Molecular cytogenetic analysis of the Vigna species distributed in Korea

Vigna plants distributed in Korea were analyzed by molecular cytogenetic fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and rDNA ITS/NTS sequences. FISH revealed that variable 45S rRNA gene loci (one to four) were localized on the terminal regions of chromosomes, while two conserved 5S rRNA gene loci from all species examined, except for rice bean (single locus), were detected. FISH and GISH showed the characteristic organization of rRNA gene loci and genomic homology on the chromosomes, indicating their cytogenetic relatationships. ITS sequence revealed that there was considerable variation in length (190–207 bp in ITS1, 205–221 bp in ITS2) and nucleotide composition (7–67 bp). The 5S rRNA gene unit comprised coding region (118 bp) and extensive sequence heterogeneity (97–221 bp). Phylogenetic analysis of the ITS and NTS sequences demonstrated that the Vigna species are divided into two groups: angularis (V. angularis, V. umbellata, V. nakashimae and V. nipponensis) and unguiculata (V. unguiculata, V. sesquipedalis and V. vexillata). Sequence data also showed that mung bean was closer to the angularis group.     

A new long terminal repeat (LTR) sequence allows to identify J genome from J<Superscript>S</Superscript> and St genomes of <Emphasis Type="Italic">Thinopyrum intermedium</Emphasis>

A repetitive sequence of 491 bp, named pMD232-500, was isolated from S. cereale cv. Kustro using wheat SSR marker Xgwm232. GenBank BLAST search revealed that the sequence of pMD232-500 was highly similar to a part of retrotransposon Nusif-1. Fluorescence in situ hybridization (FISH) analysis using pMD232-500 as probe indicated that only 14 Thinopyrum intermedium chromosomes and all the chromosomes of S. cereale cv. Kustro bear FISH signals, however, no FISH signals were observed on Dasypyrum villosum chromosomes. In addition, the FISH signals were distributed on whole arms except their terminal regions. Further genomic in situ hybridization (GISH) analysis using genomic DNA from Pseudoroegneria spicata indicated that the 14 Th. intermedium chromosomes bearing FISH signals should belong to J genome. Thereafter, the repetitive elements pMD232-500 showed the unambiguous features of genomic constitution of Th. intermedium. In addition, the results in the present study have indicated the similarity of genomes from Th. intermedium and S. cereale.     

Sequence polymorphism and chromosomal localization of 5S rDNA of three cultivated varieties of sweetpotato (Ipomoea batatas (L.) Lam.)

The 5S rDNA of plant is organized into clusters of tandem repeat units which include a coding region of 5S rRNA gene and variable sequences of nontranscribed spacer (NTS). In this study, we investigated sequence polymorphism and chromosomal localization of 5S rDNA in three cultivated varieties of sweet potato (Ipomoea batatas Lam.). Two different PCR products of 5S rDNA were amplified from all three varieties, as approximately 0.25 kb and 0.34 kb with multiples. In sequence analysis, the 5S rDNA ofI. batatas were discriminated from four consensus sequences by in reasonable sizes and molecular informative factors. Four consensus sequences were divided into three short sequences, including 263, 253, and 243 – 283 bp by sequence variation between 160 and 186 bp in NTS region, and one long sequence with 340 bp. To identify molecular relationship among varieties, phylogenetic analysis was applied. A total of 35 sequenced clones in this study were classified into four groups in phylogenetic tree. Interestingly, two varieties included all four groups, but one variety only two groups. To localize the physical map of 5S rDNA, fluorescencein situ hybridization (FISH) was performed in metaphase chromosomes of each varieties. In 90 chromosomes ofI. batatas, 6 loci of 5S rDNA were detected in chromosomes for all varieties. Our results will help to further more understand the genomic relationship inI. batatas, to investigate molecular relationship among varieties.     

Detection of a variable number of ribosomal DNA loci by fluorescent in situ hybridization in Populus species

Fluorescent in situ hybridization (FISH) was applied to related Populus species (2n = 19) in order to detect rDNA loci. An interspecific variability in the number of hybridization sites was revealed using as probe an homologous 25S clone from Populus deltoides. The application of image analysis methods to measure fluorescence intensity of the hybridization signals has enabled us to characterize major and minor loci in the 18S-5.8S-25S rDNA. We identified one pair of such rDNA clusters in Populus alba; two pairs, one major and one minor, in both Populus nigra and P. deltoides; and three pairs in Populus balsamifera, (two major and one minor) and Populus euroamericana (one major and two minor). FISH results are in agreement with those based on RFLP analysis. The pBG13 probe containing 5S sequence from flax detected two separate clusters corresponding to the two size classes of units that coexist within 5S rDNA of most Populus species. Key words : Populus spp., fluorescent in situ hybridization, FISH, rDNA variability, image analysis.     

FISH and GISH analysis of the genomic relationships amongPanax species

Ginseng is a well-known medicinal plant that has been used as an anti-aging agent for many years in East Asia. In the genusPanax, only three species,P. ginseng (Oriental ginseng),P. quinquefolius (American ginseng) andP. notoginseng (Chinese ginseng), are currently considered to be important medicinal herbs. Despite the increase in their breeding value, molecular cytogenetic information on the species is not available. To analyze the genomic relationships among thePanax species, FISH (fluorescencein situ hybridization) and GISH (genomicin situ hybridization) techniques were applied. FISH analysis revealed that the 45S and 5S rRNA genes ofP. notoginseng (2n=2x=24) andP. ginseng (2n=4x=48) cluster on a single locus on different chromosomes, whileP. quinquefolius (2n=4x=48),P. japonicus (2n=4x=48), and Korean wild ginseng (2n =4x= 48) had one locus of the 45S rRNA gene and two loci of the 5S rRNA gene, respectively. GISH analysis using genomic DNA as a probe detected strong cross-hybridization of genomes betweenP. ginseng andP. quinquefolius. GISH analysis of other species showed weak or no distinct signals on the chromosomes. Our data revealed thatP. ginseng andP. quinquefolius showed the highest degree of homology, indicating that these species diverged in most recent years.     

Physical Mapping of rRNA Gene Loci and Inter-specific Relationships in Wild <Emphasis Type="Italic">Lilium</Emphasis> Distributed in Korea

Molecular cytogenetic analyses using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were carried out to elucidate inter-specific relationships among wild Lilium species distributed in Korea. FISH revealed four to eight 45S rRNA gene loci, which are located on chromosomes 1–7, 10, and 11 among the different species. In contrast, the 5S rRNA gene locus was conserved on the long arm of chromosome 3, occasionally with two adjacent sites on the same chromosome arm in a few species. The 5S rDNA site was located adjacent to the 45S rDNA site in only three species, Lilium distichum, Lilium hansonii, and Lilium tsingtauense. GISH analysis using genomic DNA probes detected strong hybridization of genomes between diploid and triploid Lilium lancifolium species, demonstrating that triploid plants were derived from diploid L. lancifolium and not from Lilium maximowiczii. Phylogenetic analysis of the ITS and NTS sequences supported the cytogenetic data as well as Comber’s classification of the genus Lilium.     

Non-Denaturing Fluorescence in Situ Hybridization to Find Replication Origins in a Specific Genome Region on the DNA Fiber

Fluorescence in situ hybridization (FISH) is a useful method of determining the replication timing of specific genomic loci in mammals and of delineating replicon structures on DNA fibers in combination with in vivo replication labeling. In the case of simultaneous detection of a FISH probe and replicated forks, however, the DNA fibers are damaged by the DNA denaturation step for FISH detection, and the resulting fragmented fluorescence signals prevent analysis at high resolution. Here we found that hybridization of the probe to the genomic DNA was possible even under non-denaturing condition, but only at the time its genomic region replicated. Using the method designated non-denaturing FISH, we determined the replication timing of a specific BAC clone and the standard clones, and found that at least one replication origin exists within the genomic region covered by its BAC clone as an example.     

Relationships of the woody Medicago species (section Dendrotelis) assessed by molecular cytogenetic analyses

Background and Aims

The organization of rDNA genes in the woody medic species from the agronomically important Medicago section Dendrotelis was analysed to gain insight into their taxonomic relationships, to assess the levels of infraspecific variation concerning ribosomal loci in a restricted and fragmented insular species (M. citrina) and to assess the nature of its polyploidy.

Methods

Fluorescence in situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal DNA genes in the three species of section Dendrotelis (M. arborea, M. citrina, M. strasseri) and the related M. marina from section Medicago. Genomic in situ hybridization (GISH) was used to assess the genomic relationships of the polyploid M. citrina with the putatively related species from section Dendrotelis.

Key Results

The diploid (2n = 16) M. marina has a single 45S and two 5S rDNA loci, a pattern usually detected in previous studies of Medicago diploid species. However, polyploid species from section Dendrotelis depart from expectations. The tetraploid species (2n = 32) M. arborea and M. strasseri have one 45S rDNA locus and two 5S rDNA loci, whereas in the hexaploid (2n = 48) M. citrina four 45S rDNA and five 5S rDNA loci have been detected. No single chromosome of M. citrina was uniformly labelled after using genomic probes from M. arborea and M. strasseri. Instead, cross-hybridization signals in M. citrina were restricted to terminal chromosome arms and NOR regions.

Conclusions

FISH results support the close taxonomic interrelationship between M. arborea and M. strasseri. In these tetraploid species, NOR loci have experienced a diploidization event through physical loss of sequences, a cytogenetic feature so far not reported in other species of the genus. The high number of rDNA loci and GISH results support the specific status for the hexaploid M. citrina, and it is suggested that this species is not an autopolyploid derivative of M. arborea or M. strasseri. Further, molecular cytogenetic data do not suggest the hypothesis that M. arborea and M. strasseri were involved in the origin of M. citrina. FISH mapping can be used as an efficient tool to determine the genomic contribution of M. citrina in somatic hybrids with other medic species.Key words: Medicago arborea, M. citrina, M. strasseri, rRNA genes, 18S-5·8S-25-S, 5S, FISH mapping, GISH, polyploidy     

Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. genome

The chromosomal organization of two novel repetitive DNA sequences isolated from the Chenopodium quinoa Willd. genome was analyzed across the genomes of selected Chenopodium species. Fluorescence in situ hybridization (FISH) analysis with the repetitive DNA clone 18-24J in the closely related allotetraploids C. quinoa and Chenopodium berlandieri Moq. (2n = 4x = 36) evidenced hybridization signals that were mainly present on 18 chromosomes; however, in the allohexaploid Chenopodium album L. (2n = 6x = 54), cross-hybridization was observed on all of the chromosomes. In situ hybridization with rRNA gene probes indicated that during the evolution of polyploidy, the chenopods lost some of their rDNA loci. Reprobing with rDNA indicated that in the subgenome labeled with 18-24J, one 35S rRNA locus and at least half of the 5S rDNA loci were present. A second analyzed sequence, 12-13P, localized exclusively in pericentromeric regions of each chromosome of C. quinoa and related species. The intensity of the FISH signals differed considerably among chromosomes. The pattern observed on C. quinoa chromosomes after FISH with 12-13P was very similar to GISH results, suggesting that the 12-13P sequence constitutes a major part of the repetitive DNA of C. quinoa.     

Molecular Characterization of a Toluene-Degrading Methanogenic Consortium

A toluene-degrading methanogenic consortium enriched from creosote-contaminated aquifer material was maintained on toluene as the sole carbon and energy source for 10 years. The species in the consortium were characterized by using a molecular approach. Total genomic DNA was isolated, and 16S rRNA genes were amplified by using PCR performed with kingdom-specific primers that were specific for 16S rRNA genes from either members of the kingdom Bacteria or members of the kingdom Archaea. A total of 90 eubacterial clones and 75 archaeal clones were grouped by performing a restriction fragment length polymorphism (RFLP) analysis. Six eubacterial sequences and two archaeal sequences were found in the greatest abundance (in six or more clones) based on the RFLP analysis. The relative abundance of each putative species was estimated by using fluorescent in situ hybridization (FISH), and the presence of putative species was determined qualitatively by performing slot blot hybridization with consortium DNA. Both archaeal species and two of the six eubacterial species were detected in the DNA and FISH hybridization experiments. A phylogenetic analysis of these four dominant organisms suggested that the two archaeal species are related to the genera Methanosaeta and Methanospirillum. One of the eubacterial species is related to the genus Desulfotomaculum, while the other is not related to any previously described genus. By elimination, we propose that the last organism probably initiates the attack on toluene.     

AT-rich sequences containing Arabidopsis-type telomere sequence and their chromosomal distribution in Pinus densiflora

Japanese red pine Pinus densiflora has 2 n=24 chromosomes and after FISH-detection of Arabidopsis-type (A-type) telomere sequences, many telomere signals were observed on these chromosomes at interstitial and proximal regions in addition to the chromosome ends. These interstitial and proximal signal sites were observed as DAPI-positive bands, suggesting that the interstitial and proximal telomere signal sites are composed of AT-rich highly repetitive sequences. Four DNA clones (PAL810, PAL1114, PAL1539, PAL1742) localized at the interstitial telomere signals were selected from AluI-digested genomic DNA library using colony blot hybridization probed with A-type telomere sequences and characterized using FISH and Southern blot hybridization. The AT-contents of these selected four clones were 60.8–76.3%, and repeat units of the telomere sequence and degenerated telomere sequences were found in their nucleotide sequences. Except for two sites of PAL1114, FISH signals of the four clones co-localized with interstitial and proximal A-type telomere sequence signals. FISH signals a showed similar distribution pattern, but the patterns of signal intensity were different among the four clones. PAL810, PAL1539 and PAL 1742 showed similar FISH signal patterns, and the differences were only with respect to the signal intensity of some signal sites. PAL1114 had unique signals that appeared on chromosomes 7 and 10. Based on results of the Southern blot hybridization these four sequences are not arranged tandemly. Our results suggest that the interstitial A-type telomere sequence signal sites were composed of a mixture of several AT-rich repetitive sequences and that these repetitive sequences contained A-type telomere sequences or degenerated A-type telomere sequence repeats.     

Karyotype Analysis of Gossypium arboreum × G. bickii by Genome in situ Hybridization

By using genome in situ hybridization (GISH) on root somatic chromosomes of allotetraploid derived from the cross Gossypium arboreum × G. bickii with genomic DNA (gDNA) of G. bickii as a probe, two sets of chromosomes, consisting of 26 chromosomes each, were easily distinguished from each other by their distinctive hybridization signals. GISH analysis directly proved that the hybrid GarboreumxG. bickii is an allotetraploid amphiploid. The karyotype formula of the species was 2n = 4x = 52 = 46m (4sat) + 6sm (4sat). We identified four pairs of satellites with two pairs in each sub-genome. FISH analysis using 45S rDNA as a probe showed that the cross G. arboreumxG. bickii contained 14 NORs. At least five pairs of chromosomes in the G sub-genome showed double hybridization (red and blue) in their long arms, which indicates that chromatin introgression from the A sub-genome had occurred.     

Genetic assessment of the effects of self-fertilization in a Lilium L. hybrids using molecular cytogenetic methods (FISH and ISSR)

Self-fertilization (also termed selfing) is a mode of reproduction that occurs in hermaphrodites and has evolved several times in various plant and animal species. A transition from outbreeding to selfing in hermaphroditic flowers is typically associated with changes in flower morphology and functionality. This study aimed to identify genetic effects of selfing in the F2 progeny of F1 hybrid developed by crossing Lilium lancifolium with the Asiatic Lilium hybrid ‘Dreamland.’ Fluorescence in situ hybridization (FISH) and inter-simple sequence repeats (ISSR) techniques were used to detect genetic variations in plants produced by selfing. The FISH results showed that F1 hybrid were similar to the female parent (L. lancifolium) regarding the 45S loci, but F2 individuals showed variation in the number and location of the respective loci. In F2 progeny, F2-2, F2-3, F2-4, F2-5, and F2-8 hybrids expressed two strong and one weak 5S signal on chromosome 3, whereas F2-7 and F2-9 individuals expressed one strong and two weak signals. Only two strong 5S signals were detected in an F2-1 plant. The ISSR results showed a maximum similarity value of 0.6269 between the female parent and the F2-2 hybrid. Regarding similarity to the male parent, a maximum value of 0.6119 was found in the F2-1 and F2-2 hybrids. The highest genetic distance from L. lancifolium and the Asiatic Lilium hybrid ‘Dreamland’ was observed in the F2-4 progeny (0.6352 and 0.7547, respectively). Phylogenetic relationships showed that the F2 progeny were closer to the male parent than to the female parent. Self-fertilization showed effects on variation among the F2 progeny, and effects on the genome were confirmed using FISH and ISSR analyses.     

Diversified chromosomal distribution of tandemly repeated sequences revealed evolutionary trends in Secale (Poaceae)

Genomic in situ hybridization (GISH) with Secale cereale cv. ‘Jingzhou rye’ DNA as a probe to chromosomes of hexaploid triticale line Fenzhi-1 revealed that not only were all chromosomes of rye strongly hybridized along the entire chromosome length, but there were also stronger signals in terminal or subtelomeric regions. This pattern of hybridization signals is referred to as GISH banding. After GISH banding, sequential fluorescene in situ hybridizaion (FISH) with tandem repeated sequence pSc200 and pSc250 as probes showed that the chromosomal distribution of pSc200 is highly coincident with the GISH banding pattern, suggesting that GISH banding revealed chromosomal distribution of pSc200 in rye. In addition, FISH using pSc200 and pSc250 as probes to chromosomes of 11 species of the genus Secale and two artificial amphiploids (Triticum aestivum-S. strictum subsp. africanum amphiploid and Aegilops tauschii-S. silvestre amphiploid) showed that (1) the chromosomal distribution of pSc200 and pSc250 differed greatly in Secale species, and the trend towards an increase in pSc200 and pSc250 binding sites from wild species to cultivated rye suggested that pSc200 and pSc250 sequences gradually accumulated during Secale evolution; (2) the chromosomal distribution of pSc200 and pSc250 presented polymorphism on homologous chromosomes, suggesting that the same species has two heterogeneous homologous chromosomes; (3) the intensity and number of hybridization signals varied differently on chromosomes between pSc200 and pSc250, suggesting that each repetitive family evolved independently.     

Variability of ribosomal DNA sites inFestuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH

This study focuses on the variability of chromosomal location and number of ribosomal DNA (rDNA) sites in some diploid and autotetraploidFestuca pratensis andLolium perenne cultivars, as well as on identification of rDNA-bearing chromosomes in their triploid and tetraploidF. pratensis ×L. perenne hybrids. The rDNA loci were mapped using fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes, and the origin of parental genomes was verified by genomic in situ hybridization (GISH) withL. perenne genomicDNAas a probe, andF. pratensis genomic DNA as a block. FISH detected variation in the number and chromosomal location of both 5S and 45S rDNA sites. InF. pratensis mostly additional signals of 5S rDNA loci occurred, as compared with standardF. pratensis karyotypes. Losses of 45S rDNA loci were more frequent inL. perenne cultivars and intergeneric hybrids. Comparison of theF. pratensis andL. perenne genomes approved a higher number of rDNA sites as well as variation in chromosomal rDNA location inL. perenne. A greater instability ofF. pratensis-genome-like andL. perenne-genome-like chromosomes in tetraploid hybrids was revealed, indicating gains and losses of rDNA loci, respectively. Our data indicate that the rDNA loci physically mapped on chromosomes 2 and 3 inF. pratensis and on chromosome 3 inL. perenne are useful markers for these chromosomes in intergenericFestuca ×Lolium hybrids.     

Delimiting the Origin of a B Chromosome by FISH Mapping,Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei,Characiformes)

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.     

Characterization and chromosomal distribution of a novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica)

A novel satellite DNA sequence of Japanese quail (Coturnix coturnix japonica) was isolated from genomic DNA digested with restriction endonuclease, Bg/II. Sequence analysis of three different-size clones revealed the presence of a tandem array of a GC-rich 41 bp repeated element. This sequence was localized by fluorescence in situ hybridization (FISH) primarily to microchromosomes of Japanese quail (2n = 78); approximately 50 of the 66 microchromosomes showed positive signals, although hybridization signals were also detected on chromosomes 4 and W. This satellite DNA did not cross-hybridize with genomic DNA of chicken (Gallus gallus) and Chinese painted quail (Excalfactoria chinensis) under moderately stringent conditions, suggesting that this class of repetitive DNA sequences was species specific and fairly divergent in Galliformes species.     

Inter- and intra-genomic transfer of small chromosomal segments in wheat-rye allopolyploids

Newly synthesized wheat-rye allopolyploids, derived from Triticum aestivum Mianyang11 × S. cereale Kustro, were investigated by sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) using rye tandem repeat pSc200 and rye genomic DNA as probes, respectively, over the first, second and third allopolyploid generations. FISH signals of pSc200 could be observed at both telomeres/subtelomeres of all 14 chromosomes of the parental rye. In the first allopolyploid generation, there were ten rye chromosomes bearing FISH signals at both telomeres/subtelomeres and four rye chromosomes bearing FISH signals at only one telomere/subtelomere. However, in the second and the third allopolyploid generations, there were 12 rye chromosomes bearing FISH signals at both telomeres/subtelomeres and 2 rye chromosomes bearing FISH signals at only one telomere/subtelomere. Rye telomeric segments were transferred to the centromeric region of wheat chromosomes in some cells and small segments derived from non-telomeric regions of rye chromosome were transferred to the telomeric region of wheat chromosomes in some other cells. These observations indicated that the rye telomeric/subtelomeric region was unstable in newly synthesized wheat-rye allopolyploids and allopolyploidization was accompanied by rapid inter/intra-genomic exchange. The inter-genomic exchange may have occurred in somatic cells.