Sonja Siljak-Yakovlev

Nuclear DNA content, base composition, heterochromatin and rDNA in Picea omorika and Picea abies

Abstract Two closely related spruces, Picea abies and Picea omorika, a Balkan paleoendemic species, often share habitats, yet never hybridize in nature. The present study adresses their characteristics such as nuclear DNA content, base composition, heterochromatin and rDNA pattern. The genome size of P. abies was 10% larger than that of P. omorika when assessed by flow cytometry, respectively 2C=37.2 pg and 33.8 pg; although when estimated as total chromosome length it was virtually the same. The heterochromatin Chromomycin-A (CMA)/ DAPI fluorochrome banding patterns of both P. abies and P. omorika are given here for the first time. Simultaneous FISH (fluorescent in situ hybridization) using 18S-26S and 5S rDNA probes revealed 16 18S rDNA sites in P. omorika, 12 18S rDNA sites in P. abies, and a single 5S rDNA locus in both species. The genomes have about 41% GC. The number and position of CMA/DAPI bands and rDNA loci provide good chromosome markers to clarify the karyotypes of the two species.

The origin and evolution of sweet potato (Ipomoea batatas Lam.) and its wild relatives through the cytogenetic approaches

The distribution and organization of 5S and 18S-5.8S-26S (18S) rDNA were studied in 10 varieties of hexaploid Ipomoea batatas, five accessions of tetraploid Ipomeoa trifida, and six related species (five diploids, I. trifida, I. triloba, I. tiliacea, I. leucantha and I. setosa and one tetraploid, I. tabascana), by using fluorescence in situ hybridization (FISH). The FISH data obtained indicated that polyploidization was followed by decrease in the number of 18S rDNA loci in higher ploidy level and provided evidence for major genomic rearrangements and/or diploidization in polyploid I. batatas. Among the five diploid species examined, I. trifida appeared to be the most closely related to I. batatas. By contrast, I. leucantha was closed to I. tiliacea, but both species were distant from sweet potato. I. triloba and I. setosa were distantly related to the rest of Ipomoea batatas complex. The close relationship between I. trifida and I. batatas was also demonstrated by the presence of one 18S and CMA marker in these two chromosome complements only. Based on chromosome morphology, tetraploid I. trifida appeared to be more closely related to sweet potato than I. tabascana. Taking all data obtained in this study, I. trifida might be the progenitor of I. batatas, and I. tabascana, interspecific hybrid between these two species.

Molecular-cytogenetic studies of ribosomal genes and heterochromatin reveal conserved genome organization among 11 Quercus species

Abstract Genomes of 11 Quercus species were characterized using cytogenetic (Giemsa C-banding, fluorochrome banding), molecular-cytogenetic (fluorescence in situ hybridization, FISH, to ribosomal genes) and molecular (dot-blot for ribosomal gene-copy number assessment) techniques. Ribosomal genes are the first DNA sequences to be physically mapped in oaks, and the copy number of the 18S-5.8S-26 S rRNA genes is estimated for the first time. Oak karyotypes were analysed on the basis of DAPI banding and FISH patterns; five marker chromosomes were found. In addition, chromosomal organization of ribosomal genes with respect to AT- and GC-differentiated heterochromatin was studied. Fluorochrome staining produced very similar CMA/DAPI banding patterns, and the position and number of ribosomal loci were identical for all the species studied. The 18S-5.8S-26 S rRNA genes in oak complements were represented by a major locus at the subterminal secondary constriction (SC) of the only subtelocentric chromosome pair and a minor locus at paracentromeric SC of one metacentric pair. The only 5 S rDNA locus was revealed at the paracentromeric region of the second largest metacentric pair. A striking karyotypic similarity, shown by both fluorochrome banding and FISH patterns, implies close genome relationships among oak species no matter their geographic origin (European or American) or their ecophysiology (deciduous or evergreens). Dot-blot analysis gave preliminary evidence for different copy numbers of 18S-5.8S-26 S rRNA genes in diploid genomes of Q. cerris, Q. ilex, Q. petraea, Q. pubescens and Q. robur (2700, 1300, 2200, 4000 and 2200 copies, respectively) that was correlated with the size polymorphism of the major locus.

Genome size in Echinops L. and related genera (Asteraceae, Cardueae): karyological, ecological and phylogenetic implications.

Abstract Genome size was assessed by flow cytometry in 33 species belonging to seven genera of the tribe Cardueae (Asteraceae), which can be grouped in three taxonomic complexes. 2C nuclear DNA content ranged from 1.49 to 16.98 pg, which is more than elevenfold variation. Genome size correlated well with some karyological traits. Nuclear DNA amount variations also have systematic and evolutionary implications and/or are linked to adaptations to ecological conditions.

Chromosomal differentiation and genome size in three European mountain Lilium species

Abstract. Three related and taxonomically close species of the genus Lilium (L. pyrenaicum Gouan, L. pomponium L. and L. carniolicum Bernh.), all of them with 2n=24 chromosomes, have been studied for chromosomal differentiation, using fluorochrome banding and fluorescence in situhybridization (FISH), and for genome size and GC percentage using flow cytometry. The total DNA content of L. pomponium (2C=70.26 pg) was about 5% higher than that of L. pyrenaicum (2C=67.74) and L. carniolicum (2C=67.37 pg), while GC percentage was higher in this last species (36.60%) than in L. pomponium (35.56%) and lower than in L. pyrenaicum (37.92%). Silver staining, fluorochrome banding with chromomycin A3 (CMA) and fluorescence in situ hybridization (FISH) clearly pointed out the number of nucleoli, the number and position of GC-rich bands and the number and location of rDNA sites thus permitting distinction of the three species at chromosomal level. Two families of ribosomal genes, 18S-5.8S-26S (18S) and 5S rRNA genes, were separated onto different pairs in chromosome complements of examined species. Chromosome regions containing both kinds of rRNA genes were also GC-rich regions. The results revealed a clear interspecific differentiation at the chromosomal level and permitted the discussion about relationships among the species.

Extensive ribosomal DNA (18S-5.8S-26S and 5S) colocalization in the North American endemic sagebrushes (subgenus Tridentatae, Artemisia, Asteraceae) revealed by FISH

Chromomycin A3 banding and fluorescent in situ hybridization (FISH) have been performed for six Artemisia species with special emphasis on subgenus Tridentatae. Morphometrical data on karyotype characters were calculated and idiograms with the position of GC-rich regions and 18S-5.8S-26S and 5S sites of ribosomal DNA were constructed. These sites were all colocalized. To our knowledge, this is the first time in the large family Asteraceae, indeed in angiosperms in general, that colocalization of the two rDNA regions studied is found at every single marked locus. In addition, transcriptionally active nucleolar organizer regions were detected after silver nitrate staining. Tridentatae is a cytogenetically homogeneous subgenus, which suggests that evolution of these species has not been coupled with important karyotypic reorganization. However, a few species are taxonomically difficult and show substantial differences. A loss of rDNA loci has been detected in a tetraploid taxon with respect to the diploids studied. These data provide clarifying insight into interspecific relationships between the studied taxa and overall evolutionary and systematic relationships of the Tridentatae.

Karyotype evolution in South American species of Hypochaeris (Asteraceae, Lactuceae)

Abstract.The genus Hypochaeris (Asteraceae, Lactuceae) contains ten species in Europe, three in Asia, and approximately 50 in South America. Previous cytotaxonomic studies have shown two groups of taxa: (1) European species with different basic chromosome numbers and differentiated karyotypes, and (2) South American species with x=4 and uniform asymmetric and bimodal karyotypes. Karyotypic data are synthesized for South American species of Hypochaeris with new information for six Chilean species: H. acaulis, H. apargioides, H. palustris, H. spathulata, H.tenuifolia and H. thrincioides. Four main groups can be distinguished based on presence and localization of secondary constrictions – SCs (bearing Nucleolar Organizer Regions – NORs) on chromosomes 2 and 3, and 18S–25S and 5S rDNA loci number, localization, and activity. We propose karyotypic evolution of South American Hypochaeris (x=4) from H. maculata-like (x=5) European ancestors. The original South American karyotype would have possessed two SCs, one on the long arm of chromosome 2, and the other on the short arm of chromosome 3 (in terminal position). Further evolution would have involved inversion within the short arm of chromosome 3 and inactivation/loss of the SC on chromosome 2.

Molecular cytogenetics of the genus Artemisia (Asteraceae, Anthemideae): fluorochrome banding and fluorescence in situ hybridization. I. Subgenus Seriphidium and related taxa

AbstractThe distributional pattern of AT- and GC-rich regions and the physical mapping of ribosomal DNA (location of 18S-5.8S-26S and 5S rDNA) in the chromosomes of seven Artemisia species have been established by means of fluorochrome banding and fluorescence in situ hybridization (FISH). This is the first study in the large genus Artemisia using FISH. Five species (A. barrelieri, A. caerulescens subsp. gallica, A. fragrans, A. herba-alba subsp. valentina, A. herba-alba subsp. herba-alba) belong to the subgenus Seriphidium, one of the most homogeneous in the genus; one (A. tridentata susbp. spiciformis) belongs to the small subgenus Tridentatae, classically included in Seriphidium; and one (A. annua) belongs to the subgenus Artemisia, but shows some affinities with Seriphidium. Genome organization is relatively constant in all the species studied. AT- and GC-rich DNA is predominantly terminal, but some intercalary and centromeric bands also exist. The rDNA loci are also most often terminal and usually located in GC-rich regions. 5S rDNA sites are present in a lower number than 18S-5.8S-26S sites, and are always colocated with some of them. In the light of these cytogenetic features, subgenus Seriphidium is clearly placed within the genus Artemisia, so that it does not make sense to segregate it as a genus; on the other hand, subgenus Tridentatae must not be classified within Seriphidium, but kept as an independent subgenus.

Genome size variation and species relationships in the genus Hydrangea

Abstract Genome size and base composition in 16 species and subspecies of the Hydrangea, a woody ornamental genus of Hydrangeaceae, were evaluated by flow cytometry in relation to their chromosome number. This is the first such study concerning the genome size of these species together with a karyotype study of the most important species, Hydrangea macrophylla subsp. macrophylla (Hortensia), from an economical point of view. The 2C DNA content ranged from 1.95 pg in Hydrangea quercifolia to 5.00 pg in Hydrangea involucrata. The base composition ranged from 39.9% GC in Hydrangea aspera subsp. sargentiana to 41.1% in Hydrangea scandens subsp. scandens (significant difference at p < 0.05). The smallest genome sizes were those of the three species originating from North or South America. Most of the species studied presented a chromosome number of 2n = 2x = 36, except for those of the section Aspereae which showed 2n = 30, 34 and 36. A primary karyotype has been made for the first time for H. macrophylla subsp. macrophylla. Phylogenetic relationships between species, the origin of chromosome number and an exploration of the genetic diversity within the genus are discussed.

Source of resistance against Ralstonia solanacearum in fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L

Solanum aethiopicum is reported to carry resistance to bacterial wilt disease caused by Ralstonia solanacearum, which is one of the most important diseases of eggplant (Solanum melongena). These two species can sexually be crossed but the fertility of their progeny is very low. In order to transfer the resistance and improve the fertility, somatic hybrids between S. melongena cv. Dourga and two groups of S. aethiopicum were produced by electrical fusion of mesophyll protoplasts. Thirty hybrid plants were regenerated. When transferred to the greenhouse and transplanted in the field, they were vigorous and showed intermediate morphological traits. Their ploidy level was determined by DNA analysis through flow cytometry, and their hybrid nature was confirmed by examining isozymes and RAPDs patterns. Chloroplast DNA microsatellite analysis revealed that 18 hybrids had the chloroplasts of the eggplant and 12 those of the wild species. The parents and 16 hybrids were evaluated in the field for their fertility and resistance to bacterial wilt using a race 1, biovar 3 strain of R. solanacearum. All hybrids were fertile and set fruit with viable seeds. Their yield was either intermediate or as high as that of the cultivated eggplant. Both groups of S. aethiopicum were found tolerant to R. solanacearum, as about 50% of plants wilted after 8 weeks. The cultivated eggplant was susceptible with 100% of wilted plants 2 weeks after inoculation. All somatic hybrids tested were as tolerant as the wild species, except six hybrids showing a better level of resistance.