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Turczaninowia 25, 4: 5-11 (2022) DOI: 10.14258/turczaninowia.25.4.1 http://turczaninowia.asu.ru
ШШ ISSN 1560-7259 (print edition)
M TURCZANINOWIA
¡¡j| ISSN 1560-7267 (online edition)
УДК 581.9+575.1+57.087.3(235.222/.226)
DNA barcodes of the vascular flora of the Altai Mountain Country: type material of the Herbarium ALTB
A. V. Vaganov1' 2*, T. A. Sinitsyna1' 3, M. G. Kutsev1' 4, M. V. Skaptsov1' 5, E. A. Zholnerova1' 6, P. A. Kosachev1' 7, A. A. Kechaykin1' 8, S.V. Smirnov1' 9, A. I. Shmakov1' 10
1 Altai State University, Lenina Pr., 61, Barnaul, 656049, Russian Federation 2E-mail: vaganov_vav@mail.ru; ORCID iD: https://orcid.org/0000-0002-7584-5150 3ORCID iD: https://orcid.org/0000-0002-7644-9176 4ORCID iD: https://orcid.org/0000-0003-2284-6851 5ORCID iD: https://orcid.org/0000-0002-4884-0768 6ORCID iD: https://orcid.org/0000-0003-3697-4811 7ORCID iD: https://orcid.org/0000-0002-4087-6336 8ORCID iD: https://orcid.org/0000-0002-0754-4698 9ORCID iD: https://orcid.org/0000-0002-9657-3959 10ORCID iD: https://orcid.org/0000-0002-1052-4575 *Corresponding author
Keywords: Altai' biodiversity' endemics' flora' herbarium' plant DNA barcoding' sequencing.
Summary. The article presents first data of the work on DNA barcoding of type specimens of ALTB Herbarium (Barnaul' Russia). Obtained sequences of ITS and trnL-trnF, trnH-psbA markers of DNA were deposited in NCBI GenBank' and corresponding dataset was published in the GBIF (Global Biodiversity Information Facility).
ДНК-штрихкоды сосудистых растений флоры Алтайской горной страны:
типовой материал Гербария ALTB
А. В. ВаганоВ' Т. А. Синицына' М. Г. КуцеВ' М. В. СкапцоВ' Е. А. Жолнерова' П. А. КосачёВ' А. А. Кечайкин' С. В. Смирнов' А. И. Шмаков
Алтайский государственный университет, пр. Ленина, д. 61, г. Барнаул, 656049, Россия
Ключевые слова: Алтай' биоразнообразие' гербарий' ДНК-штрихкодирование растений' секвенирование' флора' эндемик.
Аннотация. В статье представлены первые результаты работы по ДНК-штрихкодированию типовых образцов' хранящихся в гербарии Алтайского государственного университета (ALTB' г. Барнаул' Россия). Полученные нуклеотидные последовательности маркерных фрагментов ДНК (ITS' trnL-trnF, trnH-psbA) внесены в базу данных NCBI GenBank' и соответствующий датасет опубликован в GBIF.
Поступило в редакцию 25.10.2022 Принято к публикации 28.11.2022
Submitted 25.10.2022 Accepted 28.11.2022
Introduction
Identification, naming, and classification of living organisms at the species level are the foundation of all biology and has become one of the indispensable criteria in biodiversity analysis and management, conservation, and breeding (Vu, Le, 2019). Genetic analysis is exclusively a DNA-based technology recognized as "DNA barcoding". In the global infrastructure of biodata, DNA barcoding plays a main role, first, to solve fundamental problems of biodiversity. This is a diagnostic technique that uses short DNA sequence(s) for effective and accurate identification of different group of organisms, as well as unknown species (Ankola et al., 2021). Using united protocols of DNA isolation and analysis allows to significantly increase the efficiency of research and, therefore, the relevance of the results obtained, as well as using of public data repositories (NCBI, EMBL-EBI, GBIF, DDBJ) makes in demand the results in other natural sciences, not only in biodiversity, ecology, and genetics.
Developing of methods of the DNA analysis led to the creation of "The Consortium for the Barcode of Life" (CBOL) and "The Barcode of Life Data System" (BOLD). These depositaries with keeping of separate markers (barcodes) are also in demand for taxa identification.
DNA barcoding has also a wide and expanding range of practical applications, including the protection of biodiversity and rare species and the prevention of their collection and illegal sale; the control of plant raw materials, herbal teas, honey, and other commercial products; the control of weeds, invasive species, and allergy-causing plants, etc. (Koltunova et al., 2019; Shneyer, Rodionov, 2019); the genotyping both cultivated (Chinnappareddy et al., 2013; Mitrova et al., 2015) and wild plants (Herden et al., 2016; Sinitsyna et al., 2016; Smirnov et al., 2017).
In global DNA barcoding, there is unresolved question regarding the approved set of markers specifically for plants (Shneyer, Rodionov, 2019). So far, the nuclear-encoded ribosomal internal transcribed spacer (ITS) region and the chloroplast intergenic spacer trnH-psbA have emerged as candidates for barcoding plants, followed by others including coding sequences from plastid genes rbcL and matK, two loci now the most commonly used for plants (Kress, Erickson, 2007; Yao et al., 2010; Loera-Sanchez et al., 2020; Guo et al., 2022). These markers can be used separately or in combination with other markers or spacers. Since a standard plant barcode
has been complicated by the trade-off that arises between the high variability of sequences and high conservation of primers, it is then recommended to simultaneously utilize more than one marker as a compromise that best matches the barcoding criteria (Lahaye et al., 2008; Shneyer, Rodionov, 2019; Guo et al., 2022).
So, DNA barcoding is considered as a strong and promising tool in the field of molecular taxonomy for the taxonomists and conservation biologists worldwide to discover new species by performing unknown DNA sequence analysis on the DNA barcode database coupled with key morphological evidence (Ankola et al., 2021).
The Altai Mountain Country (AMC, Flora Altaica. http://altaiflora.asu.ru) is the highest modern uplift amongst the continental mountain countries in Siberia, as well as in Northern and Central Asia in general (Kamelin, 1998). This area occupies about 550 000 km2 including the Chinese, Kazakh, Mongolian, and Russian Altai, as delimited by R. V. Kamelin (Kamelin, 2005; Vaganov et al., 2019). In 2002, David Olson and Eric Dinerstein singled Altai-Sayan territory as one of the 200 priority ecoregions of the world for global conservation of biodiversity in their work "The global 200 Priority ecoregions for global conservation" (Olson, Dinerstein, 2002). More than 2700 plant species, 300 of which are endemic, grow within the territory of the AMC (Vaganov et al., 2021). A list of 42 world scientific depositories containing the information on animals, plants and fungi findings of AMC placed in the Global Biodiversity Information Facility (GBIF) was obtained (Vaganov et al., 2019).
Plant biodiversity remains a potential source of novel human benefits, and the discovery of new taxa, as well as greater study of known taxa (Erst et al., 2022). Endemic species, those restricted in their distribution to a relatively small geographic area, are the most vulnerable to extinction (Chichorro et al., 2019; Erst et al., 2022). The type material of herbarium collections can play the key role in DNA barcoding in the study of new plant species, which are endemic in most cases.
The general fund of the ALTB Herbarium (South Siberian Botanical Garden, Barnaul, Russia) has more than 450 000 sheets. Of these, there are 334 items of typical material (as of date 20.11.2022). The publication of data on DNA sequences and distribution of AMC plants in gene banks and GBIF is one of the indicators of active work in the field of genetics and biodiversity informatics at the level of modern standards. In 2022, within the framework of
the RSF project "Study of Phytodiversity and Genetic Resources of the Altai Mountainous Country Based on Big Data", the process of DNA barcoding of the type material of the ALTB Foundation was started and work was carried out to digitize the collection (http://altb.asu.ru).
So, the purpose of our work was to sequence the main DNA markers as DNA barcode for type specimens of ALTB Herbarium. At the first stage, we chose 3 popular markers - ITS region of nrDNA, trnH-psbA intergenic spacer, and trnL-trnF intergenic spacer and trnL intron of plastid DNA.
Materials and methods
For molecular genetic study, we took material (little part of the dried plant) from 110 specimens of 72 type taxa of different taxonomic rank (species, subspecies, nothospecies, variations, etc.) of 16 families of the ALTB Herbarium, mainly from the territory of AMC. After revision of the type material for the analysis, the most numerous genera by number of representatives were Alchemilla L., Veronica L., Potentilla L. and Gagea Salisb.
DNA isolation and amplification were conducted in Laboratory of Bioengineering of the South Siberian Botanical Garden of Altai State University according to standard techniques (Kutsev, 2009). DNA was isolated using DiamondDNA kit (LLC "ABT", Russia) according to the manufacturer's instructions.
Amplification of the marker fragments of nuclear DNA (ITS1-5.8S-ITS) and chloroplast DNA (trnL-
intron, trnL-trnF spacer, psbA-trnH spacer) was carried out on the thermocycler TC-Plus (Techne Workbench, United Kingdom) in 30 |l reaction mix included 12 |l H2O, 15 |l HS-Taq PCR-Color (2x) mastermix (BioLabMix), 1 |l DNA, and 1 |l (10 mM) each primer. We used the following primers: ITSfor and ITSrew (Kutsev et al., 2014), trnLF-f and trnLF-r (Taberlet et al., 2007), trnH and psbA (Shaw et al., 2007) and amplification programs:
95 °C - 4 min., (95 °C - 20 sec., 56 °C - 30 sec., 72 °C - 1 min.) x 35 cycles, 72 °C - 7 min. for nuclear DNA fragments; 94 °C - 4 min., (95 °C - 30 sec., 60 °C - 30 sec., 72 °C - 1 min.) x 35 cycles, 72 °C -5 min. for plastid fragments (trnL-intron, trnL-trnF spacer); 94 °C - 4 min., (95 °C - 30 sec., 64 °C - 30 sec., 72 °C - 45 sec.) x 35 cycles, 72 °C - 7 min. for plastid fragments (trnH-psbA spacer).
Concentration of the DNA probe was determined fluorometrically by NanoPhotometer P360 Implen (Hamburg, Germany), as well as with electrophoresis in 1.5 % agarose gel using DNA ladder Step50plus (BioLabMix). PCR products were purified using magnetic buds CleanMag DNA (Evrogen, Russia) according to the manufacturer's instructions. Purified products were sequenced by Sanger-method in SB RAS Genomics Core Facility (Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia).
Obtained sequences were analyzed in Chromas 2.6.4, and then, in BLAST - for the sample confirmation. The resulted sequences were submitted in the international NCBI GenBank (see Table).
Table
Studied type herbarium specimens for DNA barcoding from the ALTB Herbarium
№ ALTB barcode (specimen voucher) Taxon ITS region NCBI accession number trnL-trnF NCBI accession number trnH-psbA NCBI accession number
1 1100036088 Erysimum kotuchovii D. A. German (Erysimum quadrangulum Desf.) 0P558070 0P644519 0P672169
2 1100036413 Erysimum mongolicum D. A. German 0P558076 0P644523 0P672174
3 1100037147 Draba czuensis Revuschkin et A. L. Ebel 0P672170
4 1100036080 Dontostemon senilis subsp. gubanovii D. A. German (Dontostemon gubanovii (D. A. German) D. A. German) 0P558071 0P672171
5 1100036137 Smelowskia calycina (Stephan ex Willd.) C. A. Mey. var. brachycarpa A. L. Ebel (Smelowskia calycina (Stephan ex Willd.) C. A. Mey.) 0P558072
6 1100036112 Leiospora exscapa (C. A. Mey.) F. Dvorak var. pilosa A. L. Ebel (Leiospora exscapa (C. A. Mey.) F. Dvorak) 0P558073 0P644521 0P672173
Table (continued)
№ ALTB barcode (specimen voucher) Taxon ITS region NCBI accession number trnL-trnF NCBI accession number trnH-psbA NCBI accession number
7 1100036421 Ptilotrichum canescens var. elongatiforme A. L. Ebel 0P644522
8 1100036145, 1100036146 Sterigmostemum schmakovii Kamelin et D. A. German 0P558074, 0P558075
9 1100036405 Thellungiella botschantzevii D. A. German (Eutrema botschantzevii (D. A. German) Al-Shehbaz et Warwick) 0P558077
10 1100036104 Veronica reverdattoi Krasnob. 0P644520 0P672172
11 1100037634 Veronica spicata subsp. kamelinii Kosachev 0P558082 0P644528 0P672179
12 1100037430 Veronica x altaica Kosachev 0P558079 0P644525 0P672176
13 1100036422 Veronica x austrosibirica Kosachev (Veronica x altaica) 0P558080 0P644526 0P672177
14 1100035080 Veronica x sapozhnikovii Kosachev 0P558081 0P644527 0P672178
15 1100036586, 1100035772 Veronica x schmakovii Kosachev 0P558083 0P644529, 0P644530 0P672180, 0P672181
16 1100036446 Veronica x smirnovii Kosachev et D. A. German 0P558078 0P644524 0P672175
17 1100036105 Astragalus lenensis Shemetova, Shaulo et Lomon. 0P558084
18 1100000011 Ranunculus schmakovii Erst 0P558085 0P644531
19 1100000001 Ranunculus tuvinicus Erst 0P558086
20 1100000120 Aconitum khanminthunii A. A. Solovjev et Shmakov 0P644535 0P672189
21 1100045130 Aquilegia synakensis Shaulo et Erst 0P558087 0P644532
22 1100044812 Aquilegia aradanica Shaulo et Erst 0P558088
23 1100000091 Gagea azutavica Kotukhov 0P558091 0P672185
24 1100000029 Gagea goljakovii Levichev 0P558090
25 1100000106 Gagea kuraiensis Levichev 0P672184
26 1100000057 Gagea shmakoviana Levichev 0P672183
27 1100000025 Gagea xiphoidea Levichev 0P558089 0P644533 0P672182
28 1100044814 Fritillaria sonnikovae Shaulo et Erst 0P558092 0P644534 0P672186
29 1100000098 Waldsteinia tanzybeica Stepanov 0P672187
30 1010000083 Polypodium x vianei Shmakov 0P672188
31 1100000022 Scorzonera veresczaginii Kamelin et S. V. Smirn. (Takhtajaniantha veresczaginii (Kamelin et S. V. Smirn.) Kamelin et S. V. Smirn.) 0P558093 0P644536 0P672190
32 1100036087 Corydalis subverticillata Lazkov 0P644537
33 1100036151 Acantholimon karabajeviorum Lazkov 0P672191
34 1100000065 Neogaillonia botschantzevii Lincz. (Plocama botschantzevii (Lincz.) M. Backlund et Thulin) 0P558094
35 1100000089 Artemisia elenae Kupr. 0P558095 0P644538 0P672192
36 1100000033 Hieracium nasimovae Stepanov 0P558096 0P644539
37 1100036071 Viola x talmensis Vl. V. Nikitin (Viola x vilnaensis W. Becker) 0P644540
38 1100000110 Elymus tzvelevii Kotukhov (Campeiostachys schrenkiana (Fisch. et C. A. Mey. ex Schrenk) Drobow) 0P672193
Table (continued)
№ ALTB barcode (specimen voucher) Taxon ITS region NCBI accession number trnL-trnF NCBI accession number trnH-psbA NCBI accession number
39 1100000112 Eritrichium alpinum Ovczinnikova 0P672194
40 1100036469 Eritrichium kamelinii Ovczinnikova 0P672195
41 1100036152 Anoplocaryum tenellum A. L. Ebel et Rudaya 0P672196
42 1100000095 Oxytropis kaspensis Krasnob. et Pshenich. 0P558097 0P644541
43 1100053760 Phlomoides hypoviridis Lazkov 0P644544 0P672209
44 1100053752 Eremurus czatkalicus Lazkov 0P672208
45 1100000034 Achillea schmakovii Kupr. 0P558098 0P644542 0P672197
46 1100037062 Saussurea revjakinae S. V. Smirn. 0P644543 0P672198
47 1100052034 Alchemilla laxescens Czkalov 0P672201
48 1100054125 Alchemilla mininzonii Czkalov 0P672204
49 1100052026 Alchemilla oirotica Czkalov 0P672200
50 1100051985 Alchemilla pseudobungeana Czkalov 0P672199
51 1100053450 Alchemilla pustynensis Czkalov 0P672205
52 1100052042 Alchemilla vorotnikovii Czkalov 0P672202
53 1100054109 Alchemilla zimoenkensis Czkalov 0P672203
54 1100042998 Potentilla friesenii Kechaykin et Shmakov 0P672212
55 1100000004 Potentilla junatovii Rudaya et A. L. Ebel 0P644545 0P672210
56 1100035874 Potentilla khanminczunii Keczaykin et Shmakov 0P672207
57 1100053474 Potentilla rudolfii Keczaykin et Shmakov 0P672206
58 1100053142 Potentilla x chemalensis Kechaykin 0P644546
59 1100053442 Potentilla x habievii Kechaykin 0P672211
60 1100054132 Potentilla x jakovlevii Kechaykin et Shmakov 0P672213
Results and discussion
The first stage result of DNA barcoding of the ALTB type material was the publication of molecular data on plant species relatively recently described in science, mainly from the AMC territory, of which a significant proportion belongs to rare and endemic ones.
In total, it was deciphered 102 nuclear and chloroplast DNA sequences of 60 taxa of vascular plants from the ALTB type material: 29 fragments (28 taxa) of nuclear-encoded ribosomal internal transcribed spacer (ITS) region, 28 fragments (27 taxa) of the chloroplast intergenic spacer trnL-trnF and trnL-intron, and 45 fragments (44 taxa) coding sequences from trnH-psbA spacer. The above data on DNA sequences were not equally successfully obtained for all taxa. In some samples, concentration of the PCR product was not enough to sequence. As
a rule, the success of DNA extraction and further amplification was depended on the quality of the herbarium material.
The length of the ITS region in the data set was from 617 bp in Neogaillonia botschantzevii Lincz. (Plocama botschantzevii (Lincz.) M. Backlund et Thulin) to 701 bp in Hieracium nasimovae Stepanov., length of the trnL-trnF fragment - from 724 bp in Oxytropis kaspensis Krasnob. et Pshenich. to 960 bp in Potentilla x chemalensis Kechaykin, length of the trnH-psbA spacer - from 204 bp in Acantholimon karabajeviorum Lazkov to 623 bp in Elymus tzvelevii Kotukhov (Campeiostachys schrenkiana (Fisch. et C. A. Mey. ex Schrenk) Drobow).
Each obtained nucleotide sequence was downloaded in Genbank and identified by BLAST. In the most cases, the percent identity was 90-100 %. If it was less, it meant this taxon was absent in the database. The results are common for barcode
evaluations of endemic species, libraries of reference sequences in GenBank are poorly covered (Hebert et al., 2004; Erst et al., 2022).
The sequences were prepared and placed in GenBank with a unique number assigned (Table).
First column of the Table is presented barcode of the type specimen in ALTB Herbarium (Virtual Herbarium ALTB. http://altb.asu.ru). The second column includes names of type specimens as they are called on the herbarium labels. If this name is obsolete and is a synonym now, then the current name under which the taxon is registered with the NCBI is given in brackets. All taxonomic nomenclature was verified by POWO service (https://powo.science. kew.org/).
The taxa, for which both nuclear and chloroplast DNA sequences were obtained, were combined into a dataset and published in the Global Biodiversity Information Facility (Vaganov et al., 2022) through the Integrated Publishing Toolkit (IPT) data publisher's operator node (http://altb.asu.ru/ipt). The dataset "DNA barcodes of the vascular flora of the Altai Mountain Country: type material of the Herbarium ALTB" has information on DNA sequences (the term "associatedSequences" of the Darwin Core specification), data on the places of collection of type material ("decimalLatitude", "decimalLongitude"), links to digitized images of the herbarium on the Internet and other information, including labels.
Conclusion
The results of the study combine molecular genetics and digital technologies, and the end-to-end number of the type collection of ALTB Herbarium is integrated into the biodata architecture of GenBank and GBIF. In the future, this approach will make it possible to obtain objective results for solving the tasks on biodiversity, evolution, and ecology of endemic and other promising plant species. General open access to the original data of the study will allow identification of taxa and trace the dynamics of their area more reasonably and accurately. In the absence of other evidence, DNA barcoding creates hypotheses regarding new species rather than outright discovering them (Taylor, Harris, 2012; Guo et al., 2022). But it should be noted that barcoding must supplement morphological data for species description (Guo et al., 2022). In the applied aspect, the identification of plant objects directly affects the solution of social problems of environmental safety, is included in the food and health agenda, and is no less significant for nature protection activities in the transboundary territory of Russia, Kazakhstan, China, and Mongolia.
Acknowledgements
The research was supported by RSF (project No. 22-24-20002, https://rscf.ru/project/22-24-20002/).
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