Regions psbA-trnH Research Articles

In Silico Evaluation of rbcL, matK, and psbA-trnH Regions on the Genus Spatholobus (Fabaceae)

Spatholobus is a genus that belongs to the Fabaceae that is known to contain various bioactive compounds and distributed across Asia, including Indonesia. However, exploration of Spatholobus in Indonesia is still rare. Therefore, DNA barcoding is used to support the exploration and conservation of Spatholobus in Indonesia. However, there is no universal marker that can be used across all plant species. In addition, there are still few studies related to DNA barcoding within the genus Spatholobus. The purpose of this study was to evaluate the rbcL, matK, and intergenic spacer psbA-trnH regions in silico that can be used as DNA barcodes for the genus Spatholobus. This study began with a sequence search on the NCBI database including the rbcL, matK, and intergenic spacer psbA-trnH genes in the genus Spatholobus and Phaseolus coccineus as the outgroup. Each sequence was then aligned with ClustalW. Then, a phylogenetic tree was constructed using the Maximum-Likelihood (ML) with 1000× bootstrap. As a result, the rbcL, matK, and psbA-trnH regions can be used as markers for DNA barcoding in the genus Spatholobus with different specifications. The rbcL and matK can be used to distinguish Spatholobus at the genus level, while the psbA-trnH can be used to distinguish Spatholobus at the species level.

The Identification and Cytotoxic Evaluation of Nutmeg (Myristica fragrans Houtt.) and Its Substituents.

The aril and seed of nutmeg, Myristica fragrans Houtt. (Myristicaceae), hold significant value in various industries globally. Our preliminary research found two morphological variations: a globose shape and an oval shape. Due to these different characteristics, the safety of consumers is of primary concern. Thus, authentication and comparative pharmacological and toxicity analyses are necessary. In this study, pharmacognostic and advanced phytochemical analyses, DNA barcoding, cytotoxicity, and the anti-nitric oxide production of commercial Thai nutmeg were examined. Via morphologic examinations and TLC fingerprinting, all the sampled aril and seed were categorized into globose and oval-shaped groups. The results of HPLC, GC-MS, and LC-MS/MS experiments revealed distinct differences between these groups. The DNA barcoding of the trnH-psbA region using the BLAST method and neighbor-joining tree analyses confirmed the globose nutmeg as M. fragrans and the oval-shaped variant as M. argentea. A comparison was then carried out between the potential toxicity and anti-inflammatory capabilities of M. fragrans and M. argentea. Cytotoxicity tests on HaCaT, 3T3-L1, Caco-2, HEK293, and RAW264.7 were performed using both methanolic extracts and volatile oil from the arils and seeds of both species. This study concludes that blending or substituting these two species maintains their therapeutic integrity without posing safety concerns.

IDENTIFICATION OF LENS CULTIVARS IN MARKET BY MOLECULAR TOOLS: DNA BARCODING AND SSRs

Substitution of plant cultivars of high commercial value with a cheaper, lower quality one is a common fraud committed against consumers and producers. Since it is one of the most widely grown legumes, lentil (Lens culinaris Medik.) is suitable for such frauds. This study aimed to identify lentil cultivars which are registered and authorized in the market in Türkiye by using current molecular methods. For this purpose, 26 lentil cultivars were analyzed for 15 SSR markers and two DNA barcode regions (trnH-psbA and matK). A high allele diversity was observed by 12 scorable SSR markers, and the average number of alleles was determined to be 16. One of the important findings was the presence of “cultivar-specific alleles” that can be used to identify each cultivar in the lentil market in Türkiye. At least one “cultivar-specific allele” was obtained for each cultivar. The lentil cultivars were also analyzed by two DNA barcode regions as trnH-psbA and matK. While it was observed that the rate of the intra-species variation for the trnH-psbA region was low and 26 varieties were divided into 7 groups, higher rate was found for matK and samples were distributed into 14 groups. Nevertheless, it was observed that intra-species discrimination can be made more effective when both loci are used together and 26 species were distributed into 18 different groups. We expect that the results of this study, especially the cultivar-specific SSR alleles and DNA barcoding sequence data may be used routinely to identify production and packaged products that are commercially available in markets.

Chloroplast genome, nuclear ITS regions, mitogenome regions, and Skmer analysis resolved the genetic relationship among Cinnamomum species in Sri Lanka.

Cinnamomum species have gained worldwide attention because of their economic benefits. Among them, C. verum (synonymous with C. zeylanicum Blume), commonly known as Ceylon Cinnamon or True Cinnamon is mainly produced in Sri Lanka. In addition, Sri Lanka is home to seven endemic wild cinnamon species, C. capparu-coronde, C. citriodorum, C. dubium, C. litseifolium, C. ovalifolium, C. rivulorum and C. sinharajaense. Proper identification and genetic characterization are fundamental for the conservation and commercialization of these species. While some species can be identified based on distinct morphological or chemical traits, others cannot be identified easily morphologically or chemically. The DNA barcoding using rbcL, matK, and trnH-psbA regions could not also resolve the identification of Cinnamomum species in Sri Lanka. Therefore, we generated Illumina Hiseq data of about 20x coverage for each identified species and a C. verum sample (India) and assembled the chloroplast genome, nuclear ITS regions, and several mitochondrial genes, and conducted Skmer analysis. Chloroplast genomes of all eight species were assembled using a seed-based method.According to the Bayesian phylogenomic tree constructed with the complete chloroplast genomes, the C. verum (Sri Lanka) is sister to previously sequenced C. verum (NC_035236.1, KY635878.1), C. dubium and C. rivulorum. The C. verum sample from India is sister to C. litseifolium and C. ovalifolium. According to the ITS regions studied, C. verum (Sri Lanka) is sister to C. verum (NC_035236.1), C. dubium and C. rivulorum. Cinnamomum verum (India) shares an identical ITS region with C. ovalifolium, C. litseifolium, C. citriodorum, and C. capparu-coronde. According to the Skmer analysis C. verum (Sri Lanka) is sister to C. dubium and C. rivulorum, whereas C. verum (India) is sister to C. ovalifolium, and C. litseifolium. The chloroplast gene ycf1 was identified as a chloroplast barcode for the identification of Cinnamomum species. We identified an 18 bp indel region in the ycf1 gene, that could differentiate C. verum (India) and C. verum (Sri Lanka) samples tested.

Qualitative identification of lonicerae japonicae flos in traditional chinese medicine using metabarcoding combined with specific mini-barcodes.

Lonicerae japonicae flos, also known as Jinyinhua (JYH), is an important component of traditional Chinese patent medicine (TCPM) products. However, the potential for adulteration and substitution with low-quality materials highlights the need for a reliable and sensitive approach to identify the species composition of TCPM products for consumer safety. We used universal ITS2 primers to amplify TCPMs containing JYH. However, the results were inconclusive, as only one operational taxonomic unit (OTU) was identified as Lonicera sp., which could not be identified at the species level. To confirm the species identification of Lonicera sp. in TCPM, we developed a short mini-barcode primer based on the psbA-trnH region, which, in combination with DNA metabarcoding technology, allowed for qualitative and quantitative analysis of artificially mixed samples. We applied the mini-barcode to distinguish TCPMs containing JYH and demonstrated its relatively accurate quantitative ability in identifying two Lonicera species. Our study presents a method for qualitative and quantitative identification of JYH, providing a promising application of DNA metabarcoding technology in the quality control of TCPM products.

Differentiation of Centella asiatica (L.) Urb. from Hydrocotyle umbellata L. using the trnH-psbA region, species-specific bands and anisocytic stomata as markers for quality control of raw materials and their products

Centella asiatica (L.) Urb. has been confused with Hydrocotyle umbellata L. in Thailand. The plants grow in the same habitat and look alike, making H. umbellata a potential adulterant species for C. asiatica. This study aimed to (i) establish reference markers of authentic C. asiatica and H. umbellata using DNA barcoding, thin-layer chromatography (TLC) and microscopic methods and (ii) determine whether H. umbellata adulterants are present in C. asiatica products in Thailand. Ribulose-bisphosphate carboxylase (rbcL), maturase K (matK), internal transcribed spacer (ITS) and chloroplast intergenic spacer (trnH-psbA) were identified as core DNA barcode regions that can be used to differentiate C. asiatica and H. umbellata. The trnH-psbA region showed the highest nucleotide variation between C. asiatica and H. umbellata and had distinct amplicon sizes of approximately 506 and 398 bp, respectively. TLC analysis revealed three distinct bands at Rf 0.20, 0.30 and 0.32 that could be used to differentiate C. asiatica and H. umbellata. Anisocytic stomata were present only in C. asiatica and were purposed as a microscopic marker. The trnH-psbA amplicon, TLC species-specific markers and anisocytic stomata were utilized to examine six Bua Bok products traded in Thailand. Agarose gel electrophoresis showed PCR amplicons at approximately 500 bp, TLC showed species-specific bands at Rf 0.20, 0.30 and 0.32, and anisocytic stomata were found in all products, confirming that all tested products were C. asiatica. These markers will benefit the herbal industry and other end users by allowing the detection of the adulterant species H. umbellata in C. asiatica raw materials and products.

A population genetic-level approach to endemic species of Oxalis section Palmatifoliae (Oxalidaceae) in Patagonia

Oxalis section Palmatifoliae (wood sorrel) includes five species that are well differentiated morphologically. A previous population study in one of the species (O. laciniata) suggested a complex phylogeographic history and highlighted the need to explore the genetic variability of all the species in the section. Thus, we performed a population level study of the five species to (1) determine the population structure within and among species, (2) model their current and paleoclimatic distributions, and (3) assess the historical processes that influenced their current genetic and morphological variation. We generated inter-simple sequence repeat (ISSR) polymorphism data and DNA sequences for the plastid region trnH-psbA and used those data to obtain a haplotype network, and used georeferenced coordinates and Maxent to obtain present and past species distribution models. Our major results included: 1) ISSR data showed a significant level of genetic differentiation among the five species and a degree of admixture within species; 2) plastid region trnH-psbA shows that one haplotype is the most frequent and widespread among all the species; 3) modelling of species present distributions showed different climatic variables for each species, whereas modelling of past distribution scenarios suggests broader distributions of all species.

COMPARISON OF rbcL AND trnH-psbA DNA BARCODES IN DIVERSE CAMELLIA SPECIES COLLECTION IN VIETNAM

Camellia is a large genus in the tea family of Theaceae. In this genus, several species serve different purposes, such as, medicinal and ornamental plants and beverage production. Thus, country-wide cultivation of various species of Camellia genus sought to alleviate poverty and promote economic development in many regions. However, studies on evaluating its genetic resources as a foundation for the conservation and development of this plant are yet to start. Currently, using DNA barcoding often serves as a highly reliable approach to identifying and characterizing numerous plants. In the presented study, a total of 10 Camellia accessions collected from Dong Bua village, Tam Quan commune, District Tam Dao, and Vinh Phuc province, Vietnam, underwent study during 2020–2022 at Ho Chi Minh City University of Food Industry, Vietnam. Evaluation of two DNA barcoding regions, namely, rbcL and trnH-psbA, transpired for their ability to distinguish the Camellia accessions belonging to different species in Vietnam. The results revealed a significant difference in the DNA sequences of the rbcL and trnH-psbA regions among the Camellia species. In addition, the trnH-psbA barcode region also showed higher effectiveness versus the rbcL region in recognizing various species of Camellia. The results authenticated the potential of DNA barcoding in the management, conservation, and development of the genetic resources of Camellia in Vietnam.

The molecular investigations on the subgenus Artemisia Less. of the genus Artemisia L. (Asteraceae) in Turkey

The genus Artemisia L. of the family Asteraceae is systematically very complex. The aim of this study was to evaluate taxonomic positions of taxa of the subgenus Artemisia belonging to the genus Artemisia in Turkey using some molecular techniques. In this molecular study, 44 individuals belong to 14 species of the subgenus Artemisia were examined. Analyses were performed on the combined dataset using maximum parsimony, maximum likelihood and Bayesian inference and Molecular parameters obtained from co-evaluations of sequences of the psbA-trnH, ITS and ETS regions of examined individuals were used in the phylogenetic tree drawing. According to the results of this study, two molecular groups have been formed based on the DNA sequence similarity of the species, but there are no obvious morphological characters corresponding to two molecular groups. There is no also agreement between the two molecular groups and the two morphological groups formed according to the hairiness condition of the receptacle of species. Due to the lack of molecular significance of their receptacles with or without hair, dividing of the subgenus Artemisia species into new subgenera or sections was not considered appropriate. Likewise, it has been found that with or without hair on the corolla lobes of the central hermaphrodite disc flowers have no molecular significance. It was found that there were no gene flow and hybridization between the 14 species of the subgenus Artemisia and these 14 species were found completed their speciation. This study is important as it is the first molecular based study relating with belong to subgenus Artemisia species growing naturally in Turkey. In addition, new haplotypes related to the populations of Turkey belonging to the subgenus Artemisia taxa were reported by us for the first time and added to the GenBank database.

Differentiation of Lingxiaohua and Yangjinhua by chloroplast genome sequencing and DNA barcoding markers

Lingxiaohua (Campsis Flos, Campsis grandiflora (Thunb.) K. Schum) is a medicinal herb used for promoting diuresis and treating blood-related disorders by the promotion of blood circulation. It also possesses anti-inflammatory and antioxidative properties. This non-poisonous plant is frequently confused with poisonous Yangjinhua (Daturae Metelis Flos, Datura metel Linnaeus) in the market, resulting in serious anticholinergic poisoning. The confusion of these two herbs is due to the similarity in their appearances. In our study, we compared the complete chloroplast genomes of the two plants and found that they are very different in terms of their gene content and gene arrangement. There were also significant differences in the number and repeating motifs of microsatellites and complex repeats. We used universal primers for the amplification of rbcL, matK, psbA-trnH, and ITS2 regions and successfully differentiated the two plants. Furthermore, we designed two pairs of primers based on the nucleotide differences in chloroplast genomes at the rps14 and rpoC1 regions to provide additional authentication markers. The universal primers and specific primers when used together can accurately discriminate Lingxiaohua and Yangjinhua.

Sideritis elica, a New Species of Lamiaceae from Bulgaria, Revealed by Morphology and Molecular Phylogeny.

Sideritis elica, from the Rhodope Mountains, is described as a species new to science. Results of a detailed morphological analysis were combined with the data of molecular analyses using DNA barcoding as an efficient tool for the genetic, taxonomic identification of plants. The combination of morphological features distinguishes the new species well: Its first three uppermost leaf pairs are significantly shorter and wider, the branchiness of the stems is much more frequent, the whole plant is much more lanate, and it looks almost white, as opposed to the other closed species of section Empedoclia, which look grayish green. The molecular analysis, based on the rbcL and trnH-psbA regions, supports the morphological data about the divergence of Sideritis scardica and Sideritis elica. The studied populations of the two taxa were found to be genetically distant (up to 6.8% polymorphism for trnH-psbA) with distinct population-specific nucleotide patterns, while no polymorphism in the DNA barcodes was detected within the Sideritis elica population. The results confirm the existence of a new species called Sideritis elica, which occurs in the nature reserve Chervenata Stena, located in the northern part of the Central Rhodope Mountains. There were only 12 individuals found in the locality, which underlines the necessity of conservation measures.

Utility of the trnH–psbA region for DNA barcoding of Aconitum anthora L. and related taxa

Aim. The genus Aconitum is represented in the Carpathian region by a significant number of endemic species. Some of these species, in particular A. pseudanthora and A. jacquinii, are included in the Red List of Ukraine. However, their taxonomic status and consequently the importance of conserving their natural resources remain uncertain. DNA barcoding techniques, e.g., the application of molecular markers based on variable regions of the chloroplast genome can help clarify these questions. Methods. DNA isolation, PCR amplification, sequencing of PCR products, phylogenetic analysis. Results. The psbA-trnH intergenic spacer region of A. pseudanthora and A. jacquinii specimens from Western Ukraine was amplified and sequenced. It is shown that the spacer sequences for these two species are identical, but differ in short indels and nucleotide substitutions from the psbA-trnH sequences of A. anthora s.l. specimens from Western Europe and China. Phylogenetic analyzes showed the phylogenetic affinity of the members of the A. anthora complex to the subgenus Lycoctonum. Conclusions. The use of the psbA-trnH intergenic spacer region for DNA barcoding makes it possible to unambiguously distinguish samples of A. jacquinii and A. pseudanthora from western Ukraine from samples of A. anthora s.l. of other geographical origins. The results obtained confirm the reticular nature of the evolution in the genus Aconitum.

Beilschmiedia bidoupensis (Lauraceae), a new species from Bidoup-Nui Ba National Park, southern highland of Vietnam

Beilschmiedia bidoupensis (Lauraceae) is described from Bidoup-Nui Ba National Park in the southern highland of Vietnam. The new species is characterized by lanceolate terminal buds that are ca. 2.5 mm long without coriaceous scales and covered with whitish brown hairs, glabrous twigs, predominantly alternate leaves evenly arranged on branches, elliptic leaf blade with acuminate apex, and depressed globose to transversely ellipsoidal fruits with not verrucose but asperously furfuraceous surface. A description, illustrations, preliminary conservation assessment, and DNA barcodes of ITS, psbA-trnH and trnL regions, are provided.

DNA Barcoding of Prunus Species Collection Conserved in the National Gene Bank of Egypt

Two intergenic spacers cpDNA barcoding regions were used to assess the genetic diversity and phylogenetic structure of a collection of 25 Prunus accessions. The trnH-psbA and trnL-trnF intergenic spacers were able to distinguish and identify only four Prunus species. The average aligned length was 316–352 bp and 701–756 bp for trnH-psbA and trnL-trnF, respectively. The overall evolutionary divergence was higher in trnH-psbA than trnL-trnF. The transition/transversion bias (R) recorded as 0.59 in trnL-trnF and 0.89 in trnH-psbA. The number of invariable sites, nucleotide diversity (Pi), and the average number of nucleotide differences (k) was higher in the trnH-psbA region. The trnL-trnF records was above the other region in the number of variable sites, number of singleton variable sites, and the parsimony informative sites. Phylogenetic relationships among the 25 accessions of Prunus species were investigated. Most of the different Prunus species clustered in a homogenized distribution in both regions, except for the plum (P. domestica) accession (African Rose) was assigned with the peach (P. persica) accessions. The two intergenic cpDNA trnH-psbA and trnL-trnF were able to distinguish and identify the four Prunus species accessions.

Blumea chishangensis sp. nov. (Asteraceae: Inuleae) from Taiwan and new insights into the phylogeny of Blumea

BackgroundBlumea plants are widely distributed in the tropical areas of Asia, Africa, and Australia, especially tropical Asia. Limited studies left the taxonomy and infrageneric phylogeny of Blumea insubstantial. Here, a new species, Blumea chishangensis S. W. Chung, Z. H. Chen, S. H. Liu & W. J. Huang, from Taiwan is described, and an extended phylogeny is reconstructed to provide new perceptions of Blumea evolution.ResultsThe new species is distinguished from B. hieraciifolia by the following features: leaf blade sparsely pilose or glabrescent (vs. silky villous), the leaves margins regularly remote mucronulate (vs. double serrate or dentate), capitula pedicelled (vs. capitula sessile or subsessile), and leaves basal rosette or sub-basal rosette and a few cauline (vs. mostly cauline). Phylogenetic analysis based on the ITS, trnL-trnF, and trnH-psbA regions places the new species in the subclade II in B. lacera clade and shows a close relationship with B. axillaris and B. oxyodonta. A key to Blumea species in Taiwan and the studied species in the subclade II is provided. Moreover, the evolutionary inferences of B. conspicua, B. linearis, and B. sinuata are first reported here. The paraphyly of B. formosana and B. sinuata are also revealed for the first time.ConclusionsBoth morphological and molecular data support that B. chishangensis is a new species. Our phylogeny highlights the need for further taxonomic and evolutionary studies on Blumea.

Evaluation of chloroplast DNA barcoding markers to individualize Papaver somniferum for forensic intelligence purposes.

The opium poppy, Papaver somniferum L., is a forensically important plant due to the medicinal and illegal uses for the milky latex stored in the pods. This latex contains the alkaloids morphine, codeine, and thebaine that are used for their analgesic properties and/or for synthesizing other opioids. However, these compounds are highly addictive and have caused a national opioid epidemic. Two other Papaver species, P. setigerum DC. and P. bracteatum Lindl., are also of forensic interest because they pose both forensic and legal issues. They are largely uncontrolled under the Controlled Substances Act, making these species a common defense strategy. Current morphological and chemical identification methods have been moderately successful but have drawbacks. There is also a lack of sequencing data available. Therefore, exploiting the genome using chloroplast DNA barcoding markers could help to accurately identify these species of interest when plant material is taken. This study screened and assessed the genetic variation both between species and within populations of P. somniferum in nine cpDNA barcode regions (ndhF-rpl32, petA-psbJ, rpl32-trnL, rps16-trnQ, trnE-trnT, trnH-psbA, trnL-trnF, rpl16 intron, and psbE-petL). Published reference genomes from the NCBI GenBank database were aligned and compared for an initial in silico screening. Additionally, ten P. somniferum seed samples from various vendors were sequenced and compared across samples and to published reference data at the various barcode regions of interest. This study showed that the regions trnH-psbA and petA-psbJ have promise for utility in individualization for both inter- and intra-species individualization of P. somniferum.

Application of DNA barcoding tool for authentication of traditionally important medicinal Dendrobium (Orchidaceae) species from Sikkim Himalaya

Dendrobium, the second largest genus of the Orchidaceae family, is equally important for ornamental value and traditional medicine. Sikkim Himalaya region is rich in traditional knowledge of medicinal plants and their diversity. However, these regions were least explored through modern molecular tools. Reference databases association with DNA Barcoding technique is a new tool for species authentication. Here, we tested the efficiency of DNA barcoding loci (ITS, matK, rbcL, trnH-psbA) based on similarity search, characters based and phylogenetic methods to authenticate the medicinally important Dendrobium species. DNA from young leaves of studied species were isolated, and ITS (∼700 bp), rbcL(∼650 bp), matK (∼ 800 bp), trnH-psbA (∼ 450 bp) were amplified and sequenced for species-level identification. ITS, rbcL and trnH-psbA regions showed easy amplification and sequencing. Among the 54 sequences, 9 sequences were first time submitted to GenBank. In case of similarity search, ITS showed most efficient to identify all Dendrobium species except two species Dendrobium thyrsiflorum and Dendrobium densiflorum. However, the characters-based method easily differentiated between Dendrobium thyrsiflorum and Dendrobium densiflorum. The similarity-based methods and ITS sequence information together may help correct species identification for medicinal plants of Dendrobium.

ORGANIZATION AND POLYMORPHISM OF THE CHLOROPLAST GENOME REGION psbA-trnH IN REPRESENTATIVES OF THE GONIOLIMON BOISS.

Four out of twenty-two species of the genus Goniolimon are common in Ukraine: G. besserianum, G. graminifoli-um, G. rubellum and G. tataricum. Two of them, G. graminifolium and G. rubellum, are listed as vulnerable species in the Red Data Book of Ukraine. Some populations of both species are sometimes reported under different species names, manly as G. desertorum (Trautv.) Klokov. and G. rubellum (S.G. Gmel.) Klokov. To clarify these taxonomic questions, the application of molecular phylogenetic methods is necessary. Recently, the fragment between the psbA and trnH genes has been particularly actively used for similar purposes. In this work, we tested the use of the psbA-trnH chloroplast genomic region for phylogenetic analysis and DNA barcoding of representatives of the genus Goni-olimon. We amplified and sequenced the psbA-trnH sequences for samples of all four species of this genus distributed in Ukraine and compared them with the psbA-trnH sequences available in the GenBank database for species of Goni-olimon and related genera. The comparison of the available sequences showed that the representatives of the genus Goniolimon differ in twelve variable nucleotide positions, seven of which are parsimony-informative. They also differ in one oligonucleotide indel and a sequence inversion corresponding to the stem-loop region in the 3΄ UTR region of psbA mRNA. The difference between the psbA-trnH regions of Goniolimon species and representatives of related gene-ra was much higher. In addition to numerous SNPs, they also differ by a polynucleotide indel. Such significant differ-ences indicate a higher rate of evolution of the psbA-trnH region during the divergence of the genus Goniolimon compared to other regions of the chloroplast genome. On the phylogenetic dendrogram based on the comparison of psbA-trnH sequences, the Goniolimon species from Central Asia form a distinct group, which is probably due to chlo-roplast genome transfer as a result of hybridization in a common distribution area. In general, the high variability of the psbA-trnH regions in representatives of the genus Goniolimon found in our study allows us to use this region to clarify the position of controversial taxa in the group.

PHYLOGENETIC RELATIONSHIPS OF UKRAINIAN ACCESSIONS OF LATHYRUS VENETUS (MILL.) WOHLF. AND L. VERNUS (L.) BERNH. BASED ON THE ANALYSIS OF THE psbA-trnH REGION OF THE CHLOROPLAST GENOME

The member of the genus Lathyrus, L. venetus is listed in the Red Data Book of Ukraine as a vulnerable species. Estimation of L. venetus abundance is complicated by the prevalence of interspecies hybrids with L. vernus. These hybrid populations are often misidentified as populations of the L. venetus due to their morphological similarity. The study of genetic resources of L. venetus requires the use of molecular markers, both nuclear (to identify hybrid forms) and plastid (to determine the direction of hybridization) localization. One of the most promising chloroplast markers, the IGS region between the psbA and trnH genes, has not yet been sequenced for L. venetus and L. vernus. In this work, we evaluated the possibility of using the psbA-trnH region of the chloroplast genome to identify the parental forms of hybrids between L. venetus and L. vernus. We amplified and sequenced the psbA-trnH IGS for the collected in Ukraine accessions of L. venetus and L. vernus. For comparison, psbA-trnH sequences from the GenBank database were used that represent species from phylogenetically distant sections of the genus Lathyrus, as well as species of the Orobus section closely related to L. venetus and L. vernus. Based on the analysis of alignment of all sequences used, we found that the level of variability of psbA-trnH exceeds that of other chloroplast DNA regions used in phylogenetic studies of the genus Lathyrus. On the phylogenetic tree constructed by us, the genus Lathyrus forms a monophyletic group with high statistical support. The sequences analysis showed significant differences between the psbA-trnH IGS of L. venetus and L. vernus and confirmed the effectiveness of using this region for molecular identification of these species and for determining the direction of hybridization in the hybrid forms.

Sequences analysis of chloroplast psbA-trnH region in Citrus L. (Rutaceae) species from the Aegean region of Turkey

In this study, sequences analysis of some Citrus species distributed in Turkey's Aegean region was based on the cpDNA psbA-trnH region. The sequences for psbA-trnH regions of the outgroups were retrieved from NCBI GenBank. Genomic DNA was isolated from healthy and green leaves. Total genomic DNA was extracted using GeneMark DNA isolation Plant Kit. The psbA-trnH region was amplified using primers psbA and trnH. DNA sequences were edited using the Sequencher 5.4.6. Sequencing data were analyzed using MEGA 6.0 software. Maximum likelihood (ML) tree was created to determine the relationships between Citrus taxa. cpDNA psbA-trnH sequences ranged between 426 and 470 nucleotides. Maximum likelihood phylogenetic tree is composed of two clades. The divergence values differed between 0.000 and 0.012. According to the results of the study, the separation of Citrus species in phylogenetic tree obtained with psbA-trnH sequence data was realized. However, it has been found that cpDNA psbA-trnH sequence populations of species belong together. In addition, the phylogenetic relationship between the sequence data of some species belonging to the Rutaceae family taken from NCBI and Citrus species was revealed.