Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA(3) ) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO (3) staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.     

Cytogenetic investigations on seven Palaearctic weevil species (Coleoptera, Curculionidae).

Cytogenetic investigations were carried out on seven species of bisexual weevils from five subfamilies. The following numbers of chromosomes were found in individual species: 2n = 22, n male = 10 + Xyp in Ptochus porcellus Boh. (Otiorhynchinae); 2n = 40, n male = 19 + Xyp in Larinodontes turbinatus Gyll. (Cleoninae); 2n = 26, n male = 12 + Xyp in Curculio pellitus (Boh.) (Curculioninae); 2n = 30, n male = 14 + Xyp in Acalles camelus (F.), Acalles echinatus (Germ.), Acalles hypocrita Boh. (Cryptorhynchinae), and 2n = 40, n male = 19 + Xyp in Cionus hortulanus (Foucr.) (Mecininae). The heterochromosomes of all the examined species form, in the first meiotic metaphase, a parachute bivalent. The chromosome number and sex determining system of six species and two genera are described for the first time.     

Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse (Equus caballus)

Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag-NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staining. The NOR foci were observed to be variable in number, size, and shape, but were usually located centrally and appeared as one or two nucleolus-like structures in the spermatozoa head. Three distinctive FISH signals identified the NOR-bearing chromosome pairs during the synaptic cell stage of meiosis I. At diakinesis/metaphase I, as well as different stages of meiosis II, FISH signals clearly depicted the NOR-bearing sister chromatids. The synaptonemal complexes of primary spermatocytes consistently showed three rDNA foci following FISH, but variably demonstrated two or three Ag-NOR bodies following silver staining. We propose rDNA loss and gain during unequal crossing-over events could be both a direct and indirect cause of variation in equine NOR foci. Additionally, our cytogenetic analysis did not confirm the presence of a fourth pair of NORs-bearing chromosomes in the horse, which is contrary to previously mitotic published data.     

Karyotypic characterization of representatives from Melolonthinae (Coleoptera: Scarabaeidae): karyotypic analysis, banding and fluorescent in situ hybridization (FISH)

Meiotic chromosomes of Phyllophaga (Phytalus) vestita, Phyllophaga (Phyllophaga) aff capillata and Lyogenys fuscus (Melolonthinae) were analyzed by conventional staining, C-banding, fluorochromes, silver nitrate and FISH. The three species had a diploid number of 2n=20 and a sex mechanism of the (Xyp; XYp) parachute type. P. (Phytalus) vestita,P. (Phyllophaga) aff capillata and Lyogenys fuscus showed pericentromeric constitutive heterochromatin (CH) in all autosomal bivalents and on X chromosomes. Staining with CMA3 and DAPI fluorochromes showed that the CH of P. (Phytalus) vestita is not specifically rich in AT and GC-base pairs, whereas in P. (Phyllophaga) aff capillata the sex bivalent and one autosomal pair were found to be enriched in GC base pairs with CMA3, and in Lyogenys fuscus CH was positive for DAPI. Silver nitrate staining revealed nucleolar remnants in all three species. However, FISH obtained a precise identification of nucleolar organizing regions with an rDNA 18S and 25S probe. A signal of hybridization was seen in each species, being detected in the X chromosome of P. (Phytalus) vestita and Lyogenys fuscus, and in a small autosomal bivalent of P. (Phyllophaga) aff capillata.     

Differences in the rDNA-bearing chromosome divide the Asian-Pacific and Atlantic species of Crassostrea (Bivalvia, Mollusca)

Karyotype and chromosomal location of the major ribosomal RNA genes (rDNA) were studied using fluorescence in situ hybridization (FISH) in five species of CRASSOSTREA: three Asian-Pacific species (C. gigas, C. plicatula, and C. ariakensis) and two Atlantic species (C. virginica and C. rhizophorae). FISH probes were made by PCR amplification of the intergenic transcribed spacer between the 18S and 5.8S rRNA genes, and labeled with digoxigenin-11-dUTP. All five species had a haploid number of 10 chromosomes. The Atlantic species had 1-2 submetacentric chromosomes, while the three Pacific species had none. FISH with metaphase chromosomes detected a single telomeric locus for rDNA in all five species without any variation. In all three Pacific species, rDNA was located on the long arm of Chromosome 10 (10q)--the smallest chromosome. In the two Atlantic species, rDNA was located on the short arm of Chromosome 2 (2p)--the second longest chromosome. A review of other studies reveals the same distribution of NOR sites (putative rDNA loci) in three other species: on 10q in C. sikamea and C. angulata from the Pacific Ocean and on 2p in C. gasar from the western Atlantic. All data support the conclusion that differences in size and shape of the rDNA-bearing chromosome represent a major divide between Asian-Pacific and Atlantic species of CRASSOSTREA: This finding suggests that chromosomal divergence can occur under seemingly conserved karyotypes and may play a role in reproductive isolation and speciation.     

Karyological notes on three weevil species from Armenia (Coleoptera, Curculionidae, Cleoninae)

Karyological studies were carried out on three Armenian weevil species from the subfamily Cleoninae. The following chromosome numbers were found in individual species: 2n = 38, n Male = 18 + Xyp in Menecleonus anxius (Gyllenhal, 1824), 2n = 40, n Male = 19 + Xyp in Conorhynchus nigrivittis (Pallas, 1781) and 2n = 44, n Male = 21 + Xyp in Lixus iridis Olivier, 1807. The heterochromosomes of all the examined species form, in the first meiotic metaphase, a typical parachute bivalent.     

Mapping of the 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1829 (Characiformes,Prochilodontidae)

A single NOR-bearing chromosome pair was identified by silver nitrate staining in a previous study of the fish Prochilodus argenteus from the S ã o Francisco River (MG, Brazil), with a third metacentric chromosome sporadically bearing active NOR. The present study focused on an analysis of the chromosomal localization of both the major (45S) and the minor (5S) rRNA genes using FISH. The use of the 18S rDNA probe confirmed the previous Ag-NOR sites interstitially located in a large metacentric pair and also identified up to three other sites located in the telomeric regions of distinct chromosomes, characterizing an interindividual variation of these sites. In addition, the 5S rDNA site was revealed adjacent to the major NOR site, identified at the end of the large Ag-NOR bearing metacentric chromosome. In a few metaphases, an additional weak hybridization signal was observed in a third chromosome, possibly indicating the presence of another 5S rDNA cluster. Despite a lower karyotype diversification (2n=54 and FN=108) often observed among species of Prochilodontidae, variations involving both 45S and 5S rRNA genes could play an important role in their chromosome diversification.     

Replication banding and FISH analysis reveal the origin of the Hyla femoralis karyotype and XY/XX sex chromosomes

The pine woods treefrog, Hyla femoralis, is unique among North American hylid frogs in having a metacentric chromosome 6 and heteromorphic sex chromosomes of the XY/XX type. The X chromosome is distinguished by having a nucleolar organizing region (NOR) in the short arm. The Y chromosome does not possess an NOR. Until the present study, it was not known if the NOR was not present on the Y chromosome or inactive and therefore not detectable by conventional cytogenetic methods like silver staining. Exclusive of its unique features the karyotype of H. femoralis closely resembles those of North American frogs with karyotypes like H. chrysoscelis. We used replication banding and fluorescence in situ hybridization (FISH) with a DNA probe to the 18S + 28S ribosomal genes, which are located at the NOR, to characterize the H. femoralis karyotype. Our analysis revealed that the 18S + 28S ribosomal genes are not present on the Y chromosome, and that the karyotype of H. femoralis was derived from an H. chrysoscelis-like karyotype by relocation of the NOR to the X chromosome from chromosome 6 and either a concurrent or subsequent pericentric inversion of chromosome 6.     

Karyological investigations on seven weevil species (Coleoptera, Curculionidae)

Karyological studies were carried out on seven Palaearctic weevils. The following chromosome numbers were found in individual species, i.e. Otiorhynchus niger (F.), Phyllobius viridearis (Laich.), Phyllobius scutellaris Redt., Phyllobius calcaratus (F.), Polydrusus cervinus (L.), and Brachyderes incanus (L.) 2n = 22, n Male = 10 + Xyp, in Lixus elegantulus (Boh.) 2n = 22, n Male = 21 + Xyp. The heterochromosomes of all the examined species form, in the first meiotic metaphase, a typical parachute bivalent.     

rDNA (18S-28S and 5S) colocalization and linkage between ribosomal genes and (TTAGGG)(n) telomeric sequence in the earthworm,Octodrilus complanatus (Annelida: Oligochaeta: Lumbricidae), revealed by single- and double-color FISH

Spermatogonial and metaphase I chromosomes of the lumbricid earthworm Octodrilus complanatus (Annelida: Oligochaeta) were examined using fluorescent in situ hybridization (FISH) with three repetitive DNA probes-5S rDNA, 18S-26S rDNA, and (TTAGGG)(n). Single-color FISH consistently mapped one chromosome pair per spread using either 5S rDNA or 18S-26S rDNA as probes. Simultaneous (18S-26S)-5S and (18S-26S)-(TTAGGG)(n) FISH demonstrated that repeated units of the two ribosomal families were overlapped and closely associated with telomeric sequences.     

FISH分析栽培高粱×拟高粱、甜高粱×拟高粱的染色体行为

采用荧光原位杂交技术对45S rDNA在栽培高粱×拟高粱、甜高粱×拟高粱F1的有丝分裂和减数分裂染色体进行定位研究。在有丝分裂中期染色体上2个杂种分别检测到2个杂交信号, 在减数分裂粗线期、终变期、中期Ⅰ染色体上45S rDNA位于一个二价体上, 说明这两个杂种携带45S rDNA的染色体为同源染色体。根据45S rDNA位点随细胞减数分裂过程的位置变化, 表明这两个杂种染色体配对行为正常, 平均构型为2n=2x=20(10Ⅱ), 证明45S rDNA可作为染色体的一个识别指标间接地观察细胞减数分裂过程染色体的变化行为。     

Differentiating sex chromosomes of the dioecious Spinacia oleracea L. (spinach) by FISH of 45S rDNA

Spinacia oleracea L. (spinach) is a dioecious species with both male and female plants having 2n = 2x = 12 chromosomes, consisting of two large metacentrics, two long subtelocentrics, two short subtelocentrics, two acrocentrics, and four submetacentrics. The location of 45S rDNA was investigated on metaphase chromosomes using fluorescence in situ hybridization (FISH). The numbers of 45S rDNA foci in diploid sets of chromosomes from females was six and from males was five. All the fluorescent foci lay in secondary constrictions and the satellites. Our results indicate that an XY-type sex chromosome system could be present in spinach where the Y chromosome lacks a 45S RNA focus.     

菠菜rDNA及端粒多色荧光原位杂交分析

以拟南芥25S rDNA、5S rDNA以及端粒序列为探针对菠菜的中期染色体进行了多色荧光原位杂交分析, 其中25S rDNA用生物素标记, 端粒序列用地高辛标记, 5S rDNA用生物素和地高辛共同标记。杂交结果显示, 菠菜中期染色体25S rDNA杂交位点为6个, 分别位于3号、5号和6号染色体的随体部位; 5S rDNA杂交位点为4个, 分别位于3号和5号染色体长臂, 端粒序列杂交位点位于6号染色体端部以及其余染色体的端部和着丝粒部位。     

Localization of rDNA sites in holocentric chromosomes of three species of triatomines (Heteroptera, Triatominae)

Chromatin organization in the holocentric chromosomes of three triatomines species was cytologically studied by fluorescent in situ hybridization with a 45S rDNA probe of Drosophila melanogaster to localize ribosomal genes. In Triatoma tibiamaculata, metaphases I showed telomeric highlights in a single, larger bivalent. In T. protacta, hybridization was detected in one of the telomeres of an autosomal chromosome. In T. platensis, there were highlights in a single, smaller chromosome (X chromosome). The results obtained did not agree with the expected localization of rDNA genes in the sex chromosomes of triatomines, as demonstrated by silver impregnation, and suggest that the chromosome reorganization that occurred in this group during evolution may be a more important mechanism involved in rDNA distribution.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Erpetoichthys calabaricus</Emphasis> (Osteichthyes,Polypteridae)

Polypterids are a group of Osteichthyan fish whose evolutionary relationships with closer basal ray-finned and lobe-finned fish have been disputed since their discovery. Very little is known about the evolutive karyology in the whole Polypteriformes group. In order to fill this gap, a cytogenetic analysis of Erpetoichthys calabaricus species was performed, using both classical and molecular techniques. Karyotype structure (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG)n and ribosomal 5S and 18S rRNA genes were examined in twenty specimens of E. calabaricus by using Ag-NOR, classical C-banding, sequential CMA3/4',6-diaminidino-2-phenylindole (DAPI) staining and fluorescent in situ hybridization (FISH). CMA3 marked all centromerical and some (no. 1 and no. 15) telomeric regions. Staining with Ag-NOR and CMA3 showed the presence of two NORs on the p arm of the chromosome pair no. 1. Hybridization with telomeric probes (TTAGGG)n showed signals at the end of all chromosomes. 5S rDNA was cloned and sequenced. After the alignment, the 5S rRNA sequences revealed an organization made up of two different classes of tandem arrays (type I and type II). FISH with 5S rDNA marked the telomeric regions of the small chromosome pair no. 15, while FISH with 18S rDNA marked the telomeric region of the pair no. 1. The results obtained were compared with cariological data on closer species now available in literature.     

Localization of 5S DNA by in situ hybridization in metaphase chromosomes of Vicia faba L.

The tritium-labelled cloned 5S DNA from Lupinus luteus was used for localization of 5S RNA genes in Vicia faba subsp. minor metaphase chromosomes. In situ hybridization sites were found to be localized in chromosomes I and VI. In chromosome I the probe hybridized to the region adjoining NOR whereas in chromosome VI silver grains were found in the median part of the long arm. After prolonged exposure the autoradiographic grains expanded in the proximal part of that chromosome arm.     

番茄的CPD带型和45S rDNA位点的鉴别

采用CPD（PI和DAPI组合）染色对番茄减数分裂粗线期和有丝分裂中期染色体进行了显带分析,随后用两种不同的45S rDNA克隆在相同的分裂相进行了荧光原位杂交定位分析。CPD染色在8条粗线期染色体上显示出了10条红色的CPD带纹,在6对有丝分裂中期染色体上显示出了12条CPD带纹。有丝分裂中期染色体上的CPD带纹与粗线期染色体上显著的带纹具有对应性。用改良的CPD染色程序清晰而稳定地显示出这些特征性的CPD带纹为番茄的染色体,特别是有丝分裂中期染色体提供了新的识别标记。用番茄的一个45S rDNA克隆进行的荧光原位杂交,不仅在位于2号染色体短臂的随体上显示了强的杂交信号,而且在粗线期染色体的5个CPD带区或有丝分裂中期染色体的4对CPD带区显示了弱的杂交信号。然而,用来自小麦的45S rDNA克隆pTa71进行的原位杂交却只在随体上显示了杂交信号。鉴于所用的两个45S rDNA克隆在序列上的差异,推断在番茄基因组中只有随体含有45S rDNA单位的编码区,即番茄只有一对45S rDNA位点。     

Differential rRNA Genes Expression in Hexaploid Wheat Related to NOR Methylation

Ribosomal RNA genes expression was analysed in 18 Portuguese bread wheat accessions (Triticum aestivum L. em Thell.), and the number of argyrophilic-nucleolar organiser regions (Ag-NORs) per cultivar was scored. Ten accessions presented six Ag-NORs per metaphase and six nucleoli per interphase, and eight accessions presented four Ag-NORs per metaphase and four nucleoli per interphase. Fluorescent in situ hybridisation with the 45S rDNA sequence pTa71 and genomic DNA from Aegilops tauschii (2n = 2x= 14, DD) confirmed the Ag-NOR location and identified the six satellited chromosomes as being the chromosome pairs 1B, 6B and 5D. The methylation pattern of the NOR region was studied by Southern blot using pTa71 as probe, which represented a complete rDNA unit of bread wheat. DNA digestions performed by MspI and HpaII resulted in different patterns revealing the high level of cytosine methylation at their recognition sequences. The total percentage values of NOR methylation indicated that wheat accessions with a maximum of four Ag-NORs were more heavily methylated at the NOR region than accessions with a maximum of six Ag-NORs.     

Ag-staining pattern, FISH and ISH with rDNA probes in the rice field eel (Monopterus albus Zuiew) chromosomes

The present paper reports the Results of rice field eel (Monopterus albus Zuiew) chromosomal analysis by Ag-staining techniques, FISH and ISH with 18S rDNA. Both the Ag-NORs and the hybridization signals were observed on the pericentromeric region of chromosome pairs 3 and 7. The Ag-NORs and the hybridization signals on homologous chromosomes 3 and 7 were corresponding to each other. And rDNA genes were mapped to bivalents 3q12-q24 and 7q14-q26. No heteromorphic sex chromosome pair was identified. The Results obtained were discussed with respect to the feature of Ag-NORs and the position of rDNA genes on chromosome pairs 3 and 7.