Genetic polymorphism of flax Linum usitatissimum based on use of molecular cytogenetic markers

Using a set of approaches based on the use of molecular cytogenetic markers (DAPI/C-banding, estimation of the total area of DAPI-positive regions in prophase nuclei, FISH with 26S and 5S rDNA probes) and the microsatellite (SSR-PCR) assay, we studied genomic polymorphism in 15 flax (Linum usitatissimum L.) varieties from different geographic regions belonging to three directions of selection (oil, fiber, and intermediate flaxes) and in the k-37 x Viking hybrid. All individual chromosomes have been identified in the karyotypes of these varieties on the basis of the patterns of differential DAPI/C-banding and the distribution of 26S and 5S rDNA, and idiograms of the chromosomes have been generated. Unlike the oil flax varieties, the chromosomes in the karyotypes of the fiber flax varieties have, as a rule, pericentromeric and telomeric DAPI-positive bands of smaller size, but contain larger intercalary regions. Two chromosomal rearrangements (chromosome 3 inversions) were discovered in the variety Luna and in the k-37 x Viking hybrid. In both these forms, no colocalization of 26S rDNA and 5S rDNA on the satellite chromosome was detected. The SSR assay with the use of 20 polymorphic pairs of primers revealed 22 polymorphic loci. Based on the SSR data, we analyzed genetic similarity of the flax forms studied and constructed a genetic similarity dendrogram. The genotypes studied here form three clusters. The oil varieties comprise an independent cluster. The genetically related fiber flax varieties Vita and Luna, as well as the landrace Lipinska XIII belonging to the intermediate type, proved to be closer to the oil varieties than the remaining fiber flax varieties. The results of the molecular chromosomal analysis in the fiber and oil flaxes confirm their very close genetic similarity. In spite of this, the combined use of the chromosomal and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.     

Karyogenomics of species of the genus Linum L

Using the molecular cytogenetic and RAPD methods of analysis, we studied genomes of 22 cultivated flax varieties and 24 wild species from six sections of the genus Linum L. The chromosome numbers were exactly determined in the karyotypes of all studied species, and all individual chromosomes were identified by the C/DAPI-banding pattern and localization of 26S rDNA and 5S rDNA. B chromosomes were identified and studied for the first time in species of the section Syllinum Griseb. According to the data obtained, the species studied were divided into eight groups on the basis of similarity of their karyotypes, which corresponded in general to their clustering based on the RAPD results. The systematic positions and phylogenetic relationships of the flax species were verified.     

Karyogenomics of species of the genus <Emphasis Type="Italic">Linum</Emphasis> L.

Using the molecular cytogenetic and RAPD methods of analysis, we studied genomes of 22 cultivated flax varieties and 24 wild species from six sections of the genus Linum L. The chromosome numbers were exactly determined in the karyotypes of all studied species, and all individual chromosomes were identified by the C/DAPI-banding pattern and localization of 26S rDNA and 5S rDNA. B chromosomes were discovered and studied for the first time in species of the section Syllinum Griseb. According to the data obtained, the species studied were divided into eight groups on the basis of similarity of their karyotypes, which corresponded in general to their clustering based on the RAPD results. The systematic positions and phylogenetic relationships of the flax species were verified.     

Intraspecific chromosomal and genetic polymorphism in Brassica napus L. detected by cytogenetic and molecular markers

The application of DNA intercalator 9-aminoacridine allowed us to increase the resolution of chromosome C-banding and DAPI-banding patterns and to investigate chromosomal polymorphism in karyotypes of seven spring and six winter rape varieties. It was shown that the pericentromeric and intercalary C-bands of most of the chromosomes in spring rape were smaller in size and less polymorphic than those of winter rape. More 26S and 5S rDNA sites were found in the winter rape karyotypes than the spring varieties. Separate or colocalized 26S and 5S rDNA sites were revealed on chromosomes 4, 5, 6, 8, 10, 14, 15, 16 and 18. Intervarietal and intravarietal polymorphism of the number and chromosomal localization of rDNA sites were detected. The generalized idiogram of chromosomes of 13 Brassica napus varieties with account of all possibilities of C-banding patterns as well as localization of 26S and 5S rDNA sites were constructed. Polymorphism of the examined molecular and cytogenetic markers as well as the heterozygosis level of FAE1.1 gene controlling erucic acid synthesis in rapeseed was higher in the winter varieties than in the spring ones. The obtained data were in a satisfactory agreement with increased tolerance to environmental stress conditions of winter rape.     

Genome comparisons of three closely related flax species and their hybrids with chromosomal and molecular markers

Chromosome C-banding patterns were analyzed in three closely related flax species (Linum usitatissimum L., 2n = 30; L. angustifolium Huds., 2n = 30; and L. bienne Mill., 2n = 30) and their hybrids. In each case, the karyotype included metacentrics, submetacentrics, and one or two satellite chromosomes. Chromosomes of the three flax species were similar in morphology, size (1-3 microns), and C-banding pattern and slightly differed in size of heterochromatic regions. In all accessions, a large major site of ribosomal genes was revealed by hybridization in the pericentric region of a large metacentric. A minor 45S rDNA site was observed on a small chromosome in L. usitatissimum and L. bienne and on a medium-sized chromosome in L. angustifolium. Upon silver staining, a nucleolus-organizing region (NOR) was detected on a large chromosome in all species. In L. angustifolium, an Ag-NOR band was sometimes seen on a medium-sized chromosome. In the karyotypes of interspecific hybrids, silver-stained rDNA loci were observed on satellite chromosomes of both parental species. RAPD analysis with 22 primers revealed a high similarity of the three species. The greatest difference was observed between L. angustifolium and the other two species. The RAPD patterns of L. bienne and L. usitatissimum differed in fewer fragments. A dendrogram of genetic similarity was constructed for the three flax species on the basis of their RAPD patterns. Genome analysis with chromosome and molecular markers showed that L. bienne must be considered as a subspecies of L. usitatissimum rather than a separate species. The three species were assumed to originate from a common ancestor, L. angustifolium being closest to it.     

Karyotype analysis of Lilium longiflorum and Lilium rubellum by chromosome banding and fluorescence in situ hybridisation.

Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red- and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.     

The Diversity of Karyotypes and Genomes within Section Syllinum of the Genus Linum (Linaceae) Revealed by Molecular Cytogenetic Markers and RAPD Analysis

The wide variation in chromosome number found in species of the genus Linum (2n = 16, 18, 20, 26, 28, 30, 32, 36, 42, 72, 84) indicates that chromosomal mutations have played an important role in the speciation of this taxon. To contribute to a better understanding of the genetic diversity and species relationships in this genus, comparative studies of karyotypes and genomes of species within section Syllinum Griseb. (2n = 26, 28) were carried out. Elongated with 9-aminoacridine chromosomes of 10 species of section Syllinum were investigated by C- and DAPI/С-banding, CMA and Ag-NOR-staining, FISH with probes of rDNA and of telomere repeats. RAPD analysis was also performed. All the chromosome pairs in karyotypes of the studied species were identified. Chromosome DAPI/C-banding patterns of 28-chromosomal species were highly similar. Two of the species differed from the others in chromosomal location of rDNA sites. B chromosomes were revealed in all the 28-chromosomal species. Chromosomes of Linum nodiflorum L. (2n = 26) and the 28-chromosomal species were similar in DAPI/C-banding pattern and localization of several rDNA sites, but they differed in chromosomal size and number. The karyotype of L. nodiflorum was characterized by an intercalary site of telomere repeat, one additional 26S rDNA site and also by the absence of B chromosomes. Structural similarities between different chromosome pairs in karyotypes of the studied species were found indicating their tetraploid origin. RAPD analysis did not distinguish the species except L. nodiflorum. The species of section Syllinum probably originated from a common tetraploid ancestor. The 28-chromosomal species were closely related, but L. nodiflorum diverged significantly from the rest of the species probably due to chromosomal rearrangements occurring during evolution.     

C-Banding/DAPI and in situ hybridization reflect karyotype structure and sex chromosome differentiation in Humulus japonicus Siebold & Zucc

Japanese hop (Humulus japonicus Siebold & Zucc.) was karyotyped by chromosome measurements, fluorescence in situ hybridization with rDNA and telomeric probes, and C-banding/DAPI. The karyotype of this species consists of sex chromosomes (XX in female and XY1Y2 in male plants) and 14 autosomes difficult to distinguish by morphology. The chromosome complement also shows a rather monotonous terminal distribution of telomeric repeats, with the exception of a pair of autosomes possessing an additional cluster of telomeric sequences located within the shorter arm. Using C-banding/DAPI staining and 5S and 45S rDNA probes we constructed a fluorescent karyotype that can be used to distinguish all autosome pairs of this species except for the 2 largest autosome pairs, lacking rDNA signals and having similar size and DAPI-banding patterns. Sex chromosomes of H. japonicus display a unique banding pattern and different DAPI fluorescence intensity. The X chromosome possesses only one brightly stained AT-rich terminal segment, the Y1 has 2 such segments, and the Y2 is completely devoid of DAPI signal. After C-banding/DAPI, both Y chromosomes can be easily distinguished from the rest of the chromosome complement by the increased fluorescence of their arms. We discuss the utility of these methods for studying karyotype and sex chromosome evolution in hops.     

The karyotype of Clivia mirabilis analyzed by differential banding and fluorescence in-situ hybridization

The karyotype of the recently described species Clivia mirabilis was analyzed by differential chromosome staining with Giemsa, chromomycin, and DAPI and by fluorescence in-situ hybridization with 5S and 45S rDNA probes. Like the other five Clivia species it was shown to have a unique karyotype, although its karyotype was similar in several respects to that of C. gardenii, differing in having only one pair of chromosomes with CMA bands compared with two pairs in C. gardenii and lacking any DAPI-positive bands. The evolutionary relationships of the species and their karyotypes are discussed.     

Chromosomal organization of the genomes of small-chromosome plants

An effective approach to study the chromosome organization in genomes of plants with small chromosomes and/or with low-informative C-banding patterns was developed in the course of investigation of the karyotypes of cotton plant, camomile, flax, and pea. To increase the resolving power of chromosome analysis, methods were worked out for revealing early replication patterns on chromosomes and for artificial impairment of mitotic chromosome condensation with the use of a DNA intercalator, 9-aminoacridine (9-AMA). To estimate polymorphism of the patterns of C-banding of small chromosomes on preparations obtained with the use of 9-AMA, it is necessary to choose a length interval that must not exceed three average sizes of metaphase chromosomes without the intercalator. The use of 9-AMA increases the resolution of differential C- and OR-banding and the precision of physical chromosome mapping by the FISH method. Of particular importance in studying small chromosomes is optimization of the computer-aided methods used to obtain and process chromosome images. The complex approach developed for analysis of the chromosome organization in plant genomes was used to study the karyotypes of 24 species of the genus Linum L. It permitted their chromosomes to be identified for the first time, and, in addition, B chromosomes were discovered and studied in the karyotypes of the species of the section Syllinum. By similarity of the karyotypes, the studied flax species were distributed in eight groups in agreement with the clusterization of these species according to the results of RAPD analysis performed in parallel. Systematic positions and phylogenetic relationships of the studied flax species were verified. Out results can serve as an important argument in favour of the proposal to develop a special program for sequencing the genome of cultivated flax (L. usitatissimum L.), which is a major representative of small-chromosome species.     

Karyotype of Mesembryanthemum crystallinum (Aizoaceae) studied by chromosome banding,FISH with rDNA probes and immunofluorescence detection of DNA methylation

The karyotype of the common ice plant Mesembryanthemum crystallinum L. (Aizoaceae) was studied using Chromomycin A₃ (CMA)/4′,6-diamidino-2-phenylindole (DAPI) staining, fluorescence in situ hybridization with 5S and 18S–5.8S–25S rDNA probes, DAPI/C-banding and immunodetection of 5-methylcytosine. A single bright CMA-band was revealed on the satellite chromosome, whose location was coincided with a position of a site of 18S–5.8S–25S rRNA genes. A site of 5S rRNA genes was observed on one of the other chromosomes. Relatively large DAPI/C-bands were mainly localized in the pericentromeric regions of the chromosomes. DAPI/C-banding patterns allowed us to identify all the chromosomes in the karyotype of M. crystallinum. The methylation of euchromatic chromosome regions was weaker as compared with heterochromatic DAPI/C-bands, which were hypermethylated. The obtained results may provide opportunities for investigating, at the chromosomal level, the genomic changes occurring in M. crystallinum either under salinization or under the action of other stress factors.     

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.     

Comparative study of genomes of two species of flax by C-banding of chromosomes

C-banding patterns of the karyotypes of two closely related wild flax species, Linum austriacum L. (2n = 18) and Linum grandiflorum Desf. (2n = 16), were studied. The karyotypes of both species were similar in the chromosome morphology and size. In each species, metacentric and acrocentric chromosomes (1.7-4.3 microns) and one satellite chromosome were observed. In the karyotypes of the species studied, all homologous chromosome pairs were identified, and quantitative ideograms were constructed. Eight chromosome pairs in the two species had similar C-banding patterns. A low level of intraspecific polymorphism in the intercalary and telomeric C-bands was shown in both species. The results indicate that the genomes of two flax species originated from one ancestral genome with the main chromosome number of 8 or 9. Apparently, the doubling of chromosome number or loss of one chromosome with subsequent redistribution of the chromosome material in the ancestral form resulted in the divergence into two species, L. austriacum L. and L. grandiflorum Desf. A considerable similarity of chromosomes in these species provides evidence for their close phylogenetic relatedness, which makes it possible to place them in one section within the Linum genus.     

Chromosomal organization of ribosomal genes and NOR-associated heterochromatin, and NOR activity in some populations of Allium commutatum Guss. (Alliaceae)

The position and the number of 18S-5.8S-26S and 5S rDNA loci, characterization of nucleolar organizing region (NOR)-associated heterochromatin and NOR activity assessment are given for six south-eastern Adriatic populations of Allium commutatum Guss. The karyotype characteristics were identical for all the populations studied, even those of distant islands. Diploid karyotypes (2 n = 16) always possessed two NOR-bearing chromosome pairs with pericentric and median secondary constrictions (SCs) on the short arm of the chromosomes VII and VIII. Fluorescent in situ hybridization (FISH) confirmed that these were the only sites of 18S-5.8S-26S rRNA genes. NOR-associated heterochromatin was of the constitutive character as shown after C-banding. Differential fluorochrome banding with Chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) revealed that this heterochromatin comprises both GC- and AT-rich DNA segments. Heteromorphism of C- and CMA-bands was noticed between homologous NOR-bearing chromosomes. The maximum number of four active NORs was correlated with the maximum number of four nucleoli in interphase. Variability of NOR-activity, expressed as number and size of silver stained NORs, existed between cells and between individuals of the same population. The different size of homologous and nonhomologous silver stained NORs was correlated with the extension of SCs. The only 5S rDNA locus was in an intercalary position on short arm of the chromosome VI, at the region of AT-rich constitutive heterochromatin. Dimorphism of C-bands and DAPI/Hoechst(H)-fluorescent bands was noticed between homologous chromosomes VI. © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 99–108.     

Cytogenetic characterization of the bay scallop, Argopecten irradians irradians, by multiple staining techniques and fluorescence in situ hybridization

The chromosomes of Argopecten irradians irradians were studied by various cytogenetic approaches. Conventional chromosome characterization built on C-banding, DAPI-staining, and silver staining was complemented by the physical mapping of ribosomal DNA and telomeric sequence (TTAGGG)n by FISH. Results showed that the constitutive heterochromatin revealed by C-banding was mainly distributed at telomeric and centromeric regions. However, interstitial C-bands were also observed. The pattern of DAPI banding was almost consistent with that of C-banding. Silver staining revealed that NORs were located on the short arms of chromosome 3 and 10, and this was further confirmed by FISH using 18S-28S rDNA. 5S rDNA was mapped as two distinguishable loci on the long arm of chromosome 11. 18S-28S and 5S rDNA were located on different chromosomes by sequential FISH. FISH also showed that the vertebrate telomeric sequence (TTAGGG)n was located on both ends of each chromosome and no interstitial signals were detected. Sequential 18S-28S rDNA and (TTAGGG)n FISH demonstrated that repeated units of the two multicopy families were closely associated on the same chromosome pair.     

Chromosomal localization of 5S and 45S ribosomal DNA in species of Linum L. section Linum (syn=Protolinum and Adenolinum)

Fluorescence in situ hybridization (FISH) was for the first time used to study the chromosomal location of the 45S (18-2.5S-26S) and 5S ribosomal genes in the genomes of five flax species of the section Linum (syn. Protolinum and Adenolinum). In L. usitatissimum L. (2n = 30), L. angustifolium Huds. (2n = 30), and L. bienne Mill. (2n = 30), a major hybridization site of 45S rDNA was observed in the pericentric region of a large metacentric chromosome. A polymorphic minor locus of 45S rDNA was found on one of the small chromosomes. Sites of 5S rDNA colocalized with those of 45S rDNA, but direct correlation between signal intensities from the 45S and 5S rDNA sites was observed only in some cases. Other 5S rDNA sites mapped to two chromosomes in these flax species. In L. grandiflorum Desf. (2n = 16) and L. austriacum L. (2n = 18), large regions of 45S and 5S rDNA were similarly located on a pair of homologous satellite-bearing chromosomes. An additional large polymorphic site of 45S and 5S rDNA was found in the proximal region of one arm of a small chromosome in the L. usitatissimum. L. angustifolium, and L. bienne karyotypes. The other arm of this chromosome contained a large 5S rDNA cluster. A similar location of the ribosomal genes in the pericentric region of the pair of satellite-bearing metacentrics confirmed the close relationships of the species examined. The difference in chromosomal location of the ribosomal genes between flax species with 2n = 30 and those with 2n = 16 or 18 testified to their assignment to different sections. The use of ribosomal genes as chromosome markers was assumed to be of importance for comparative genomic studies in cultivated flax, a valuable crop species of Russia, and in its wild relatives.     

Karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) using C-banding and bicolor fluorescence in situ hybridization

An intensive karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) was carried out with three different methods. These included Feulgen staining, Giemsa C-banding, and fluorescence in situ hybridization (FISH). The mitotic chromosomes of the cucumber (2n = 2x = 14) were characterized, based on the length and arm ratio values. A C-banding analysis showed dark stains on the centromeric, telomeric, and intercalary regions of the chromosomes, except that chromosome 2 had a heavy staining in the long arm. Bicolor FISH, using 45S and 5S rDNA probes, provided additional information to identify cucumber chromosomes. The signals for 45S rDNA were detected on the pericentromeric regions of chromosomes 1, 2, and 4. The signals for 5S rDNA were on the short arm of chromosome 5. Similar band patterns (as the C-banding) were observed when the chromosomes were counter-stained with 4',6-diamidino-2-phenyoindole (DAPI). The data implied that the karyotype of the Korean cucumber cultivar is peculiar and different from previous reports.     

The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): A populational and cytomolecular analysis

The genus Nothoscordum Kunth comprises approximately 20 species native to South America. Karyologically, the genus is remarkable for its large chromosomes and Robertsonian translocations. Variation in chromosome number has been recorded in a few polyploid species and it is unknown among diploids. This study presents the chromosome number and morphology of 53 individuals of seven populations of N. arenarium Herter (2n = 10). In addition, karyotype analyses after C-banding, staining with CMA and DAPI, and in situ hybridization with 5S and 45S rDNA probes were performed in six individuals from one population. All individuals exhibited 2n = 10 (6M + 4A), except for one tetraploid (2n = 20, 12M + 8A) and one triploid (2n = 15, 9M + 6A) plant. C-banding revealed the presence of CMA(+) /DAPI (-) heterochromatin in the short arm and in the proximal region of the long arm of all acrocentric chromosomes. The 45S rDNA sites co-localized with the CMA (+) regions of the acrocentrics short arms, while the 5S rDNA probe only hybridized with the subterminal region of a pair of metacentric chromosomes. A change in the pattern of CMA bands and rDNA sites was observed in only one individual bearing a reciprocal translocation involving the long arm of a metacentric and the long arm of an acrocentric chromosome. These data suggest that, despite isolated cases of polyploidy and translocation, the karyotype of N. arenarium is very stable and the karyotypic instability described for other species may be associated with their polyploid condition.