USE OF SEQUENCING METHODS FOR SPECIES IDENTIFICATION EXEMPLIFIED BY PHYLOGENETIC RELATIONSHIPS WITHIN GENUS HEDYSARUM L

For citation: D.R. Imachueva, F.K. Serebryanaya, E.M. Machs, V.V. Kotseruba. Use of sequencing methods for species identification exemplified by phylogenetic relationships within genus Hedysarum L. Pharmacy & Pharmacology. 2021;9(6):506-518. DOI: 10.19163/2307-9266-2021-9-6506-518 © Д.Р. Имачуева, Ф.К. Серебряная, Э.М. Мачс, В.В. Коцеруба, 2021 Для цитирования: Д.Р. Имачуева, Ф.К. Серебряная, Э.М. Мачс, В.В. Коцеруба. Использование методов секвенирования для идентификации видов на примере филогенетических связей в пределах рода Hedysarum L. Фармация и фармакология. 2021;9(6):506-518. DOI: 10.19163/2307-9266-2021-9-6-506-518 USE OF SEQUENCING METHODS FOR SPECIES IDENTIFICATION EXEMPLIFIED BY PHYLOGENETIC RELATIONSHIPS WITHIN GENUS HEDYSARUM L.

As for the study of the genus Hedysarum L., the works by Chennaoui H., Marghali S., Marrakchi M., Trifi-Farah N.
should be notified. The phylogenetic relationships within the genus Hedysarum L. have been studied on the basis of its morphological and biochemical characters 2 [12][13][14]. Nafisi H., Ranjbar M., Wojciechowski M. et al. have carried out molecular genetic studies based on a comparative analysis of ribosomal genes of the genus Hedysarum L. species growing in Southeast Asia [15,16].
The H. chaiyrakanicum and H. theinum species, for which the authors notified a pronounced polymorphism of the internal transcribed ITS spacers of the 5.8S rRNA gene and the traces of a phylogenetic relationship with Mediterranean species of the genus, have been studied. A relationship is notified between the Asian and European species H. chaiyrakanicum and H. gmelinii, which belong to the related Subacaulia and Multicaulia sections [17][18][19].
In addition to well-known foreign authors, a significant contribution to the genus Hedysarum L. was made by domestic authors, including I.A. Shantser and Suprun N.A., who had studied the genetic variation of H. grandiflorum Pall., H. biebersteinii and H. argyrophyllum [4,[20][21][22][23]. To study the genetic polymorphism of Hedysarum L., the analysis of ISSR markers had been used, which made it possible to analyze more than 100 DNA fragments [24][25][26][27].
THE AIM of the research is to study the possibility of using molecular genetic research methods in carrying out complex pharmacognostic studies, to study the intra-and interpopulation variability of three species of the genus Hedysarum L. collected in the North Caucasus (Hedysarum caucasicum Bieb.(H 1 ), Hedysarum grandiflorum Pall. (H 2 ), Hedysarum daghestanicum Rupr. ex Boiss.(H 0 )), to determine possible phylogenetic relationships between the species of the genus Hedysarum L.

MATERIALS AND METHODS
The material for the study was the samples of the genus Hedysarum L. species gathered in the territory of the North Caucasus: H. caucasicum Bieb., collected in the fruiting phase in 2017 in the Alibek gorge, the Dombai section in the territory of the KChR; H. grandiflorum Pall., collected in the fruiting phase in 2018 in the village of Kondrashi, the Ilovlinsky district of the Volgograd region; H. daghestanicum Rupr. ex Boiss, collected in the flowering phase in 2015 in the village of Andi in the Republic of Dagestan [28]. In this work, sequencing was performed according to the Sanger method determining the sequence of ITS1-5.8S-ITS2 rRNA [29]. The DNA sequencing was performed on an AbiPrism 3130 genetic analyzer (Applied Biosystems, USA) at the Biosystematics and Cytology Laboratory in Komarov Botanical Institute of the Russian Academy of Sciences.
The DNA sequence analysis was performed using the MEGA 10.0 software, USA. The isolation of genomic DNA was carried out by the CTAB method from the leaves of the herbarium samples [14]. For the amplification, Dream Taq PCR Master Mix reagents (Thermo Scientific, USA) were used. The polymerase chain reaction was carried out on a C1000 Thermal Cycler (Bio-Red, USA). The amplification cycle parameterswere: 3 min 98°C; 35 cycles: (1 min 98°C; 30 seconds 54°C; 30 seconds 72°C); 10 min 72°С.
The Big Dye Terminator Kit v. 2.0 (Perkin Elmer Life Sciences, Inc., USA) and the ABI Prizm 3130 sequencer (Applied Biosystems, UK) were used for sequencing. The DNA isolation from the leaves or herbarium samples was performed using the CTAB technique and included the following stages: the leaves of the studied samples were ground into a fine powder for 10 seconds using a Tissue-Lyser hemogenizer (QIAGEN, USA). Then 700 μl of prewarmed extraction buffer EB was added and vigorously shaken; it was incubated at 65°C for 1, 2 or more hours; purified with an equal volume of a chloroform mixture: isoamyl alcohol (24:1) was shaken for 5 minutes, the samples were centrifuged for 10 minutes at room temperature at 14000 rpm. The upper phase was transferred into a new 1.5 ml tube, the DNA was precipitated with 2/3 of the isopropanol volume (5 min at room temperature), centrifuged at room temperature for 10 minutes at 14,000 rpm; the supernantant was removed and the supernantant was washed twice with Wash Buffer (WB). The precipitate was air dried and dissolved in 300 μl of TE buffer; 3 μl of RNase L (10 mg/ml) was added and incubated for 30 min at 37°C. The concentration was adjusted with 2M sodium chloride solution; precipitated again by adding 2 volumes of 96% ethyl alcohol, washed with 500 μl of 70% ethyl alcohol, then the granules were air dried and dissolved in TE buffer. For a direct PCR analysis, the Phire Plant Direct PCR Master Mix (Thermo Scientific, USA) was used, which was directly intended for plant leaves and seeds without any preliminary DNA purification.
The amplified DNA fragments were purified using a standard agarose electrophoresis method. The control was carried out visually using a UV transilluminator, since the bands of the DNA stained with fluorescent dyes, formed by molecules of the same size when moving through the pores of the gel, are visible in the UV light. Ethidium bromide (λ max = 590 nm) which intercalates into DNA molecules (embedded between adjacent pairs of nucleotides), was used as a DNA dye. The intensity of this fluorescence is 20 times higher. The gel strip containing the necessary DNA fragment, was excised. To isolate DNA from the gel, a QIAquick Gel Extraction Kit (QIAGEN, USA) was used.
The ITS1-5.8S-ITS2 marker region of the 5.8S rRNA gene was sequenced in the representatives of the genus Hedysarum. The phylogenetic reconstruction was based on a comparison of this marker region from the sequenced samples of different geographic origins and the data from the Genbank NCBI 3 . To construct phylogenetic trees, the maximum likelihood method was used in the MEGA 10.0 program.

RESULTS AND DISCUSSION
A comparative molecular study of Hedysarum caucasicum M. Bieb., Hedysarum daghestanicum Rupr. ex Boiss, Hedysarum grandiflorum Pall. samples represented in the flora of the Caucasus, were carried out. The ITS1-5.8S-ITS2 sequences of the 5.8S rRNA gene in Hedysarum caucasicum were compared with the data presented in Genbank 4 [30][31][32][33][34]. The resulting phylogenetic tree is shown in Fig. 2.   long; the flowers are 5-30 in the amount, somewhat drooping, purple, violet-red. In the arctic and adjacent forest zone in the tundra, on sandy islands, in larch sparse forests, in riverside shrubs; in the alpine zone. 3

Hedysarum austrosibiricum
B.Fedtsch. 13 The flower spikes are apical, the racemes are loose, the floral bracts are brown, linear-lanceolate, glabrous or slightly pubescent; calyx is short-campanulate, short-fluffy; the corolla is lilac, purple-lilac. In the alpine zone, in descends into the forests; on subalpine, alpine, forest meadows, rocky slopes, in sparse larch forests. 8
The flower spikes are longer than the leaves; the racemes have long stems, 1.5-2 times longer than the leaves, not very dense; the corolla is dark purple or crimson [13]. Alpine meadows 1500-3500 m above the sea level, on subalpine and alpine meadows, on moraines, on slide rocks, in crooked forests, on rock ledges. 9

Hedysarum Semenovii
Rgl. et Herd. 21 the stipules are accreted in the form of a vagina; the leaflets are 8-9-paired, linear-oblong, glabrous above, scarcely pubescent along the mid-rib and edges below. The racemes are loose, 15-20 cm long; the flowers are in the amount of 10-30; the calyx is glabrous; the corolla is pale yellow, 10-12 mm long.
In river valleys, on the meadows, on riverine gravels, in larch and pine forests.
The racemes are elongated; the calyx teeth are subulate from the lanceolate base; the corolla is yellow, 13-15 mm long, pale [13].

Russian 29
Ibid. 30 Stepanov NV.  [13]. The racemes are dense, equal to or longer than the leaves; the corolla yellow; its purple carina is at the apex [13].

Hedysarum Gmelinii
Ldb. 41,42 A perennial plant; the stems are few, almost upright,, pubescent, with appressed hairs, below 2-3 mm in diameter, the stipules are membranous, accrete, the leaflets are 5-11-paired, oblong, hairy below, sometimes glabrous above, 7-30 mm long. The flower spike is longer than leaves; the racemes, 15-30 in number, are flowering, dense, elongated at the end; the floral bracts are lanceolate, the calyx is appressed-hairy; the corolla is pink-purple.
On meadows and in stony steppes. 20

Hedysarum songaricum
Bong. 43,44 A perennial plant; the stems are well developed, numerous, almost glabrous, 25-60 cm high; the stipules are membranous, the lower ones are fused at the base, slightly colored, hairy; the leaflets are 5-8 paired, lanceolate or oblong-elliptical, glabrous above, about 20 mm long The racemes are multi-flowered, oblong; the floral bracts are scarious, lanceolate; the calyx is campanulate-shaped, with lanceolate-subulate teeth, the corolla is pink-violet In steppes, on rank soil and finely earthy slopes in the middle and lower mountain belts 17,20 21

Hedysarum monophyllym
Boriss. 47 A perennial sericeous hairy plant 5-12 сm high; the stipules are imbricate at the base of leaves, the stipules are sericeous with strigose; the leaflets are simple, bottom sand, with a rounded-ovoid plate, sericeous with strigose, rounded or acute leaf apexes, sometimes sinuated, rounded at the base, 10-40 mm long. The inflorescence is rounded-cephalanthium, compact, the floral bracts are ovoid, white-hairy outside, the calyx is wide campanulate-shaped, the corolla is dry, yellowish. On stony and clay slopes of mountains, at an altitude of up to 2500 m. 38 Ibid. 39 Ibid. 40 Grossheim AA.  [13]. The racemes are multi-flowered, with deviated flowers; the floral bracts are lanceolate, light brown, hairy, the calyx is campanulate-shaped; the corolla is yellow or purple-violet [13]. Forest-steppe, stony and thyme steppes, rubble and clay-limestone slopes, limestones, chalk steppes, on outcrops of chalk and marl [13]. 25

Hedysarum ferganense
Korsh. 49,50 A perennial acaulous plant; 10-30 cm high, the leaves are 4-7-paired, oblong or elliptical, covered with short white, appressed hairs, on the upper surface of the leaves the pubescence is not so dense, 10-18 mm long. The floral bracts are brownish, lanceolate; the raceme is dense; the calyx is campanulate-shaped, its teeth are linear, 2-3 times longer than the tube, covered with adpressed or somewhat divaricately hairs; the corolla is lilac-violet. On mountain meadows, on stony and gravelly slopes. 26
In solonized steppes and gravelly terraces.
ex Boiss. 54,55,56,57 A perennial plant; gray in color because of appressed pubescence; the stipules accreted; the leaves are covered with sericeous pubescence on both sides; the leaflets are 2-3-paired, oblong or ovate-lanceolate, acute, the apical leaflet is larger, up to 18 mm long. The raceme is pauciflorous, dense; the flowers are big, creamy-white or purple [13].
Calcareous and dry grassy slopes, rocky places, at an altitude of from 800 to 1500 m [13].