Inventory of some Tulipa species from Uzbekistan using DNA barcoding

Climate fluctuations in the Quaternary period in si gnificantly influenced the evolution and distribution of plants , which caused to a serious genetic effects and speciation activity [1] . Thus, Central Asia is distinguished by a wide variety of flora, including 9520 species, while 20% are endemics [2]. Great phenotypic diversity of the wild flora in Uzbekistan, numbering about 4400 species of vascula r plants is determined by the peculiar climatic conditions, a variety of z oning, altitudinal zones [3]. At the same time, being one of the centers of early diversification of monocotyledonous geophytes, taxonomically complex r epresentatives of this group are not sufficiently involved in phyloge netic studies. This study is a pilot study of the flora of Uzbekistan using D NA barcoding method, that based on sequencing of phylogenetically signif icant sequences. Four plastid (rbcL, psbA-trnH, matK, trnL-F) and one nuc lear (ITS) markers were used for species identification of the genus T lipa. The results of molecular phylogenetic analysis for 15 species of T ulipa (Liliaceae) using the marker sequence encoding ITS fragment are prese nted. The successful usefulness of DNA technology for genetic inventory has been demonstrated.

Species of the genus Tulipa L. (Liliaceae), occupying a leading position in the flora of Uzbekistan, were used as model objects in this molecular phylogenetic analysis. It is one of the largest genus of Liliaceae family, it includes about 100 species distributed in the southern and eastern Europe, North Africa, the Middle East, Central Asia and China [5]. In recent years, the classification has changed dramatically, based on the classical and modern molecular phylogenetic studies [4][5][6]. This study allows to express an opinion about the taxonomic position selected objects. Thus, in previously published works on taxonomic studies of the genus Tulipa, the species number is various: from 76 [6], 87 species [5] to up 114 species [7]. Currently 34 species of Tulipa have been recorded in Uzbekistan, united into six sections (Tulipanum, Kolpakovskiana, Lanatae, Vinistriatae, Spiranthera, Biflores) [8], 19 of them are listed in the Red Book of Uzbekistan (2019) [9]. The species investigated in this study (9 taxa) are representatives of five sections.
Termination reaction performed using a commercial kit Brilliant Dye Terminator v3.1 Cycle Sequencing Kit (Nimagen, Netherlands). Determination of the nucleotide sequence was performed on a genetic analyzer ABI 3500 DNA Analyzer (Applied Biosystems, USA) in a forward and reverse directions. Sequence chromatograms viewed in Sequence Scanner 1.0. Alignment of pairs of sequences conducted in the BioEdit program. The resulting consensus DNA sequences are saved in FASTA format.
To maximize the efficiency of identification and high reliability of the results, 3-4 locus genotyping was performed for all species. As a result of research, 31 consensus sequences of phylogenetically significant markers (DNA barcodes) were obtained for nine representatives of the genus Tulipa. Nucleotide sequence data were submitted in the international Genbank database with the assignment of ID numbers (GenBank: http://www.ncbi.nlm.nih.gov) (Table). Consensus sequences were compared with DNA sequences of similar species in international databases NCBI BLAST (Basic Local Alignment Search Tool) and BOLD (Barcode of Life Data Systems). Good reproducibility of amplification results was demonstrated for all markers. So, DNA sequences were compared to known and well characterized sequence in the BLAST database. The similarity with sequences was at least 98%. Marker sequences for the species Tulipa mogoltavica, Tulipa ferganica, Tulipa intermedia, Tulipa affinis, Tulipa dubia are presented to the database for the first time.
The choose of one or another molecular marker, the variability of which is a basis for the work, is one of the important tasks, since the random of evolutionary changes in a specific DNA segment determines the level phylogenetic resolution and may be different in different organisms.
Many researchers have noted the high efficiency of the ITS nuclear region as a DNA barcode in both cases, using independently (up to 92.7% of all studied species) and in combination with chloroplast markers.
Nuclear DNA regions are also under interest due to a high variability and a high discrimination rate compared to mitochondrial and chloroplast genes. Therefore, the second internal transcribed spacer (ITS) of nuclear ribosomal DNA represents the most suitable region for molecular genetic studies in plant taxonomy at the "genus, species, subspecies" taxon levels, for DNA barcoding applications. As a rule, the lengths of the combined internal transcribed spacer ITS region (ITS1 is located between 18S and 5.8S rRNA genes, ITS2 is between 5.8S and 28S) are given in the NCBI databases.
A phylogenetic analysis of 8 tulip species was performed based on the obtained nucleotide sequences of the ITS nuclear region. A fragments of target marker ranged from ~ 400 to ~ 700 bp were obtained by amplification of plant DNA. Seven sequences of the ITS region used for a phylogenetic dendrogram construction were download from the GenBank database. The region for comparing sized 329 b.p.
The model choose was estimate using the jModelTest 2.1.10 program [10]. The nucleotide sequences alignment was performed using the ClustalW algorithm [11].
The results of hierarchical clustering are presented by a constructed dendrogram obtained by the Maximum Likelihood (ML) method in the MEGA5 program; the reliability assess of the resulting phylogenetic tree topology was obtained with Bootstrap analysis (1000 replicates). The resulting graphic image was visualized using the FigTree v1.4.0 program. Thus, hierarchical clustering showed results consistent with the classical classification of the genus Tulipa L., proving the taxonomic position of taxa according to their morphological characters [8]. This division also coincides with the molecular phylogenetic studies of genus Tulipa L. by other authors [5,6,12].
Thus, the results obtained in this research make possible to compile a DNA barcode national reference library for rare and endangered plant species growing on the territory of Uzbekistan. This work demonstrates the effectiveness of using DNA barcoding as a tool for the diversity assessing of rare plant species.