Phylogenetic relationships among cultivated Allium species from restriction enzyme analysis of the chloroplast genome

Summary The genus Allium contains many economically important species, including the bulb onion, chive, garlic, Japanese bunching onion, and leek. Phylogenetic relationships among the cultivated alliums are not well understood, and taxonomic classifications are based on relatively few morphological characters. Chloroplast DNA is highly conserved and useful in determining phylogenetic relationships. The size of the chloroplast genome of Allium cepa was estimated at 140 kb and restriction enzyme sites were mapped for KpnI, PstI, PvuII, SalI, XbaI, and XhoI. Variability at restriction enzyme sites in the chloroplast DNA was studied for at least three accessions of each of six cultivated, old-world Allium species. Of 189 restriction enzyme sites detected with 12 enzymes, 15 mutations were identified and used to estimate phylogenetic relationships. Cladistic analysis based on Wagner and Dollo parsimony resulted in a single, most-parsimonious tree of 16 steps and supported division of the species into sections. Allium species in section Porrum were distinguished from species in sections Cepa and Phyllodolon. Two species in section Rhiziridium, A. schoenoprasum and A. tuberosum, differed by five mutations and were placed in separate lineages. Allium cepa and A. fistulosum shared the loss of a restriction enzyme site and were phylogenetically closer to each other than to A. schoenoprasum. This study demonstrates the usefulness of restriction enzyme site analysis of the chloroplast genome in the elucidation of phylogenetic relationships in Allium.     

Antioxidant and xanthine oxidase inhibitory activities of total polyphenols from onion

Onions (Allium cepa L.) comprise a valuable vegetable crop in many countries. Modern scientific research has shown that onions possess many biological activities, including antibacterial, anticancer, hypoglycemic, hypolipidemic, antiplatelet aggregation, and antioxidant activities. The goal of this study was to investigate the impact of total onion polyphenols on antioxidant and xanthine oxidase (XO) inhibitory activities. Total onion polyphenols showed significant antioxidant activity in DPPH, FRAP, and OH-assays (IC₅₀ [µg/mL]), 43.24, 560.61, and 12.97, respectively). In a X/XO system, antioxidant properties of these polyphenols significantly inhibited XO activity (IC₅₀ [µg/mL], 17.36). These results indicated that total onion polyphenols showed promising antioxidant and anti-gout properties and might be used as potential, natural drugs against oxidative diseases after successful studies in vivo as well as clinical trials.     

Chromosomal location of rDNA in Allium: in situ hybridization using biotin- and fluorescein-labelled probe

Summary A biotin- and fluorescein-labelled probe of Helianthus argophyllus has been used to map specific repeated rDNA sequences by in situ hybridization on mitotic chromosomes of Alliwn cepa, Allium fistulosum, a diploid interspecific (Allium fistulosum x Allium cepa) F₁ hybrid, and a triploid interspecific (2 x = A. cepa, 1 x = A. fistulosum) shallot. Hybridization sites were restricted to satellited and smallest pairs of chromosomes in both A. cepa and A. fistulosum. The number, size, and position of the hybridization sites distinguish homologous chromosomes and identify the individual chromosomes carrying the nucleolus organizing region (NOR) at the secondary constriction, as well as the individual chromosomes carrying an additional NOR. This in situ hybridization technique is the first reported in a plant species and offers new cytogenetic markers in Allium.     

In situ hybridization confirms jumping nucleolus organizing regions in Allium

In situ hybridization with a ¹²⁵I-rDNA clone from Vicia faba was performed against Allium cepa and three strains of top onion, which represent hybrids between A. cepa and A. fistulosum. In principle, the labelling patterns correspond to the patterns of the silver-stained nucleolus organizing regions (NORs) in the same species. This strongly supports the inference drawn from the Ag-NOR patterns that NORs can jump between terminal heterochromatin blocks of different Allium chromosomes in the parental species A. cepa as well as in their interspecific hybrids.     

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.     

Identification of alliin,a constituent of Allium cepa with an inhibitory effect on platelet aggregation

A component of Allium cepa which inhibits platelet aggregation in vitro was isolated. The active compound was identified as alliin, (+)-S-allyl-L-cysteine sulfoxide. Alliin was synthesized and found to exert the same activity on platelet aggregation as the natural compound.     

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     

AFLP marking of the genotypes of leek (<Emphasis Type="Italic">Allium porrum</Emphasis>) varieties

The results of the AFLP analysis of 16 leek (Allium porrum) accessions and related species of the sections of the genus Allium are presented. Restriction enzymes and primer combinations for the identification of the genotypes of the A. porrum accessions were chosen. As a result, 265 polymorphic AFLP fragments were amplified for 25 analyzed genotypes, and specific spectra of DNA fragments were obtained for each accession. A total of 24 fragments specific for the A. porrum genome was detected, of which only two characterized the genotypes of individual accessions. A wide range of genetic diversity (0.11–0.32) was revealed for the A. porrum varieties and lines used in the analysis. The highest level of similarity in the analyzed set of accessions was found between A. porrum and sand leek (A. scorodoprasum).     

Random amplified polymorphic DNA (RAPD) markers for genetic analysis inAllium

RAPD analysis was applied to onion (Allium cepa) and otherAllium species in order to assess the degree of polymorphism within the genus and to investigate if this approach was suitable for genetic studies of onion. Seven cultivars ofA. cepa, including shallot, and single cultivars of Japanese bunching onion (A. fistulosum), chive (A. schoenoprasum), leek (A. ampeloprasum), and a wild relative of onion (A. roylei), were evaluated for variability using a set of 20 random 10-mer primers. Seven out of the twenty primers revealed scorable polymorphisms between cultivars ofA. cepa and these will be further evaluated for use in genetic mapping. Wide variations in banding profiles between species were observed with nearly every primer tested. These were assessed for use in systematic studies within the genus. Ninety-one band positions were scored (+/-) for all the cultivars studied. Genetic distances between each of the cultivars were calculated and cluster analysis was used to generate a dendrogram showing phylogenetic relationships between them. The resulting analysis was in broad agreement with previous classifications of the species studied, confirming the validity of the method. However, amongst the species studied, it placedA. roylei as the closest relative ofA. cepa, questioning the current classification of the former species in the section Rhizideum.     

Restriction fragment length polymorphisms reveal considerable nuclear divergence within a well-supported maternal clade in allium section Cepa (Alliaceae)

Maternal phylogenies estimated by restriction fragment length polymorphisms (RFLPs) in the chloroplast DNA (cpDNA) delineated a well-supported clade containing Allium altaicum, A. cepa (bulb onion), A. fistulosum (Japanese bunching onion), A. galanthum, and A. vavilovii. Few polymorphic restriction-enzyme sites were detected among the wild and cultivated species within this clade, and relationships could not be confidently estimated. Random nuclear RFLPs revealed considerable variation among these species and three distinct groups were identified (Altaicum, Cepa, and Galanthum). Relationships were estimated using principal components and cluster analyses of the Jaccard's similarity matrix. For five out of six analyses, nuclear phylogenies estimated by UPGMA and neighbor joining of the Jaccard's similarity matrix produced a weakly supported monophyletic lineage for A. altaicum, A. fistulosum, and A. galanthum, disagreeing with maternal phylogenies that produced a weakly supported monophyletic lineage for A. altaicum, A. fistulosum, A. cepa, and A. vavilovii. Allium oschaninii was closely related to A. galanthum and these two species may represent the progenitor types. Overall, restriction-enzyme analyses of the nuclear and cpDNA produced few synapomorphies among closely related species in Allium section Cepa.     

Variation in economically and ecologically important traits in onion plant organs during reproductive development

The spatial distribution of organosulphur compounds throughout the onion (Allium cepa L.) plant body during reproduction is of ecological and horticultural interest. These secondary metabolites are associated with both pest resistance and many of the vegetable's culinary and medicinal properties, including the ability to inhibit platelet aggregation. Inhibition of platelet aggregation can be of benefit to human cardiovascular health. Organosulphur compound concentrations are associated with elemental sulphur, pungency, soluble solids and effect on human platelet aggregation. These parameters were evaluated in extracts collected separately from bulb scales, leaf blades, scapes and umbels biweekly throughout the reproductive phase of the life cycle of the onion. Significant variation in pungency, platelet inhibition, total sulphur content and soluble solids existed among samples of organs and within organs over time during reproductive growth. Furthermore, some extracts from leaf, scape and bulb induced rather than inhibited platelet aggregation.     

Enhanced seed development in the interspecific cross Allium cepa x A. sphaerocephalon through ovary culture

Allium sphaerocephalon pollen tubes grew into styles and penetrated micropyles of Allium cepa, but ovules started to degenerate about 16 days after pollination and no seeds developed. Seeds developed in vitro in ovaries excised from flowers 4 and 7 days after pollination. Seven weeks after culture initiation, seeds had grown in 4 of 96 excised ovaries, cultured on BDS medium supplemented with GA₃. Although the culture medium supported seed maturation within excised ovaries of self-pollinated A. cepa flowers, no viable hybrid seeds were recovered from crosses with A. sphaerocephalon. Extended post-fertilization barriers may have restrained development of hybrid embryos in vitro. Ovary culture followed by in ovulo embryo rescue may be feasible for distant-species hybridization in Allium.     

Bioactive polar natural compounds from garlic and onions

Bioactive natural compounds from garlic and onions have been the focus of researches for decades, firstly due to their pharmacological effects, and secondly due to their defence properties against plant diseases. In fact, garlic and onion, belonging to Allium genus, are among the oldest food plants known since ancient times and used as ingredient of many recipes and for therapeutic properties. These plants are well known to produce bioactive apolar sulphur compounds but less is known about their polar natural compounds, such as phenols, sapogenins and saponins, that are more stable to cooking, So, we continued our work on the discovery of polar bioactive metabolites from Allium with the isolation of a number of sapogenins and saponins from the wild onion species Allium elburzense, Allium hirtifolium, Allium atroviolaceum, and Allium minutiflorum, and, more recently, from the cultivated white onion, Allium cepa, and garlic, Allium sativum. In particular, the sapogenins and saponins isolated from A. elburzense and A. hirtifolium, named elburzensosides and hirtifoliosides respectively, exhibited significant antispasmodic properties. In addition, the saponins named minutosides isolated from A. minutiflorum showed promising antimicrobial activity. More recently the phytochemical analysis of A. cepa and A. sativum has been undertaken and afforded the characterization of saponins, phenols and N-cynnamic amides which showed significant antifungal activity.     

Interspecific crosses of onion with distant Allium species and characterization of the presumed hybrids by means of flow cytometry, karyotype analysis and genomic in situ hybridization

Interspecific crosses were made by hand-pollination of Allium cepa with pollen of 19 species belonging to nine sections of two subgenera of the genus Allium. In all cases viable plantlets were obtained from ovary culture. The efficiency depended on the relationship of the pollen donor to A. cepa. The hybrid character of the regenerants was checked by morphological comparisons with the parents and/or by one or more cytological methods such as flow cytometric DNA measurement, karyotype analysis, and genomic in situ hybridization (GISH). Hybrids were confirmed for 18 new species combinations. The viable hybrid of the most distant cross resulted from crossing A. cepa with A. sphaerocephalon. The relevance of the verification methods and the potential use of the hybrids for breeding purposes are demonstrated.     

Isolation and identification of soluble polysaccharides in epidermal tissue of Allium cepa

Because starch is absent from Allium-guard cells, another polysaccharide was sought that, in connection with stomatal opening, could be a source of organic anions. Analysis of isolated polysaccharides revealed xylose, arabinose, glucose, galactose, and galacturonic acid (3.4:1:1.6:0.7) to be components of the water-soluble mucilage of the epidermal strips of Allium cepa. However, the experiments gave no indication that the mucilage is the malate donator during the stomatal opening. After ¹⁴CO₂ fixation the following substances were labeled: organic acids, especially malate and citrate, amino acids and the polysaccharide mentioned above; radioactive 3-phosphoglyceric acid and sugar phosphates were not found. Therefore we conclude that the Calvin cycle does not operate in the guard cells of Allium cepa.Abbreviations ABA Abscisic acid     

High frequency somatic embryogenesis and plant regeneration from zygotic embryo-derived callus cultures of three Allium species

The plant regeneration ability of zygotic embryo-derived callus cultures was studied for 12 A. cepa varieties and accessions, two A. fistulosum varieties, one A. fistulosum x A. cepa interspecific hybrid and two A. porrum varieties. Compact embryogenic callus was induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid. The embryogenic calluses of all three Allium species were similar in appearance. For all accessions tested plants could be regenerated at a high frequency from this compact callus through somatic embryogenesis, when using kinetin supplemented MS medium (regeneration medium). Addition of abscisic acid to the regeneration medium stimulated the formation of both somatic embryos and shoots for a number of varieties. Concerning shoot regeneration from callus cultures, significant differences existed between genotypes of all accessions except one.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - VDH Van Der Have Seed company     

Studies on fungi in the root region

Summary Aqueous extracts of adventitious roots ofAllium sativum L. (garlic), seedling roots ofAllium porrum L. (leek), and both adventitious and seedling roots ofAllium cepa L. (onion), were tested for antibiotic activity against three root-surface fungi,Cylindrocarpon radicicola Wollenw.,Gliocladium roseum (Link) Thom andFusarium culmorum (W. G. Smith) Sacc. by means of two different techniques.With a filter-paper disc technique, root extracts sterilised by membrane-filtration produced zones of inhibition of the test fungi, whereas root extracts sterilised by autoclaving showed no activity. Garlic root extract produced inhibition zones with all the test fungi, whereas extracts of onion adventitious roots and leek seedling roots produced inhibition zones with only one of the test fungi. The extract of onion seedling roots produced no inhibition zones. Root extracts of all theAllium species, when sterilised by membrane filtration, generally inhibited spore-germination of all the test fungi.     

Onion microsatellites for germplasm analysis and their use in assessing intra- and interspecific relatedness within the subgenus Rhizirideum

We have identified a set of informative STMS markers in onion (Allium cepa L.) and report on their application for genotyping and for determining genetic relationships. The markers have been developed from a genomic library enriched for microsatellites. Integrity of the microsatellite polymorphism was confirmed by amplicon sequencing. The microsatellite genotypes of 83 onion accessions and landraces from living onion collections were compared. As few as four primer pairs were sufficient to assign unique microsatellite patterns to the 83 accessions. Some of the microsatellite markers can be used for interspecific taxonomic analyses among close relatives of Allium cepa. Generally, our data support and extend results obtained from recently performed analyses using ITS, RAPD and morphology. Received: 8 October 1999 / Accepted: 3 November 1999     

Karyological studies of Iranian <Emphasis Type="Italic">Allium</Emphasis> L. (Amaryllidaceae) species with focus on sect. <Emphasis Type="Italic">Acanthoprason</Emphasis>. 1. Mitotic chromosomes

Eighteen species and subspecies (34 accessions) of Allium sect. Acanthoprason and 11 species (17 accessions) belonging to other subgenera and sections of Allium were karyologically investigated and include first reports for 12 species. The examined plants of 47 accessions were diploid, three accessions of two species were tetraploid, and in the A. bisotunense accession, we found a mix of di- and triploid individuals. B chromosomes were found in 10 accessions. A basic chromosome number of x = 8 was confirmed for all investigated members of subg. Melanocrommyum and subg. Allium, and x = 9 for Allium tripedale of subg. Nectaroscordum. Idiograms were drawn for each accession, and metaphase images are presented illustrating observed chromosomal variations. Also, karyotype features and asymmetry parameters were calculated for all accessions. Chromosomal aberrations, e.g. aneuploid cells or loss of whole or parts of chromosome arms, were rarely observed. In general, the karyotypes showed low variation in inter- and intrachromosomal asymmetry especially inside of the taxonomic groups, though satellited chromosomes were good markers for subgenera and even specific for two studied sections of subg. Allium. Six different types of satellites were recognized, two of them were newly described: Type P was prevalent in subg. Melanocrommyum, and type O in sect. Codonoprasum. Statistical analyses were performed on five karyological parameters to test correct relationships and also to test previous grouping hypotheses. Although our data confirm distinct karyological characters for the subgenera investigated, the remarkable morphological diversity inside of subg. Melanocrommyum is not mirrored by striking karyological differences.