Somatic Metaphase Chromosomes Research Articles

Molecular cytogenetic characterization of a new wheat-Thinopyrum intermedium homoeologous group-6 chromosome disomic substitution line with resistance to leaf rust and stripe rust.

Thinopyrum intermedium (JJJsJsStSt, 2n = 6x = 42), a member of tertiary gene pool of hexaploid wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), provides several beneficial genes for wheat improvement. In this study, line CH51 was developed from the BC1F8 progeny of a partial wheat-Th. intermedium amphiploid TAI8335 (2n = 56) and wheat cultivar (cv.) Jintai 170. Somatic metaphase chromosome counting showed that CH51 had stable 42 chromosomes. Genomic in situ hybridization (GISH) analysis showed that CH51 had 40 wheat chromosomes and two Th. intermedium chromosomes involving translocation between Js- and St-genome chromosomes. Non-denaturing fluorescence in situ hybridization (ND-FISH) analysis revealed that CH51 lacked a pair of wheat chromosome 6B. Wheat 55K SNP array analysis verified that chromosome 6B had the highest percentage of missing SNP loci in both CH51 and Chinese Spring (CS) nullisomic 6B-tetrasomic 6D (CS-N6BT6D) and had the highest percentage of polymorphic SNP loci between CH51 and cv. Jintai 170. We identified that CH51 was a wheat-Th. intermedium T6StS.6JsL (6B) disomic substitution line. Disease resistance assessment showed that CH51 exhibited high levels of resistance to the prevalent Chinese leaf rust and stripe rust races in the field. Therefore, the newly developed line CH51 can be utilized as a potential germplasm in wheat disease resistance breeding.

Molecular cytogenetic analysis of genome-specific repetitive elements in Citrus clementina Hort. Ex Tan. and its taxonomic implications

BackgroundClementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine.ResultsA graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG)3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin.ConclusionsThe composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future.

Chromosome painting and comparative physical mapping of the sex chromosomes in Populus tomentosa and Populus deltoides.

Dioecious species accounted for 6% of all plant species, including a number of crops and economically important species, such as poplar. However, sex determination and sex chromosome evolution have been studied only in few dioecious species. In poplar, the sex-determining locus was mapped to chromosome 19. Interestingly, this locus was mapped to either a peritelomeric or a centromeric region among different poplar species. We developed an oligonucleotide (oligo)-based chromosome painting probe based on the sequence of chromosome 19 from Populus trichocarpa. We performed chromosome painting in P. tomentosa and P. deltoides. Surprisingly, the distal end on the short arm of chromosome 19, which corresponds to the location of the sex-determining locus reported in several species, was not painted in both species. Thus, the DNA sequences associated with this region have not been anchored to the current chromosome 19 pseudomolecule, which was confirmed by painting of somatic metaphase chromosome 19 of P. trichocarpa. Interestingly, the unpainted distal ends of the two chromosome 19 did not pair at the pachytene stage in 22-24% of the meiotic cells in the two species, suggest that these regions from the sex chromosomes have structurally diverged from each other, resulting in the reduced pairing frequency. These results shed light on divergence of a pair of young sex chromosomes in poplar.

Chromosomal characterization based on repetitive DNA distribution in a tetraploid cytotype of Chrysanthemum zawadskii

The aim of this study was to gain insight into the basic genetics of Chrysanthemum zawadskii Herbich ssp. lucidum (NAK.) Y. Lee, a Chrysanthemum subspecies from Ulleung Island, South Korea, by investigating its karyotypic and cytogenetic characteristics, and to contribute to the karyosystematic study of the genus Chrysanthemum (tribe: Anthemideae). Fluorescence in situ hybridization (FISH) was used to characterize the ploidy level, chromosome constitution, and distribution of 5S and 18S ribosomal DNA (rDNA) and Arabidopsis-type telomere repeats (TTTAGGG)n in C. zawadskii ssp. lucidum chromosomes. The somatic metaphase chromosome number of C. zawadskii ssp. lucidum was tetraploid (2n = 4x = 36). Signals from four 5S rDNA loci were detected in the middle of the long arms of some chromosomes, whereas signals from eight 18S rDNA loci were located at the termini of the short arms. The two rDNA markers localized to different chromosomes with no co-localization. The Arabidopsis-type telomeric repeats, however, co-localized with 18S rDNA sites at the ends of both chromosomal arms. This co-localization of telomeric sequences and 18S rDNA has also been observed in other Chrysanthemum species with various levels of ploidy, indicating that it is a general feature of the genus. DAPI (4′,6-diamidino-2-phenylindole) bands incidentally detected after FISH are also illustrated. These preliminary data contribute towards understanding the karyotypic trends and role of polyploidy in the evolution of the genus Chrysanthemum.

Karyotypes and Distribution of Tandem Repeat Sequences in Brassica nigra Determined by Fluorescence in situ Hybridization

Whole-genome shotgun reads were analyzed to determine the repeat sequence composition in the genome of black mustard, Brassica nigra (L.) Koch. The analysis showed that satellite DNA sequences are very abundant in the black mustard genome. The distribution pattern of 7 new tandem repeats (BnSAT13, BnSAT28, BnSAT68, BnSAT76, BnSAT114, BnSAT180, and BnSAT200) on black mustard chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of BnSAT13 and BnSAT76 provided useful cytogenetic markers; their position and fluorescence intensity allowed for unambiguous identification of all 8 somatic metaphase chromosomes. A karyotype showing the location and fluorescence intensity of these tandem repeat sequences together with the position of rDNAs and centromeric retrotransposons of Brassica (CRB) was constructed. The establishment of the FISH-based karyotype in B. nigra provides valuable information that can be used in detailed analyses of B. nigra accessions and derived allopolyploid Brassica species containing the B genome.

Karyomorphology and nuclear DNA content of sixteen Ophrys L. taxa from Turkey

Karyotypes in 16 representative taxa of the Ophrys genus are compared, based on Feulgen-stained somatic metaphase chromosomes. The karyotypes of O. omegaifera subsp. israelitica, O. ulupinara, O. lycia, O. argolica subsp. lucis, O. argolica subsp. lesbis, O. climacis and O. reinholdii subsp. reinholdii are described for the first time. Karyological analyses indicate relationships among the species with respect to their asymmetry indices. All Ophrys taxa studied were diploid with 2n = 2x = 36 chromosomes. One B chromosome has been detected among the chromosomes of O. argolica subsp. lucis. All karyotypes are symmetrical, consisting of metacentric and submetacentric chromosomes. The longest chromosomes of all the investigated specimens contain a secondary constriction. It is determined that there is a correlation between the total number of chromosomes having secondary constrictions and the evolutionary development order of the taxa. Based on nuclear DNA content, analysis was carried out by flow cytometer using propodium iodide as fluorochrome, 2C nuclear DNA content of 16 Ophrys species varying between 20.80 pg (O. argolica subsp. lucis) and 23.11 pg (O. omegaifera subsp. israelitica). Karyotype asymmetry relationships are discussed according to the bidimensional scatter plots of A1–A2, CVCL–CVCI, CVCL–MCA and CVCI–MCA.

Karyological studies of six taxa of the genusAlliumL. (Amaryllidaceae) from Turkey

AbstractKaryotype structures in representative taxa of the genus Allium L. are compared, based on Feulgen-stained somatic metaphase chromosomes. The karyotypes of Allium arlgirdense, A. anacoleum and A. microspathum (in subg. Allium), and A. rhetoreanum and A. shirnakense (in subg. Melanocrommyum) are described for the first time. The karyological analyses indicate the relationships among the species with respect to asymmetry indices. All Allium taxa studied are diploid with 2n = 2x = 16 chromosomes. One B chromosome is detected in the chromosomes of A. microspathum. All chromosome sets of the studied taxa consist of seven median chromosomes and one submedian chromosomes and all submetacentric pairs bear a satellite on the short arms. The lengths of the chromosomes show remarkable differences particularly at the subgenus level. The three subgenus of genus Allium show a clear tendency to have karyotypes distinct on asymmetry grounds. Karyotype asymmetry relationships are discussed according to the bidimens...

Repetitive sequence analysis and karyotyping reveals centromere-associated DNA sequences in radish (Raphanus sativus L.)

BackgroundRadish (Raphanus sativus L., 2n = 2x = 18) is a major root vegetable crop especially in eastern Asia. Radish root contains various nutritions which play an important role in strengthening immunity. Repetitive elements are primary components of the genomic sequence and the most important factors in genome size variations in higher eukaryotes. To date, studies about repetitive elements of radish are still limited. To better understand genome structure of radish, we undertook a study to evaluate the proportion of repetitive elements and their distribution in radish.ResultsWe conducted genome-wide characterization of repetitive elements in radish with low coverage genome sequencing followed by similarity-based cluster analysis. Results showed that about 31% of the genome was composed of repetitive sequences. Satellite repeats were the most dominating elements of the genome. The distribution pattern of three satellite repeat sequences (CL1, CL25, and CL43) on radish chromosomes was characterized using fluorescence in situ hybridization (FISH). CL1 was predominantly located at the centromeric region of all chromosomes, CL25 located at the subtelomeric region, and CL43 was a telomeric satellite. FISH signals of two satellite repeats, CL1 and CL25, together with 5S rDNA and 45S rDNA, provide useful cytogenetic markers to identify each individual somatic metaphase chromosome. The centromere-specific histone H3 (CENH3) has been used as a marker to identify centromere DNA sequences. One putative CENH3 (RsCENH3) was characterized and cloned from radish. Its deduced amino acid sequence shares high similarities to those of the CENH3s in Brassica species. An antibody against B. rapa CENH3, specifically stained radish centromeres. Immunostaining and chromatin immunoprecipitation (ChIP) tests with anti-BrCENH3 antibody demonstrated that both the centromere-specific retrotransposon (CR-Radish) and satellite repeat (CL1) are directly associated with RsCENH3 in radish.ConclusionsProportions of repetitive elements in radish were estimated and satellite repeats were the most dominating elements. Fine karyotyping analysis was established which allow us to easily identify each individual somatic metaphase chromosome. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of satellite and centromere-specific retrotransposon at centromeres. Our study provides a valuable basis for future genomic studies in radish.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0480-y) contains supplementary material, which is available to authorized users.

Morphological characterization under different ecological habitats and physical mapping of 5S and 45S rDNA in Lilium distichum with fluorescence in situ hybridization

This study was performed to investigate the phenotypic, karyomorphological, and habitat environment characteristics of Lilium distichum that grows naturally in South Korea. Currently, this species follows limited distribution areas and its natural populations are at the brink of extinction mainly due to fragmentation or destruction its natural habitat. This species was distributed between approximately 1,000 and 1,500 m above sea level with an average temperature of 22°C. The soil characteristics surrounding the natural habitats included loamy and silt loam soils having organic matter content (10.82%), pH (5.22), electrical conductivity (EC) (0.37 dS/m), total nitrogen (0.45%), and cation exchange capacity (34.3 cmol+/kg). The peak period of blossoming was between 27 July and 1 August. The maximum number of flowers was observed in Mount Deogyu (2.8), whereas the minimum number of flowers was observed in Mount Jiri (1.3) and Mount Seorak (1.2). Results regarding the number of verticillate leaf, bract counts, and verticillate leaf length and width were highest in Mount Odae, while lowest in the Mount Seorak region. The chromosome complement of L. distichum is 2n = 2x = 24; the length of somatic metaphase chromosomes ranges from 17.01 ± 0.32 μm (chromosome 10) to 32.06 ± 0.35 μm (chromosome 1) with a total length/genome of 261.92 μm. In L. distichum, the presence of 1 pair (two loci) of 5S ribosomal DNA (rDNA) and 8 pairs (16 loci) of 45S rDNA was revealed on metaphase chromosomes. One pair of 5S rDNA signal was observed in interstitial region of long arm of chromosome 3 which co-occurred with 45S rDNA. Among the eight pairs of 45SrDNA, three pairs of 45S rDNA signals were observed in short arm of chromosome (chromosome 2, 6, and 7) which were located close to centromere. The other five pairs of 45S rDNA signals were positioned in the interstitial region of long arm (chromosome 3, 4, 5, 10, and 11). This study provides baseline information regarding the effective exploitation and use of L. distichum resources for breeding research to be used as cut flower and potted plants.

Physical localization of molecular markers and assignment of the 15th linkage group to chromosome 11 of the karyotype in cassava (Manihot esculenta Crantz) by primed in situ labeling.

Physical localization of molecular markers and assignment of the 15th linkage group to chromosome 11 of the karyotype in cassava (Manihot esculenta Crantz) were achieved using primed in situ labeling. Amplified signals for both the EST507-1 and SSRY13-5 markers were consistently observed in different stages of cell division. A comparison of the length, arm ratio, and other morphological characteristics of somatic metaphase chromosomes in karyotype analysis indicated that the EST507-1 and SSRY13-5 markers were localized on the short and long arm of cassava chromosome 11 with the relative map positions of 41.67 and 23.07, respectively. The physical localization of the 2 markers on chromosome 11 of the karyotype corresponds to their positions on the 15th linkage group in cassava.

Karyotypes in Ethiopian Aloe species (Xanthorrhoeaceae: Asphodeloideae)

A cytogenetic survey of 17 species in the succulent-leaved genus Aloe L. (Xanthorrhoeaceae subfamily Asphodeloideae) in Ethiopia was undertaken towards a more complete genetic characterisation of the genus throughout its distribution in Africa, Arabia and Madagascar. Somatic metaphase chromosomes of all species studied showed the same diploid chromosome number of 2n = 14, consisting of four pairs of long and three pairs of short chromosomes. Symmetry was characterised as Stebbins type 2B and the asymmetry indices AsK%, TF%, A1 and A2 revealed minor karyotypic variation common in closely related species. The metrics showed that A. benishangulana has a marginally higher degree of asymmetry than is typical, and the pubescent-flowered A. trichosantha has a more symmetrical karyotype than most other species. We suggest that the stable karyotype morphology in Aloe indicates that structural genome changes and ecological factors play a more prominent role in speciation in Aloe.

정선황기의 세포유전학적 연구

This study was carried out to determine the karyotype and chromosomal localizations of 45S and 5S rDNAs using FISH in Astragalus koraiensis. The somatic metaphase chromosome number of this species was 2n

Centromere Repeat DNA Originated from Brassica rapa is Detected in the Centromere Region of Raphanus sativus Chromosomes

Fluorescence in situ hybridization (FISH) is a powerful tool for the detection of DNA sequences in the specific region of the chromosomes. As well as for the integrated physical mapping, FISH karyotype analysis has to be preceded. Karyotype of Raphanus sativus 'Wonkyo 10039' was analyzed by a dual-color FISH technique; using various repetitive DNA probes, including 5S rDNA, 45S rDNA, and centromere retrotransposon. The length of the somatic metaphase chromosome ranged from 1.35 to <TEX>$2.06{\mu}m$</TEX> with a total length of <TEX>$15.29{\mu}m$</TEX>. The chromosome complements comprised of eight pairs of metacentrics and one pair of submetacentric. Bleached DAPI Band analysis revealed a heterochromatin region, covering 28.6% to 50.4% each chromosomes. 5S and 45S rDNA sequences were located on two and three pairs of chromosomes, respectively. The centromere retrotransposon of Brassica (CRB) is a major component in Brassica related species that has been maintained as a common centromere component. CRB signals were detected on the centromere and pericentromeric region of R. sativus 'Wonkyo 10039' and three basic Brassica species (B. rapa, B. nigra, and B. oleracea). These results will provide a valuable background for physical mapping and elucidation of the evolutionary relationship among the Brassica related species.

주요 장미 7품종의 FISH 핵형분석

효율적인 교배를 위해서는 모본 또는 부본으로 사용하게 될 개체의 배수성 검정이 선행되어야 한다. 이에 본 연구에서는 국내 장미 신품종 육성 프로그램에 이용되고 있는 장미 7품종의 FISH 핵형분석을 통하여 배수성을 확인하고 이를 육종에 있어서 기초 자료로 이용하고자 수행되었다. 배수성 검경 결과, 7품종 모두 4배체(2n = 4x = 28)인 것으로 관찰되었다. FISH 핵형분석 결과, 45S rDNA는 4개의 signal이 7번 염색체 단완의 말단부위에서 관찰되었다. 염색체의 길이 관찰 결과, '알렉산드라'는 <TEX>$1.67-2.67{\mu}m$</TEX>, '프로이트'는 <TEX>$1.40-2.04{\mu}m$</TEX>, '리틀실버'는 <TEX>$1.64-2.24{\mu}m$</TEX>, '테레사'는 <TEX>$1.69-2.26{\mu}m$</TEX>, '티네케'는 <TEX>$1.70-2.65{\mu}m$</TEX>, '비탈'은 <TEX>$1.35-2.08{\mu}m$</TEX>, '옐로우미미'는 <TEX>$1.39-2.04{\mu}m$</TEX>로 관찰되었다. 염색체 전체 길이의 합은 프로이트 품종이 <TEX>$11.23{\mu}m$</TEX> 로 가장 작았으며 알렉산드라 품종이 <TEX>$15.05{\mu}m$</TEX>로 가장 크게 관찰되었다. 염색체의 조성은 중부동원체형, 차중부동원체형, 차단부동원체형으로 구성되었으나 차단부염색체는 관찰되지 않았다. Determination of ploidy level for the mother plant is prerequisite for effective breeding. The study was carried out to determine the ploidy level in 7 different plant materials by FISH karyotype analysis. Among the seven plant varieties analyzed, all exhibit tetraploid (2n = 4x = 28) based on the results observed in chromosome analysis. Four signals of 45S rDNAs were detected on the terminal region of the short arm of chromosome 7. The length of somatic metaphase chromosomes ranges from 1.67 to <TEX>$2.67{\mu}m$</TEX> in 'Alexandra', 1.40 to <TEX>$2.04{\mu}m$</TEX> in 'Freud', 1.64 to 2.24 in 'Little Silver', 1.69 to <TEX>$2.26{\mu}m$</TEX> in 'Teresa', 1.70 to <TEX>$2.65{\mu}m$</TEX> in 'Tineke', 1.35 to <TEX>$2.08{\mu}m$</TEX> in 'Vital', 1.39 to <TEX>$2.04{\mu}m$</TEX> in 'Yellow Mimi'. Total length of the chromosome ranges from <TEX>$11.23{\mu}m$</TEX> in 'Freud' as minimum to <TEX>$15.05{\mu}m$</TEX> in 'Alexandra' as maximum. The karyotypes were composed of metacentric, submetacentric, and subtelocentric chromosome but there is no subtelocentric chromosome.

Karyotype analysis using FISH (fluorescence in situ hybridization) in Fragaria

The karyotypes of Fragaria species, diploid (Fragaria vesca ‘UC-01’, ‘Hawaii-30’, Fragaria nilgerrensis ‘Whiteberry’) and octoploid (Fragaria×ananassa ‘Akihime’), were analyzed using a method based on fluorescence in situ hybridization (FISH), using ribosomal DNA probes (5S rDNA and 45S rDNA). All diploid species of F. vesca and F. nilgerrensis had 2n=14 chromosomes, while F.×ananassa, an octoploid species, were 2n=56. The total length of somatic metaphase chromosome was measured as 12.22, 13.61, and 11.19μm for ‘UC-01’, ‘Hawaii-30’, and ‘Whiteberry’, respectively, and ‘Akihime’ measured 94.23μm. The karyotypes were composed of 6 pairs of metacentric and 1 pair of submetacentric in ‘UC-01’ and ‘Whiteberry’, and 5 pairs of metacentric and 2 pairs of submetacentric in ‘Hawaii-30’. 5S rDNA loci were detected in 1 pair from ‘UC-01’, ‘Hawaii-30’ and ‘Whiteberry’, and 45S rDNA loci were detected in 3 pairs from ‘UC-01’ and ‘Hawaii-30’, whereas 45S rDNA loci were detected in 2 pairs from ‘Whiteberry’. In octoploid strawberry ‘Akihime’, 45S rDNA signals were observed at 12 chromosomes, and 5S rDNA signals were observed at 2 chromosomes.

Intraspecific polymorphism of ribosomal DNA loci number and morphology in Brachypodium pinnatum and Brachypodium sylvaticum

The genus Brachypodium has become the target of extensive cytomolecular studies since one of its representatives, B. distachyon, has been accepted as a model plant for temperate cereals and forage grasses. Recent preliminary studies suggested that intraspecific rDNA polymorphism can occur in at least two members of the genus, B. sylvaticum and B. pinnatum, so the aim of this study was to further analyse this phenomenon. FISH with 25S rDNA and 5S rDNA probes was performed on somatic metaphase chromosomes, supplemented by the silver staining technique which distinguishes transcriptionally active from inactive 18S-5.8S-25S rDNA loci. The number, size and chromosomal distribution of 5S rDNA loci were very constant: two loci were invariably observed in all studied diploid accessions of both species, while four 5S rDNA loci were present in the tetraploid B. pinnatum. In contrast to 5S rDNA loci, those of the 35S rDNA were more variable. Two or three loci were observed in the diploid B. pinnatum and four in tetraploid accessions. In chromosome complements of B. sylvaticum accessions from two to six 35S rDNA sites were detected. Regardless of total rDNA locus number, only two were transcriptionally active in diploid accessions of both species, while two or four were active in the tetraploid B. pinnatum. Additionally, the fluorescent CMA/DAPI banding method was used to identify the relation between rDNA sites and CMA+ bands. It was revealed that the number and chromosomal distribution of CMA+ bands are in congruence only with 35S rDNA loci which gave strong FISH signals.

Comparison of the distribution of the repetitive DNA sequences in three variants of Cucumis sativus reveals their phylogenetic relationships

Repetitive DNA sequences with variability in copy number or/and sequence polymorphism can be employed as useful molecular markers to study phylogenetics and identify species/chromosomes when combined with fluorescence in situ hybridization (FISH). Cucumis sativus has three variants, Cucumis sativus L. var. sativus, Cucumis sativus L. var. hardwickii and Cucumis sativus L. var. xishuangbannesis. The phylogenetics among these three variants has not been well explored using cytological landmarks. Here, we concentrate on the organization and distribution of highly repetitive DNA sequences in cucumbers, with emphasis on the differences between cultivar and wild cucumber. The diversity of chromosomal karyotypes in cucumber and its relatives was detected in our study. Thereby, sequential FISH with three sets of multi-probe cocktails (combined repetitive DNA with chromosome-specific fosmid clones as probes) were conducted on the same metaphase cell, which helped us to simultaneously identify each of the 7 metaphase chromosomes of wild cucumber C. sativus var. hardwickii. A standardized karyotype of somatic metaphase chromosomes was constructed. Our data also indicated that the relationship between cultivar cucumber and C. s. var. xishuangbannesis was closer than that of C. s. var. xishuangbannesis and C. s. var. hardwickii.

Karyotyping in Melon (Cucumis melo L.) by Cross-Species Fosmid Fluorescence in situ Hybridization

Chromosome identification is critical for cytogenetic research and will accelerate studies on genetic variation and breeding, especially for those species with relatively little sequence information. So far, no reliable cytological landmarks have been developed to distinguish individual chromosomes in melon. In this study, using FISH (fluorescence in situ hybridization) combined with comparative genome information, we selected 21 cucumber fosmids anchored by SSR markers as chromosome-specific cytological markers for melon chromosomes. Moreover, with the help of melon centromeric satellite DNA repeats CentM, 45S rDNA and 5S rDNA, sequential FISH with 3 sets of multi-fosmid cocktails were conducted on the same metaphase cell, which allowed us to simultaneously identify each of the 12 metaphase chromosomes of melon and a standardized melon karyotype of somatic metaphase chromosomes was constructed. Finally, we compared the distribution of 21 FISH-mapped fosmids between melon and cucumber chromosomes, which allows a better understanding of the evolutionary process shaping these 2 species. Our study provides a basis for cytological characterization of the melon genome and comparative genomics of Cucurbitaceae.

PHYSICAL MAPPING OF 5S AND 45S RDNAS BY FLUORESCENT IN SITU HYBRIDIZATION IN MANGO (MANGIFERA INDICA L.)

Mango (Mangifera indica L.) is thought to be diploid despite the many numbers of chromosomes (2n=2x=40). Although the allopolyploid nature of M. indica is reported by Mukherjee (1950), no chromosome studies are available to elucidate genome composition of mango until now. We have applied fluorescent in situ hybridization (FISH) technique using 5S and 45S ribosomal DNAs (rDNAs) as probes on the somatic metaphase chromosome of mango for the first step to elucidate genome composition of mango. The digoxigenin (DIG)-labeled 5S or 45S probe was hybridized onto the chromosomes separately, and both of them were visualized by incubation with anti-DIG-fluorescein isothiocyanate (FITC). Strong signals of 5S rDNA were observed on two chromosomes, and these two seemed to be homologous chromosomes due to their size and identical chromosomal location of 5S rDNA loci. On the other hand, six chromosomes were demonstrated to carry 45S rDNA loci. Among these six chromosomes, two chromosomes seemed to be homologous from their size and the location of rDNA loci in addition to signal intensity. But, another four chromosomes could not be identified whether or not these four form two pairs of chromosomes, since no clear images of these chromosomes were observed. In addition, multicolor FISH using 5S and 45S rDNA probes differently labeled with DIG and biotin, revealed separate localization of the two rDNA genes on different chromosomes of mango. We could distinguish eight chromosomes among forty chromosomes of mango in this study, but no evidence was obtained for a possibility of heterozygotic karyotype in mango not like in sweet orange.

Cytogenetic analysis of Hieracium transylvanicum (Asteraceae)

Abstract Hieracium transylvanicum is a relic species which occurs in refugial areas of south-eastern Europe. The structure of its karyotype, including determination of morphometric features of somatic metaphase chromosomes as well as the number and distribution of ribosomal DNA loci, was attempted using both classical chromosome staining and the FISH method with 25S rDNA and 5S rDNA probes. The cytotypes under study were collected from eight sites and did not vary significantly in terms of their karyotype organisation. All 11 specimens had the same chromosome number, 2n=2x=18, and 9 similar chromosomal types, whose total length ranged from 4.62 to 2.64 μm. The karyotype of the species is highly symmetrical. Two chromosomal types, 3 and 6, have a 45S rDNA locus in distal parts of their short arms. In addition, the short arm of type 6 has interstitial 5S rDNA sequences. No intraspecific variation in number and chromosomal distribution of ribosomal DNA loci was observed. The very uniform structure of the kar...