5S rDNA Loci Research Articles

Molecular cytogenetics studies inReichardia tingetana: Physical mapping of heterochromatin, telomere repeats, and 5S and 45S rDNA by 4′, 6-diamidino-2-phenylindole and fluorescencein situhybridization

Molecular cytogenetics studies of A-T-rich regions, telomeres, and 5S and 45S rDNA sites on the chromosomes of Reichardia tingetana Roth (2n= 16; diploid) were done using 4′, 6-diamidino-2-phenylindole (DAPI) and fluorescence in situ hybridization (FISH). The species were collected from three geographically isolated populations at Borg El Arab (salt marsh habitat), and Rashed and Shosha (sandy clay habitats) in Egypt. The three populations showed the chromosome number of all plants are diploid except for two tetraploid samples from Shosha. Plants from both Rashed and Shosha showed similarity in the distribution of six DAPI bands on six chromosomes, whereas those of Borg El Arab showed a distribution of 16 bands on 14 chromosomes. The FISH signals of the telomeres, and 5S and 45S rDNA, were at the telomeres of all chromosomes, two interstitial, and four terminal, respectively. The combination of DAPI and FISH showed colocalization of the DAPI bands with two 5S and two 45S rDNA loci. The increased number of DAPI bands in the cytotypes from the salt marsh habitat could indicate natural genetic adaptation through increasing the heterochromatin of A-T-rich regions.

Physical mapping of 5S and 18S-5.8S-26S RNA gene families in polyploid series of Cenchrus ciliaris Linnaeus, 1771 (Poaceae)

The Buffelgrass (Cenchrus ciliaris L., Poaceae) is one of the most important pasturage grasses due to its high productivity and good forage qualities. This species possess a high adaptability to bioclimatic constraints of arid zones and may be used for the restoration of degraded arid ecosystems. Tunisian populations present three ploidy levels (4x, 5x and 6x) with a basic chromosome number x=9. This study reported for the first time the distribution of the ribosomal genes (rRNA) for pentaploid and hexaploid cytotypes of Cenchrus ciliaris. Molecular cytogenetic study using double fluorescence in situ hybridization has shown that the two rDNA families, 5S and 18S-5.8S-26S (18S), displayed intraspecific variation in number of loci among different ploidy levels. Each ploidy level was characterized by specific number of both 5S and 18S rDNA loci (two loci in tetraploid, five in pentaploid and six in hexaploid level). For three studied cytotypes (4x, 5x and 6x) all 5S rDNA loci were localized on the subcentromeric region of chromosomes, while 18S loci were situated on the telomeric region of short chromosome arms. Data of the FISH experiments show proportional increase of ribosomal loci number during polyploidization processes.

Karyotype variation is indicative of subgenomic and ecotypic differentiation in switchgrass

BackgroundKaryotypes can provide information about taxonomic relationships, genetic aberrations, and the evolutionary origins of species. However, differentiation of the tiny chromosomes of switchgrass (Panicum virgatum L.) and creation of a standard karyotype for this bioenergy crop has not been accomplished due to lack of distinguishing features and polyploidy.ResultsA cytogenetic study was conducted on a dihaploid individual (2n = 2X = 18) of switchgrass to establish a chromosome karyotype. Size differences, condensation patterns, and arm-length ratios were used as identifying features and fluorescence in-situ hybridization (FISH) assigned 5S and 45S rDNA loci to chromosomes 7 and 2 respectively. Both a maize CentC and a native switchgrass centromeric repeat (PviCentC) that shared 73% sequence identity demonstrated a strong signal on chromosome 3. However, only the PviCentC probe labeled the centromeres of all chromosomes. Unexpected PviCentC and 5S rDNA hybidization patterns were consistent with severe reduction or total deletion of these repeats in one subgenome. These patterns were maintained in tetraploid and octoploid individuals. The 45S rDNA repeat produced the expected number of loci in dihaploid, tetraploid and octoploid individuals. Differences observed at the 5S rDNA loci between the upland and lowland ecotypes of switchgrass provided a basis for distinguishing these subpopulations.ConclusionCollectively, these results provide a quantitative karyotype of switchgrass chromosomes. FISH analyses indicate genetic divergence between subgenomes and allow for the classification of switchgrass plants belonging to divergent genetic pools. Furthermore, the karyotype structure and cytogenetic analysis of switchgrass provides a framework for future genetic and genomic studies.

Swarm of terminal 35S in Cheirolophus (Asteraceae, Centaureinae)

Island radiation constitutes a playground for species diversification, which has long fascinated researchers and still does today. Because only a small subset of taxa within the pool of island colonizers is concerned by this process, the question is raised on whether some factors could make a taxon prone to radiate. Cheirolophus is the only genus of Centaureinae subtribe to have experienced a radiation in the Canary Islands. Cytogenetic characterization through FISH of 5S and 35S ribosomal RNA genes in eight Cheirolophus species from continent and Canary Islands revealed an unusually high number of 35S predominantly at terminal position, together with a single interstitial 5S rDNA locus in all the studied taxa. Such an abundance of 35S rDNA signals is unique among Centaureinae and predates Cheirolophus arrival in Canary Islands. The possible link of the rDNA profile with radiation process is discussed through a comparison with two other case studies, the closely related Rhaponticum group and the genus Centaurea.

A dynamic evolution of chromosome in subgenus Potamogeton revealed by physical mapping of rDNA loci detection

In this study, we analyzed 12 species in the subgenus Potamogeton (8 tetraploids and 2 diploids, 2 putative tetraploid hybrids) at the chromosomal level, including counting the chromosome number and physically mapping the rDNA. The extent of variation in the chromosome number and rDNA loci was determined in the Potamogeton species on both the inter- and intra-specific level. Moreover, one of the hybrid sets (P. perfoliatus a (TM), x P. wrightii a (TM) Euro) was picked for performing artificial pollination for producing F1 generation adults and for sequential morphological character analysis and FISH detection. After comparing the parental species and natural hybrids from three different geographic locations, the intraspecific variations of rDNA loci were revealed in sampled plants; in addition, comparisons between artificial F1 and natural hybrids (P. x intortusifolius) showed a rapid change of 45S rDNA loci in response to the interspecific hybridization. We also compared our results concerning rDNA patterns with phylogenetic documents to derive complementary clues to karoytype evolution and interspecific relationships. Based on frequent hybridizations and active clonal reproduction with weak genetic selection, the rDNA chromosomal repatterning, such as the gain or loss of rDNA loci together with rDNA movements, might be one trend of chromosome evolution in this genus.

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.

Cytotaxonomical Study of the Chenopodium album and its Related Species in Korea

The purpose of this study was to analyze the interspecific relationships of Chenopodium album and its related taxa collected in Korea. The 18S-26S ribosomal DNA (45S rDNA) loci were detected directly on mitotic chromosomes by fluorescence in situ hybridization (FISH) and the chromosome numbers were examined using aceto-orcein methods. The chromosomal numbers of Chenopodium album var. album and C. album var. centrorubrum were 2n = 6x = 54, whereas for C. album var. stenophyllum, this number was 2n = 4x = 36. The basic chromosome number was x = 9. The biotin labeled 18S-26S rDNA probe exhibited eight yellow fluorescent signals on the metaphase chromosome of C. album var. album and var. centrorubrum respectively, while two yellow signals of C. album var. stenophyllum were noted. All of the signals on the chromosomes were located at the terminal regions. The chromosome number and FISH findings suggest that C. album var. centrorubrum is merged into var. album and that it is clearly distinguished from C. album var. stenophyllum.

Cytogenetic and phylogenetic studies of diploid and polyploid members of Tribe Anemoninae (Ranunculaceae)

The ancestry, phylogenetic differentiation and systematic classification of the worldwide-distributed genus Anemone have been debated for many years. In this paper 11 Anemone, three Pulsatilla species and Hepatica nobilis were subjected to detailed karyotype analysis with the aim of obtaining new cytogenetic data that will contribute to karyotype evolutionary studies of the tribe Anemoninae. The results are interpreted in a phylogenetic context, established from the intergenic nontranscribed spacer (NTS) of 5S rDNA and internal transcribed spacer (ITS) of 35S rDNA. One to three 35S and one to three 5S rDNA loci are present in diploid and polyploid taxa. The 35S rDNA loci are located terminally on the short arm of acrocentric chromosomes, while for 5S rDNA there is no preferential chromosomal position as it exhibits terminal, subterminal, interstitial or pericentromeric positions, and is located either on acrocentric or metacentric chromosomes. The karyotype of hexaploid A. baldensis (2n = 6x = 48) is presented for the first time, and A. sylvestris is proposed as one of its putative parental species. Chromosome fusion/translocation is proposed as the key mechanism involved in reduction of the basic chromosome number from 8 in the Anemone subgenus to 7 in the Anemonidium subgenus. The cytogenetic data obtained are mainly supported by ITS and NTS phylogeny. Diversification of the genus Anemone was accompanied by a large reduction of heterochromatin, from the Mediterranean anemones that have large amounts of heterochromatin to the New World anemones without any detectable heterochromatic blocks.

Heterochromatin patterns and ribosomal DNA loci distribution in diploid and polyploid Crotalaria species (Leguminosae, Papilionoideae), and inferences on karyotype evolution

Most Crotalaria species display a symmetric karyotype with 2n = 16, but 2n = 14 is found in Chrysocalycinae subsection Incanae and 2n = 32 in American species of the section Calycinae. Seven species of the sections Chrysocalycinae, Calycinae, and Crotalaria were analyzed for the identification of heterochromatin types with GC- and AT-specific fluorochromes and chromosomal location of ribosomal DNA loci using fluorescent in situ hybridization (FISH). A major 45S rDNA locus was observed on chromosome 1 in all the species, and a variable number of minor ones were revealed. Only one 5S rDNA locus was observed in the species investigated. Chromomycin A(3) (CMA) revealed CMA(+) bands colocalized with most rDNA loci, small bands unrelated to ribosomal DNA on two chromosome pairs in Crotalaria incana, and CMA(+) centromeric bands that were quenched by distamycin A were detected in species of Calycinae and Crotalaria sections. DAPI(+) bands were detected in C. incana. The results support the species relationships based on flower specialization and were useful for providing insight into mechanisms of karyotype evolution. The heterochromatin types revealed by fluorochromes suggest the occurrence of rearrangements in repetitive DNA families in these heterochromatic blocks during species diversification. This DNA sequence turnover and the variability in number/position of rDNA sites could be interpreted as resulting from unequal crossing over and (or) transposition events. The occurrence of only one 5S rDNA locus and the smaller chromosome size in the polyploids suggest that DNA sequence losses took place following polyploidization events.

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.

Diversity and evolution of the Hordeum murinum polyploid complex in Algeria

Population diversity and evolutionary relationships in the Hordeum murinum L. polyploid complex were explored in contrasted bioclimatic conditions from Algeria. A multidisciplinary approach based on morphological, cytogenetic, and molecular data was conducted on a large population sampling. Distribution of diploids (subsp. glaucum) and tetraploids (subsp. leporinum) revealed a strong correlation with a North-South aridity gradient. Most cytotypes exhibit regular meiosis with variable irregularities in some tetraploid populations. Morphological analyses indicate no differentiation among taxa but high variability correlated with bioclimatic parameters. Two and three different nuclear sequences (gene coding for an unspliced genomic protein kinase domain) were isolated in tetraploid and hexaploid cytotypes, respectively, among which one was identical with that found in the diploid subsp. glaucum. The tetraploids (subsp. leporinum and subsp. murinum) do not exhibit additivity for 5S and 45S rDNA loci comparative with the number observed in the related diploid (subsp. glaucum). The subgenomes in the tetraploid taxa could not be differentiated using genomic in situ hybridization (GISH). Results support an allotetraploid origin for subsp. leporinum and subsp. murinum that derives from the diploid subsp. glaucum and another unidentified diploid parent. The hexaploid (subsp. leporinum) has an allohexaploid origin involving the two genomes present in the allotetraploids and another unidentified third diploid progenitor.

The plant-specific family of DNA-binding proteins containing three HMG-box domains interacts with mitotic and meiotic chromosomes

• The high mobility group (HMG)-box represents a DNA-binding domain that is found in various eukaryotic DNA-interacting proteins. Proteins that contain three copies of the HMG-box domain, termed 3 × HMG-box proteins, appear to be specific to plants. The Arabidopsis genome encodes two 3 × HMG-box proteins that were studied here. • DNA interactions were examined using electrophoretic mobility shift assays, whereas expression, subcellular localization and chromosome association were mainly analysed by different types of fluorescence microscopy. • The 3 × HMG-box proteins bind structure specifically to DNA, display DNA bending activity and, in addition to the three HMG-box domains, the basic N-terminal domain contributes to DNA binding. The expression of the two Arabidopsis genes encoding 3 × HMG-box proteins is linked to cell proliferation. In synchronized cells, expression is cell cycle dependent and peaks in cells undergoing mitosis. 3 × HMG-box proteins are excluded from the nuclei of interphase cells and localize to the cytosol, but, during mitosis, they associate with condensed chromosomes. The 3 × HMG-box2 protein generally associates with mitotic chromosomes, while 3 × HMG-box1 is detected specifically at 45S rDNA loci. • In addition to mitotic chromosomes the 3 × HMG-box proteins associate with meiotic chromosomes, suggesting that they are involved in a general process of chromosome function related to cell division, such as chromosome condensation and/or segregation.

Identification of novel trypanosome genotypes in native Australian marsupials

In the present study, the occurrence and molecular phylogeny of trypanosome parasites were studied in both wild and captive marsupials from Western Australia and Queensland. Blood samples were screened by PCR at the 18S rDNA locus, and the glycosomal glyceraldehyde phosphate dehydrogenase gene.Overall, 5.3% of the blood samples were positive at the 18S rDNA locus. All positives belonged to wild-captured Western Australian individuals, where trypanosome-specific DNA was detected in 9.8% of the screened samples from wild marsupials, in common brushtail possums, and woylies. The detection rate of trypanosome DNA in these two host species was 12.5% and 20%, respectively.Phylogenetic analyses based on two loci, indicated that the possum-derived trypanosome isolates were genetically distinct, and most closely related to the Australian marsupial trypanosomes H25 from a kangaroo, and BRA2 from a bush rat. This is the first study to genetically characterise trypanosome isolates from possums.The analysis of the woylie-derived isolates demonstrated that this marsupial host can harbour multiple genotypes within the same geographical location and furthermore multiple genotypes within the same host, indicative of mixed infections. All the woylie-derived genotypes grouped with trypanosomes found in Australian marsupials, suggesting that they are more likely to belong to an endemic or Australasian trypanosome species.This is the first study to genetically characterise trypanosome isolates from possums (Trichosurus vulpecula). Although the clinical significance of these infections is currently unknown, the identification of these novel sequences may support future investigations on transmission, threats to endangered wildlife, and evolutionary history of the genus Trypanosoma.

Karyotype and AFLP data reveal the phylogenetic position of the Brazilian endemic Hypochaeris catharinensis (Asteraceae)

The genus Hypochaeris offers an excellent model for studies of recent adaptive radiation in the South American continent. We used karyotype analysis with chromomycin A3 (CMA3)/4’,6-diamidino-2-phenylindole (DAPI) banding and fluorescence in situ hybridization (FISH), and amplified fragment length polymorphism (AFLP) fingerprinting to investigate for the first time the Brazilian endemic H. catharinensis and define its position within the South American group of species. Strong CMA-positive signals were seen at the end of both arms of chromosome 3 and at the end of the long arm of chromosome 4. DAPI bands were only detected in subterminal position on short arm of chromosome 4. FISH with 5S and 35S ribosomal DNA (rDNA) probes revealed a single 5S rDNA locus on short arm of chromosome 2, typical for all other South American Hypochaeris taxa analyzed to date. The 35S rDNA locus was identified at subterminal position on the short arm of chromosome 3, as reported so far for only two of the known species (H. lutea and H. patagonica). The AFLP study included 55 individuals, comprising nine species of the South American Hypochaeris plus their putative ancestor H. angustifolia. Eleven AFLP primer combinations generated a total of 401 fragments, of which 388 (96.7%) were polymorphic. High genetic similarities were observed among taxa, with all South American Hypochaeris species falling into one main cluster [100% bootstrap (BS)]. Hypochaeris catharinensis is closely related to H. lutea (82% BS), forming a well-separated subcluster within the South American species. Taken together, the karyological and AFLP data contribute to the placement of H. catharinensis within the phylogenetic framework of South American species of Hypochaeris and allow the definition of a novel and well-resolved phylogenetic group (the Lutea group).

Evolution of rDNA FISH patterns in the Fagaceae

The Fagaceae is one of the most important plant families in European forest ecosystems, and it includes several genera distributed in the Northern hemisphere. In this work we studied the genome organization and evolution within the family, by karyotyping and physically mapping rDNA in ten European and Asian species of the genera Fagus, Quercus, and Castanea. All of the species studied had a chromosome number of 2n=2x=24, except for the first report of a single individual of Quercus suber which proved to be triploid (2n=3x=36). The rDNA physical mapping revealed several patterns: the dominant one is present in European and Asian Quercus subgenus Quercus, and in Castanea sativa and Castanea crenata, consisting of two 18S–25S rDNA loci (one subterminal major and one pericentromeric minor) and one 5S rDNA pericentromeric locus. In Fagus sylvatica and in Quercus sessilifolia, different patterns were observed: four terminal 18S–25S rDNA loci and two 5S rDNA pericentromeric loci in the former, and five 18S–25S rDNA loci (three terminal and two intercalary) and one 5S rDNA pericentromeric locus in the latter. In Castanea mollissima a distinct rDNA distribution pattern with two intercalary 18S–25S rDNA loci and two 5S rDNA was found. These findings suggest rDNA loci restructuring during Castanea evolution, and variability of 18S–25S loci between Quercus and Cyclobalanopsis subgenera.

Karyotype analysis of Lilium tigrinum by FISH

The detailed karyotypes of diploid (2n = 2x = 24) and triploid (2n = 3x = 36) of L. tigrinum were constructed based on fluorescence in situ hybridization (FISH) using 5S rDNA and 45S rDNA probes. The lengths of mitotic metaphase chromosomes ranged from 15.92 to 30.18 um, with a total length/genome of 250.46 um in diploid, and 15.24 to 28.16 um, with a total length/genome of 233.42 um in triploid. The chromosome composed of two pairs of metacentrics (Chromosomes 1 and 2), four pairs of subtelocentrics (Chromosomes 3, 4, 5, and 6) and six pairs of telocentrics (Chromosomes 7, 8, 9, 10, 11, and 12) in diploid L. tigrinum, two pairs of metacentrics (Chromosomes 1 and 2), six pairs of subtelocentrics (Chromosomes 3, 4, 5, 6, 7, and 11) and four pairs of telocentrics (Chromosomes 8, 9, 10, and 12) in triploid L. tigrinum. Ten (Chromosomes 1, 2, 6, 7, and 11) or twelve (Chromosomes 1, 2, 4, 6, 7, and 11) loci of 45S rDNA were detected in the diploid chromosomes whereas fifteen loci were detected in triploid chromosomes (Chromosomes 1, 2, 6, 7, and 11). Two loci of 5S rDNAs were located close to each other at the both diploid and triploid chromosome 3. New cytotypes of secondary constriction were observed in diploid (1-1-1-0) and triploid (3-2-1-1).

Chromosomal Localization of the 5S and 45S rDNA Sites and 5S rDNA Sequence Analysis in Nelumbo Species

The physical location of 45S and 5S rDNA sites by double-target fluorescence in situ hybridization (FISH) and 5S rDNA non-transcribed spacer (NTS) sequences was studied in five accessions of Nelumbo nucifera Gaertn. and one accession of N. nucifera subsp. lutea (Willd.). The chromosome number of all tested materials was 2n=16. The loci of 5S rDNA were close to the centromere regions of chromosome 3, and the loci of 45S rDNA were found on chromosomes 7 and 8 in all the six tested accessions, respectively. Similarly, 45S rDNAs were also located on chromosome 4 in four tested accessions. The 5S rRNA gene sequences were conserved and the NTS sequences were variable among the samples. N. nucifera subsp. lutea was the longest branch in the phylogenetic tree and clustered with N. nucifera cv. Liangzihu. N nucifera cv. Heilongjiang and N nucifera cv. Weishanhu showed a close relationship with each other. N nucifera cv. Dahe Lian was closer to N nucifera cv. Nihelu Lian than to other tested accessions. Analysis of the molecular karyotype and the 5S rRNA gene spacer sequence suggested the genetic diversity was limited within Nelumbo species and it seems suitable that American lotus was considered as the subspecies of N nucifera.

Comparative analysis of the N-genome in diploid and polyploid Aegilops species

The genetic classification for the N-genome chromosomes has been developed on the basis of C-banding analysis on the set of Triticum aestivum × Aegilops uniaristata single chromosome addition lines and examination of A. uniaristata (2n = 2 × = 14, NN), Aegilops ventricosa (2n = 4 × = 28, DDNN) and Aegilops recta (2n = 6 × = 42, UUX(n)X(n)NN) accessions carrying intergenomic translocations using fluorescence in situ hybridisation with probes for three repetitive DNA sequences as well as the 5S and 45S rDNA families. The N-genome chromosomes of the tetraploid A. ventricosa show significant changes relative to the diploid progenitor species, while those of the hexaploid A. recta are similar to A. uniaristata with regard to the distribution of C-bands, 45S and 5S rDNA loci and hybridisation sites of all the three families of tandem repeats. The possible mechanisms of N-genome evolution are discussed.

Molecular and cytogenetic assessment of transgenic hot peppers resistant to Cucumber mosaic Virus

In this study, we have analyzed Cucumber mosaic virus (CMV)-resistant transgenic hot pepper (Capsicum annuum) plants harboring coat protein gene (CMVP0-CP) of the virus as a framework for molecular and cytogenetic assessment. PCR, Southern blot, RT-PCR, and Western blot techniques were applied to identify the existence and expression of the transgene in two independent transgenic hot pepper events in T4 generation, H15 and B20. Fluorescence in situ hybridization (FISH) was also conducted to detect physical location of the transgene on the chromosome of the plants. The amplified products of the inserted genes, 35S promoter with CMVP0-CP, CMVP0-CP, and NPT II were obtained from the genomic DNA of the transgenic hot pepper lines by PCR. A single band, indicative of a single copy insertion, was observed by Southern blot analysis. Transcription of the transgene was confirmed from RT-PCR fragments CMVP0-CP and NPT II genes in transgenic plants. Translated protein of the target gene, CMV-CP, was detected in the transgenic hot pepper events but not in nontransgenic control hot pepper lines by Western blot analysis. FISH analysis of transgenic line H15 revealed that the transgene was physically located at the sub-terminal portion of the long arm of chromosome d where the 5S rDNA locus is positioned. Conclusively, we observed that transgenes were maintained stably in T4 generation of transgenic hot peppers via cytogenetic investigation as well as molecular detection. These results can be useful in future studies conducted to evaluate breeding techniques and to assess genetically modified hot peppers.