Exploitation of the chloroplast trnL (UAA) intron polymorphisms for the authentication of plant oils by means of a lab-on-a-chip capillary electrophoresis system

Methods to discriminate plant oils facilitate the detection of either deliberate or accidental adulteration. To this direction, the variability in length among plant species of the chloroplast trnL intron was exploited for the authentication of edible and cosmetic plant oils, with an extra emphasis on olive oil. The methodology was based on the combinatorial use of a PCR assay with a capillary electrophoresis system such as the lab-on-a-chip technology. Application of the assay on DNA extracted from different oil producing plant species, including olive oil and sesame oil, indicated the ability of the trnL intron to be used as an analytical target. Furthermore, this assay could be used for the detection of adulteration of olive oil with various other plant oils, with the exception of avocado and sesame oil.     

Internal transcribed spacer sequences of nuclear ribosomal DNA resolving complex taxonomic history in the genus Vicia L.

The ITS1-5.8S-ITS2 region of the 18S-28S nrDNA was sequenced in 49 Vicia species and subspecies selected from nine sections of the two subgenera to resolve taxonomic contradictions. Phylogenies derived from the internal transcribed spacer (ITS) sequences strongly support the view that both Vicia faba and Vicia bithynica are distant from the Narbonensis species complex (NSC) in section Faba. V. faba is more closely aligned with sections Peregrinae and Hypechusa, whereas V. bithynica is more closely aligned with section Vicia. Six synapomorphic substitutions unique to the NSC support a separate section for the Narbonensis complex. Vicia johannis subsp. johannis was the most diverged species within the NSC with high genetic distance between V. johannis subsp. johannis and remaining species and subspecies of the NSC. These results suggest that Vicia johannis subsp. johannis should be given species rank. The neighbor-joining and parsimony based trees supported the sections Vicia (after the exclusion of Vicia lathyroides) and Cracca (after the exclusion of Vicia vicioides and Vicia hirsuta). The section Hypechusa is polyphyletic and paraphyletic based on the present ITS sequence cladistic analysis. The x = 7 as the ancestral number for the genus is supported by the present study.     

Germplasm Sources for Improvement of Forage Quality in Lotus corniculatus L. and L. uliginosus Schkuhr (Fabaceae)

Extensive germplasm resources are present in collections of Lotus corniculatus and L. uliginosus, two forage species that are valuable in situations where other legumes are not persistent. Naturalised populations in many regions are the primary gene pool for genetic improvement of these species and interspecific hybrids are an important source of variation for agronomic and forage quality traits. Although previous research has provided evidence that selection for optimal levels of condensed tannins and herbage productivity are feasible breeding objectives, less is known about correlations of condensed tannin with other parameters of nutritive value. A germplasm collection of 38 accessions in both species from 9 geographic regions was used as a data base to determine these correlations. Forage of each accession was sampled in two successive years from a field site in south-eastern Australia and analysed for condensed tannin, in vitro digestible dry matter, nitrogen, acid detergent fibre and neutral detergent fibre. In both species condensed tannin was negatively correlated with in vitro digestible dry matter and nitrogen. This result helped to identify potentially valuable accessions with low condensed tannin (<4% of dry weight) and high in vitro digestible dry matter (>70%). One of these was an interspecific hybrid of L. uliginosus and L. corniculatus, confirming the view that interspecific hybridization would be a valuable avenue for improvement of agronomic and forage quality characters in Lotus species.     

Vicia sativa L. aggregate (Fabaceae) in the flora of former USSR

Morphological differences and ecogeographical characteristics of five species from Vicia sativa agg. in the area of former USSR were investigated. The key to V. sativa agg. species and subspecies as well as maps of their areals are presented. A complementary study of polymorphism at the phenotypical level of 58 local weedy and wild populations from different geographical regions was conducted to determine areas with higher variability of morphological characters used for intraspecific taxonomy and cultivar identification. The regions of greatest interest for curators and collectors of V. sativa agg. germplasm are some regions of Transcaucasus (Nagornyi Karabakh, Talysh range, Lenkoran valley) and South of Crimea.     

Molecular phylogeny of Chinese vegetable mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) based on the internal transcribed spacers (ITS) of nuclear ribosomal DNA

Sequence variation of nuclear internal transcribed spacer regions of ribosomal DNA (ITS1, 5.8S rRNA and ITS2) from Chinese vegetable mustards (AB-genome) and its putative parents Brassica rapa (the A-genome) and Brassica nigra (the B-genome) were used to investigate the molecular phylogeny and the probable evolutional pattern of this amphidiploid species that uniquely formed in China. Totally, 16 accessions of Chinese vegetable mustard those covering nearly all the diverse variations were included in this study, and together with three accessions of B. rapa and one accession of B. nigra. The results disclosed two strongly supported clades, one containing four accessions of vegetable mustard which have closer relationship with B-genome species “B.nigra” lineage and the other containing 12 accessions of B. juncea and three A-genome accessions. This classification was in disagreement with the evidence from chloroplast DNA, mitochondrial DNA, nuclear DNA restriction fragment length polymorphism (RFLP), which suggested that B. juncea was closely related to the A-genome type. For the incongruence, we speculated that the B. juncea crops derived from Chinese have evolved through different recombined events of the diploid morphutypes and evolved unidirectional concerted evolution. The traditional phenotypic classification of B. juncea was not wholly supported by ITS results, and hence the phylogenetic relationships among these subspecies need to be reconsidered on molecular level.     

Molecular differentiation in Indian Citrus L. (Rutaceae) inferred from nrDNA ITS sequence analysis

Molecular differentiation in 24 accessions representing 19 taxa of Indian Citrus has been examined through sequence analysis of Internal Transcribed Spacer (ITS) region of nrDNA. Sequence length in the 24 accessions of Citrus taxa ranged from 512 to 665 bp (ITS1 & ITS2 partial and 5.8S complete sequence). The ITS sequences were very rich in G+C content ranging from 61.40 to 66.60% with an average of 64.2%. Genetic distance within Citrus group ranged from 0 to 13.4% with an average of 4.6%, showing moderate rate of nucleotide divergence. The phylogeny was inferred using the Maximum parsimony (MP) and Neighbor-Joining (NJ) methods. Both MP and NJ trees separated all the 24 accessions of Citrus into six distinct clusters. The disposition of all the accessions of Citrus in separate clusters in ITS-derived dendrograms was partly in accordance with the morpho-taxonomic affinities of the target taxa. This study supports the concept of Citrus medica (citron), C. reticulata (mandarin), and C. maxima (pummelo) as the basic species of the genus. However, ITS marker could not find any clear cut differentiation between subgenera Citrus and Papeda as proposed in Swingle’s Citrus classification system. The present study also supports the distinctiveness of C. indica (Indian wild orange), C. latipes (Khasi papeda) and C. hystrix (Melanesian papeda) as true species, besides elucidating the probable hybrid origin and relationships among the cultivated species/biotypes, such as Citrus ×aurantiifolia (sour lime) C. ×limon (lemon), C. ×taitensis (Indian rough lemon), C. limettioides (sweet lime), C. ×aurantium (including sour and sweet oranges and grapefruit), and other indigenous varieties of Indian origin: C. megaloxycarpa (sour pummelo), C. karna (karna orange), C. pseudolimon (Hill lemon), ‘Memang athur’, ‘Pummelo-lemon’ and ‘Kathairi nimbu’.     

Hybridization Between Wild and Domesticated Types of Phaseolus vulgaris L. (Fabaceae) in Argentina

A polymorphic population of Phaseolus vulgaris L. var. aborigineus (Burkart) Baudet growing in northwestern Argentina was studied. In order to know the origin of this polymorphism, plants belonging to the var. aborigineus, others showing floral dimorphism and individuals with weedy type characters were collected. Their seeds, obtained after fieldwork treatments of autogamy and free pollination in order to know their reproductive system, were sown in a greenhouse. Information of their growth was recorded and several numerical analyses were performed. With the results obtained, we concluded that those individuals that showed floral dimorphism are probably a result of hybridization and/or introgression between the var. aborigineus and cultivated forms that grow in the area. This hypothesis is supported by the presence of divergent segregation in the offspring. Also, the offspring of the F1 was followed and allowed us to conclude the possible existence of degeneration of the hybrid progeny. This degeneration indicates that an unwanted gene flow in the area could lead to a decline in the wild bean population. The vigour and high reproductive success of the offspring belonging to the individuals corresponding to the var. aborigineus, whose progenitor was treated for outcrossing, and the existence of plants with weedy type characteristics, are indicative of the necessity to preserve this germplasm in order to evaluate its agronomic potential as brief as possible. The population here studied constitutes a wild-weed–crop complex growing in Argentina.     

Influence of lupin (Lupinus luteus L. cv. 4492 and Lupinus angustifolius L. var. zapaton) and fenugreek (Trigonella foenum-graecum L.) germination on microbial population and biogenic amines

Microbial population and bioactive amine profile and levels of two lupin species (Lupinus luteus L. cv. 4492 and Lupinus angustifolius L. var. zapaton) and fenugreek (Trigonella foenum-graecum L.) seeds as affected by germination were investigated. Microbial population increased considerably mainly in the first stage of germination (2 days), then small changes in bacterial numbers were observed up to 5 days to levels between 7.8 and 8.9 log colony-forming units/g. Microorganisms belonging to the Enterobacteriaceae family were dominant for the legumes tested. Ungerminated legume seeds contained putrescine, cadaverine, histamine, tyramine, spermidine, and spermine. Bioactive amine levels found in fenugreek seeds were between 3- and 4-fold higher than those found in lupin seeds. The highest total amine levels were found in fenugreek seeds [162 mg/kg of dry weight (dw)], followed by L. angustifolius var. zapaton seeds (84 mg/kg of dw) and, finally, L. luteus cv. 4492 (46 mg/kg of dw) seeds. The concentration of individual amines showed a gradual rising trend during the germination period in all tested sprouts, reaching levels >3 times higher than those found in ungerminated seeds. After 5 days of germination, the fenugreek sprouts contained the highest amount of total bioactive amines. Tyramine was the predominant amine in both lupin varieties, whereas cadaverine was the main bioactive amine detected in fenugreek. The results of this work thus indicated that microbial population and biogenic amine levels in the studied lupin and fenugreek sprouts are not a risk for healthy consumers or for individuals with restricted activity of detoxification enzymes.     

Assessment of genetic diversity, and phylogenetic relationships based on ribosomal DNA repeat unit length variation and Internal Transcribed Spacer (ITS) sequences in chickpea (Cicer arietinum) cultivars and its wild species

Repeat unit length variation and internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA were used to assess genetic diversity, and phylogenetic relationships in chickpea (C. arietinum) cultivars, and its related wild species. Total genomic DNAs of 76 accessions of 10 Cicer species, belonging to three sections of the genus, were restricted with seven enzymes and the restriction fragments were hybridized to heterologous ribosomal clones of wheat pTa71 and Vicia faba probes Ver 6-5 and Ver18-6. A single repeat unit length class of 11.4 kb or 10.5 kb was recognized across Cicer accessions with pTa71. The intraspecific variation was negligible in those species where more than one accession was studied, except the four C. judaicum accessions, which were different from the rest. EcoRI and DraI digests gave two and one-two fragments, respectively. All the accessions produced three and three-five bands with BamHI and SacI, respectively. Both the accessions of C. yamashitae differed in their rDNA repeat unit length as well as restriction site variation. Maximum likelihood tree with rDNA RFLP recognized five clades which were more or less congruent with the previous data. Length of ITS-1 region was more variable (235–239 bp) than the ITS-2 region (212–213 bp). Cladistic analysis of ITS data revealed two major clades, clade I consisting of C. arietinum, C. reticulatum and C. echinospermum, and clade II comprised of C. judaicum, C. chorassanicum, C. bijugum and C. cuneatum. C. microphyllum grouped with the above four species. C. pinnatifidum was present as a separate branch. C. yamashitae emerged as the most distinct species.     

Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoflavones and estrogenic activity

Red clover (Trifolium pratense L., Fabaceae) dietary supplements are currently used to treat menopausal symptoms because of their high content of the mildly estrogenic isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds are estrogenic in vitro and in vivo, but little information exists on the best time to harvest red clover fields to maximize content of the isoflavones and thus make an optimal product. Samples of cultivated red clover above-ground parts and flower heads were collected in parallel over one growing season in northeastern Illinois. Generally, autohydrolytic extracts of above-ground parts contained more isoflavones and had more estrogenic activity in Ishikawa endometrial cells as compared with extracts of flower heads. Daidzein and genistein contents peaked around June to July, while formononetin and biochanin A contents peaked in early September. Flower head and total above-ground parts extracts exhibited differential estrogenic activity in an Ishikawa (endometrial) cell-based alkaline phosphatase induction assay, whereas nondifferential activity was observed for most extracts tested in an MCF-7 (breast) cell proliferation assay when tested at the same final concentrations. Ishikawa assay results could be mapped onto the extracts' content of individual isoflavones, but MCF-7 results did not show such a pattern. These results suggest that significant metabolism of isoflavones may occur in MCF-7 cells but not in Ishikawa cells; therefore, caution is advised in the choice of bioassay used for the biological standardization of botanical dietary supplements.     

Genetic diversity and phylogeny of pearl millets [Pennisetum glaucum (L.) R. Br.] based on chloroplast trnL-F region

Local varieties of pearl millets in Saudi Arabia are heat- and drought-tolerant and are grown under various environments. Here, we evaluated the diversity of six local varieties based on sequencing and analysis of the trnL-F region of their chloroplast DNA that includes the genes of tRNA^Leu and tRNA^Phe and the intergenic spacer between them, trnL-F. The obtained sequences were 1171–1172 bp in length, including the complete sequences of the tRNA^Leu, trnL-F and tRNA^Phe regions. Three variable nucleotide sites were observed in the sequences of the Indian variety, whereas for each of the three varieties, Tihamah, Jazan, and Sayah, a single variable site was observed. The varieties, Yemeni and Baydhan, were identical for the entire analyzed sequence. The secondary structure of tRNA^Leu intron of Indian variety was distinct from the other five varieties, while Tihamah and Jazan were distinct in the secondary structure of trnL-F spacer. Additionally, Indian was distinct in the secondary structure of tRNA^Leu whereas Sayah and Jazan were distinct in the secondary structure of tRNA^Phe. Phylogenetic analysis with NCBI-retrieved accessions of the Pennisetum glaucum revealed close relationships between the investigated varieties with an exception of the Indian, which clustered separately from the other five varieties. The nucleotide polymorphism analysis revealed higher nucleotide diversity in the spacer between tRNA^Leu and tRNA^Phe than in the tRNA^Leu intron across the six local varieties. This study provides insights into the genetic diversity within local pearl millets, which is important for the conservation of the local varieties that exhibit exceptional adaptation to the harsh climate conditions in the Arabian Peninsula.

     

Effects of vermicompost and vermiwash biofertilizers on fenugreek (Trigonella foenum) plant

ABSTRACT

In order to evaluate the effect of vermicompost and vermiwash biofertilizers on the traits of fenugreek (Trigonella foenum) plant, a field experiment of a randomized complete block design with 8 treatments and 3 replications was conducted. The treatments consisted of control without fertilizer, vermicompost at 5, 10, and 15 tons per hectare, vermiwash spraying with 3,000, 6,000, and 12,000 ppm, and a combined application of 10 tons of vermicompost with 6,000 ppm vermiwash. In this research, traits of crop growth rate (CGR), pod length, pod width, grain yield, chlorophyll a and b, essential oil percentage, and essential oil yield were evaluated. The results revealed that the use of vermicompost biofertilizer increased the measured quantitative traits more than that of the vermiwash. The most and the least effects on all traits, except for the length of the pod, were 15 and 5 tons vermicompost per hectare, respectively. The treatment of 10 tons of vermicompost with 6,000 ppm vermiwash did not show significant differences in 10 and 15 tons vermicompost per hectare in most traits. It was observed that the application of vermicompost fertilizer leads to weaker results than the vermiwash fertilizer. With the increase of vermiwash concentration, the effect of this fertilizer on the measured traits reduced; however, in most traits, the results of the application of 3,000 and 6,000 ppm vermiwash had a statistically significant effect on the treatment with 5 tons of vermicompost per hectare. Overall, this experiment showed that all the evaluated traits in this study have a positive reaction to the application of vermicompost and vermiwash biofertilizers. Also, the response of the fenugreek plant to the vermicompost is much greater than the vermiwash fertilizer.     

Molecular phylogeny of the apterygotan insects based on nuclear and mitochondrial genes

Basal hexapodan orders (Apterygota) are traditionally divided in two well defined taxonomic groups, Entognatha and Ectognatha. Entognathy (the enclosed mouthparts condition) is considered the most distinctive character joining the Ellipura (Protura+ Collembola) with the order Diplura, whereas the presence of exposed mouthparts (ectognathy) is the feature shared by Microcoryphia and Zygentoma with the pterygote insects. In spite of the growing interest for the evolutionary history of the Apterygota, there is no complete agreement among the general phylogenetic hypotheses based on the study of morphological characters. In this study we analyzed the DNA sequence of segments of the nuclear Elongation Factor-1α(EF-1α) and of the mitochondrial 12S rRNA genes, and used different methods of phylogenetic reconstruction. The 12S gene seems to be more suited than the EF-1α to resolve some of the most outstanding systematic disputes, whereas the lack of resolution at the deeper nodes does not allow to assess the phylogenetic relationships within Microcoryphia and between ectognathan orders. We have obtained a fairly high support for the monophyly of the orders Diplura and Zygentoma. In the 12S analysis, the Ellipura and the Entognatha form monophyletic assemblages. In addition, the study of the distribution of introns in the EF-1α suggests a relationship between Collembola and Diplura.     

New evidence on the origin of mangosteen (Garcinia mangostana L.) based on morphology and ITS sequence

Mangosteen (Garcinia mangostana L.), known as one of the most desirable tropical fruits of Southeast Asia, has been considered as an obligate agamospermous hybrid, thought to have arisen from two wild species, G. celebica L. (syn. G. hombroniana Pierre) and G. malaccensis Hook. f. However, this putative origin was based on a misidentification of G. malaccensis, which was confused for G. penangiana Pierre. Intensive field studies and molecular investigations based on internal transcribed spacer (ITS) sequence data of 22 samples were conducted, which included six samples of true G. malaccensis. Morphological observation shows that mangosteen highly resembles G. malaccensis, particularly in its vegetative and fruit characters, even sharing similar taste of ripe fruits. ITS data revealed that mangosteen shared more than 99 % of its sequence with G. malaccensis with a few accessions identical with wild populations in Peninsular Malaysia. Phylogenetic analysis revealed that clades of mangosteen are paraphyletic per se, but monophyletic if both mangosteen and G. malaccensis are grouped together. This show that mangosteen and G. malaccensis are so closely related that they should be combined together as one species. I propose two theories on the origin of mangosteen, first, that it is a hybrid of different varieties of G. malaccensis, and second, that it may be a product of multiple, superior selections from different populations of female trees of G. malaccensis originating in Peninsular Malaysia.     

Phylogenetic relationships among Triticum L. and Aegilops L. species as genome progenitors of bread wheat based on sequence diversity in trnT-F region of chloroplast DNA

Cultivated wheat, (Triticum aestivum L.), is one of the most important food crops in the world. The Aegilops L. genus is frequently utilized by plant breeders for improving the current wheat cultivars due to their close relationships. Therefore, understanding the phylogenetic relationships among the species of these genera is not only valuable for plant taxonomy, but also for plant breeding efforts. The presented phylogenetic analysis was based on the sequences of trnT-F chloroplast DNA containing three non-coding sub-regions. Twelve genotypes belonging to four species of Triticum L. genus and twenty-four genotypes belonging to eight species of Aegilops genus were used in the current study. The results postulated a close genetic relationship between diploid Aegilops species containing the BB genome and polyploid Triticum species. With the exception of Aegilops cylindrica Host (CCDD), all other Aegilops species having the CC genome were alienated from Aegilops speltoides Tausch (BB) and clustered together. These two clusters joined by a third cluster including the AA genome containing diploid Triticum species.     

杀虫剂对甘蓝蚜与七星瓢虫的毒力及选择性研究

试验研究杀虫剂对甘蓝蚜及其天敌七星瓢虫幼虫的毒力及选择性结果表明,15种供试杀虫剂中以吡虫啉对七星瓢虫幼虫和甘蓝蚜的选择性最高,其选择性毒力比值(瓢虫LD50/甘蓝蚜LD50)为174.29。5种有机磷类杀虫剂中以马拉硫磷的选择性最高,其次为敌敌畏,其选择性毒力比值分别为35.73和25.32;7种菊酯类杀虫剂中以氟氯氰菊酯和氯氰菊酯的选择性最高,其选择性毒力比值分别为55.16和30.00。故吡虫啉、氟氯氰菊酯和马拉硫磷等选择性较高的杀虫剂对甘蓝蚜相对毒力高,而对其天敌瓢虫相对安全,为防治菜田甘蓝蚜的理想品种。