis of 21 samples of 11 species of Eleocharis ( Cyperaceae) from 10 localities in Brazil, showed the presence of chromosomes without primary constrictions and parallel movement of chromatids at metaphase-anaphase transition. Only the terminal nucleolar constrictions ( satellites) were visualised. The chromosome numbers varied from 2n=6 in E. subarticulata to 2n=54 in E. acutangula, but the chromosome basic number x=5 was confirmed. Generally, C-CMA(3)(+) bands appear mostly in the extremities of the chromosomes, associated to NOR, and interstitial C-CMA(3) bands were found only in E. geniculata and E. acutangula. C-DAPI(+) bands were not found. Fluorescence in situ hybridisation ( FISH) with the 45S rDNA probe was performed in five species. The results showed from four to eight hybridisation signals, always terminal. The analysed species include representatives of the following three subgenera of Eleocharis that occur in Brazil: Limnochloa, Scirpidium and Eleocharis. Species from the subgenus Limnochloa have small and numerous chromosomes. The remaining species, belonging to subgenera Eleocharis and Scirpidium, possess fewer and larger chromosomes. In subgenus Eleocharis, karyotypes of the section Eleocharis were differentiated by symploidy, agmatoploidy and polyploidy, whereas species of the section Eleogenus were all polyploids. Polyploidy seems to be the most frequent event in the karyotype differentiation in Eleocharis, but changes in the chromosome size and repetitive DNA sites were also observed.