Genome Comparisons with Chromosomal and Molecular Markers for Three Closely Related Flax Species and Their Hybrids

Chromosome C-banding patterns were analyzed in three closely related flax species (Linum usitatissimumL., 2n = 30; L. angustifolium Huds., 2n = 30; and L. bienneMill., 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 m), 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. InL. 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 thatL. 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. angustifoliumbeing closest to it.     

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.     

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.     

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.     

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.     

Genetic polymorphism of flax Linum usitatissimum based on the 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 flax) and in the k-37 × 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 chromosome rearrangements (chromosome 3 inversions) were detected in the variety Luna and in the k-37 × 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 chromosome analysis in the fiber and oil flax confirm their very close genetic similarity. In spite of this, the combined use of the chromosome and molecular markers has opened up unique possibilities for describing the genotypes of flax varieties and creating their genetic passports.     

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.     

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.     

Chromosome Localization of 5S and 45S Ribosomal DNA in the Genomes of Linum L. Species of the 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 (18S–5.8S–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 were 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.     

RAPD analysis of the flax (Linum usitatissimum L.) varieties and hybrids of various productivity

Genetic polymorphism in varieties and hybrids of cultivated flax (Linum usitatissimum L.) has been investigated by RAPD-PCR. Analysis with 15 primers has revealed varietal specificity and hybrid inheritance of RAPD alleles. This allows genetic certification of the original varieties and their hybrids for breeding purposes. Polymorphic amplification products were obtained in RAPD analysis of DNA from two cultivated flax varieties with the use of 10-11 nucleotide primers.     

Molecular cytogenetic analysis of Aegilops cylindrica host.

The genomic constitution of Aegilops cylindrica Host (2n = 4x = 28, DcDcCcCc) was analyzed by C-banding, genomic in situ hybridization (GISH), and fluorescence in situ hybridization (FISH) using the DNA clones pSc119, pAs1, pTa71, and pTA794. The C-banding patterns of the Dc- and Cc-genome chromosomes of Ae. cylindrica are similar to those of D-and C-genome chromosomes of the diploid progenitor species Ae. tauschii Coss. and Ae. caudata L., respectively. These similarities permitted the genome allocation and identification of the homoeologous relationships of the Ae. cylindrica chromosomes. FISH analysis detected one major 18S-5.8S-25S rDNA locus in the short arm of chromosome 1Cc. Minor 18S-5.8S-25S rDNA loci were mapped in the short arms of 5Dc and 5Cc. 5S rDNA loci were identified in the short arm of chromosomes 1Cc, 5Dc, 5Cc, and 1Dc. GISH analysis detected intergenomic translocation in three of the five Ae. cylindrica accessions. The breakpoints in all translocations were non-centromeric with similar-sized segment exchanges.     

Comparative Genome Analysis in Two Flax Species by C-Banding Patterns

C-banding patterns of the karyotypes of two closely related wild flax species, Linum austriacumL. (2n= 18) and Linum grandiflorumDesf. (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 m) and one satellite chromosome were observed. In the karyotypes of the species studied, all homologous chromosome pairs were identified, and quantitative idiograms 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 basic chromosome number of 8 or 9. Apparently, the duplication or loss of one chromosome with subsequent redistribution of the chromosome material in the ancestral form resulted in the divergence into two species,L. austriacumL. and L. grandiflorumDesf. 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 Linumgenus.     

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.     

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.     

Karyological evidence for interspecific hybridization between Cichla monoculus and C. temensis (Perciformes, Cichlidae) in the Amazon

Cichla monoculus, Cichla temensis (peacock bass or tucunare), and its presumed hybrids, were cytogenetically analyzed. The fish were collected at three distinct sites in the central Amazon basin, namely in the Uatuma (C. monoculus, C. temensis and their natural hybrid), Jau (C. temensis), and Solimoes rivers (C. monoculus). The two species and the natural hybrid showed the same diploid number, 2n=48 acrocentric chromosomes. Single NORs were detected in the distal region of the long arm in all three species. However, in C. monoculus, the NOR was found on the second pair of the complement, in C. temensis, on the third pair and in the hybrid two NOR patterns were found, one on the second pair and the other on the third pair of chromosomes. The two species and the hybrid have their constitutive heterochromatin located in the pericentromeric region of all chromosomes and an interstitial C-band located on the largest chromosome pair. The great similarity in the chromosome number and morphology, chromosome size class differences, the NOR patterns and C-banding suggested chromosomal stasis during speciation and hybridization of Cichla.     

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.     

Chromosomal studies on five species of the genus Leptodactylus Fitzinger, 1826 (Amphibia, Anura) using differential staining

Cytogenetic studies were carried out on five species of Leptodactylus, namely L. fuscus, L. notoaktites, L. labyrinthicus, L. ocellatus, and L. podicipinus, after standard staining, Ag-NOR and C-banding as well as BrdU incorporation for three of them. The species had 2n = 22 chromosomes and two basic karyotype patterns. Chromosome 8 was a marker bearing a secondary constriction. In all species, this secondary constriction corresponded to the Ag-NOR site. The species had centromeric C-bands in all chromosomes of the complement, but some interstitial or telomeric bands seemed to differentiate some karyotypes, either at the species or the population level. In L. ocellatus, the C-banding pattern confirmed the occurrence of a heteromorphic pericentric inversion in chromosome 8 in specimens from one of the populations. The BrdU incorporation technique showed no detectable difference in the replication patterns of the major bands in the chromosomes of L. notoaktites, L. labyrinthicus, and L. ocellatus.