Karyotypes, constitutive heterochromatin, and genomic DNA values in the blowfly genera Chrysomya, Lucilia, and Protophormia (Diptera: Calliphoridae).

The karyotypes and C-banding patterns of Chrysomya species C. marginalis, C. phaonis, C. pinguis, C. saffranea, C. megacephala (New Guinean strain), Lucilia sericata, and Protophormia terraenovae are described. All species are amphogenic and have similar chromosome complements (2n = 12), including an XY-XX sex-chromosome pair varying in size and morphology between species. Additionally, the C-banding pattern of the monogenic species Chrysomya albiceps is presented. The DNA contents of these and of further species Chrysomya rufifacies, Chrysomya varipes, and Chrysomya putoria were assessed on mitotic metaphases by Feulgen cytophotometry. The average 2C DNA value of the male genomes ranged from 1.04 pg in C. varipes to 2.31 pg in C. pinguis. The DNA content of metaphase X chromosomes varied from 0.013 pg (= 1.23% of the total genome) in C. varipes to 0.277 pg (12.20%) in L. sericata; that of Y chromosomes ranged from 0.003 pg (0.27%) in C. varipes to 0.104 pg (5.59%) in L. sericata. In most species, the corresponding 5 large chromosome pairs showed similar relative DNA contents. The data suggest that the interspecific DNA differences in most species are mainly due to quantitative variation of (repetitive) sequences lying outside the centromeric heterochromatin blocks of the large chromosomes. The results are also discussed with regard to phylogenetic relationships of some species.     

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea)

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.     

Studies on sex chromosomes of four hamster species: Cricetus cricetus, Cricetulus griseus, Mesocricetus auratus, and Phodopus sungorus.

In this paper, we present an analysis of the sex chromosomes of four hamster species after application of different staining techniques. The mitotic X chromosomes show a striking similarity in G-banding pattern but rather great differences in their C-banding patterns. A presumably homologous euchromatic segment that exhibits two distinct G-bands appears in the X chromosome of each species. The Y chromosome of Cricetus cricetus is in contrast to those of the other species, because it reveals a relatively well-differentiated G- and C-banding pattern. In meiotic metaphase I, interstitial chiasmata can be found in the sex bivalents of Cricetus cricetus and Cricetulus griseus, whereas the gonosomes of Mesocricetus auratus and Phodopus sungorus sungorus are terminally associated. The regions that are involved in pairing or association are always heterochromatic.     

Cytological investigation of Haplopappus gracilis (Nutt.) Gray: 5-methylcytosine-rich regions, fluorochrome banding and chromatin sensitivity to DNase I digestion

Haplopappus gracilis (Nutt.) Gray, one of the five known higher plants with a chromosome number of 2n = 4, was studied from a cytological point of view. The chromosome complement of this species was characterized by means of automated karyotype analysis. Moreover, the DNA methylation pattern and fluorochrome banding were determined and compared with cytological data present in the literature. DNA methylation distribution along metaphase chromosomes involved all chromosome territories evidenced by C-banding. Other methylated bands correlated positively with aceto-orcein-positive heterochromatic portions and/or with late replicating bands and/or fluorochrome bands. Some methylated bands showed differences between homologous chromosomes. These bands belonged partly to certain heterochromatic domains and partly to intercalary sites not defined by other standard banding techniques. Differences between the homologues were also indicated by our DNA content data obtained after DNase I digestion.     

Cytogenetics of Hylodes and Crossodactylus Species (Anura, Leptodactylidae) with Comments on Hylodinae/Dendrobatidae Relationships

The karyotype, nucleolar organizer region (NOR) location and C-banding pattern of two species of Hylodes (H. phyllodes and H. asper) and two of Crossodactylus (Crossodactylus sp. n. and Crossodactylus cf. caramaschi) were studied. All species had a diploid number of 2n = 26, with differences in the chromosomal morphology of the Hylodes species while the two Crossodactylus species were cytogenetically indistinguishable. The NOR was located on pair 1 in both species of Hylodes, and on pair 8 in the Crossodactylus species. In the latter, the NOR was heteromorphic between the homologues. The NOR was coincident with a secondary constriction in the four species. Except to H. phyllodes, such secondary constrictions were clearly seen strongly stained after C-banding treatment. The C-banding pattern varied between the two species of Hylodes, but was identical in the Crossodactylus species. The results from conventionally stained karyotypes confirmed the uniformity within the genus Crossodactylus, and the relatively conserved karyotypes within Hylodes, in agreement with other literature reports. We conclude that the cytogenetic data do not provide further evidence which could be useful to corroborate the supposed relationships between the hylodines and dendrobatids since there are no unambiguous homeologies between the karyotypes of these groups.     

Cytogenetic analysis of three species of the genus <Emphasis Type="Italic">Haemulon</Emphasis> (Teleostei: Haemulinae) from Margarita Island,Venezuela

This paper describes the karyotype analysis of Haemulon aurolineatum, Haemulon bonariensis and Haemulon plumierii, by Giemsa staining, C-banding, Ag-staining and fluorescent in situ hybridization (FISH), to locate the 18S and 5S rRNA genes. Diploid modal count in the three species was 2n = 48 acrocentric elements. Except for pair 24, which exhibited an unmistakable secondary constriction in all three species, it was not possible to classify them as homologous to each other because differences in chromosome size were too slight between adjacent pairs within a size-graded series. Ag-NOR clusters were located in pair 24 in the three species with signal located on the secondary constriction of these chromosomes. C-banding demonstrated that the three species share the same distribution pattern of the constitutive heterochromatin with centromeric heterochromatic blocks in the 23 chromosome pairs and a pericentromeric block in pair 24 which is coincident with the NORs. FISH experiments showed that 18S rDNA sequences were located coincident with the Ag-NOR site in the three species; however, differences in both the number and chromosome distribution of 5S-rDNA cluster were detected among them. Our data suggest that chromosome evolution of Haemulon has been preserved from major changes in the karyotypic macrostructure, whereas microstructural changes have occurred.     

Computer assisted karyotype analysis of Pyrrhopappus carolinianus

With the help of Computer Aided Karyotyping procedures, Ag-NOR staining and C-banding techniques, the karyotype of Pyrrhopappus carolinianus (Asteraceae, Lactuceae) has been studied. The species has 2n=12 chromosomes. Silver staining reveals that the two shortest pairs of chromosomes possess NOR's. On the basis of chromosome length and centromere position, only the longest chromosome pair and the satellite chromosomes can be identified. Two types of C-banding can be obtained, dependent on the temperature of the hydrochloric acid hydrolysis of the root tips. Hydrolysis at 60°C results exclusively in centromeric bands, whereas a treatment at room temperature reveals a pattern of intercalary bands. A computer assisted analysis of the intercalary banding pattern resulted in the construction of schematic representation of the average C-banding pattern. This banding pattern allows an easy identification of each of the chromosome pairs.     

Mitochondrial DNA sequence divergence between two cryptic species of Lethenteron, with reference to an improved identification technique

Genetic divergences and population structures were examined in the cryptic Lethenteron sp. N and sp. S, based on mitochondrial DNA (mtDNA) cytochrome oxidase subunit I (CO I) region sequences. An improved method of discrimination between L . sp. N and sp. S was found using PCR, with diagnostic primers for each species-specific sequence in the mtDNA CO I region. Identification of 50 individuals of each species by this analysis was consistent with that by allozyme analysis of nuclear DNA. L . sp. N and sp. S, identified on the basis of diagnostic alleles at five allozymic loci, were independently grouped in a neighbour-joining (NJ) tree, with a large sequence difference (mean ±  s . d . = 9·10 ± 0·36%) between them. Within each species, the values of sequence divergence among localities were significantly higher in L . sp. S (1·61 ± 0·44%) than in L . sp. N (1·10 ± 0·48%). On the tree and nested clade analyses, several phylogenetic groups comprising geographically close localities were detected in the former, although scarcely detected in the latter, probably resulting from dispersal pattern differences between them.     

Influence of the C-banding procedure on DNA and proteins of mouse chromosomes: I. A microdensitometric study

Cytochemical quantitative methods were used to investigate DNA protein contents of mouse metaphase plates during an alkaline C-banding procedure ( Sumner et al., 1971). Cytochemical stains and reactions for DNA and for total protein content were used to quantitatively assess the sequential involvement (losses) of DNA and protein during the appearance of the classic C-banding pattern which was monitored with Giemsa staining. The data point the preferential loss of DNA from euchromatic regions of chromosomes as the main cause of the C-banding pattern appearance. The effect of chromosomal protein is more likely indirect and perhaps tied to some specific interaction with centromeric DNA that contributes to DNA retention in C-bands. Following the C-banding procedure it was possible to differentially stain the centromeric area with Feulgen and GCA and even with non-fully specific stain for DNA such as methylene blue.     

Karyotypic conservatism in five species of Prochilodus (Characiformes,Prochilodontidae) disclosed by cytogenetic markers

The family Prochilodontidae is considered a group with well conserved chromosomes characterized by their number, morphology and banding patterns. Thence, our study aimed at accomplishing a cytogenetic analysis with conventional methods (Giemsa staining, silver staining of the nucleolus organizer regions-AgNOR, and C-banding) and fluorescence in situ hybridization (FISH) with 18S and 5S ribosomal DNA probes in five species of the Prochilodus genus (Prochilodus argenteus, Prochilodus brevis, Prochilodus costatus, Prochilodus lineatus and Prochilodus nigricans) collected from different Brazilian hydrographic basins. The results revealed conservatism in chromosome number, morphology, AgNORs 18S and 5S rDNAs location and constitutive heterochromatin distribution patterns. The minor differences observed in this work, such as an Ag-NOR on a P. argenteus chromosome and a distinct C-banding pattern in P. lineatus, are not sufficient to question the conservatism described for this group. Future work using repetitive DNA sequences as probes for FISH will be interesting to further test the cytogenetic conservatism in Prochilodus.     

Identifying species of moths (Lepidoptera) from Baihua Mountain,Beijing, China,using DNA barcodes

DNA barcoding has become a promising means for the identification of organisms of all life‐history stages. Currently, distance‐based and tree‐based methods are most widely used to define species boundaries and uncover cryptic species. However, there is no universal threshold of genetic distance values that can be used to distinguish taxonomic groups. Alternatively, DNA barcoding can deploy a “character‐based” method, whereby species are identified through the discrete nucleotide substitutions. Our research focuses on the delimitation of moth species using DNA‐barcoding methods. We analyzed 393 Lepidopteran specimens belonging to 80 morphologically recognized species with a standard cytochrome c oxidase subunit I (COI) sequencing approach, and deployed tree‐based, distance‐based, and diagnostic character‐based methods to identify the taxa. The tree‐based method divided the 393 specimens into 79 taxa (species), and the distance‐based method divided them into 84 taxa (species). Although the diagnostic character‐based method found only 39 so‐identifiable species in the 80 species, with a reduction in sample size the accuracy rate substantially improved. For example, in the Arctiidae subset, all 12 species had diagnostics characteristics. Compared with traditional morphological method, molecular taxonomy performed well. All three methods enable the rapid delimitation of species, although they have different characteristics and different strengths. The tree‐based and distance‐based methods can be used for accurate species identification and biodiversity studies in large data sets, while the character‐based method performs well in small data sets and can also be used as the foundation of species‐specific biochips.     

Cytogenetic Analysis of A- and B-Chromosomes of Prochilodus Lineatus (Teleostei, Prochilodontidae) Using Different Restriction Enzyme Banding and Staining Methods

Different cytogenetic techniques were used to analyse the chromosomes of Prochilodus lineatus with the main objective of comparing the base composition of A- and B-chromosomes. The results of digestion of chromosomes with 10 different restriction endonucleases (REs), silver staining, CMA₃ staining and C-banding indicated the existence of different classes of highly repetitive DNA in the A-set and also suggested the existence of compositional differences between the chromatin of A- and B-chromosomes. The 5-BrdU incorporation technique showed a late replicating pattern in all B-chromosomes and in some heterochromatic pericentromeric regions of A-chromosomes. The cleavage with RE BamHI produced a band pattern in all chromosomes of P. lineatus which permitted the tentative pairing of homologues in the karyotype of this species. We concluded that the combined use of the above techniques can contribute to the correct identification of chromosomes and the karyotypic analysis in fishes. On the basis of the results, some aspects of chromosome structure and the origin of the B-chromosomes in P. lineatus are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

A comparative study of mitotic and meiotic chromosomes of the Montandon's newt,Triturus montandoni (Urodela: Salamandridae) from Poland and Rumania

A karyological analysis was carried out on two populations ofTriturus montandoni, one from Poland and another from Rumania. For both samples, morphometric characteristics and C-banding pattern of mitotic chromosomes are provided. Only slight differences between specimens from two geographic localities were found. Data on chiasma frequency and distribution are presented for male meiosis. No sex-related heteromorphism was found and for none of the chromosomal arms was a consistent absence of chiasmata recorded. There was a relatively high proportion of spermatocyte metaphases I with chiasmata on both arms of all chromosomes in all specimens studied. It is concluded that there are no well-defined sex chromosomes in the chromosomal complement of the maleT. montandoni. The findings are compared with previous studies on chromosome morphology, C-banding pattern, and meiosis in closely related species,T. vulgaris andT. helveticus.     

Polymorphism of heterochromatic regions of flax chromosomes

The C-banding technique was used to study flax chromosomes (Linum usitatissimum L., 2n = 30). Heterochromatin was located mainly in pericentromeric regions of chromosomes. In spite of small size (1.5-3.5 microm), all 15 pairs of homologous chromosomes were identified on the basis of the C-banding pattern and morphology. An idiogram of C-banded chromosomes of L usitatissimum L. is presented. Polymorphism of chromosomal heterochromatic regions was studied in karyotypes of three flax samples: L usitatissimum L., accession K-603 (L usitatissimum var. usitatissimum), and accession K-594 (L. usitatissimum var. humile (Mill.)). A common C-banding pattern was observed in all forms studied, although there were some distinctions in the individual band size. The fibre flax (accession K-603) karyotype had the C-banding pattern similar to that of L usitatissimum L., but some intercalary and telomeric C-bands were somewhat larger, and a satellite (NOR) was observed in the short arm of chromosome I. In crown flax, (K-594) chromosomal C-banding pattern exhibited smaller pericentromeric and larger intercalary bands; telomeric bands were present on almost all chromosomes. Thus, the intraspecies polymorphism revealed in the chromosomal C-banding pattern makes possible the use of C-bands as chromosome markers in the studies of genetic and genomic polymorphism of this species.     

The phylogenetic significance of peptidoglycan types: Molecular analysis of the genera Microbacterium and Aureobacterium based upon sequence comparison of gyrB, rpoB, recA and ppk and 16SrRNA genes

The type strains of 27 species of the genus Microbacterium, family Microbacteriaceae, were analyzed with respect to the phylogeny of the housekeeping genes coding for DNA gyrase subunit B (gyrB), RNA-polymerase subunit B (rpoB), recombinase A (recA) and polyphosphate kinase (ppk). The resulting gene trees were compared to the 16S rRNA gene phylogeny of the same species. The topology of neighbour-joining and maximum parsimony phylogenetic trees based upon nucleic acid sequences and protein sequences of housekeeping genes differed among each other and no gene tree was identical to that of the 16S rRNA gene tree. Only some species showed consistent clustering by all genes analyzed, but the majority of species branched with different neighbours in most gene trees. The failure to phylogenetically cluster type strains into two groups based upon differences in the amino acid composition of peptidoglycan on the basis of 16S rRNA gene sequence similarity, once leading to the union of the genera Microbacterium and Aureobacterium, was also seen in the analysis of recA, rpoB and gyrB gene and protein phylogenies. Analysis of the pkk gene and protein as well as of a concatenate tree, combining sequences of all five genes (total of 3.700 nucleotides), sees members of the former genus Aureobacterium and other type strains with lysine as diagnostic diamino acid to form a coherent cluster that branches within the radiation of Microbacterium species with ornithine in the peptidoglycan.     

Comparative cytogenetic analysis on four tree frog species (Anura, Hylidae, Hylinae) from Brazil

A comparative cytogenetic analysis was carried out on four Hylinae tree frogs from Brazil ((Aparasphenodon brunoi, Corythomantis greeningi, Osteocephalus langsdorffii, and Scinax fuscovarius) using Giemsa staining, BrdU replication banding, Ag-NOR staining, C-banding, DAPI and CMA(3) fluorochrome staining, and fluorescence in situ hybridization (FISH) with an rDNA probe. All the species share closely similar 2n = 24 karyotypes, almost indistinguishable by standard staining. The technique of BrdU incorporation allowed the identification of each pair of homologs and the establishment of extensive homeology for the great majority of the chromosomes, mainly of A. brunoi, C. greeningi, and O. langsdorffii. Despite highly conserved replication banding patterns, the use of the other banding techniques disclosed some minor differences, which reinforces the importance of extensive cytogenetic analyses for the karyotypic characterization of Anuran species. The present cytogenetic data confirm the closer proximity of A. brunoi, C. greeningi, and O. langsdorffii, whereas S. fuscovarius is phylogenetically more distant.     

DNA似近距离及进化时间的估算

在似近分析和Ｎｅｉ氏遗传距离的基础上,给出了ＤＮＡ似近距离计算公式,并以ＤＮＡ似近距离估算类群间的分歧时间（进化时间）,应用１０种限制内切酶对猕猴属（ｇｅｎｕｓ Ｍａｃａｃａ）内５个种ｍｔＤＮＡ的切点数据计算了这５个种的ＤＮＡ似近距离和进化时间,比较由ＤＮＡ似近距,遗传距离构建的歧化树和Ｆｏｏｄｅｎ及Ｄｅｌｓｏｎ的形态歧化树表明,除遗传距离的歧化树外,其它三种歧化树都有一个共同点,就是熊猴（Ｍ．ａ     

Chromosome study of Amazona amazonica and A. aestiva (Aves: Psittaciformes): determination of chromosome number and identification of sex chromosomes by C-banding methods

Two parrot species, Amazona amazonica and A. aestiva, submitted to cytogenetic analysis presented a diploid chromosome number of 2n=70 (20M+50m). With the C-banding pattern, the cells of female speciments showed an almost totally heterochromatic W chromosome. No chromosome differences were observed in the two species studied.     

香蕉束顶病植原体16S rDNA片段的RFLP和序列分析

香蕉束顶病（ＢＢＴ）是一种发生在蕉类作物的严重病害。从带有典型香蕉束顶病症状的香蕉植株中按照检测植原体的方法提取ＤＮＡ,扩增患病植株中植原体１６ＳｒＤＮＡ片段,证明香蕉束顶病中有植原体存在。对此扩增片段进行限制性酶切片段长度多态性（ＲＦＬＰ）分析和核酸序列分析,并与已知植原体的序列进行同源性比较,构建进化树。结果显示该片段与Ｇｒ１的亲缘关系最近。