Glucosinolates,flea beetle resistance,and leaf pubescence as taxonomic characters in the genus Barbarea (Brassicaceae)

Glucosinolate content of leaves and roots, diversity in leaf pubescence, and resistance to two near-isogenic lines of the flea beetle Phyllotreta nemorum with or without an R-gene, were determined for 27 accessions of 7 Barbarea taxa, i.e. B. stricta, B. orthoceras, B. intermedia, B. verna, B. vulgaris var. vulgaris, the G-type of B. vulgaris var. arcuata and the P-type of B. vulgaris var. arcuata. Four variable glucosinolate biosynthetic characters were deduced. For (formally) homophenylalanine-derived glucosinolates: (1). Presence or absence of 2-hydroxylation, and if present, R- or S-configuration of 2-hydroxylation; (2). presence or absence of p-hydroxylation; and for tryptophan-derived glucosinolates: (3). presence or absence of N-methoxyglucobrassicin; and (4). presence or absence of 1,4-dimethoxyglucobrassicin. Three phenotypes of leaf-pubescence were observed; (1). glabrous to glabrate leaves; (2). glabrous to glabrate leaves with hairs along the edge; (3). pubescent leaves. The hairs were characterized as simple by scanning electron microscopy. Full resistance to a flea beetle line (ST) was found in B. vulgaris var. vulgaris and in the G-type of var. arcuata; partial resistance was found in B. verna and B. intermedia, while the remaining taxa were fully susceptible to the ST line. All investigated Barbarea taxa were susceptible to larvae from another line containing an R-gene, indicating a similar flea beetle resistance mechanism in the three resistant species. Most Barbarea taxa could be characterized by a particular combination of the investigated characters. The most aberrant was the P-type of B. vulgaris var. arcuata, and the taxonomic status of this type should be reconsidered.     

Genetics of the ability of Phyllotreta nemorum larvae to survive in an atypical host plant, Barbarea vulgaris ssp. arcuata

A polymorphism in host plant exploitation has been discovered in the flea beetle, Phyllotreta nemorum L. (Coleoptera: Chrysomelidae: Alticinae) where one resistant population is able to use Barbarea vulgaris R.Br. ssp. arcuata (Opiz.) Simkovics (Brassicaceae) as a host plant while a susceptible population is not. Crosses (F1, F2, and backcrosses) between the two flea beetle populations were made, and survival of the progeny on B. v. ssp. arcuata was measured. The ability of P. nemorum larvae to survive in this plant species depended on the presence of major, dominant genes (R-genes). The two most abundant R-genes in the resistant flea beetle population were X- and Y-linked, respectively. The use of B. v. ssp. arcuata as a natural host plant by the resistant population of P. nemorum seems to be an extension of the host plant range of the species. The role of sex-linked genes in the evolution of host range is discussed.     

Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species

Thirty-nine cultivars of cassava and eight related wild species of Manihot were analyzed in this work for number, morphology and size of chromosomes, prophase condensation pattern and the structure of the interphase nucleus. In four accessions, the chromosome size was measured and in some others, the number of secondary constrictions, meiotic behavior, C-band pattern, CMA/DAPI bands, nucleoli number and the location of 5S and 18S-5.8S-28S rDNA sites were also observed. All investigated accessions showed a similar karyotype with 2n = 36, small metacentric to submetacentric chromosomes. Two pairs of terminal secondary constrictions were observed in the chromosome complement of each accession except Manihot sp. 1, which presented two proximal secondary constrictions. The prophase chromosome condensation pattern was proximal and the interphase nuclei structure was areticulate to semi-reticulate. The meiosis, investigated in seven cultivars and four wild species, was regular, displaying 18 bivalents. C-banding revealed heterochromatin in 9 or 10 chromosomes. The analysis with fluorochromes frequently showed four chromosome pairs with a single CMA+ terminal or subterminal band and a few other chromosomes with DAPI+ unstable bands. Six 45S rDNA sites were revealed by FISH, which seemed to colocalize with six CMA+ bands. Only one chromosome pair presented a 5S rDNA site. The maximum nucleoli number observed per nucleus was also six. These data suggest that all Manihot species present a very similar chromosome complement.     

Differential rRNA Genes Expression in Hexaploid Wheat Related to NOR Methylation

Ribosomal RNA genes expression was analysed in 18 Portuguese bread wheat accessions (Triticum aestivum L. em Thell.), and the number of argyrophilic-nucleolar organiser regions (Ag-NORs) per cultivar was scored. Ten accessions presented six Ag-NORs per metaphase and six nucleoli per interphase, and eight accessions presented four Ag-NORs per metaphase and four nucleoli per interphase. Fluorescent in situ hybridisation with the 45S rDNA sequence pTa71 and genomic DNA from Aegilops tauschii (2n = 2x= 14, DD) confirmed the Ag-NOR location and identified the six satellited chromosomes as being the chromosome pairs 1B, 6B and 5D. The methylation pattern of the NOR region was studied by Southern blot using pTa71 as probe, which represented a complete rDNA unit of bread wheat. DNA digestions performed by MspI and HpaII resulted in different patterns revealing the high level of cytosine methylation at their recognition sequences. The total percentage values of NOR methylation indicated that wheat accessions with a maximum of four Ag-NORs were more heavily methylated at the NOR region than accessions with a maximum of six Ag-NORs.     

Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae

BACKGROUND AND AIMS: The Brassicaceae family encompasses numerous species of great agronomic importance, belonging to such genera, as Brassica, Raphanus, Sinapis and Armoracia. Many of them are characterized by extensive intraspecific diversity of phenotypes. The present study focuses on the polymorphism of number, appearance and chromosomal localization of ribosomal DNA (rDNA) sites and, when possible, in relation to polyploidy, in 42 accessions of Brassica species and ten accessions of Diplotaxis, Eruca, Raphanus and Sinapis species. METHODS: Chromosomal localization of ribosomal DNA was carried out using dual colour fluorescence in situ hybridization (FISH) with 5S rDNA and 25S rDNA sequences as probes on enzymatically digested root-tip meristematic cells. KEY RESULTS: Loci for 5S and 18S-5.8S-25S rDNA were determined for the first time in six taxa, and previously unreported rDNA constellations were described in an additional 12 accessions. FISH revealed frequent polymorphism in number, appearance and chromosomal localization of both 5S and 25S rDNA sites. This phenomenon was most commonly observed in the A genome of Brassica, where it involves exclusively pericentromeric sites of 5S and 25S rRNA genes. The intraspecific polymorphism was between subspecies/varieties or within a variety or cultivar (i.e. interindividual). CONCLUSIONS: The number of rDNA sites can differ up to 5-fold in species with the same chromosome number. In addition to the eight previously reported chromosomal types with ribosomal genes, three new variant types are described. The extent of polymorphism is genome dependent. Comparing the A, B and C genomes revealed the highest rDNA polymorphism in the A genome. The loci carrying presumably inactive ribosomal RNA genes are particularly prone to polymorphism. It can also be concluded that there is no obvious polyploidization-related tendency to reduce the number of ribosomal DNA loci in the allotetraploid species, when compared with their putative diploid progenitors. The observed differences are rather caused by the prevailing polymorphism within the diploids and allotetraploids. This would make it difficult to predict expected numbers of rDNA loci in natural polyploids.     

Variation in chromosome numbers, CMA bands and 45S rDNA sites in species of Selaginella (Pteridophyta)

BACKGROUND AND AIMS: Selaginella is the largest genus of heterosporous pteridophytes, but karyologically the genus is known only by the occurrence of a dysploid series of n=7-12, and a low frequency of polyploids. Aiming to contribute to a better understanding of the structural chromosomal variability of this genus, different staining methods were applied in species with different chromosome numbers. METHODS: The chromosome complements of seven species of Selaginella were analysed and, in four of them, the distribution of 45S rDNA sites was determined by fluorescent in situ hybridization. Additionally, CMA/DA/DAPI and silver nitrate staining were performed to investigate the correlation between the 45S rDNA sites, the heterochromatic bands and the number of active rDNA sites. KEY RESULTS: The chromosome numbers observed were 2n=18, 20 and 24. The species with 2n=20 exhibited chromosome complement sizes smaller and less variable than those with 2n=18. The only species with 2n=24, S. convoluta, had relatively large and asymmetrical chromosomes. The interphase nuclei in all species were of the chromocentric type. CMA/DA/DAPI staining showed only a weak chromosomal differentiation of heterochromatic bands. In S. willdenowii and S. convoluta eight and six CMA+ bands were observed, respectively, but no DAPI+ bands. The CMA+ bands corresponded in number, size and location to the rDNA sites. In general, the number of rDNA sites correlated with the maximum number of nucleoli per nucleus. Ten rDNA sites were found in S. plana (2n=20), eight in S. willdenowii (2n=18), six in S. convoluta (2n=24) and two in S. producta (2n=20). CONCLUSIONS: The remarkable variation in chromosome size and number and rDNA sites shows that dramatic karyological changes have occurred during the evolution of the genus at the diploid level. These data further suggest that the two putative basic numbers of the genus, x=9 and x=10, may have arisen two or more times independently.     

Karyotypes of Elymus scabrifolius (Poaceae: Triticeae) from South America studied by banding techniques and in situ hybridization

Karyotypes of 4 accessions of Elymus scabrifolius (2n = 4x = 28) were investigated by Giemsa C- and N-banding, GAA-banding (one accession), AgNO3-staining and in situ hybridization with the rDNA probe pTa71. Two additional accessions were studied in less detail. The chromosomes were large (9-14 microns). The complements included 11 pairs of metacentrics, one with conspicuous satellites on the short arms, and 3 pairs of submetacentrics. Two of 4 accessions from Eastern Argentina and Uruguay had minute or small satellites on a submetacentric pair. No such satellites were observed in the other two accessions. In two accessions from the Cordoba province, a non-homologous submetacentric pair had very long satellites. AgNO3-staining established the presence of 4 nucleoli, two larger and two small ones, in 5 accessions. The C-banding patterns comprised from one to 12 conspicuous bands per chromosome at no preferential positions. The amount of constitutive heterochromatin (19-21%) was the highest hitherto established in the Triticeae. Similarities in banding patterns and chromosome morphology identified homologous and discriminated between non-homologous chromosomes within and, except for two chromosomes, between plants. Heteromorphic chromosome pairs were identified in satellite-carrying chromosomes only. N-banding produced conspicuous bands overall at the same positions as C-banding. GAA-banding patterns were similar to N-banding patterns. The rDNA probe hybridized to chromosome segments at nucleolar constrictions only. The production of C- and N-banding patterns in both genomes of E. scabrifolius suggests the presence of two H genomes and the absence of the pivotal St genome of Elymus. On account of the uncertain identity of one genome, and the overall similar gross morphology of E. scabrifolius and other tetraploid South American species referred to Elymus, E. scabrifolius is retained in Elymus.     

Genetic diversity and similarity in the Barbarea vulgaris complex (Brassicaceae)

Two subspecies of Barbarea vulgaris are taxonomically recognized as ssp. vulgaris and ssp. arcuata. In addition, two types of Barbarea vulgaris ssp. arcuata occurs in Denmark. The G‐type is resistant to an herbivorous flea beetle (Phyllotreta nemorum) whereas the P‐type is susceptible. A previous study suggested that the P‐type evolved by a loss of resistance from a resistant progenitor. We analyzed the genetic relatedness among eight Barbarea taxa: B. vulgaris spp. vulgaris, B. vulgaris ssp. arcuata G‐ and P‐types, hybrids between the types, B. verna, B. intermedia, B. stricta, B. orthoceras and B. australis, using AFLP and SSR markers. A clear distinction between the G‐ and P‐types was revealed. Both were distinct from B. vulgaris ssp. vulgaris, the G‐type less so than the P‐type. Barbarea verna and B. intermedia formed unambiguous clusters, whereas the remaining taxa produced less discrete groupings. Possible evolutionary scenarios for flea‐beetle resistance and susceptibility are discussed, including lineage sorting from a polymorphic ancestral population, and de novo loss of resistance in the P‐type of B. vulgaris ssp. arcuata.     

Seasonal variation in leaf glucosinolates and insect resistance in two types of Barbarea vulgaris ssp. arcuata

Leaves from natural populations of Barbarea vulgaris ssp. arcuata (Brassicaceae) in Denmark were examined for glucosinolate content and resistance to the crucifer specialist flea beetle Phyllotreta nemorum. Two types of the plant (P- and G-type) could be recognized. Leaves of the G-type contained the glucosinolates (only side chains mentioned): (S)-2-hydroxy-2-phenylethyl- (2S), indol-3-ylmethyl- (4) and in trace amount (R)-2-hydroxy-2-phenylethyl- (2R), 2-phenylethyl- (1) and 4-methoxyindol-3-ylmethyl- (5). Leaves of the P-type were dominated by 2R and 4, and had only trace amounts of 1, 2S, and 5 but contained in addition the previously unknown (R)-2-hydroxy-2-(4-hydroxyphenyl)ethyl- (3R). The epimer, (S)-2-hydroxy-2-(4-hydroxyphenyl)ethyl- (3S) was found in populations believed to be hybrids, and in B. orthoceras. 2S, 2R, desulfo 2S,-2R, -3S and -3R were isolated and identified by NMR and MS. Acylated glucosinolates or allylglucosinolate were not detected in leaves. The glucosinolate content in August was variable, 3-46 micromol/g dry wt, but was low in most populations, 3-15 micromol/g dry wt. In general, the glucosinolate content increased during the autumn, to 35-75 micromol/g dry wt in November. The G-type was resistant to neonate larvae of Phyllotreta nemorum in August and September (survival in 3-day bioassay typically 0%), and gradually lost the resistance in October and November (survival in 3-day bioassay 40-90%), and there was no correlation between glucosinolate content and resistance. Neither did glucosinolates explain the difference in resistance between the P-type (always susceptible) and the G-type (resistant in the summer season).     

Genetic differentiation in the Bladder campions, Silene vulgaris and S. uniflora (Caryophyllaceae), in Sweden

Allozyme variation was studied in Swedish populations of Silene vulgaris (a widespread weed), S. uniflora ssp. uniflora (restricted to coastal habitats) and S. uniflora ssp. petraea (endemic to Sweden and confined to open limestone habitats). The taxa are diploid, gynodioecious, perennial herbs and showed high levels of within-taxon and within-population gene diversity at four polymorphic loci. Within-taxon diversity was highest (H_tax=0.52) in S. vulgaris and lowest (H_tax=0.36) in S. uniflora ssp. uniflora. The weedy S. vulgaris has more alleles than either of the other two taxa and 5 out of a total of 27 alleles are unique to S. vulgaris. Most of the gene diversity within each of the taxa is accounted for by within-population diversity. The between-population component of diversity is 10% in S. vulgaris , and 24% and 5%, respectively, in S. uniflora ssp. uniflora and ssp. petraea. Hybrids may occur between S. vulgaris and S. uniflora , but introgression is limited by the species' ecology. Neither allozyme nor distributional data support the suggestion that ssp. petraea is a recent hybrid between S. vulgaris and S. uniflora ssp. uniflora , although an older hybrid origin for ssp. petraea is possible. Patterns of allele frequency variation suggest that there has been some historical gene flow between taxa, outside their present areas of sympatry. It is likely that the two subspecies of S. uniflora , which occur in naturally open habitats, were able to colonize Sweden during the Late Glacial or early post-glacial, whereas S. vulgaris followed the spread of agriculture into Sweden.     

Localization of ribosomal genes and nucleolar activity in lymphocytes of swine (Sus scrofa domestica) stimulated by phytohemagglutinins

The Pig chromosomes that contain rDNA sites displayed a polymorphism in the distribution of the genes among the nucleolar organizers located on pairs Nos. 8 and 10. Two, or more often three, active sites were observed in the chromosomes of lymphocytes stimulated with phytohemagglutinin. Only 5% of the metaphases showed a 4th small active site. At the onset of stimulation most cells contained one-two nucleoli; four nucleoli were never observed. After prolonged stimulation, the number of nuclei containing three nucleoli increased. A 4th small nucleolus appeared in a few cells, presumably formed by activation of the smallest rDNA site.     

Chloroplast DNA phylogeny of Lens (Leguminosae): origin and diversity of the cultivated lentil

A restriction-site analysis of chloroplast DNA (cpDNA) variation in Lens was conducted to: (1) assess the levels of variation in Lens culinaris ssp. culinaris (the domesticated lentil), (2) identify the wild progenitor of the domesticated lentil, and (3) construct a cpDNA phylogeny of the genus. We analyzed 399 restriction sites in 114 cultivated accessions and 11 wild accessions. All but three accessions of the cultivar had identical cpDNAs. Two accessions exhibited a single shared restriction-site loss, and a small insertion was observed in the cpDNA of a third accession. We detected 19 restriction-site mutations and two length mutations among accessions of the wild taxa. Three of the four accessions of L. culinaris ssp. orientalis were identical to the cultivars at every restriction site, clearly identifying ssp. orientalis as the progenitor of the cultivated lentil. Because of its limited cpDNA diversity, we conclude that either the cultivated lentil has passed through a genetic bottleneck during domestication and lost most of its cytoplasmic variability or else was domesticated from an ancestor that was naturally depauperate in cpDNA restriction-site variation. However, because we had access to only a small number of populations of the wild taxa, the levels of variation present in ssp. orientalis can only be estimated, and the extent of such a domestication bottleneck, if applicable, cannot be evaluated. The cpDNA-based phylogeny portrays Lens as quite distinct from its putative closest relative, Vicia montbretii. L. culinaris ssp. odemensis is the sister of L. nigricans; L. culinaris is therefore paraphyletic given the current taxonomic placement of ssp. odemensis. Lens nigricans ssp. nigricans is by far the most divergent taxon of the genus, exhibiting ten autapomorphic restriction-site mutations.     

Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan

Endosymbiotic green algae of Japanese Paramecium bursaria were phylogenetically analyzed based on DNA sequences from the ribosomal DNA operon (18S rDNA, ITS1, 5.8S rDNA, and ITS2). Phylogenetic trees constructed using 18S rDNA sequences showed that the symbionts belong to the Chlorella sensu stricto (Trebouxiophyceae) group. They are genetically closer to the C. vulgaris Beijerinck group than to C. kessleri Fott et Nováková as proposed previously. Branching order in C. vulgaris group was unresolved in 18S rDNA trees. Compared heterogeneities of 18S rDNA, ITS1, 5.8S r, and ITS2 among symbionts and two Chlorella species, indicated that the ITS2 region (and probably also ITS1) is better able to resolve phylogenetic problems in such closely related taxa. All six symbiotic sequences obtained here (approximately 4000-bp sequences of 18S rDNA, ITS1, 5.8S rDNA, and ITS2) were completely identical in each, strongly suggesting a common origin.     

Physical mapping of rRNA genes in Medicago sativa and M. glomerata by fluorescent in situ hybridization

Fluorescent in situ hybridization (FISH) was applied to diploid and tetraploid subspecies of alfalfa (Medicago sativa L.) to investigate the distribution of rRNA genes and to utilize the sites of 18S-5.8S-25S rDNA and 5S rDNA sequences as markers for studying the genome evolution within the species. Medicago glomerata Balb., the species considered to be the ancestor of alfalfa, was included in this study in order to obtain more information on the phylogenetics of alfalfa. Simultaneous in situ hybridization was performed with the probes pTa71 and pXVI labeled with digoxigenin and biotin, respectively. In the diploid taxa, M. glomerata, M. sativa ssp. coerulea Schmalh and ssp. falcata Arcangeli, the 18S-5.8S-25S rDNA sequences were mapped to two sites corresponding to the secondary constrictions of the nucleolar chromosome pair, while 5S rDNA appeared to be distributed in two pairs of sites. Chromosomes carrying 5S loci could be distinguished on the basis of their morphological characteristics. The number of rDNA sites detected in the tetraploid M. sativa ssp. falcata and ssp. sativa (L.) L. & L. were twice the number found in the respective diploid ssp. falcata and ssp. coerulea. The results of this study show that the distribution of ribosomal genes was maintained during the evolutionary steps from the primitive diploid to the cultivated alfalfa. Modifications of the number of rRNA loci were not observed. The importance of in situ hybridization for improving karyotype analysis in M. sativa L. is discussed.     

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.     

Impact of gene flow from cultivated beet on genetic diversity of wild sea beet populations

Gene flow and introgression from cultivated plants may have important consequences for the conservation of wild plant populations. Cultivated beets (sugar beet, red beet and Swiss chard: Beta vulgaris ssp. vulgaris) are of particular concern because they are cross-compatible with the wild taxon, sea beet (B.vs. ssp. maritima). Cultivated beet seed production areas are sometimes adjacent to sea beet populations; the numbers of flowering individuals in the former typically outnumber those in the populations of the latter. In such situations, gene flow from cultivated beets has the potential to alter the genetic composition of the nearby wild populations. In this study we measured isozyme allele frequencies of 11 polymorphic loci in 26 accessions of cultivated beet, in 20 sea beet accessions growing near a cultivated beet seed production region in northeastern Italy, and 19 wild beet accessions growing far from seed production areas. We found one allele that is specific to sugar beet, relative to other cultivated types, and a second that has a much higher frequency in Swiss chard and red beet than in sugar beet. Both alleles are typically rare in sea beet populations that are distant from seed production areas, but both are common in those that are near the Italian cultivated beet seed production region, supporting the contention that gene flow from the crop to the wild species can be substantial when both grow in proximity. Interestingly, the introgressed populations have higher genetic diversity than those that are isolated from the crop. The crop-to-wild gene flow rates are unknown, as are the fitness consequences of such alleles in the wild. Thus, we are unable to assess the long-term impact of such introgression. However, it is clear that gene flow from a crop to a wild taxon does not necessarily result in a decrease in the genetic diversity of the native plant.     

Distribution of highly repeated DNA sequences in species of the genus Lens Miller.

Multiple-target fluorescence in situ hybridization (FISH) was applied on mitotic chromosomes of seven Lens taxa using two highly repetitive sequences (pLc30 and pLc7) isolated from the cultivated lentil and the multigene families for the 18S-5.8S-25S (pTa71) and 5S rRNA (pTa794) from wheat simultaneously as probes. The number and location of pLc30 and pLc7 sites on chromosomes varied markedly among the species, whereas the hybridization pattern of 5S rDNA and 18S-5.8S-25S rDNA was less variable. In general, each species showed a typical FISH karyotype and few differences were observed among accessions belonging to the same species, except for the accessions of Lens odemensis. The most similar FISH karyotype to the cultivated lentil is that of Lens culinaris subsp. orientalis, whereas Lens nigricans and Lens tomentosus are the two species that showed the most divergent FISH patterns compared with all taxa for number and location of pLc30 and 18S-5.8S-25S rDNA sites.