Бюллетень науки и практики /Bulletin of Science and Practice Т. 7. №11. 2021
https://www.bulletennauki.com https://doi.org/10.33619/2414-2948/72
БИОЛОГИЧЕСКИЕ НАУКИ/BIOLOGICAL SCIENCES
UDC 582.824.3 https://doi.org/10.33619/2414-2948/72/02
AGRIS F70
MOLECULAR-PHYLOGENETIC RESEARCH OF THE GENUS Hypericum L. IN FLORA OF AZERBAIJAN
©Fatdayeva A., Institute of Botany Azerbaijan NAS, Baku, Azerbaijan, ayten.fetdayeva@mail.ru
МОЛЕКУЛЯРНО-ФИЛОГЕНЕТИЧЕСКОЕ ИССЛЕДОВАНИЕ РОДА Hypericum L. ВО ФЛОРЕ АЗЕРБАЙДЖАНА
©Фатдаева А., Институт ботаники НАНАзербайджана, г. Баку, Азербайджан, ayten.fetdayeva@mail.ru
Abstract. 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.
Аннотация. Зверобой — один из 100 крупнейших родов цветковых растений, образующих семейство Hypericaceae Juss., которое принадлежит к кластероидкладам Malpighiales. Зверобой представлен во флоре Азербайджана 19 аборигенными видами и 1 подвидом, относящимися к 7 таксономическим разделам. ДНК хлоропластов 8 видов этого рода исследовали методом ПЦР-ПДРФ Тотальную геномную ДНК извлекали из ткани листа с помощью набора DNeasyPlantMini (Qiagen Inc.; Валенсия, Калифорния, США) в соответствии с прилагаемым протоколом и полем количества с использованием спектрофотометра Nanodrop (Nanodrop Technologies; Уилмингтон, Делавэр, США). Статья является частью экспериментального исследования, включающего молекулярно-филогенетические исследования этого рода во флоре Азербайджана.
Keywords: Hypericum, species, subspecies, molecular-phylogenetic research.
Ключевые слова: Hypericum, виды, подвиды, молекулярно-филогенетические исследования.
Introduction
Hypericum is one of nine genera and represents approximately 80%of the diversity of the family Hypericaceae Juss. This genus is originated from Eurasia and widely distributed in tropical and subtropical regions. Species of this genus grow on damp soils, meadows, and swamps.
Morphologically Hypericum genus are characterized by the presence of different kinds of secretory glands and channels, including transparent, dark glands. The secretory structures are the
Бюллетень науки и практики /Bulletin of Science and Practice Т. 7. №11. 2021
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accumulation of biologically active substances, and their various configurations are important in the classification of the genus.
Species of this genus are annual and perennial herbaceous, semi-shrubs, and shrubs. The leaves are opposite, rarely whorled, whole-edged, sessile, or with short petioles on the surface and at the edges often with transparent, sometimes with black point glands. Flowers are collected in the corymbose inflorescence.
Plant material and DNA extraction Fieldwork was conducted during the expeditions (2015-2018) between May September, at different stages of plant development (Table 1). Dried herbarium specimens deposited in the Herbarium fond of the Institute of Botany ANAS (BAK) and were examined according to standard procedures.
Table 1.
PLACES OF COLLECTION OF MATERIAL
Species Collecting data
AZ0001 Hypericum Shach-buz (Kuku village) 2260 m
helianthemoides
AZ0002 H. perforatum subsp. Gabala503 m
veronense.
AZ0003 H. elongatum Shach-buz(mountainofYellica ) 2300 m
AZ0004 H. tetrapterum Lankaran (Dasdatuk village) 800 m; Gadabay(Soyudluvillage) 1473 m
AZ0005 H. lydium Shach-buz (Kuku village, Safdara) 2270 m
AZ0006 H. androsaemum Zagatala (Gabizdaravillage) 643 m, Gakh (Lakit village) 1571 m,
Gabala (Vandam village) 563 m
AZ0008 H. perforatum Zagatala (Gabizdara village) 562 m, Car666 m
Also, the Herbarium specimens stored in the Herbarium fond of the Institute of Botany Azerbaijan NAS were used in this study. Classic comparative morphological and results of the molecular-phylogenetic analysis were used for the identification of species. From each sample of 100-200 mg of young leave plants put in 2 ml tubes. Leaf material was obtained from three individual plants per accession, flash-frozen in liquid nitrogen, and stored at -200 °C.
The buffer is added to the dried DNA and stored in the refrigerator for 1 night. Amplification reactions were shown in Table 2.
Table 2.
AMPLIFICATION REACTIONS
Components Stock Cons. Reac. Cons.
PCR Buffer 10X 1X
MgCl2 25mM 1,5 mM
dNTP mix 20 mM 0,2 mM
F. Primer 10 цМ 0,3 цМ
R. Primer 10 цМ 0,3 цМ
Taq DNA Polymerase 5U/ ц1 2 U
DNA template 3 ц1
PCR grade with H2O 35 ц1
Бюллетень науки и практики /Bulletin of Science and Practice Т. 7. №11. 2021
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Amplifications were performed as follows: first denaturation (3 min at 94 °C), 35 cycles of denaturation (15 s at 94 °C), elongation (at 72 °C), and final extension of (5 min at 72 °C). The amplified products were precipitated with ice-cold ethanol, washedwith70% ethanol, and dissolved in water. PCR products were verified byelectrophoresison1.5% agarose gels containing ethidium bromide in this-acetate EDTA (TAE) buffer and detected under UV light (Table 2).
The restriction fragments with 100 bladders (Gene Ruler TM 100 bp ladder, Fermentas) as a size marker were separated on 1% agarose gels in Tris-acetate EDTA (TAE) buffer (Figure 1).
Table 2.
CYCLES AND THE DURATION OF THE POLYMERASE CHAIN REACTION
Temperature of PCR Duration Cycle
94 °C 3 min 1
94 °C 15 sec 1
50 °C 15 sec 35 J
72 °C 30 sec j
72 °C 5 min 1
Checking the amount and purity of DNA The amount of DNA is determined by a spectrophotometer (260 and 280 nm) (Nano Drop 200 °C UV-VisSpectrophotometer-Thermo Scientific). The mixture was used to determine the amount of 20 |l extracted DNA and 1980 |l of DD H2O. The density of theDNAsolutioniscalculated as follows: DNA density (NG/|l) = (0S260H100 (dilution factor) x 50 ng/ml) / 1000. The optical densityratiobetween260and280 nm (OS26 0/0S280) shows the purity of sound acids. The optimum cleaning speed for the PCR is 8-2.0. After determining the amount of DNA, from each sample, are prepared 50 ng / [j.1 of DNA for each PCR reaction.
100 BP DNA LADDER
bp ng/10ul
3,000 100 2,000 134 1,500 100
1,000 104
900 93
800 83
700 73
600 63
500 104
400 42
300 31
200 42
100 31
100 bp DNA Ladder 1% agarose, lxTBE
Figure 1. 100bp_DNA_Ladder
Бюллетень науки и практики / Bulletin of Science and Practice Т. 7. №11. 2021
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PCR reaction with its primers The total volume of reactionfor1sampleis20 |il (2 |il of DNA sample + 18 |il of the reaction mixture). The whole reaction should be arried outside the ice and the unit needs to be centrifuged. The tags added to theology measure action mixture, stirring, and the total reaction mix tourist thoroughly vortex. The amount of DNA is pre-placed in a tube or RDA (plate) and then stop resonance. Then, 18 |il is poured from the reaction mixture onto each sample and swirled again. After the reaction tube is placed in the apparatus PCR (Gene Amp System 2720, Applied Biosystems Foster City, CA and BigDyeTerminator v3.1 Cycle) and the programs are compiled in the following sequence.
Results
Nucleotide sequence reading (Sequencing) Be for the sequencing of the resulting products PCRQIA quick Gel Extraction Kit was cleaned by using a kit (Qiagen, Germany). Then PCR sequence lingua automated ABI 3730 XL with the head edition of primers is placing rid and nucleotides equine cesarean.
Table 2.
THE (5-3) NUCLEOTIDES SEQUENCE OF IT SPRIMERS
DNA region Primer Primer sequence 5-3 Reference
ITS ITS 1 TCC GTA GGT GAA CCT GCG G White and other, 1990
ITS 4 TCC TCC GCT TAT TGA TAT GC White, and other 1990
Trn L intron trnL C CGA AAT CGG TAG ACG CTA CG Taberlet et al., 1991
trnLD GGG GAT AGA GGG ACT TGA AC Taberlet et al., 1991
The obtained nucleotides equine care orted bytes of aware Clustal W [8]. Results of some kinds of America Gen Banker and data-fornication were taken. The nucleotide sequences were included in the Molecular Evolutionary Genetics Analysis program (MEGA 6.0). The phylogenetic tree according to the model of Tamura-Nei [9] was constructed with 500 bootstrap-sample using the Maximum Likelihood (ML) method (Figure 2).
69
71
16
10.9
0.0
0,0
0,1
0.1
0.3
0.5
99
0.4
0.0
0.0
0.5
2.5
0,4
0.2
2.7
14.0
AZDQ01 Hypericum helianthemoides
AZDD06 Hypericum androsaemum
AZD005 Hypericum lydium
AZD008 Hypericum perforatum
AZD002 Hypericum perforatum subsp.veronense
HE653671.1 Vismia cayennensis
AZD007 Hypericum tetrapterum
MK995180.1 Cratoxylum cochinchinense
AZDD03 Hypericum eiongatum
Figure 2. Phylogenetic tree of the genus Hypericum based on the Maximum Likelihood (ML) method
Бюллетень науки и практики / Bulletin of Science and Practice Т. 7. №11. 2021
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The obtained nucleotide sequences were compared with the data centers of the USA General Bank and the following results were obtained:
Sample AZ0001 with the data of Gen Bank with the type of Hypericum helianthemodes Spach.
Sample AZ0002 with the data of Gen Bank with the type of Hypericum perforatum subsp. veronense. Schrank.
Sample AZ0003 with the data of Gen Bank with the type of Hypericum elongatum Ledeb.
Sample AZ0005 with the data of Gen Bank with the type of Hypericum lydium Boiss.
Sample AZ0006 with the data of Gen Bank with the type of Hypericum androsaemum God.
Sample AZ0007 with the data of Gen Bank with the type of Hypericum tetrapterum Fries.
Sample AZ0008 with the data of Gen Bank with the type of Hypericum perforatum L.
References:
1. Clapham, A. R., Tutin, T. G., & Moore, D. M. (1990). Flora of the British Isles. CUP Archive.
2. Flora of Turkey (1967). Ed. by P. H. Davis. Edinburg, II, 355-401.
3. Robson, N. B. (1987). Studies in the genus Hypericum L. (Guttiferae). VII: Section 29. Brathys (part 1). Bulletin of the British Museum. Natural History. Botany, 16(1), 1-106.
4. Gorshkova, S. (1949). Rod Hypericum L. In Flora SSSR Leningrad, 15, 203-258. (in Russian).
5. Rzazade, R. (1955). Rod Hypericum L. In Flora Azerbaidzhana. Baku, 6, 248-259.
6. Bondarenko, S. V. (2012). Konspekt flory Kavkaza. 3(2), Moscow. 308-314. (in Russian).
7. Crockett, S. L., Douglas, A. W., Scheffler, B. E., & Khan, I. A. (2004). Genetic profiling of Hypericum (St. John's Wort) species by nuclear ribosomal ITS sequence analysis. Planta medica, 70(10), 929-935. https://doi.org/10.1055/s-2004-832619
8. Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic acids research, 22(22), 4673-4680. https://doi.org/10.1093/nar/22.22.4673
9. Felsenstein, J. (1985). Phylogenies and the comparative method. The American Naturalist, 125(1), 1-15. https://doi.org/10.1086/284325
Список литературы:
1. Clapham A. R., Tutin T. G., Moore D. M. Flora of the British Isles. CUP Archive, 1990.
2. Flora of Turkey. Ed. by P. H. Davis. 1967. Edinburg, Vol II. P. 355-401.
3. Robson N. K. B. Studies in the genus Hypericum L. (Guttiferae). VII: Section 29. Brathys (part 1) // Bulletin of the British Museum. Natural History. Botany. 1987. V. 16. №1. P. 1-106.
4. Горшкова С. Род Hypericum L. // Флора СССР. Ленинград. АН СССР, 1949. Т. 15. С. 203-258.
5. Рзазаде Р. Род Hypericum L. // Флора Азербайджана. 1955. Баку: Изд-во АН Азерб. ССР. Т. 6. С. 248-259.
6. Бондаренко С. В. Конспект флоры Кавказа. Т. 3(2) : в 3 т. М.: Товарищество научных изданий КМК, 2012. С. 308-314.
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Т. 7. №11. 2021 https://doi.org/10.33619/2414-2948/72
7. Crockett S. L., Douglas A. W., Scheffler B. E., Khan I. A. Genetic profiling of Hypericum (St. John's Wort) species by nuclear ribosomal ITS sequence analysis // Planta medica. 2004. V. 70. №10. P. 929-935. https://doi.org/10.1055/s-2004-832619
8. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice // Nucleic acids research. 1994. V. 22. №22. P. 4673-4680. https://doi.org/10.1093/nar/22.22.4673
9. Felsenstein J. Phylogenies and the comparative method // The American Naturalist. 1985. V. 125. №1. P. 1-15. https://doi.org/10.1086/284325
Работа поступила в редакцию 11.10.2021 г.
Принята к публикации 14.10.2021 г.
Ссылка для цитирования:
Fatdayeva A. Molecular-Phylogenetic Research of the Genus Hypericum L. in Flora of
Azerbaijan // Бюллетень науки и практики. 2021. Т. 7. №11. С. 22-27. https://doi.org/10.33619/2414-2948/72/02
Cite as (APA):
Fatdayeva, A. (2021). Molecular-Phylogenetic Research of the Genus Hypericum L. in Flora of Azerbaijan. Bulletin of Science and Practice, 7(11), 22-27. https://doi.org/10.33619/2414-2948/72/02