High macro-collinearity between lima bean (Phaseolus lunatus L.) and the common bean (P. vulgaris L.) as revealed by comparative cytogenetic mapping

Common bean (P. vulgaris) and lima bean (P. lunatus) are the most important crop species from the genus Phaseolus. Both species have the same chromosome number (2n = 22) and previous cytogenetic mapping of BAC clones suggested conserved synteny. Nevertheless, karyotype differences were observed, suggesting structural rearrangements. In this study, comparative cytogenetic maps for chromosomes 3, 4 and 7 were built and the collinearity between the common bean and lima bean chromosomes was investigated. Thirty-two markers (30 BACs and 2 bacteriophages) from P. vulgaris were hybridized in situ on mitotic chromosomes from P. lunatus. Nine BACs revealed a repetitive DNA pattern with pericentromeric distribution and 23 markers showed unique signals. Nine of these markers were mapped on chromosome 3, eight on chromosome 4 and six on chromosome 7. The order and position of all analyzed BACs were similar between the two species, indicating a high level of macro-collinearity. Thus, although few inversions have probably altered centromere position in other chromosomes, the main karyotypic differences were associated with the repetitive DNA fraction.     

The first karyogram of a Bromeliaceae species: an allopolyploid genome

The Bromeliaceae family has been traditionally distributed in the subfamilies Bromelioideae, Tillandsioideae and Pitcairnioideae. However, phylogenetic studies have provided other classifications, highlighting the need for analyses in order to characterize the genome of different species from this family. In this sense, the present work aimed to determine nuclear 2C-value and base composition, characterize the chromosomes and establish the karyogram of Pitcairnia flammea. Flow cytometry yielded 2C = 1.44 pg, AT = 64.28 % and GC = 35.72 % for this species, indicating its relatively small genome size. Despite reduced length and morphological similarity of the chromosomes, P. flammea metaphases presented well-spread chromosomes, with well-defined primary constriction, without chromatin damage and cytoplasmic background. These aspects allowed morphometric chromosomal characterization and assembly of the first karyogram of a Bromeliaceae species. The karyogram displayed 2n = 50 chromosomes, of which all were submetacentric. Karyomorphological analysis revealed grouped pairs of cytogenetically identical chromosomes (2–3, 4–5, 6–9, 10–17, 18–19, 20–23 and 24–25), plus one isolated chromosome (1), not identical to any other. This result suggests an allopolyploid origin for the P. flammea genome. Thus, the present investigation contributed with karyotype data for taxonomic and evolutionary aspects of this group.     

Karyotype of mitotic metaphase and meiotic diakinesis in non-heading Chinese cabbage

Non-heading Chinese cabbage [Brassica rapa L. ssp. chinensis (L.) Hanelt] is one of the most popular leafy vegetables. Despite the economic importance of non-heading Chinese cabbage, little attention has been given to its cytogenetic profile. This study reveals the karyotype of non-heading Chinese cabbage. Fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes was performed on mitotic metaphase complementary regions. We located 45S rDNA on the centromeric or adjacent region of chromosomes A1 and A2, with the largest on the satellite of chromosome A5. Meanwhile, 5S rDNA co-localized with 45S rDNA on chromosomes A2 and A5, and on the telomeric region of chromosome A10. We performed DAPI fluorescence banding on the same metaphase chromosomes to identify homologous chromosomes. The DAPI fluorescence pattern was observed mainly on the centromeric heterochromatin regions of each chromosome. However, the lengths of chromosomes A2 and A6 were completely stained, except for their telomeric regions. Meiotic diakinesis chromosomes as new substrates in FISH-developed karyotype were revealed for the first time. The karyotype of non-heading Chinese cabbage reveals that it contains eight submetacentric chromosomes, one subtelocentric chromosome (bearing satellite), and one telocentric chromosome. Diakinetic chromosome pairing can overcome the difficulty of unlabeled chromosome identification. This study provided valuable information for cytogenetic research and molecular breeding of non-heading Chinese cabbage by using the combination of FISH and DAPI fluorescence patterns on mitotic and meiotic chromosomes.     

First Coffea arabica karyogram showing that this species is a true allotetraploid

Evolutive studies have verified that Coffea arabica (2n = 44) is a natural segmental allopolyploid originated from a cross between two diploid (2n = 22) Coffea species. Data obtained by classical cytogenetic analyses showed that C. arabica chromosomes are small and morphologically similar, which hampers the karyogram assembly with well-identified homologue pairs. In the present study, the C. arabica complement was reanalysed using an improved cytogenetic protocol that allowed the obtention of high-quality prometaphasic and metaphasic chromosomes. The results showed that chromosomes are cytogenetically distinct (1, 2, 19, 20, 21 and 22) and identical (3–4, 5–6, 7–8, 9–10, 11–12, 13–14, 15–16 and 17–18), with regard to their total length, short and long arm sizes or chromosome classes. Our work suggests that C. arabica is a true non-segmental allotetraploid but originated from different species exhibiting similar and distinct chromosomes.     

Integration of the FISH pachytene and genetic maps of Medicago truncatula

A molecular cytogenetic map of Medicago truncatula (2n = 2x = 16) was constructed on the basis of a pachytene DAPI karyogram. Chromosomes at this meiotic prophase stage are 20 times longer than at mitotic metaphase, and display a well differentiated pattern of brightly fluorescing heterochromatin segments. We describe here a pachytene karyogram in which all chromosomes can be identified based on chromosome length, centromere position, heterochromatin patterns, and the positions of three repetitive sequences (5S rDNA, 45S rDNA and the MtR1 tandem repeat), visualized by fluorescence in situ hybridization (FISH). We determined the correlation between genetic linkage groups and chromosomes by FISH mapping of bacterial artificial chromosome (BAC) clones, with two to five BACs per linkage group. In the cytogenetic map, chromosomes were numbered according to their corresponding linkage groups. We determined the relative positions of the 20 BACs and three repetitive sequences on the pachytene chromosomes, and compared the genetic and cytological distances between markers. The mapping resolution was determined in a euchromatic part of chromosome 5 by comparing the cytological distances between FISH signals of clones of a BAC contig with their corresponding physical distance, and showed that resolution in this region is about 60 kb. The establishment of this FISH pachytene karyotype, with a far better mapping resolution and detection sensitivity compared to those in the highly condensed mitotic metaphase complements, has created the basis for the integration of molecular, genetic and cytogenetic maps in M. truncatula.     

Mitotic evidence for the tetraploid nature of Glycine max provided by high quality karyograms

The high number, very small size and morphological similarity of the chromosomes, and low metaphasic indexes obtained in root meristems have hindered the progress in cytogenetic and evolutionary studies of Glycine max. In order to contribute to the solving of these problems, we have developed a method based on the use of DNA synthesis inhibiting and anti-microtubule solutions and enzymatic maceration and air-drying techniques. Besides, we have employed a digital image analysis system tool. This method provided prometaphasic and metaphasic chromosomes showing well-defined primary and secondary constrictions, which facilitated the pairing of homologues and assembly of the first karyogram for G. max. This species possesses twenty chromosome pairs, being six metacentric and fourteen submetacentric. The karyograms support its tetraploid nature (4x = 40), specifically for the presence of chromosomes with identical morphology, and suggest that chromosome rearrangements may have occurred during the speciation of G. max.     

Repetitive DNA Sequences and Evolution of ZZ/ZW Sex Chromosomes in Characidium (Teleostei: Characiformes)

Characidium constitutes an interesting model for cytogenetic studies, since a large degree of karyotype variation has been detected in this group, like the presence/absence of sex and supernumerary chromosomes and variable distribution of repetitive sequences in different species/populations. In this study, we performed a comparative cytogenetic analysis in 13 Characidium species collected at different South American river basins in order to investigate the karyotype diversification in this group. Chromosome analyses involved the karyotype characterization, cytogenetic mapping of repetitive DNA sequences and cross-species chromosome painting using a W-specific probe obtained in a previous study from Characidium gomesi. Our results evidenced a conserved diploid chromosome number of 2n = 50, and almost all the species exhibited homeologous ZZ/ZW sex chromosomes in different stages of differentiation, except C. cf. zebra, C. tenue, C. xavante and C. stigmosum. Notably, some ZZ/ZW sex chromosomes showed 5S and/or 18S rDNA clusters, while no U2 snDNA sites could be detected in the sex chromosomes, being restricted to a single chromosome pair in almost all the analyzed species. In addition, the species Characidium sp. aff. C. vidali showed B chromosomes with an inter-individual variation of 1 to 4 supernumerary chromosomes per cell. Notably, these B chromosomes share sequences with the W-specific probe, providing insights about their origin. Results presented here further confirm the extensive karyotype diversity within Characidium in contrast with a conserved diploid chromosome number. Such chromosome differences seem to constitute a significant reproductive barrier, since several sympatric Characidium species had been described during the last few years and no interespecific hybrids were found.     

A comparative cytogenetic analysis of five pine species from North America, Pinus banksiana, P. contorta, P. monticola, P. resinosa, and P. strobus

The genus Pinus comprises more than 100 species, which are widely distributed in the Northern hemisphere. Cytogenetic information on North American pines is very limited despite their economic importance. In the present study, a detailed comparative cytogenetic analysis is presented for five pine species from North America, P. resinosa, P. monticola, P. contorta, P. banksiana, and P. strobus. Morphometric analysis and physical mapping of rDNA probes were performed. The karyotype of P. monticola was considered ancestral with small difference in relative chromosome lengths. P. banksiana, P. contorta, and P. strobus karyotypes were considered semi-asymmetrical and less ancestral type. P. banksiana showed five secondary constrictions, P. strobus six, and P. contorta eight. P. resinosa karyotype was semi-asymmetrical and derived with 14 secondary constrictions identified on eight different chromosomes. Karyological data were consistent with molecular cytogenetic information. A significant association was observed between the number and locations of secondary constrictions and the 18S-5.8S-26S rDNA sites detected using fluorescence in situ hybridization. The two methods were used to establish a reliable comparative karyotype of the selected pines. In general, karyotype and chromosome evolution were not related to genetic relationships among pine species studied.     

Molecular cytogenetic analysis and the establishment of a cell culture in the fish species Hollandichthys multifasciatus (Eigenmann & Norris, 1900) (Characiformes,Characidae)

Hollandichthys is a fish genus of the family Characidae that was until recently considered to be monotypic, with cytogenetic, morphological, and molecular data being restricted to a few local populations. In the present study, the karyotype of a population of Hollandichthys multifasciatus was analyzed using classical and molecular cytogenetic approaches for the investigation of potential markers that could provide new perspectives on the cytotaxonomy. H. multifasciatus presented a diploid number of 2n=50 chromosomes and a karyotype formula of 8m+10sm+32st. A single pair of chromosomes presented Ag-NORs signals, which coincided with the 18S rDNA sites visualized by FISH, whilst the 5S rDNA sequences were mapped in two chromosome pairs. The distribution of the U snRNA genes was mapped on the Hollandichthys chromosomes for the first time, with the probes revealing the presence of the U1 snDNA on the chromosomes of pair 20, U2 on pairs 6 and 19, U4 on pair 16, and U6 on the chromosomes of pair 11. The results of the present study indicated karyotypic differences in comparison with the other populations of H. multifasciatus studied previously, reinforcing the need for further research to identify isolated populations or the potential existence of cryptic Hollandichthys species.     

Identification of the linkage group of the Z sex chromosomes of the sand lizard (Lacerta agilis,Lacertidae) and elucidation of karyotype evolution in lacertid lizards

The sand lizard (Lacerta agilis, Lacertidae) has a chromosome number of 2n?=?38, with 17 pairs of acrocentric chromosomes, one pair of microchromosomes, a large acrocentric Z chromosome, and a micro-W chromosome. To investigate the process of karyotype evolution in L. agilis, we performed chromosome banding and fluorescent in situ hybridization for gene mapping and constructed a cytogenetic map with 86 functional genes. Chromosome banding revealed that the Z chromosome is the fifth largest chromosome. The cytogenetic map revealed homology of the L. agilis Z chromosome with chicken chromosomes 6 and 9. Comparison of the L. agilis cytogenetic map with those of four Toxicofera species with many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) showed highly conserved linkage homology of L. agilis chromosomes (LAG) 1, 2, 3, 4, 5(Z), 7, 8, 9, and 10 with macrochromosomes and/or macrochromosome segments of the four Toxicofera species. Most of the genes located on the microchromosomes of Toxicofera were localized to LAG6, small acrocentric chromosomes (LAG11–18), and a microchromosome (LAG19) in L. agilis. These results suggest that the L. agilis karyotype resulted from frequent fusions of microchromosomes, which occurred in the ancestral karyotype of Toxicofera and led to the disappearance of microchromosomes and the appearance of many small macrochromosomes.     

Imaginal discs--a new source of chromosomes for genome mapping of the yellow fever mosquito Aedes aegypti

Background

The mosquito Aedes aegypti is the primary global vector for dengue and yellow fever viruses. Sequencing of the Ae. aegypti genome has stimulated research in vector biology and insect genomics. However, the current genome assembly is highly fragmented with only ∼31% of the genome being assigned to chromosomes. A lack of a reliable source of chromosomes for physical mapping has been a major impediment to improving the genome assembly of Ae. aegypti.

Methodology/Principal Findings

In this study we demonstrate the utility of mitotic chromosomes from imaginal discs of 4^th instar larva for cytogenetic studies of Ae. aegypti. High numbers of mitotic divisions on each slide preparation, large sizes, and reproducible banding patterns of the individual chromosomes simplify cytogenetic procedures. Based on the banding structure of the chromosomes, we have developed idiograms for each of the three Ae. aegypti chromosomes and placed 10 BAC clones and a 18S rDNA probe to precise chromosomal positions.

Conclusion

The study identified imaginal discs of 4^th instar larva as a superior source of mitotic chromosomes for Ae. aegypti. The proposed approach allows precise mapping of DNA probes to the chromosomal positions and can be utilized for obtaining a high-quality genome assembly of the yellow fever mosquito.     

Comparative cytogenetics of opisthorchid species (Trematoda, Opisthorchiidae)

In the present study karyotypes and chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus (Rivolta, 1884), O. viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), M. bilis (Braun, 1893), and Clonorchis sinensis (Cobbold, 1875)) were compared. Karyotypes of O. felineus, M. xanthosomus, M. bilis and C. sinensis consist of two pairs of large meta- and submetacentrics and five pairs of small chromosomes (2n = 14). The karyotype of O. viverrini is 2n = 12, which indicates a fusion of two chromosomes of opisthorchid ancestral karyotype. Analysis of mitotic and meiotic chromosomes was performed by heterologous in situ hybridization of microdissected DNA probes obtained from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining (C- and AgNOR-banding) and FISH of telomeric probes and ribosomal DNA probe on opisthorchid chromosomes were used for chromosome comparison. Data on chromosome number in opisthorchid species were also discussed.     

Cytogenetics for the model system Arabidopsis thaliana

A detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 μm, which is 20–25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm-ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.     

Karyotype of maize (Zea mays L.) mitotic metaphase chromosomes as revealed by fluorescencein situ hybridization (FISH) with cytogenetic DNA markers

Here we demonstrate fluorescencein situ hybridization (FISH) of chromosome-specific cytogenetic DNA markers for chromosome identification in maize using repetitive and single copy probes. The fluorescently labeled probes, CentC and pZm4–21, were shown to be reliable cytogenetic markers in the maize inbred line KYS for identification of mitotic metaphase chromosomes. The fluorescent strength of CentC signal, relative position, knob presence, size and location were used for the karyotyping. Based on direct visual analysis of chromosome length and position of FISH signals, a metaphase karyotype was constructed for maize inbred line KYS. All chromosomes could be identified unambiguously. The knob positions in the karyotype agreed well with those derived from traditional cytological analyses except chromosomes 3, 4 and 8. One chromosome with a telomeric knob on the short arm was assigned to 3. A chromosome with a knob in the middle of the long arm was assigned number 4 by simultaneous hybridization with a knob-specific probe pZm4–21 and a chromosome 4-specific probe Cent 4. On chromosome 8, we found an additional small telomeric knob on the short arm. In addition, chromosome-specific probes were employed to identify chromosome 6 (45S rDNA) and chromosome 9 (single-copy probeumc105a cosmid).     

Comparison of the <Emphasis Type="Italic">Coffea canephora</Emphasis> and <Emphasis Type="Italic">C. arabica</Emphasis> karyotype based on chromosomal DNA content

Nuclear genome size has been measured in various plants, seeing that knowledge of the DNA content is useful for taxonomic and evolutive studies, plant breeding programs and genome sequencing projects. Besides the nuclear DNA content, tools and protocols to quantify the chromosomal DNA content have been also applied, expanding the data about genomic structure. This study was conducted in order to calculate the Coffea canephora and Coffea arabica chromosomal DNA content, associating cytogenetic methodologies with flow cytometry (FCM) and image cytometry (ICM) tools. FCM analysis showed that the mean nuclear DNA content of C. canephora and C. arabica is 2C = 1.41 and 2.62 pg, respectively. The cytogenetic methodology provided prometaphase and metaphase cells exhibiting adequate chromosomes for the ICM measurements and karyogram assembly. Based on cytogenetic, FCM and ICM results; it was possible to calculate the chromosomal DNA content of the two species. The 1C chromosomal DNA content of C. canephora ranged from 0.09 (chromosome 1) to 0.05 pg (chromosome 11) and C. arabica from 0.09 (chromosome 1) to 0.03 pg (chromosome 22). The methodology presented in this study was suitable for DNA content measuring of each chromosome of C. canephora and C. arabica. The cytogenetic characterization and chromosomal DNA content analyses evidenced that C. arabica is a true allotetraploid originated from a cross between Coffea diploid species. Besides, the same analyses also reinforce that C. canephora is a possible progenitor of C. arabica.     

Genomic content and new insights on the origin of the B chromosome of the cichlid fish <Emphasis Type="Italic">Astatotilapia latifasciata</Emphasis>

B chromosomes are additional chromosomes widely studied in a diversity of eukaryotic groups, including fungi, plants and animals, but their origin, evolution and possible functions are not clearly understood. To further understand the genomic content and the evolutionary history of B chromosomes, classical and molecular cytogenetic analyses were conducted in the cichlid fish Astatotilapia latifasciata, which harbor 1–2 B chromosomes. Through cytogenetic mapping of several probes, including transposable elements, rRNA genes, a repeated DNA genomic fraction (C ₀ t − 1 DNA), whole genome probes (comparative genomic hybridization), and BAC clones from Oreochromis niloticus, we found similarities between the B chromosome and the 1st chromosome pair and chromosomes harboring rRNA genes. Based on the cytogenetic mapping data, we suggest the B chromosome may have evolved from a small chromosomal fragment followed by the invasion of the proto-B chromosome by several repeated DNA families.     

A new insight into Serjania Mill. (Sapindaceae, Paullinieae) infrageneric classification: a cytogenetic approach

Serjania (Sapindaceae, Paullinieae) comprises about 230 species, and currently two infrageneric classifications have been proposed but both are difficult to apply. This work tested which infrageneric classification fitted better in relation to cytogenetic traits added to the main morphological features used by the authors of the subgenus arrangements to gain an insight into the evolutionary karyotype relationships. In order to test the relationship between karyotypes and the systematics of this genus, the karyotypes of five species of Serjania belonging to five different sections (sensu Radlkofer) were described. Known karyological information on 26 species was used to complement the results. With these data, a cluster analysis was set up to test which infrageneric classification fitted better. In addition, a principal component analysis (PCA) was performed to examine the relevance of the traits in the subgenus classification. All the karyotypes analyzed (including new as well as previous records) had 2n?=?24 chromosomes, and the karyotypes were asymmetrical: submetacentric and metacentric chromosomes were common, whereas telocentric chromosomes were rare. The PCA revealed seven principal components, the first two explained 52?% of the total variation, and the last ones were related to all the karyotypic features studied. The phenogram obtained reflected a scarce fitting into both infrageneric classifications, with only 3 sections of the 12 proposed by Radlkofer and two of the six sections proposed by Acevedo-Rodríguez being represented. Finally, regarding karyotype evolution, the constancy of chromosome number and the variation in the length of the complement suggest that structural chromosome changes would have played a leading role.     

美丽硬仆骨舌鱼核型分析

美丽硬仆骨舌鱼(Scleropages formosus)是一种古老的具有很高经济价值的观赏鱼类。为了解该鱼的细胞遗传背景,采用胸腔注射植物血球凝集素(PHA)和秋水仙素的方法,以头肾为材料,空气干燥法制片,对美丽硬仆骨舌鱼的染色体进行了分析研究。结果表明,美丽硬仆骨舌鱼的二倍体染色体数目为50,核型公式为2n=2m+8sm+8st+32t,臂数(NF)为60。这一核型符合低等鱼类的基本特征,研究结果可为美丽硬仆骨舌鱼的种质标准和系统演化等提供基础数据。