Potentialities of Six Plant Species on Phytoremediation Attempts of Fuel Oil-Contaminated Soils

A field experiment investigating the phytoremediation potential of six plant species—Goosegrass (Eleusine indica), Bermuda grass (Cynodon dactylon), Sessile joyweed (Alternanthera sessilis), Benghal dayflower (Commelina benghalensis), Lovanga (Cleome ciliata), and Chinese violet (Asystasia gangetica)—on soil contaminated with fuel oil (82.5 ml/kg of soil) have been conducted from March to August 2016. The experiments consider three modalities—Tn: unpolluted planted soils, To: unplanted polluted soils, and Tp: polluted planted soil—randomized arranged. Only three (E. indica, C. dactylon, and A. sessilis) of the six species survived while the others died 1 month after the beginning of experimentations. The relative growth indexes showed a strong similarity between the growth parameters of E. indica and C. dactylon, each on polluted and control soils, unlike A. sessilis. Total petroleum hydrocarbons (TPHs) removal efficiency were 82.56, 80.69, and 77% on soil planted with E. indica, C. dactylon, and A. sessilis, respectively; and 57.25% on non-planted soil. According to the bioconcentration and translocation factors, E. indica and A. sessilis are involved on rhizodegradation and phytoextraction of hydrocarbons whereas C. dactylon is only involved into rhizodegradation. Overall, E. indica and C. dactylon out-yielded A. sessilis in the phytoremediation capacity of fuel oil-contaminated soils.     

Collection and evaluation of wild tulip (<Emphasis Type="Italic">Tulipa</Emphasis> spp.) resources in China

China is known throughout the world as one of the most diverse centres of wild Tulipa L. resources. There are 17 wild Tulipa species distributed in China, and 12 species are only distributed in Xinjiang Province. In this paper, total 83 accessions were collected from Xinjiang, Liaoning and several other provinces. Their distribution, collection, classification and evaluation were described. According to morphological characteristics, they were classified into eight species, which included T. sinkiangensis Z. M. Mao, T. altaica Pall. ex Spreng., T. iliensis Regel, T. heterophylla Baker, T. buhseana Boiss., T. thianschanica Regel, T. schrenkii Regel and T. edulis (Miq.) Baker. Their phenotypic genetic relationships were analysed and showed that the eight Tulipa species were divided into two groups: Group I included one species T. edulis, and Group II included the other seven species. Bulb renewal was observed in eight wild Tulipa species; T. iliensis and T. edulis showed that one or more long fleshy stolons formed horizontally or vertically at the basal plate of the mother bulbs, new bulblets (dropper) appeared at the top of each fleshy stolon, and the mother bulbs eventually disappeared. The analytic hierarchy process was used to evaluate the ornamental value, utilization potential and ecological adaptability of the eight identified species. The results showed that T. iliensis, T. buhseana and T. thianschanica had better ornamental value and utilization potential than any other species. The distribution, collection, classification, and evaluation of wild Tulipa species could be helpful in creating novel tulip germplasms in China.     

<Emphasis Type="Italic">Panax</Emphasis> species identification with the assistance of DNA data

Panax species have been used as medicinal plants in China and adjacent areas, but the difficulty in wild species identification based on morphological characters prevents the exploration and utilization of these resources. To assist in identifying the species, three plastid intergenic spacers and four nuclear genes of 129 samples from 20 different localities were sequenced to conduct the clustering analyses. P. stipuleanatus and P. notoginseng were relatively easy to be distinguished from other species based on morphological characters and clustering results. Three samples collected from Laos grouped together with those collected from Jinping (China), and they were all identified as P. vietnamensis. However, the fourth one LW4 had closer genetic relationship to P. notoginseng than to P. vietnamensis. Although they were morphologically different in petiolule length, the samples preliminarily identified as P. zingiberensis grouped together in the clustering analyses, and the results of STRUCTURE analysis suggested that samples collected from Zhenyuan shared some genetic component with P. vietnamensis. As for the samples identified as P. japonicus and P. wangianus, there were so many variations in leaves, rhizomes and rootstocks that it was difficult to differentiate them from each other. Molecular data also showed that these taxa were genetically admixed and the samples identified as P. wangianus could be classified into P. japonicus. The nucleotide diversity results indicated that the wild relatives had more substantial genetic diversity compared to P. notoginseng.     

Phylogenetic relationships within <Emphasis Type="Italic">Lactuca</Emphasis> L. (Asteraceae), including African species,based on chloroplast DNA sequence comparisons

Lettuce (Lactuca sativa L.) belongs to the genus Lactuca L. and is an important vegetable worldwide. Over the past decades, there have been many controversies about the phylogeny of Lactuca species due to their complex and diverse morphological characters and insufficient molecular sampling. In this study we provide the most extensive molecular phylogenetic reconstruction of Lactuca, including African wild species, using two chloroplast genes (ndhF and trnL-F). The sampling covers nearly 40 % of the total endemic African Lactuca species and 34 % of the total Lactuca species. DNA sequences from all the subfamilies of Asteraceae in Genebank and those generated from Lactuca herbarium samples were used to establish the affiliation of Lactuca within Asteracaeae. Based on the subfamily tree, we selected 33 ndhF sequences from 30 species and 79 trnL-F sequences from 48 species to infer relationships within the genus Lactuca using randomized axelerated maximum likelihood and Bayesian inference analyses. Biogeographical, chromosomal and morphological character states were reconstructed over the Bayesian tree topology. We conclude that Lactuca contains two distinct phylogenetic clades—the crop clade and the Pterocypsela clade. Other North American, Asian and widespread species either form smaller clades or mix with the Melanoseris species. The newly sampled African endemic species probably should be treated as a new genus.     

Phylogenetic implications of the internal transcribed spacers of nrDNA and chloroplast DNA fragments of <Emphasis Type="Italic">Musa</Emphasis> in deciphering the ambiguities related to the sectional classification of the genus

The genus Musa L. comprises of economically important bananas and has been divided into five sections based on their chromosome number and morphological characteristics viz., Australimusa, Callimusa, Eumusa, Rhodochlamys and Ingentimusa. However, this sectional classification has long been disputed. In this study, we present data on sequenced multiple DNA fragments from the maternally inherited chloroplast genome (atpB-rbcL spacer, trnK-matK intron and psbK-psbI spacer) and the biparentally inherited internal transcribed spacers of nuclear ribosomal DNA of three species of Musa along with data available in GenBank database to understand their implications on the sectional relationship and phylogeny of the genus Musa. In our findings, none of the five sections of Musa previously defined based on morphology was recovered in the molecular phylogeny analysis using cpDNA and nrDNA. Instead, the results corroborate with the suggestion that sectional classification in the genus Musa should be reviewed by combining together the section Eumusa and Rhodochlamys as one and section Australimusa and Callimusa as a single different section, along with that of Ingentimusa.     

Diversity and differentiation of <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis> in Indonesia

Analysis of the genetic structure of Indonesian Oryza sativa and O. rufipogon using neighbour-joining trees based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers revealed that O. sativa in Indonesia is separated from O. rufipogon. Accessions of O. sativa in this study were differentiated into two major groups, indica and tropical japonica, excluding some varieties. SSR and SNP markers revealed the high value of differentiation (F _ST) and genetic distance (D) between indica and tropical japonica and we discovered four loci by SNP markers and one locus by SSR markers that play a role in differentiation between indica and tropical japonica. Interestingly, genetic diversity (H) in O. rufipogon was lower than that in O. sativa, however H in O. rufipogon was the highest and H in tropical japonica was the lowest when O. sativa was divided into two groups. Inbreeding coefficient (Fst) showed evidences that gene flow (Nm) between species and within species might be one of the mechanisms related to the diversification and differentiation of Indonesian rice germplasm by asymmetric pattern between species and within O. sativa as revealed by SSR and SNP markers. In addition, we found evidences on stabilizing selection in Indonesian rice germplasm and they might be the reasons why Indonesian rice germplasm did not differentiate due to source location of landrace. However, we found a weak relation between SSR and SNP markers probably due to highly polymorphic in SSR and the different properties of both markers.     

A new taxonomic treatment for some wild relatives of mungbean (<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilcz.) based on their molecular phylogenetic relationships and morphological variations

Mungbean (Vigna radiata (L.) Wilcz.) is an important leguminous crop cultivated mainly in Asia. Its wild relatives are considered useful genetic resources for mungbean breeding. However, the taxonomic history of mungbean and its wild relatives is complicated and some confusion is still present in recent publications. In this study, we examined the rDNA-ITS sequences and morphological characteristics of 83 gene bank accessions closely related to mungbean. As a result, we classified the 83 accessions into five species and one unclassified accession. The proper taxon name for each species was determined (Vigna grandiflora (Prain) Tateishi et Maxted, Vigna mungo (L.) Hepper, V. radiata, Vigna subramaniana (Babu ex Raizada) Raizada, and Vigna trinervia (Heyne ex Wight et Arn.) Tateishi et Maxted) based on a review of their taxonomic history and morphological comparisons between gene bank accessions and type specimens. A new taxonomic treatment is proposed and a morphological key has been prepared. In this treatment, Vigna radiata (L.) Wilcz. var. setulosa (Dalz.) Ohwi et Ohashi is treated as a synonym of Vigna radiata (L.) Wilcz. var. sublobata (Roxb.) Verdc., and Vigna hainiana Babu, Gopinathan et Sharma is a synonym of V. subramaniana. Accession ‘NI1135’ was revealed to be most closely related to, but is considerably differentiated from, mungbean (V. radiata) based on its rDNA-ITS sequences. It also has distinguishing morphological characters. Plants with similar morphologies and DNA profiles might be distributed in the Indian Himalaya. However, since only one accession is available at present, the taxonomic classification of ‘NI1135’ needs to be reconsidered in the future.     

Phenetic and genetic diversity in Indian <Emphasis Type="Italic">Luffa</Emphasis> (Cucurbitaceae) inferred from morphometric,ISSR and DAMD markers

Genetic variations and relationships among cultivated and wild genotypes of five taxa of Indian Luffa were examined using inter simple sequence repeats (ISSR), directed amplification of minisatellite DNA (DAMD) markers and morphological characterization. Morphometric evaluation of 21 discrete characters in 51 representative accessions segregated the five taxa of Luffa in three main clusters: the two wild species (L. echinata, L. graveolens) in the first, and the cultivated L. aegyptiaca (smooth gourd) and L. acutangula (ridged gourd)/L. hermaphrodita (Hermaphrodite luffa) in the second and third clusters, respectively. Cumulative data analysis of 15 ISSR and seven DAMD markers revealed high percentage polymorphism (97.67 %), moderate genetic distance (0.06–0.72, avg. 0.51), and low heterozygosity and Shannon index values (H = 0.15; I = 0.22) across all the 76 genotypes assayed. A UPGMA dendrogram, based on the combined marker data, resolved the five taxa in two main clusters with high bootstrap support. The morphological and molecular trees showed incongruence in the number of main clusters resolved and in the disposition of the wild and cultivated taxa in different sub-clusters. The cluster analyses and PCoA plots revealed a nested grouping of the hermaphrodite luffa within the ridge gourd group. The Bayesian STRUCTURE analysis identified three genetic clusters for the five assumed taxa. Outcrossing test revealed a mixed mating system in Indian Luffa. This is the first ever report on the mating system in Luffa using molecular markers. The study also demonstrates the utility of using more than one DNA marker in the assessment of molecular diversity in a widely cultivated crop genus like Luffa with a narrow genetic base.     

Remarks on genetic diversity and relationship of <Emphasis Type="Italic">Punica protopunica</Emphasis> and <Emphasis Type="Italic">P. granatum</Emphasis> assessed by molecular analyses

Here, two Punica species, viz., P. protopunica Balf. fil., reported as native to Socotra, and P. granatum L., were compared for the first time. Analysis of one P. protopunica and eleven P. granatum accessions was performed using three molecular markers, i.e., sequence related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), and intron targeted amplified polymorphism (ITAP), along with analysis of pgWD40 sequences, a gene involved in anthocyanin biosynthesis. All markers revealed the relationship between the two species and placed them at 33% similarity. SRAP, TRAP, and ITAP generated a total of 299, 260, and 160 bands, respectively. Of these, 78, 74, and 41 bands were specific for P. protopunica, and 92, 85, and 57 bands, respectively, were shared between both species. Sequence analysis of pgWD40~870 bp amplicons exhibited 100% identity among P. granatum accessions and 98% identity to that of P. protopunica. Phylogenetic analysis of WD40 sequences from monocot and dicot species, including both Punica species confirmed the relation between P. protopunica and P. granatum, supporting earlier reports that P. protopunica could be an ancestral species of P. granatum. Furthermore, the genetic diversity among and within P. granatum accessions from Egypt (3), Mexico (5), and Yemen (3) was assessed. Molecular marker-based relationships among region-bulked accessions was approximately the same (~90% similarity), whereas the degree of genetic variation was altered within each region. Specific bands (alleles) for accessions of each region along with those shared among them were identified. Thus, these bands could be used for pomegranate genotyping and breeding programs.     

Polymorphism of <Emphasis Type="Italic">Got2</Emphasis> DNA sequences sheds light on <Emphasis Type="Italic">Aegilops tauschii</Emphasis> Coss. intraspecies divergence and origin of <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

DNA sequences of nuclear gene Got2 was studied in 60 accessions of Aegilops tauschii, 29 of subsp. tauschii and 31 of subsp. strangulata. It was found that Got2 allozyme polymorphism in Ae. tauschii is due to a single, unique, mutation which led to replacement of glutamic acid by isoleucine in residue 256 of the enzyme molecule, encoded by Got2. As revealed by Got2 DNA sequences variation, initially in its history Ae. tauschii was presented by subsp. strangulata, and among phylogenetic lineages of subsp. strangulata, the lineage “t-9¹s” (TauL3) is the most ancient, a relict one. Subspecies tauschii is relatively “young”. Initially it was presented by the lineage marked by combination of allozyme alleles Got2 ¹⁰⁵ and Acph1 ¹⁰⁰. In the past it inhabited the Continental area from Caucasia to Pakistan, but later on it was forced out by newly originated, now—a major lineage of subsp. tauschii, marked by Got2 ¹⁰⁰. This lineage extended the Continental area of the species up to Kirgizstan, but actually failed to penetrate into pre-Caspian area, occupied by subsp. strangulata. These results essentially differ from those obtained previously, using chloroplast DNA (cpDNA) sequences polymorphism. As revealed by cpDNA, the major, “usual”, subsp. strangulata (TauL2) is “younger” than subsp. tauschii, which resided on phylogenetic tree between relict lineage “t-9¹s”of subsp. strangulata—and major subsp. strangulata. But both cpDNA and Got2 DNA sequences indicate that the level of genetic variation in subsp. tauschii is much lower than in subsp. strangulata. According to Got2 DNA sequences variation, it was Ae. tauschii subsp. strangulata lineage “k-109″ which donated genome D to Triticum aestivum L. This lineage includes accessions: k-109 from South-Eastern Precaspian Azerbaijan; KU-2105, KU-2159 from Western Precaspian Iran; KU-2080 from Eastern Precaspian Iran.     

Diversity of <Emphasis Type="Italic">Citrus</Emphasis><Emphasis Type="Italic">depressa</Emphasis> Hayata (Shiikuwasha) revealed by DNA analysis

Citrus depressa Hayata is an indigenous mandarin species on the Ryukyu Islands located in the subtropical region of Japan. We deduced its phylogenetic relationships by evaluating accessions grown on various Ryukyu Islands via cleaved amplified polymorphic sequence analysis of cpDNA and sequence-related amplified polymorphism (SRAP). The cpDNA results indicated that C. depressa could be classified into two types. SRAP revealed patterns of diversity within C. depressa consistent with our cpDNA results. These results indicate that maternal origin may influence or is correlated with the constitution of the nuclear genome of C. depressa. Another Japanese mandarin species, Citrus tachibana (Makino) Tanaka was distinguished from C. depressa by SRAP markers. Moreover, both C. depressa and C. tachibana could be distinguished from other Citrus species. Our results suggest that Japanese mandarin possesses a characteristic genome with the genus Citrus.     

Electrophoretic variation in seed proteins and interrelationships of species in the genus <Emphasis Type="Italic">Oryza</Emphasis>

Comparative studies of two cultivated and sixteen wild species of the genus Oryza were carried out using one- and two-dimensional gel electrophoresis for variation in their seed proteins for interrelationships of these species. A number of polypeptides in the range of molecular weight 13–110 kDa were seen to vary. Under reducing conditions, polypeptides spread over the regions of mol. wt. 33–40.5, 25–27 and 19–21.5 kDa exhibited maximum variation in their patterns. Two-dimensional gel electrophoresis revealed the occurrence of disulphide-linked glutelin polypeptide pairs of mol. wt. 60, 58, 52 and 25 kDa breaking into a large and a small subunit each in the range of mol. wt. 18–40.5 and 16–25 kDa respectively in Oryza sativa. The number of such polypeptide pairs varied from 2 to 6 in different species and also in O. sativa showed variation in mol. wts. of their constituent subunits. The UPGMA dendrogram revealed that most of the Oryza species occurred in different clusters and subclusters and thus did not share very close relationships. The undisputed and closest relationship observed was that of cultivated rice O. sativa with the O. rufipogon followed by that with O. nivara. The African cultivated O. glaberrima occurring on the nearest branch of the same subcluster, thereby, supporting the phylogenetic of these species suggested in earlier studies. Eight diploid species and seven tetraploid species were included in one part of the dendrogram while the remaining two species with AA genome i.e. O. glumaepatula and O. meridionalis and one with FF i.e. O. brachyantha stood separately from these as scattered in the group of seven tetraploid species with BBCC, CCDD and HHJJ genomes. The tetraploids O. alta, O. latifolia and O. grandiglumis with CCDD genomes which occurred on the farthest part were distantly related with O. sativa. The cyanogen bromide peptide maps and two dimensional gel electrophoresis also supported the closest relationship between O. sativa and O. rufipogon.     

Geographical distribution of wild <Emphasis Type="Italic">Daucus</Emphasis> species in the natural grasslands of the Argentinian pampas

Three wild carrot species have been reported in the argentinian flora: Daucus montanus Humb. et Bonpl. ex Schult., D. montevidensis Link ex Sprengel and D. pusillus Michx. There is a discrepancy among authors about the distinctive morphological traits of the last two species; thus, it is difficult to ascertain if they are truly two distinct taxa. Based on the available literature and in the search of a paradigmatic site, four collection trips were carried out in 2004 and 2005 in Buenos Aires and Southern Entre Ríos provinces. Populations were sampled at 30 sites, and local environmental parameters and associated plant species were recorded. Morphological observations and chromosome counts were carried out on 10 plants/population. Three morphological phenotypes were distinguished: one in 18 populations, all with 2n = 2x = 18, and two in the remaining 12, with 2n = 2x = 22 or aneusomaty (2n = 2x = 20, 22). Populations of the first phenotype were assigned to wild D. carota and the rest, tentatively, to D. pusillus (D. montevidensis?), till further evaluations are carried out in test sites to verify this tentative conclusion.     

Genotyping-by-sequencing reveals the origin of the Tunisian relatives of cultivated carrot (<Emphasis Type="Italic">Daucus carota</Emphasis>)

Tunisia, other countries in northwestern Africa, and the Iberian Peninsula, represent a center of diversity of Daucus. The genus traditionally has included about 20–25 species worldwide, but a recent molecular study redefined and expanded Daucus to include representatives from nine other genera. By this classification, Daucus now contains about 40 species, with some of them having winged fruits in addition to its traditionally recognized spiny fruits. The taxonomy of Daucus in Tunisia has recently been studied with morphological data, concluding that D. carota subsp. capillifolius is a subspecies that co-occurs and crosses with subsp. carota. The present study extends these findings with additional data from Genotyping-by-Sequencing (GBS) from 33 Tunisian accessions of D. carota (93 individuals with two to three replicates per accession), including the first collections of subsp. gummifer from Tunisia. Placed in the context of additional GBS data, D. carota subsp. gummifer has separate origins from other collections of subsp. carota in Tunisia and/or immediately adjacent areas to the north in Italy or its surrounding islands. Our results add support to the utility of large SNP datasets for species-level phylogenetic studies in Daucus.     

Cross-transferability of SSR markers in <Emphasis Type="Italic">Osmanthus</Emphasis>

Developing a molecular tool kit for hybrid breeding of Osmanthus species and related genera is an important step in creating a systematic breeding program for this species. To date, molecular resources have been aimed solely at Osmanthus fragrans with little work to develop markers for other species and cultivars. The objectives of this study were to (1) determine cross-transferability of O. fragrans and Chionanthus retusus derived SSRs in diverse Osmanthus taxa, (2) quantify the influence of locus-specific factors on cross-transferability, and (3) determine the genetic relationships between accessions. We tested 70 SSR markers derived from O. fragrans and C. retusus in 24 accessions of Osmanthus. Sixty-seven markers showed transfer to at least one other Osmanthus species with an overall transfer rate of 84% of loci across taxa. Genotyping with 42 microsatellite markers yielded a total of 367 loci. Number of alleles per locus ranged from 2 to 17 with a mean of 8.7 ± 4.8. Mean observed and expected heterozygosities were 0.560 ± 0.225 and 0.688 ± 0.230, respectively. Percent of polymorphic loci ranged from 40% in Osmanthus delavayi to 100% in O. fragrans. Osmanthus fragrans had the highest mean number of alleles per locus (4.2) while O. delavayi had the lowest (1.1). A reduced suite of eight-markers can distinguish between accessions with non-exclusion probabilities of identity from 3.91E?⁰⁴ to 2.90E?⁰⁷. The SSR markers described herein will be immediately useful to characterize germplasm, identify hybrids, and aid in understanding the level of genetic diversity and relationships within the cultivated germplasm.     

Investigation and taxonomy of wild <Emphasis Type="Italic">Fragaria</Emphasis> resources in Tibet,China

Tibet is one of the main distribution centers of wild Fragaria species in China. There are abundant, precious and rare wild strawberry resources. This paper focused on the classification, distribution and evaluation of the 65 wild strawberry accessions collected from 17 counties in Tibet. The main taxonomical characters, such as stolon branching, leaflet type, flower sex, fruit shape, status of achene and sepal, and chromosome number were observed for each accession. All accessions were identified and classified into seven Fragaria species and two forms, including F. daltoniana J. Gay (2x), F. nilgerrensis Schlecht. (2x), F. nubicola Lindl. (2x), F. pentaphylla Losinsk. (2x), F. gracilis A. Los. (4x), F. moupinensis (French.) Card. (4x) and F. tibetica Staudt et Dickoré. (4x). The two white-fruited forms were F. nubicola f. alba (2x) and F. moupinensis f. alba (4x). The analytic hierarchy process (AHP) method was used to perform a comprehensive assessment on the value of seven Fragaria species and two forms. The results showed that F. nilgerrensis had better fruit quality, disease resistance and waterlogging tolerance than any other species in Tibet. The collection and identification of wild strawberry resources in Tibet would be helpful to improve the fruit quality and stress resistance of modern strawberry cultivars.     

Diversity of the composition and content of soluble carbohydrates in seeds of the genus <Emphasis Type="Italic">Vicia</Emphasis> (Leguminosae)

Low molecular weight carbohydrates of seeds of 10 species of Vicia, namely: V. angustifolia, V. articulata, V. cordata, V. ervilia, V. johannis, V. macrocarpa, V. monantha, V. narbonensis, V. pannonica and V. sativa were analyzed by the high resolution gas chromatography method. Seeds of the investigated species contain common (glucose, fructose, myo-inositol, sucrose, galactinol, di-galactosyl myo-inositol and raffinose family oligosaccharides—RFOs) and species-specific carbohydrates (d-pinitol and its α-d-galactosides—in V. articulata, V. monantha and V. pannonica or d-ononitol and its galactoside—in V. ervilia). Among the species containing in seeds RFOs as the main α-d-galactosides (V. angustifolia, V. cordata, V. johanensis, V. macrocarpa, V. narbonensis and V. sativa), an additional subgroup can be separated, which contains a set of unknown compounds (found in V. angustifolia, V. cordata and V. macrocarpa). Moreover, several other unidentified carbohydrate-containing compounds were detected exclusively in seeds of V. ervilia. The concentrations of total soluble carbohydrates (TSCs), including sugars, RFOs, cyclitols and galactosyl cyclitols and unknown compounds, in seeds differ significantly (P < 0.05) among the species. RFOs and sucrose are the main fractions in TSCs, with the exception of V. pannonica seeds, containing more galactosyl pinitols, and V. ervilia, in which unknown compounds account for 50% of TSCs. In V. ervilia RFOs occur at the lowest concentration (9.32 mg g^?1 seeds), and the concentration of DGMI (di-galactosyl myo-inositol) is comparable with that of RFOs, which is unusual among all the investigated Vicia species.