Restriction enzyme analysis of the chloroplast and nuclear 45s ribosomal DNA ofAllium sectionsCepa andPhyllodolon (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of molecular characters. Restriction enzyme analysis of the chloroplast DNA (cpDNA) of the bulb onion (Allium cepa), Japanese bunching onion (A. fistulosum), wildAllium species in sect.Cepa andPhyllodolon, and the outgroupsA. ampeloprasum andA. tuberosum detected 39 polymorphisms.Allium cepa andA. vavilovii were identical for all characters. Cladistic analysis generated three most-parsimoniousWagner trees of 44 steps differing only in a zero-length branch.Allium fistulosum andA. altaicum (sect.Phyllodolon) comprised a monophyletic lineage separated from theA. cepa andA. vavilovii of sect.Cepa. The unresolved node was composed ofA. galanthum, A. roylei, and the lineage containingA. cepa, A. vavilovii, A. fistulosum, andA. altaicum. The clade containingA. altaicum, A. cepa, A. fistulosum, A. galanthum, A. roylei, andA. vavilovii remained resolved for strict consensus ofWagner trees of 48 steps or less.Allium pskemense andA. oschaninii were increasingly distant.Allium oschaninii has been proposed as the progenitor of the bulb onion, but was more closely related to the common progenitor of all species in sect.Cepa andPhyllodolon. Phylogenies estimated from cpDNA characters usingDollo parsimony resulted in a single most-parsimonious tree of 46 steps and agreed with phylogenies based onWagner parsimony. Polymorphic restriction enzyme sites in the 45s ribosomal DNA were not used to estimate phylogenies because of uncertain homologies, but are useful for identifying interspecific hybrids. The maternal phylogenies estimated in this study help to distinguish wildAllium species closely related to the bulb onion. Although not in agreement with classifications based on morphology, the phylogenies closely reflected crossability among species in sect.Cepa andPhyllodolon.     

Effect of a gap on gene flow between otherwise adjacent transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> crops

The chloroplast DNA diversity of 33 accessions belonging to 16 species of five sections in Allium subgenus Rhizirideum was studied by analysing the sequence of three fragments: the trnL-F intergenic spacer, the rps 16 intron and rbcL (rubisco large subunit). The three sections Cepa, Schoenoprasum and Rhizirideum, representing the majority of the included species, each possess a separate clade after phylogenetic analysis. Exceptions to this general rule are the placement of Allium pskemense (section Cepa) connected to Allium senescens (section Rhizirideum) and Alium roylei, taking an intermediate position between sections Cepa and Schoenoprasum. Both species were located in their own section after nuclear DNA analysis. A range of crossing experiments has been carried out. The different position of A. roylei when comparing cpDNA and nDNA diversity was not confirmed with the production of hybrid seeds after crossing A. roylei with species other than those of section Cepa. The different position of A. pskemense in the cpDNA and the nDNA tree can not be compared to its crossability, since only a few crossing experiments are reported for this species. The hypothesis that a shorter distance between two species in a cpDNA tree compared to their distance in a nDNA tree will indicate interfertility at a certain level, is neither confirmed nor rejected by the currently available results.Communicated by R. Hagemann     

Closely relatedAllium species (Alliaceae) share a very similar satellite sequence

A satellite sequence repeat ofAllium cepa was tested by fluorescent in situ hybridization (FISH) for cross-hybridization to chromosomes of 27 species (in 37 accessions) belonging to 14 sections of four subgenera ofAllium. All investigated species of sect.Cepa, with the two subsects.Cepa andPhyllodolon, revealed clear satellite-specific hybridization signals mainly at their chromosome termini. The tested species belonging to other sections/subgenera revealed no hybridization signals. An exception wasA. roylei, assigned to sect.Oreiprason. Its chromosomes also showed strong terminal hybridization signals. This and other features suggest a close relationship ofA. roylei to the species of sect.Cepa in spite of deviating morphological characters. The divergence between the satellite repeats to species to which theA. cepa repeat cross-hybridized was determined and revealed high degrees of similarity. Therefore, we conclude that this satellite sequence had evolved already in progenitor forms of sect.Cepa and remained unusually well conserved during speciation. This might indicate selection pressure exerted on a secondarily acquired telomere function of the satellite sequence.Dedicated to Dr habil.Peter Hanelt on the occasion of his 65th birthday.     

Mobile nucleolus organizing regions (NORs) inAllium (Liliaceae s. lat.)? — Inferences from the specifity of silver staining

NORs and interphase nucleoli have been silver stained inAllium cepa, A. fistulosum, reciprocal crosses between both species, and in different strains of top onions which originated from hybridization betweenA. cepa andA. fistulosum. The variability observed in size, number, and position of active NORs and correspondingly in number (and size) of interphase nucleoli is at least in part strain-specific. These data are taken to indicate that NORs inAllium behave like movable genetic elements.—With respect to the staining specifity of silver nitrate, it was found that AgNO₃ labels (1) nucleoli, (2) NORs (i.e., actively transcribed ribosomal genes) inside the achromatic secondary constrictions, and (3) sometimes (but less pronounced) centromeres; Giemsa banding labels heterochromatin surrounding the NOR but not the nucleolus organizing secondary constriction.     

Genetic diversity in the top onion,Allium ×proliferum (Alliaceae), analysed by isozymes

Polymorphisms for six enzyme systems (GPI, IDH, PDG, PGM, SKD, and TPI) were analysed in the top onion,Allium ×proliferum. Five multilocus isozyme genotypes were found. The banding patterns of top onions were compared with those ofA. ×wakegi, A. cepa, A. fistulosum, A. altaicum, and artificial hybrids between these three species. One top onion type and one artificial hybrid had identical banding patterns. Shallots andA. altaicum, the wild progenitor ofA. fistulosum, cannot be distinguished from the common onion andA. fistulosum, respectively; these species are also potential contributors to the top onion's gene pool.     

Serological comparisons of seed proteins of some representatives of the genusAllium

Results obtained by comparison of similarity of seed protein spectra of 15 species of the genusAllium (in 22 samples) by means of immunochemical methods more or less correspond to the contemporary division of this genus. Species belonging to the sectionCepa (according to Vvedenskiy) andPhyllodolon have very similar spectra, which suggests that they either should be allocated to one sectionCepa or to two subsections (Cepa andPhyllodolon). Species belonging to the subgenusRhizirideum considerably differ in seed proteins. The investigated species of the subgenusMelanocrommyum show the most distinct spectrum of all the other analyzed representatives, less marked differences were found in the case of the representatives of the subgenusAllium.     

Evolution of genome size inAllium (Alliaceae)

The 4C DNA amounts of 86 species fromAllium subgg.Allium, Rhizirideum, Bromatorrhiza, Melanocrommyum, Caloscordum andAmerallium show a 8.35-fold difference ranging from 35.60 pg (A. ledebourianum, 2n = 16) to 297.13 pg (A. validum 2n = 56). At diploid level the difference is 3.57-fold betweenA. ledebourianum (35.60 pg) andA. ursinum (127.14 pg). This shows that a significant loss and/or gain of DNA has occurred during evolution. On average subgg.Rhizirideum andAllium have less DNA amount than subgg.Melanocrommyum andAmerallium. The distribution of nuclear DNA amounts does not show discontinuous pattern and regular groups. The evolution of genome size has been discussed in relation to polyploidy and genomes, heterochromatin, adaptive changes in morphological characteristics, phenology and ecological factors, and infrageneric classification.     

Sequence analysis of a chloroplast intergenic spacer for phylogenetic estimates in Allium section Cepa and a PCR-based polymorphism detecting mixtures of male-fertile and male-sterile cytoplasmic onion

Restriction-enzyme analysis of the chloroplast (cp) DNA yielded maternal phylogenies supporting a close phylogenetic relationship among normal (N) male-fertile and male-sterile (S) cytoplasmic bulb onion (Allium cepa), Allium altaicum, Allium fistulosum, Allium galanthum, Allium roylei, and Allium vavilovii. The S cytoplasm of onion is most likely an alien cytoplasm introduced in antiquity into onion populations. We previously showed that size differences in an intergenic spacer in the cp DNA distinguish N and S cytoplasms of onion. We cloned and sequenced this intergenic spacer from the N and S cytoplasms of onion, A. altaicum, A. fistulosum, A. galanthum, Allium pskemense, Allium oschaninii, A. roylei, and Allium ampeloprasm (outgroup) to identify the nature of previously described RFLPs and to develop a PCR-based marker revealing N-cytoplasmic contamination of S-cytoplasmic hybrid seed lots. Phylogenies based on restriction-enzyme analysis of the entire cp DNA were similar, but not identical, to those based on sequence divergence in this intergenic region. Received: 29 November 1999 / Accepted: 28 April 2000     

Amounts of ribosomal DNA inAllium (Liliaceae)

Eight species ofAllium subgen.Allium sect.Allium have been studied at the cytological level by means of karyological analyses and at the biochemical level with regard to the proportions of ribosomal DNA. All the species have a basic genome of x = 8.A. sativum, A. commutatum, A. ampeloprasum, andA. vineale possess approximately 0.050% rDNA and two nucleolus organizer regions per basic chromosome set.A. sphaerocephalon andA. arvense have two nucleolus organizers, andA. amethystinum three nucleolus organizers per haploid (n = x) genome: the three species possess approximately 0.075% rDNA.A. acutiflorum has five nucleolus organizer regions per haploid genome and 0.121% rDNA. An attempt to relate these differences with functional and ecological characteristics indicates that evolutionary variation of rDNA proportions is not casual. Such data also can help to define systematic affinities and circumscribe infrageneric taxa.     

A new species and a new combination of <Emphasis Type="Italic">Allium</Emphasis> sect. <Emphasis Type="Italic">Rhizirideum</Emphasis> (Alliaceae) from northeastern China and Korea

A new species, Allium pseudosenescens, belonging to sect. Rhizirideum (Alliaceae), is described from northeastern China. It is easily distinguished from A. senescens by the slender pedicels, pale pink perianths, narrower tepals and ovaries, yellowish anthers, and sometimes toothed subulate filaments. Also, A. senescens var. minus in sect. Rhizirideum is raised to the rank of species, as A. minus. This Korean endemic taxon is shown to be a biologically distinct species based on morphological and cytological characters. Taxonomic keys for the species of Allium sect. Rhizirideum in northeastern China and Korea are provided.     

Phylogeny reconstruction and hybrid analysis in Allium subgenus Rhizirideum

Amplified fragment length polymorphisms (AFLPs) for assessing nuclear DNA diversity have been used for the reconstruction of the phylogeny and evolution of several sections of Allium subgenus Rhizirideum. A dataset of 355 characters for 33 accessions belonging to 20 species has been compiled. The band-sharing of five interspecific hybrids and of an F₂ population between Allium cepa and Allium roylei with their parents indicated a heterozygosity level between 6 and 14%, which allows the use of dominant markers such as AFLPs for phylogeny reconstruction. A majority rule consensus tree based on 56 most-parsimonius trees (CI = 0.528) revealed a separate clade for each of the sections, Cepa, Rhizirideum and Schoenoprasum, and one clade combining the sections Oreiprason and Petroprason. An unweighted pair group mean average (UPGMA)-based dendrogram showed the same subdivision. The trees and the ’Hybrid Distance’ approach both supported the assumption of a hybrid origin for A. roylei with considerable subsequent secondary evolution. The establishment of three alliances in the section Cepa and the close relationship of sections Oreiprason and Petroprason are now confirmed. The predictions of the Soybean domestication scenario, i.e. selection of a crop from one progenitor with subsequent narrowing of the genetic diversity of the crop, which applies to the cultigens A. cepa and Allium fistulosum, is supported by the Hybrid Distance approach. Received: 23 August 1999 / Accepted: 14 September 1999     

Isolation of Allium pollen protoplasts

Studies on protoplasts isolation were carried out with mature pollen grains of 29 samples of species of Allium aflatunense, A. cepa, A. fistulosum, A. karataviense, A. longicuspis, A. nutans, A. odorum, A. sativum and A. schoenoprasum. Surface sterilized pollen grains drifted from crushed anthers were incubated in an enzyme solution containing 1% (w/v) cellulase Onozuka R-10, 1% (w/v) Macerozyme R-10, 0,5 mol l^-1 sucrose and the basal salts of Nitsch medium. Protoplasts were released within 3 to 120 min, either from the pollen grain, through a slightly disturbed germination pore (narrow aperture), or through a wider aperture, when the exine surrounding the germination pore was disturbed. For the first time, protoplasts were obtained from 13 genotypes of 6 Allium species, at a rate of 1 to 30% of the digested intact pollen grains, depending on the genotype.     

AFLP linkage group assignment to the chromosomes of Allium cepa L. via monosomic addition lines

Two complete sets of Allium fistulosum L.– A. cepa monosomic addition lines (2n=2x+1=17) together with an AFLP linkage map based on a cross between A. cepa and A. roylei Stearn were used to re-evaluate the eight A. cepa linkage groups identified in the mapping study. The linkage groups could be assigned to individual, physical chromosomes. The low level of molecular homology between A. cepa and A. fistulosum enabled the identification of 186 amplified fragment length polymorphisms (AFLP™ markers) present in A. cepa and not in A. fistulosum with ten different primer combinations. With the monosomic addition lines the distribution of the markers over the eight chromosomes of A. cepa could be determined. Of these 186 AFLP markers 51 were absent in A. roylei and consequently used as markers in the mapping study (A. cepa ×A. roylei cross). Therefore, these 51 AFLP markers could be used to assign the eight A. cepa linkage groups identified in the mapping study to physical chromosomes. Seven isozyme and three CAPS markers were also included. Two of the linkage groups had to be split because they included two sets of markers corresponding to different chromosomes. A total of 20 (approx. 10%) of the A. cepa-specific AFLP markers were amplified in more than one type of the monosomic addition lines, suggesting unlinked duplications. The co-dominant isozyme and CAPS markers were used to identify the correspondence of linkage groupsoriginating from A. cepa or from A. roylei. Received: 16 April 1999 / Accepted: 13 August 1999     

Cytogenetical evidences for hybrid structure and origin of diploid and triploid shallots (Allium cepa var.viviparum,Liliaceae) from Dalmatia (Croatia)

Two viviparous strains of common onion (Allium cepa) denoted asAllium cepa var.viviparum (syn.Allium ×proliferum), traditionally cultivated in the seaside regions of Croatia were found to be diploid (2n = 2x = 16) and triploid (2n = 3x = 24), respectively. The triploid cultivated onion Ljutika has not been previously reported in Europe. Using Feulgen and Giemsa C-banding methods both karyotypes were shown to be of hybrid constitution. Results obtained in this work indicate that Dalmatian viviparous onions arose from spontaneous hybridization ofA. cepa withA. fistulosum.     

Taxonomic evaluations of the genusPinus (Pinaceae) based on electrophoretic data of salt soluble and insoluble seed storage proteins

Electrophoretic studies on seed storage proteins of various gymnosperm species showed that both salt soluble and insoluble protein fractions give taxonomic information. Among species of the genusPinus evident differences are found between the subgeneraHaploxylon (Strobus) andDiploxylon (Pinus). In sect.Strobus the two subsectt.Strobi andCembrae are readily distinguished fromP. bungeana (sect.Parrya) which appears to fall in an intermediate position between haploxyl and diploxyl pines. Among the species of subg.Diploxylon, mountain pines show very similar protein patterns in agreement with their recent speciation (Quaternary).     

Monoterpene und Systematik der GattungArtemisia (Asteraceae—Anthemideae)

Leaf monoterpenes from 166 provenances representing 71 species of the genusArtemisia were identified by gas-liquid (using 3 columns with different polarity) and thin layer co-chromatography with authentic samples. Cineol and thujane and camphane derivatives together with small quantities of pinanes, monocyclic and acyclic monoterpenes apparently characterize sect.Artemisia and sect.Abrotanum (pro parte), and probably also the North American sect.Tridentatae. The subg.Dracunculus and the ser.Laciniatae andLatifoliae of sect.Abrotanum are clearly distinguished by only small amounts of pinanes, monocyclic and acyclic monoterpenes together with a nearly complete reduction of all major components, i.e. cineol and thujane and camphane derivatives. Different trends towards -thujone or myrcene accumulation appear to be of some systematic relevance within the ser.Frigidae of sect.Absinthium.

     

A trnL-F based phylogeny for species ofPelargonium (Geraniaceae) with small chromosomes

Phylogenetic analysis was performed of 921 positions of trnL (UAA) 5 exon — trnF (GAA) exon chloroplast DNA regions from 68 representatives ofPelargonium sectt.Campylia, Cortusina, Glaucophyllum, Hoarea, Isopetalum, Ligularia, Otidia, Pelargonium, Peristera, Polyactium, andReniformia, together with five putative outgroup species from sectionsCiconium, Chorisma andJenkinsonia. The total data set therefore comprised 67.2 kb of DNA sequence. Two main ingroup clades were identified: one clade contains sectionsPeristera, Reniformia, andIsopetalum, the other contains sectionsCampylia, Cortusina, Glaucophyllum, Hoarea, Ligularia, Otidia, Pelargonium, Polyactium and two species currently grouped in sect.Peristera. Branching order among five main clades within the latter clade was not resolved. The trnL-F sequence data support monophyly only for sectionsReniformia andHoarea, the remainder of the currently recognized sections ofPelargonium being either paraphyletic or polyphyletic. The data further suggest that sect.Polyactium is diphyletic and that sect.Glaucophyllum is nested within sect.Pelargonium. One relatively derived clade, which represents half of the genus, contains predominantly geophytic and succulent species, occurring in the geographically restricted winter rainfall region of the South African Cape. This pattern is interpreted as reflecting explosive radiation, possibly as an adaptive response to recent aridification in the western Cape.     

Phylogenetic relationships inSecale (Poaceae): An isozymatic study

The genetic frequencies of 9 isozyme loci have been estimated in 23 samples of 4 species ofSecale by means of starch gel electrophoresis. The populations ofS. silvestre andS. vavilovii were monomorphic and uniform within each species, those ofS. montanum andS. cereale were polymorphic for most of the isozyme loci. On the basis of isozyme patterns as well as allelic and genotypic frequencies of isozyme loci,S. silvestre can be distinguished fromS. vavilovii, and both fromS. cereale andS. montanum; but there is no clear differentiation between the two latter species. Clusters constructed from genetic distances separateS. silvestre andS. vavilovii, whereasS. cereale andS. montanum were grouped together. The isozymatic data presented here, along with cytogenetic and life habit data, agree with the generally admitted existence of 4 species inSecale, and support the relationships suggested byKhush & Stebbins (1961).     

Phylogeny of Allium L. subgenus Rhizirideum (G. Don ex Koch) Wendelbo according to dot blot hybridization with randomly amplified DNA probes

 Among 341 randomly amplified DNA sequences generated from 11 Allium species, 55 were purified by gel excision and subsequent reamplification by PCR. These were then used as probes in dot blot analysis to evaluate the relationships between 44 Allium accessions classified under the subgenus Rhizirideum. The hybridization signals were standardized and converted to Euclidean taxonomic distances. Unweighted Pair Group Mean analysis of the distance data generated a phyllogram which basically conformed to the classification system proposed by the Gatersleben (Germany) group. However, there was insufficient evidence to suppport the proposal to join A. chinense G. Don with A. virgunculae F. Maek. et Kitam. into sect. Sacculiferum or the recent suggestion to re-establish sect. Phyllodolon. Received: 26 June 1997 / Accepted: 22 July 1997