Pachytene Bivalents Research Articles

The highly rearranged karyotype of the hangingfly Bittacus sinicus (Mecoptera, Bittacidae): the lowest chromosome number in the order.

Cytogenetic features of the hangingfly Bittacus sinicus Issiki, 1931 were investigated for the first time using C-banding and DAPI (4',6-diamidino-2-phenylindole) staining. The karyotype analyses show that the male B. sinicus possesses the lowest chromosome number (2n = 15) ever observed in Mecoptera, and an almost symmetric karyotype with MCA (Mean Centromeric Asymmetry) of 12.55 and CVCL (Coefficient of Variation of Chromosome Length) of 19.78. The chromosomes are either metacentric or submetacentric with their sizes decreasing gradually. Both the C-banding and DAPI+ patterns detect intermediate heterochromatin on the pachytene bivalents of B. sinicus, definitely different from the heterochromatic segment at one bivalent terminal of other bittacids studied previously. The male meiosis of B. sinicus is chiasmate with two chiasmata in metacentric bivalents and one in the submetacentric bivalent. The sex determination mechanism is X0(♂), which is likely plesiomorphic in Bittacidae. Two alternative scenarios of karyotype origin and evolution in Bittacus Latreille, 1805 are discussed.

An idiogram on pachytene bivajents with high resalution multiple bands of Epinephelus malabaricus

Epinephelus malabaricus (Perciformes, Serranidae), a rare marine commercial fish inhabits in the warm-water reef along the southeast coast of China. In the present study, well-spread pachytene bivalents with multiple high-resolution bands were obtained after the testes were treated with a hypotonic alkaline solution and a high-chloroform fixative solution, showing that the number and characteristics of band in each bivalent were stable. According to karyotype analysis, the haploid chromosome set was n=24 (24t), and no heteromorphic bivalent was found. The relative length of the longest and shortest bivalent were 5.86 and 1.66, and the length ratio was 3.53, which was much higher than the length ratio of the exact metaphase mitotic karyotype (1.5) and premetaphase endonuclease karyotype (1.75). An idiogram with 447 bands was constructed, and the zone of each bivalent was divided, respectively. The detailed description of the landmark system and the band positions were preliminary given. This study aims to provide a theoretical reference for the further research on cytogenetics and the physical map construction of chromosome fine frame of E. malabaricus.

Diverse stages of sex-chromosome differentiation in tinamid birds: evidence from crossover analysis in Eudromia elegans and Crypturellus tataupa

All extant birds share the same sex-chromosome system: ZZ males and ZW females with striking differences in the stages of sex-chromosome differentiation between the primitive palaeognathus ratites and the large majority of avian species grouped within neognaths. Evolutionarily close to ratites is the neotropical order Tinamiformes that has been scarcely explored regarding their ZW pair morphology and constitution. Tinamous, when compared to ratites, constitute a large group among Palaeognathae, therefore, exploring the extent of homology between the Z and W chromosomes in this group might reveal key features on the evolution of the avian sex chromosomes. We mapped MLH1 foci that are crossover markers on pachytene bivalents to determine the size and localization of the homologous region shared by the Z and W chromosomes in two tinamous: Eudromia elegans and Crypturellus tataupa. We found that the homologous (pseudoautosomal) region differ significantly in size between these two species. They both have a single recombination event on the long arm of the acrocentric Z and W chromosomes. However, in E. elegans the pseudoautosomal region occupies one-fourth of the W chromosome, while in C. tataupa it is restricted to the tip of the long arm of the W. The W chromosomes in these two species differ in their heterochromatin content: in E. elegans it shows a terminal euchromatic segment and in C. tataupa is completely heterochromatic. These results show that tinamous have ZW pairs with more diversified stages of differentiation compared to ratites. Finally, the idea that the avian proto-sex chromosomes started to diverge from the end of the long arm towards the centromere of an acrocentric pair is discussed.

Heterochromatin and rDNA 5S and 45S sites as reliable cytogenetic markers for castor bean ( Ricinus communis, Euphorbiaceae)

The increasing need for renewable energy resources has led to higher demands for biofuel, a scenario where the castor bean ( Ricinus communis L.) seed oil represents a promising source of raw material. Despite that, information regarding the genome organization of R. communis is still scarce, impairing the application of modern biotechnological and breeding procedures. The present work brings the first evaluation of the mitotic chromosomes of this species, including 10 potentially interesting accessions for cultivation in semi-arid environments aiming at the biofuel production. The approach included standard staining, fluorochrome staining (CMA/DAPI), fluorescent in situ hybridization (FISH) with rDNA 5S and 45S, as well as silver impregnation. All accessions were diploid with 2 n = 2 x = 20, displaying mainly metacentric chromosomes, with CMA-positive bands (GC-rich) in all pairs of the complement. After silver impregnation, one to 14 nucleoli were observed, while the FISH with rDNA 45S revealed two large sites and a variety of minor dots, and the DNAr 5S hybridized in a single pair. The observed features were discussed and compared with literature data regarding pachytene bivalents.

Cytology of hexaploid cranberry with special reference to chromosomal fibres

The hexaploid cranberry species, Vaccinium hagerupii, was studied cytologically. In the meiotic prophase of this plant some hitherto undescribed structures were demonstrated, which obviously play a part in chromosome pairing. Secondary pairing was shown to occur between pachytene bivalents. The intertelomeric fibres were visible at most stages of both mitosis and meiosis. A brief botanical comment is made, and the origin of this polyploid species is discussed.

Cytogenetic studies on Rubus arcticus, Rubus stellatus and their hybrids

A new cytological technique has been developed which makes it possible to study the meiosis, both in micro- and megasporogenesis, of Rubus arcticus L., R. stellatus Sm. and their hybrids in more detail than before, especially the prophase. In the prophase, a bouquet is observed. Its formation begins in the leptotene, when the telomeres gather in a restricted area and the heterochromatic centromere regions form a kind of chromocenter. In the zygotene there is a synizesis, i.e. a form of bouquet, whereas, in the pachytene, the chromosomes are paired and the bouquet is at its height. After the pachytene “schizonema” is observed, the phase where homologous chromosomes begin to separate forming a starlike figure, and chiasmata are not seen. The chromocenter is still extant, but it breaks in the diplotene, and the chiasmata become visible. Subsequently, the meiosis proceeds as usual. Only two of the hybrid individuals displayed any disturbances. The pachytene bivalents are divided into three groups: one nucleolus-organising bivalent, three subacrocentric and three submetacentric bivalents. The same groups are obtained when the meiotic chromosomes are grouped.

Pairing and synapsis in wild type Arabidopsis thaliana

A spreading technique was used to perform a structural analysis of prophase I nuclei in pollen mother cells (PMCs) of wild-type Arabidopsis thaliana. In leptotene, all chromosomes developed fully axial elements before a presynaptic alignment was observed. Pairing and synapsis start in regions close to the telomeres at early zygotene. Interstitial synaptonemal complex (SC) stretches were found to occur at several sites per bivalent at mid zygotene. Within individual bivalents, extensive regions of SC formation often existed at the same time as other extensive regions that were unsynapsed. Also in the same nucleus, one bivalent might have several SC segments, while other bivalents have only a few. The classical bouquet was not so evident as in other plant species. Length measurements of the five pachytene bivalents have allowed the elaboration of a pachytene karyotype. Pachytene chromatin compaction in Arabidopsis was significantly less than that observed in the other species analysed and this is paralleled with a higher recombination rate (centimorgans per megabase).

Comparative study of the grasshopperZoniopoda tarsata(Romaleide) andPaulinia acuminata(Pauliniidae) based on chromosome and C-banding analysis.

Abstract In most Acridoidea the basic karyotype is composed by 11 telocentric chromosomes and a telocentric X (2n=23, X0) in males and 12 telocentric chromosomes in females (2n=24, XX). In this paper we comparatively analyzed meiosis of Z. tarsata (Acrididae, Romaleinae) and P. acuminata (Pauliniidae) after C-banding and DAPI staining in order to investigate differences due to heterochromatin amount and localization. Z. tarsata and P. acuminata showed a karyotype composed by 11 somatic telocentric chromosomes and a telocentric X, corresponding to 2n=23, X0 diploid karyotype in males. After C-banding procedure Z. tarsata pachytene bivalents showed 12 densely stained spots including the X chromosome, 4 of these were observed in interstitial locations and the remaining in pericentric position of the telocentric bivalents. In P. acuminata 4 C-banded spots were observed, 2 of these in interstitial locations and the remaining in pericentric position. After pachytene DAPI staining, bivalents showed specific bright stained areas corresponding to those detected after C-banding in Z. tarsata, the same was observed in P. acuminata. These differences demonstrate that despite chromosome similarity between these taxa heterochromatin evolution seems to follow a different path, allowing a clear differentiation between these two karyotypes.

Conserved synteny of genes between chromosome 15 of Bombyx mori and a chromosome of Manduca sexta shown by five-color BAC-FISH

The successful assignment of the existing genetic linkage groups (LGs) to individual chromosomes and the second-generation linkage map obtained by mapping a large number of bacterial artificial chromosome (BAC) contigs in the silkworm, Bombyx mori, together with public nucleotide sequence databases, offer a powerful tool for the study of synteny between karyotypes of B. mori and other lepidopteran species. Conserved synteny of genes between particular chromosomes can be identified by comparatively mapping orthologous genes of the corresponding linkage groups with the help of BAC-FISH (fluorescent in situ hybridization). This technique was established in B. mori for 2 differently labeled BAC probes simultaneously hybridized to pachytene bivalents. To achieve higher-throughput comparative mapping using BAC-FISH in Lepidoptera, we developed a protocol for five-color BAC-FISH, which allowed us to map simultaneously 6 different BAC probes to chromosome 15 in B. mori. We identified orthologs of 6 B. mori LG15 genes (RpP0, RpS8, eIF3, RpL7A, RpS23, and Hsc70) for the tobacco hornworm, Manduca sexta, and selected the ortholog-containing BAC clones from an M. sexta BAC library. All 6 M. sexta BAC clones hybridized to a single M. sexta bivalent in pachytene spermatocytes. Thus, we have confirmed the conserved synteny between the B. mori chromosome 15 and the corresponding M. sexta chromosome (hence provisionally termed chromosome 15).

Molecular divergence of the W chromosomes in pyralid moths (Lepidoptera)

Most Lepidoptera have a WZ/ZZ sex chromosome system. We compared structure of W chromosomes in four representatives of the family Pyralidae--Ephestia kuehniella, Cadra cautella, Plodia interpunctella, and Galleria mellonella--tracing pachytene bivalents which provide much higher resolution than metaphase chromosomes. In each species, we prepared a W-chromosome painting probe from laser-microdissected W-chromatin of female polyploid nuclei. The Ephestia W-probe was cross-hybridized to chromosomes of the other pyralids to detect common parts of their W chromosomes, while the species-specific W-probes identified the respective W chromosome. This so-called Zoo-FISH revealed a partial homology of W-chromosome regions between E. kuehniella and two other pyralids, C. cautella and P. interpunctella, but almost no homology with G. mellonella. The results were consistent with phylogenetic relationships between the species. We also performed comparative genomic hybridization, which indicated that the W chromosome of C. cautella is composed mainly of repetitive DNA common to both sexes but accumulated in the W chromosome, whereas E. kuehniella, P. interpunctella, and G. mellonella W chromosomes also possess a large amount of female specific DNA sequences, but differently organized. Our results support the hypothesis of the accelerated molecular divergence of the lepidopteran W chromosomes in the absence of meiotic recombination.

Identification of all pachytene bivalents in the common shrew using DAPI-staining of synaptonemal complex spreads

A major problem in studies of synaptonemal complexes (SC) is the difficulty in distinguishing individual chromosomes. This problem can be solved combining SC immunostaining with FISH of chromosome-specific sequences. However, this procedure is expensive, time-consuming and applicable only to a very limited number of species. In this paper we show how a combination of SC immunostaining and DAPI staining can allow identification of all chromosome arms in surface-spreads of the SC of the common shrew (Sorex araneus L.). Enhancement of brightness and contrast of the images with photo editing software allowed us to reveal clear DAPI-positive and negative bands with relative sizes and positions similar to DAPI landmarks on mitotic metaphase chromosomes. Using FISH with DNA probes prepared from chromosome arms m and n we demonstrated correct recognition of the chromosomes mp and hn on the basis of their DAPI pattern. We show that the approach we describe here may be applied to other species and can provide an important tool for identification of individual bivalents in pachytene surface-spreads.

Genome evolution in Arabidopsis/Brassica: conservation and divergence of ancient rearranged segments and their breakpoints

Since the tetraploidization of the Arabidopsis thaliana ancestor 30-35 million years ago (Mya), a wave of chromosomal rearrangements have modified its genome architecture. The dynamics of this process is unknown, as it has so far been impossible to date individual rearrangement events. In this paper, we present evidence demonstrating that the majority of rearrangements occurred before the Arabidopsis-Brassica split 20-24 Mya, and that the segmental architecture of the A. thaliana genome is predominantly conserved in Brassica. This finding is based on the conservation of four rearrangement breakpoints analysed by fluorescence in situ hybridization (FISH) and RFLP mapping of three A. thaliana chromosomal regions. For this purpose, 95 Arabidopsis bacterial artificial chromosomes (BACs) spanning a total of 8.25 Mb and 81 genetic loci for 36 marker genes were studied in the Brassica oleracea genome. All the regions under study were triplicated in the B. oleracea genome, confirming the hypothesis of Brassica ancestral genome triplication. However, whilst one of the breakpoints was conserved at one locus, it was not at the two others. Further comparison of their organization may indicate that the evolution of the hexaploid Brassica progenitor proceeded by several events, separated in time. Genetic mapping and reprobing with rDNA allowed assignment of the regions to particular Brassica chromosomes. Based on this study of regional organization and evolution, a new insight into polyploidization/diploidization cycles is proposed.

Chromosomal localization of the telomeric (TTAGGG)n sequence in four species of Armadillo (Dasypodidae) from Argentina: an approach to explaining karyotype evolution in the Xenarthra

The distribution of the vertebrate telomeric sequence (TTAGGG)(n) in four species of armadillos (Dasypodidae, Xenarthra), i.e. Chaetophractus villosus (2n = 60), Chaetophractus vellerosus (2n = 62), Dasypus hybridus (2n = 64) and Zaedyus pichiy (2n = 62) was examined by FISH with a peptide nucleic acid (PNA) probe. Besides the expected telomeric hybridization, interstitial (centromeric) locations of the (TTAGGG)n sequence were observed in one chromosome pair of Chaetophractus vellerosus and Zaedyus pichiy, suggesting chromosome fusion of ancestral chromosomes occurring during the evolution of Dasypodidae. In addition, all the species analysed showed one to four apparently telocentric chromosomes, exhibiting only two telomeric signals. However, the immunodetection study of kinetochore proteins on synaptonemal complex spreads from C. villosus showed that the apparently telocentric chromosomes have a tiny short arm that can be resolved only in the more elongated pachytene bivalents. This finding suggests that none of the species of armadillos possess true telocentric chromosomes. Our present results support a reduction in the diploid number by fusion of acrocentrics with loss of chromosome material as a tendency in Dasypodidae.

Satellite repeats in the functional centromere and pericentromeric heterochromatin of Medicago truncatula.

Most eukaryotic centromeres contain long arrays of tandem repeats, with unit lengths of 150-300 bp. We searched for such repeats in the functional centromeres of the model legume Medicago truncatula (Medicago) accession Jemalong A17. To this end three repeats, MtR1, MtR2 and MtR3, were identified in 20 Mb of a low-pass, whole genome sequencing data set generated by a random shotgun approach. The nucleotide sequence composition, genomic organization and abundance of these repeats were characterized. Fluorescent in situ hybridization of these repeats on chromosomes at meiosis I showed that only the MtR3 repeat, encompassing stretches of 450 kb to more than 1.0 Mb, is located in the functional portion of all eight centromeres. MtR1 and MtR2 occupy distinct regions in pericentromeric heterochromatin. We also studied the presence and distribution of MtRs in Medicago accession R108-1, a genotype with a genome that is 20% smaller than that of Jemalong A17. We determined that while MtR3 is also centromeric on all pachytene bivalents in R108-1, MtR1 and MtR2 are not present in the R108 genome.

Cytological evaluation of global DNA methylation in mouse testicular genome.

Global methylation of DNA from different testicular cell types has been studied by DNA end-labeling and nick translation of fixed chromatin (in situ), following digestion with cytosine methylation-sensitive restriction enzymes. Both at the level of chromatic (chromosome) and naked DNA, there is extensive methylation of the genome. Although the extent of methylation was nearly the same among different cell types in the MspI, HpaII, and HhaI digested end-labelled DNA, in the chromosome preparations the digestion patterns varied in cell type-specific manner, pachytene being the most sensitive and spermatids and sperm the most resistant. The differential sensitivity is attributable to the difference in the chromatin organisation in different testicular cell types though no specific region could be identified as particularly more sensitive or resistant to the enzymes. Pachytene bivalents do not reveal a consistent segmental pattern of digestion, but the perichiasmate regions of diplotene/diakinesis and metaphase I chromosomes show hypersensitivity to the enzymes.

The Synaptonemal Complex Complement of the Wax Moth, Galleria Mellonella

Synaptonemal complexes (SCs) of pachytene bivalents from oocytes and spermatocytes of the wax moth, Galleria mellonella, were studied with a microspreading technique. In both sexes 30 bivalents were found. Male SCs in contrast to female SCs contained recombination nodules. No specific bivalent could be recognized in male complements. In female complements, the WZ bivalent was recognized among the three longest bivalents by its delayed pairing and/or unequal lengths of the lateral elements. Pairing of WZ bivalents is complete and lateral elements are nearly equalized in a few cells, presumably at the end of pachytene.

Recombination nodules in coleopteran species: Palembus dermestoides (Tenebrionidae) and Epicauta atomaria (Meloidae)

This work describes the first report about the occurrence of recombination nodules (RNs) in spread pachytene cells of two species of Coleoptera: Palembus dermestoides (Tenebrionidae) and Epicauta atomaria (Meloidae). The RNs were observed in preparations contrasted with phosphotungstic acid. Considering RN morphology and its occurrence in pachytene bivalents (one per autosome bivalent) these structures were interpreted to be late RNs. P. dermestoides and E. atomaria have 2n = 20 chromosomes including an Xy_p sex determination system. In spite of most frequently subtelocentric morphology observed in the autosomes of both species, the occurrence of RNs is limited only to the synaptonemal complex (SC) structure of the long arms. These findings are in agreement with those obtained using light microscopy analysis in which only one chiasma or terminalization event is observed per autosomal bivalent in early or late metaphase I cells. The RNs have the same average width of the SC of each analyzed species, a circular shape, strong electron density, and are observed mainly between the lateral elements of the SC. The RNs of P. dermestoides and E. atomaria have approximately the same average size (width), 180 ± 20 nm and 160 ± 80 nm, respectively. The absence of RNs in the short arms and its occurrence in the long arms are discussed considering the short arm pericentromeric and pro-centric heterochromatin.

The meiotic chromosomes of maleDrosophila willistoni

The male meiotic chromosomes of Drosophila willistoni are described and compared to Giemsa stained mitotic chromosomes. Meiosis was examined in males from two wild type stocks: WIP-4, an almost monomorphic stock, and 17A2, a high-inversion polymorphic stock. Hybrids between these stocks were also examined. Stocks and hybrids were cultured at 25oC and at 29oC, a tem- perature condition thought to induce male recombination associated with trans- posable element(s) mobilisation. Chromatin in primary spermatocytes is seen as pachytene and early diplotene bivalents, representing the two autosomal bivalents and a steady association between X and Y. Bivalents remain relatively extended from diplotene through anaphase I, which is followed by the conventional stages of meiosis II. Neither chiasmata nor chromatid bridges and fragments were detected in both control and experimental males. Thread-like bubble structures on the distal region of the X chromosome were observed in early diplotene cells from F1 male offspring between WIP-4 and 17A2 reared at 29oC; their functional meaning is unknown.

Telomeric and interstitial telomeric sequences in holokinetic chromosomes of Lepidoptera: telomeric DNA mediates association between postpachytene bivalents in achiasmatic meiosis of females.

Telomeres, besides their main role in the protection and maintenance of chromosome ends, have several other vital functions in the cell cycle. We studied their role in the achiasmatic meiosis of female Lepidoptera, insects with holokinetic chromosomes. By fluorescence in-situ hybridization (FISH) with the insect telomeric probe, (TTAGG)n, we mapped the distribution of telomeric and interstitial telomeric sequences (ITS) in female meiotic chromosomes of two species, Orgyia antiqua with a reduced chromosome number (2n = 28) and Ephestia kuehniella mutants, possessing a radiation-induced chromosome fusion in the genome (2n = 59). In addition to the strong typical telomeric signals, O. antiqua displayed weaker hybridization signals in interstitial sites of pachytene bivalents. The observed ITS most probably reflect remnants of chromosomal rearrangements and support the hypothesis that the Orgyia karyotype had arisen by multiple fusions of ancestral chromosomes. On the other hand, the absence of ITS in the chromosome fusion of Ephestia indicated the loss of telomeres before the two original chromosomes fused. When the telomeric probe was amplified by enzymatic reaction with tyramid, the number of ITS observed increased in Orgyia, and a few ITS were also observed in several chromosomes of Ephestia but not in the fused chromosome. This suggests that the genomes of both species also contain ITS other than those originating from chromosome fusions. The analysis of female meiotic prophase I revealed non-homologous associations of postpachytene bivalents mediated by telomeric DNA, which were not observed in the pachytene stage. Surprisingly, in early postpachytene nuclei the telomeric associations also involved ITS, whereas later postpachytene nuclei displayed chains of bivalents interconnected only by true telomeres. This finding favours a hypothesis that telomeric associations between bivalents play a role in chromosome segregation in the achiasmatic meiosis of female Lepidoptera.

Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping.

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromosome 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed. Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.