Modeling the technology of identification of wine materials and wines by PCR analysis of microsatellite loci of grapevine chloroplast DNA

Использование полиморфных микросателлитных локусов ДНК является одним из подходов к аутентификации виноматериалов и вин. При этом SSR-маркеры хлоропластной ДНК имеют большую копийность мишени на клетку и менее подвержены деградации из-за содержания в органеллах с двойной мембраной. Целью настоящей работы являлось моделирование технологии идентификации виноматериалов и вин ПЦР-анализом микросателлитных локусов хлоропластной ДНК винограда. Подобраны условия экстракции нуклеиновых кислот, постановки ПЦР с соответствующими наборами праймеров и электрофоретической детекции, направленные на практическое воспроизведение генетического тестирования пробоподготовленного биоматериала из осаждаемого винного дебриса. Представлены наглядные результаты выравнивания частичных нуклеотидных последовательностей аллелей микросателлитных локусов хлоропластной ДНК Vitis vinifera L. Проанализирована разделяющая способность метода горизонтального электрофореза в геле «Spreadex EL 300» in silico моделированием генерируемых аллельспецифичных фрагментов, позволяющая идентифицировать известные хлоротипы винограда даже при постановке ПЦР с ограниченными наборами праймеров, нацеленных на локусы cpSSR3, cpSSR5, cpSSR10, NTCP12 и ccSSR9. The use of polymorphic microsatellite DNA loci is one of the approaches to the authentication of wine materials and wines. At the same time, SSR markers of chloroplast DNA have a large target copy number per cell and are less susceptible to degradation due to their content in organelles with a double membrane. The aim of this work was to simulate the technology of identification of wine materials and wines by PCR analysis of microsatellite loci of grapevine chloroplast DNA. The conditions for the extraction of nucleic acids, PCR with the corresponding sets of primers and electrophoretic detection were selected, aimed at the practical reproduction of genetic testing of the sample prepared biomaterial from the precipitated wine debris. Illustrative results of the alignment of partial nucleotide sequences of alleles of microsatellite loci of Vitis vinifera L. chloroplast DNA are presented. The separating ability of the method of horizontal electrophoresis in «Spreadex EL 300» gel by in silico modeling the generated allele-specific fragments, which makes it possible to identify the known chlorotypes of grapevine with a limited set of primers targeting loci (cpSSR3, cpSSR5, cpSSR10, NTCP12 and ccSSR9) has been analyzed.

An investigation on the chloroplast and nuclear genomes of taxa belong to the subgenus Dracunculus (Bess.) Rydb. of Artemisia L. (Asteraceae) in Turkey

Artemisia is one of the biggest genera in the family Asteraceae, with around 500-600 taxa at specific and sub-specific levels and organised in 5 subgenera. Due to the high number of taxa, a lot taxonomists are trying to solve the problem of its classification and phylogeny but its natural classification still hasn’t been achieved. In this research, 60 individuals belonging to 4 taxa of the subgenus Dracunculus of Artemisia L. in Turkey were examined. For all the examined individuals from both the same and different populations belonging to the taxa of the subgenus Dracunculus, the sequences of the regions both psbA-trnH of chloroplast DNA and ITS of nuclear DNA were determined. Also, the gene regions obtained were recorded in the NCBI GenBank database and an accession number was taken. It was found that there was no gene flow and hybridization between the four studied taxa of the subgenus Dracunculus, and these 4 taxa also completed their speciation. According to the results of this molecular study, A. campestris var. campestris, A. campestris var. marschalliana and A. campestris var. araratica were proposed to be raised from the variety level to the species level. This research is important as it is the first molecular based study relating with the subgenus Dracunculus growing in Turkey.

Phylogenetic study of the genus Pohlia (Mielichhoferiaceae, Bryophyta) based on chloroplast DNA sequences

The phylogenetic circumscription and taxonomic status of the genus Pohlia in the Mniaceae sensu lato was investigated based on chloroplast DNA sequences (rbcL, rps4, and trnL-F), with a focus on species occurring in Japan. The maximum likelihood and Bayesian inference analyses of sequences obtained from 34 species of Mniaceae s.l., including 13 Pohlia species, suggested that the genus Pohlia and the family Mielichhoferiaceae are not monophyletic in their present circumscription, but confirmed that the family Mniaceae is monophyletic in its traditional sense. These results are congruent with previous molecular phylogenetic studies. Three distinct clades were recognized in the Mielichhoferiaceae, almost corresponding to three sections of Pohlia (Pohlia, Cacodon and Apalodictyon). One of them branched off first within the Mniaceae s.l., and the other two were sister to the remainder of the Mniaceae s.l. The single included Schizymenium formed a monophyletic group with Pohlia sect. Pohlia and Epipterygium with Pohlia sect. Apalodictyon, confirming the results of previous studies. The results indicate that the taxonomic status of the genus Pohlia and family Mielichhoferiaceae are in need of revision. Phylogenetic analyses nested the accessions of P. camptotrachela within P. annotina and P. flexuosa clades, highlighting the need for taxonomic revision of Japanese propaguliferous Pohlia species.

Genetic Analysis of Hexaploid Wheat (Triticum aestivum L.) Using the Complete Sequencing of Chloroplast DNA and Haplotype Analysis of the Wknox1 Gene

The aim of the presented study is a genetic characterization of the hexaploid wheat Triticum aestivum L. Two approaches were used for the genealogical study of hexaploid wheats—the complete sequencing of chloroplast DNA and PCR-based haplotype analysis of the fourth intron of Wknox1d and of the fifth-to-sixth-exon region of Wknox1b. The complete chloroplast DNA sequences of 13 hexaploid wheat samples were determined: Free-threshing—T. aestivum subsp. aestivum, one sample; T. aestivum subsp. compactum, two samples; T. aestivum subsp. sphaerococcum, one sample; T. aestivum subsp. carthlicoides, four samples. Hulled—T. aestivum subsp. spelta, three samples; T. aestivum subsp. vavilovii jakubz., two samples. The comparative analysis of complete cpDNA sequences of 20 hexaploid wheat samples (13 samples in this article plus 7 samples sequenced in this laboratory in 2018) was carried out. PCR-based haplotype analysis of the fourth intron of Wknox1d and of the fifth-to-sixth exon region of Wknox1b of all 20 hexaploid wheat samples was carried out. The 20 hexaploid wheat samples (13 samples in this article plus 7 samples in 2018) can be divided into two groups—T. aestivum subsp. spelta, three samples and T. aestivum subsp. vavilovii collected in Armenia, and the remaining 16 samples, including T. aestivum subsp. vavilovii collected in Europe (Sweden). If we take the cpDNA of Chinese Spring as a reference, 25 SNPs can be identified. Furthermore, 13–14 SNPs can be identified in T. aestivum subsp. spelta and subsp. vavilovii (Vav1). In the other samples up to 11 SNPs were detected. 22 SNPs are found in the intergenic regions, 2 found in introns, and 10 SNPs were found in the genes, of which seven are synonymous. PCR-based haplotype analysis of the fourth intron of Wknox1d and the fifth-to-sixth-exon region of Wknox1b provides an opportunity to make an assumption that hexaploid wheats T. aestivum subsp. macha var. palaeocolchicum and var. letshckumicum differ from other macha samples by the absence of a 42 bp insertion in the fourth intron of Wknox1d. One possible explanation for this observation would be that two Aegilops tauschii Coss. (A) and (B) participated in the formation of hexaploids through the D genome: Ae. tauschii (A)—macha (1–5, 7, 8, 10–12), and Ae. tauschii (B)—macha M6, M9, T. aestivum subsp. aestivum cv. ‘Chinese Spring’ and cv. ‘Red Doly’.

Molecular-Phylogenetic Research of the Genus Hypericum L. in Flora of Azerbaijan

Hypericum is one of the 100 largest flowering plant genera forming the family Hypericaceae Juss., which belongs to the clusioid clade of the Malpighiales. Hypericum is represented in Azerbaijan flora by 19 native species and 1 subspecies belonging to 7 taxonomic sections. The chloroplast DNA of 8 species from the genus was studied by PCR-RFLP analysis. Total genomic DNA was extracted from leaf tissue using the DNeasyPlantMini kit. (Qiagen Inc.; Valencia, CA, USA) following the supplied protocol and quanti field using a Nanodrop (Nanodrop Technologies; Wilmington, DE, USA) spectrophotometer. The article is part of an experimental study that comprises molecular-phylogenetic research of this genus in the flora of Azerbaijan.

A DNA barcode database for the woody plants of Japan

DNA barcode databases are increasingly available for a range of organisms facilitating the wide application of DNA barcode –based pursuits. Here we announce the development of a comprehensive DNA barcode database of the Japanese woody flora representing 43 orders, 99 families, 303 genera and 834 species and comprising 77.3% of genera and 72.2% of species of woody plants in Japan. A total of 6,216 plant specimens were collected from 223 sites (municipalities, i.e. city, town, village) across the subtropical, temperate, boreal and alpine biomes in Japan with most species represented by multiple accessions. This database utilised three chloroplast DNA regions (rbcL, trnH –psbA and matK) and consists of 14,404 barcode sequences. Individual regions varied in their identification rates with species-level and genus-level rates for rbcL, trnH –psbA and matK being 57.4%/ 96.2%, 78.5%/ 99.1 % and 67.8%/ 98%, respectively. Identification rates were higher using region combinations with total species level rates for two region combinations (rbcL & trnH, rbcL & matK, and trnH –psbA & matK) ranging between 90.6 —95.8%, and for all three regions equal to 98.6%. Genus level identification rates were even higher ranging between 99.7 —100% for two region combinations and being 100% for the three regions. These results indicate that this DNA barcode database is an effective resource for investigations of woody plants in Japan using DNA barcodes and provides a useful template for development of libraries for other components of the Japanese flora.

Population genetic structure of pedunculate oak (Quercus robur L.) in Belarus according to the analysis of chloroplast DNA

Pedunculate oak (Quercus robur L.) is one of the main forest forming species in the Republic of Belarus. Its population genetic structure was formed under the influence of various migration processes. Six chloroplast DNA loci (µdt1, µdt3, µdt4, µcd4, µcd5 and µkk4) were used for the genogeographic study. The material for the analysis was collected in 100 oak forest stands (2325 samples); 18 allelic variants were identified, which are grouped into 17 different combinations (haplotypes). Five of them are widespread (the proportion of occurrence varies from 7 to 48 %, totalling 85 %). The remaining 12 are rare (the proportion of occurrence varies from 1 to 3 %, totalling 15 %). Phylogenetic trees constructed using the nearest neighbour and maximum likelihood methods show the presence of two groups (branches) of haplotypes. One of it comprises 8 variants including 2 dominant haplotypes and the other comprises 9 variants including 3 dominant haplotypes. PCR-RFLP analysis of chloroplast DNA showed that the pedunculate oak in Belarus originates from the Balkan refugium. Haplotype No. 1 (µdt189, µdt3123, µdt4142, µcd494, µcd574, µkk4109) is found almost everywhere in Belarus with the exception of the southwest and northeast, while haplotype No. 8 (µdt189, µdt3121, µdt4142, µcd494, µcd574, µkk4109) is mainly localised in the southwest and northeast. Haplotypes No. 3 (µdt189, µdt3120, µdt4141, µcd494, µcd575, µkk4109) and No. 7 (µdt189, µdt3122, µdt4142, µcd494, µcd574, µkk4109) predominantly found in the west of the country. Haplotype No. 2 (µdt190, µdt3120, µdt4141, µcd495, µcd574, µkk4109) is typical for the southeast.

Predominance of clonal propagation conceals extinction risks of the highly endangered floodplain herb Cnidium dubium

Habitat loss and degradation due to human-induced landscape alterations are considered to be a major threat to biodiversity. The decline of biodiversity may occur with a time delay leading to a so called extinction debt. Therefore, determining extinction risks and conservation status is not always straightforward. Several life history traits might play a role for the accumulation of an extinction debt. Thus, perennial plant species capable of vegetative propagation might be able to persist temporarily in degraded habitats even though sexual and evolutionary processes are effectively halted. We studied Cnidium dubium, which occurs in scattered patches along river corridors in Central Europe and is critically endangered in Germany. It is a perennial species which is able to propagate clonally. Our aims were to reconstruct demographic processes regarding clonal propagation and gene flow along 400 km of river stretch and with respect to the position in the flooplain, i.e. before or behind dykes. We also wanted to determine whether there is evidence for an extinction debt in C. dubium and to use our insights for conservation recommendations. For this, we used nuclear microsatellites and maternally inherited chloroplast DNA markers and applied a systematic grid based sampling strategy for small scale geographic structures. We observed a high level of clonal propagation. In 935 analysed plants we observed only 121 different genotypes and of 50 studied patches of C. dubium the majority (31 patches) consisted of one single genotype each. Patch size and position were correlated with clonal diversity. Large patches and patches behind dykes exhibited higher clonal diversity. There was no evidence for a large scale genetic substructuring of the study area and no differences in overall genetic diversity between upstream and downstream patches as well as between patches before and behind the dykes. High levels of heterozygosity and a high number of 18 chloroplast DNA haplotypes togetherwith a slightly elevated inbreeding coefficient (Fis) point toward a high level of ancestral polymorphism in an out of equilibrium population due to high levels of clonal propagation and low levels of gene flow and recombination. Therefore, we assume that an extinction debt is present in C. dubium. As a management strategy, we propose to transplant ramets between multiple patches to increase the number of mating partners and therefore restore sexual reproduction.

Taxonomic Composition of Iris Subser. Chrysographes (Iridaceae) Inferred from Chloroplast DNA and Morphological Analyses

The species of Iris subser. Chrysographes are herbaceous perennials found mainly in southwestern and central China and also in the Eastern Himalayas. To date, six species have been recognized in this group. In the framework of its taxonomic revision, we have carried out molecular and morphological studies. For this, we have sequenced four chloroplast DNA regions (trnS–trnG, trnL–trnF, rps4–trnSGGA, and psbA–trnH) for 25 samples across the major distribution ranges of the six species. Our phylogenetic analyses evidence that I. subser. Chrysographes is indeed a monophyletic group, which is sister to I. subser. Sibiricae. Within I. subser. Chrysographes, we have recovered four divergent lineages further supported by diagnosable morphological traits and geographical distributions. In this context, our data confirm the recognition of I. clarkei, I. delavayi, and I. wilsonii in their traditional concepts. Furthermore, both molecular and morphological data support the close affinities and similar distribution ranges of I. bulleyana, I. chrysographes, and I. forrestii, which suggests including I. chrysographes and I. forrestii as color forms in I. bulleyana. A revised taxonomic treatment for the group, including the notes on the species distributions and habitats, and also an identification key to the species are provided.