Перший прогрес у виділенні та ампліфікації ДНК з матеріалу, що зберігається у гербарії LWS
The isolation of DNA from the herbarium specimens deposited at the LWS herbarium (State Museum of Natural History of the NAS of Ukraine, Lviv, Ukraine) has been tested using the column-based protocol. The isolated DNA has been amplified using different nuclear and plastid primers. The yield of obtai...
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Plant Introduction| _version_ | 1860145158460276736 |
|---|---|
| author | Novikov, Andriy Nachychko, Viktor |
| author_facet | Novikov, Andriy Nachychko, Viktor |
| author_sort | Novikov, Andriy |
| baseUrl_str | https://www.plantintroduction.org/index.php/pi/oai |
| collection | OJS |
| datestamp_date | 2025-02-12T12:22:44Z |
| description | The isolation of DNA from the herbarium specimens deposited at the LWS herbarium (State Museum of Natural History of the NAS of Ukraine, Lviv, Ukraine) has been tested using the column-based protocol. The isolated DNA has been amplified using different nuclear and plastid primers. The yield of obtained total DNA showed no significant dependence from the year of collection and plant family of studied specimens. In general, the obtained DNA of LWS specimens had medium yield (mean – 56.47 ng/µL) but relatively low purity (mean 260/230 value – 0.85 units and mean 260/280 value – 1.66 units). The success of DNA amplification for old herbarium material varied from 12.5 % to 91.1 % depending on applied primers. The trnL P6 Loop primers demonstrated the best performance (91.1 % successful amplification), but due to short resulted DNA fragments, it was not possible to purify the product for further processing. UniPlant primers performed the worst, and only 12.5 % of samples taken from the LWS herbarium (excluding controls) were successfully amplified. In general, nuclear primers, except for UniPlant, demonstrated a better success rate (mean – 31.5 %) during the work with samples taken from the LWS herbarium. Meanwhile, the plastid primers, except for trnL P6 Loop, showed slightly lower amplification success (mean – 26.8 %). |
| doi_str_mv | 10.46341/PI2024011 |
| first_indexed | 2025-07-17T12:54:24Z |
| format | Article |
| fulltext |
Plant Introduction, 103/104, 31–42 (2024)
© The Authors. This content is provided under CC BY 4.0 license.
RESEARCH ARTICLE
Pilot progress in DNA isolation and amplification from the material stored
at the LWS herbarium
Andriy Novikov 1, Viktor Nachychko 2
1 State Museum of Natural History, National Academy of Sciences of Ukraine, Teatralna str. 18, 79008 Lviv, Ukraine;
novikoffav@gmail.com
2 Ivan Franko National University of Lviv, Hrushevskoho str. 4, 79005 Lviv, Ukraine
Received: 12.11.2024 | Accepted: 15.12.2024 | Published online: 16.12.2024
Abstract
The isolation of DNA from the herbarium specimens deposited at the LWS herbarium (State Museum of
Natural History of the NAS of Ukraine, Lviv, Ukraine) has been tested using the column-based protocol. The
isolated DNA has been amplified using different nuclear and plastid primers. The yield of obtained total
DNA showed no significant dependence from the year of collection and plant family of studied specimens.
In general, the obtained DNA of LWS specimens had medium yield (mean – 56.47 ng/µL) but relatively
low purity (mean 260/230 value – 0.85 units and mean 260/280 value – 1.66 units). The success of DNA
amplification for old herbarium material varied from 12.5 % to 91.1 % depending on applied primers. The
trnL P6 Loop primers demonstrated the best performance (91.1 % successful amplification), but due to short
resulted DNA fragments, it was not possible to purify the product for further processing. UniPlant primers
performed the worst, and only 12.5 % of samples taken from the LWS herbarium (excluding controls) were
successfully amplified. In general, nuclear primers, except for UniPlant, demonstrated a better success
rate (mean – 31.5 %) during the work with samples taken from the LWS herbarium. Meanwhile, the plastid
primers, except for trnL P6 Loop, showed slightly lower amplification success (mean – 26.8 %).
Keywords: herbarium specimens, plant DNA barcoding, DNA extraction methods, degraded DNA, LWS herbarium
https://doi.org/10.46341/PI2024011
UDC 577.2.08 : 582.32/.998
Authors’ contributions: Andriy Novikov: conceptualization, project administration, supervision, funding acquisition, formal analysis,
visualization, writing – original draft. Viktor Nachychko: resources, validation, writing – original draft.
Funding: This work has been realized in the frames of the project “Digitization of natural history collections damaged as a result of
hostilities and related factors: development of protocols and implementation on the basis of the State Museum of Natural History
of the National Academy of Sciences of Ukraine” (Nr 2022.01/0013), financed by the National Research Foundation of Ukraine in the
grant call “Science for the Recovery of Ukraine in the War and Post-War Periods”.
Competing Interests: The authors declared no conflict of interest.
Introduction
Despite the development of modern
approaches to biodiversity data gathering by
community science, herbarium collections
remain a key point for different studies,
including biogeographic, phylogenetic, and
taxonomic (e.g., Nualart et al., 2017; Besnard et
al., 2018; Martin et al., 2018; Lang et al., 2019;
Rosche et al., 2022, 2025). It was shown that
herbarium collections outperform community
science platforms (i.e., iNaturalist) by providing
the data with lower spatial, taxonomic,
phylogenetic, and functional bias (Daru &
https://creativecommons.org/licenses/by/4.0/
https://orcid.org/0000-0002-0112-5070
https://orcid.org/0000-0001-6756-2823
32 Plant Introduction • 103/104
Novikov & Nachychko
Rodriguez, 2023; Eckert et al., 2024). Providing
well-documented specimens preserved for
decades or even hundreds of years, the herbaria
have attracted the attention of molecular
biologists for many years (Savolainenet al.,
1995; Särkinen et al., 2012; Bakker et al., 2020;
Bieker & Martin, 2018; McAssey et al., 2023).
However, the DNA in long-stored herbarium
material is often highly degraded (Adams &
Sharma, 2010; Staats et al., 2011). It was shown
that DNA degradation and fragmentation tend
to increase over time, resulting in shorter
lengths of reads and extensive accumulation
of cytosine-to-thymine substitutions (Weiß
et al., 2016; Quatela et al., 2023). Forrest et al.
(2019) demonstrated considerable variation in
the read length for Begonia L. depending on
the preservation method. They suggested that
such differences can be even higher at higher
taxonomic levels. Despite all mentioned
problems, the undoubted value of the material
stored at the herbaria prompts the search
for special techniques for DNA isolation and
amplification, as well as the reconstruction
of short reads (Drábková et al., 2002; Ribeiro
& Lovato, 2007; Tarieiev et al., 2011; Drábková,
2014; Höpke et al., 2018; Kurt et al., 2022;
Quatela et al., 2023).
Considering that the molecular laboratory
was established at the State Museum of
Natural History of the NAS of Ukraine in 2024,
it was decided to test the protocols of DNA
isolation and amplification on the material
stored at the institutional herbarium with
the acronym LWS. The herbarium LWS is one
of the oldest and richest in Ukraine, hosting
ca. 120,000 specimens of vascular plants and
ca. 26,000 specimens of non-vascular plants
(Novikov et al., 2024). However, for many
years, the specimens at LWS were regularly
exposed to high temperatures (ca. 90 °C)
during anti-fungal and anti-insect treatment,
significantly increasing the chances of DNA
degradation and, therefore, the possibility
of poor amplification success (Staats et al.,
2011; Forrest et al., 2019). Meanwhile, there
was already reported success in CTAB-based
isolation and 5S rDNA intergenic spacer
amplification using the material stored at the
LWS herbarium (Tynkevich et al., 2022).
Höpke & Albach (2018) demonstrated
that column-based DNA extraction from
the herbarium material generally results in
higher DNA yield and purity. They also pointed
out that column-based DNA extraction is
preferred for work with herbarium specimens
as it requires less sampling material and
consequently causes less damage to the
collection. Hence, here we share our pilot
experience on column-based DNA extraction
and amplification of four nuclear and five
plastid regions, based on the example of 63
specimens of vascular plants (56 stored at
the herbarium LWS and seven controls – see
Appendix A).
Material and methods
The leaf fragments ca. 0.5–1 сm2 were sampled
from randomly selected herbarium specimens
representing different species and collected
at various years (Appendix A). Additionally,
seven positive controls were implemented –
six silica-dried and one herbarized (without
heating) leaf samples of Staphylea pinnata L.
(Staphyleaceae) collected in 2023.
The total DNA was isolated in July–August
2024 using the Macherey-Nagel NucleoSpin
Plant II kit following a slightly modified
protocol. Leaf samples were ground in the
ceramic mortars with a direct addition of
500 µL of PL1 lysis buffer. Homogenizing
the material would have been problematic
without adding the lysis buffer to the mortar.
Moreover, due to the small amount of the
sampled material, taking it out from the
mortar in case of dry homogenization would
be problematic, too – the homogenate stuck
to the mortar walls. The resulting suspension
has been carefully transferred (avoiding macro
fragments) to a new tube with the addition
of 10 µL of RNase A solution and vortexed
thoroughly. Then, the mixture was incubated
at 65 ° for 30 minutes in a thermoshaker.
Further steps followed the standard protocol.
The concentration and purity (260/230
and 260/280 ratios) of total DNA have been
measured spectrophotometrically using
DeNovix DS-11 FX spectrophotometer/
fluorometer. After that, eluted DNA was
diluted 10 times and stored in the fridge until
further processing.
Different regions of eluted DNA were
amplified in Applied Biosystems 2720 thermal
cycler using four nuclear and five plastid
primers (Table 1). Thermo Scientific DreamTaq
Green PCR Master Mix (2X) has been applied
Plant Introduction • 103/104 33
Pilot progress in DNA isolation and amplification from the LWS herbarium
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34 Plant Introduction • 103/104
Novikov & Nachychko
R² = 0.0078
0
20
40
60
80
100
120
140
160
180
200
1940 1950 1960 1970 1980 1990 2000 2010 2020
DN
A
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el
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(n
g/
µL
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Collection year
R² = 0.0327
0
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0.8
1
1.2
1.4
1.6
1.8
1940 1950 1960 1970 1980 1990 2000 2010 2020
26
0/
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0
0.5
1
1.5
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1940 1950 1960 1970 1980 1990 2000 2010 2020
26
0/
28
0
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tio
Collection year
Figure 1. The dependence of DNA yield (A) and purity (B, C) from the collection year. The control samples
and three outlet samples representing 1853, 1904, and 1906 years are excluded from the graphs for
better visual representation.
A
B
C
Plant Introduction • 103/104 35
Pilot progress in DNA isolation and amplification from the LWS herbarium
to prepare the samples for amplification. The
amplification success has been evaluated
by electrophoresis realized in agarose gel
prepared with ×10 TBE buffer and stained by
BentoLab GelGreen nucleic acid stain. The
electrophoresis has been run on BentoLab
portable PCR workstation at 50 V for 30
minutes.
The statistics have been performed in
Microsoft Excel 2016 and Past 4.14 (Hammer
et al., 2001) environments.
Results and discussion
Due to the limitation of the sample number and
size, we were not able to statistically assess
the full range of plant families and collection
years, as well as other factors that could
influence the result. The studied specimens do
not allow delimiting any significant difference
between the samples regarding the DNA yield
(mean ± standard deviation – 56.47 ± 43.92 ng/
µL; coefficient of variation – 77.79 %) and purity
(discussed further). There was only a slightly
insignificant trend in increasing DNA yield
and purity jointly with collection year (Fig. 1).
Regression analysis proved insignificant
dependence between the collection year
and DNA extraction characteristics. Similar
to our results, the DNA yield did not show a
significant dependence from the collection
year during the application of CTAB DNA
isolation protocol and column-based DNA
isolation (with the same NucleoSpin Plant II
mini kit) applied for herbarium material in
other studies (Höpke & Albach, 2018; Höpke
et al., 2018). However, these conclusions and
our outcomes contradict the reports of Zeng
et al. (2018) and Marinček et al. (2022), who
applied column-based isolation kits (Tiangen
DNAsecure Plant Kit and Qiagen DNeasy Plant
Mini Kit, respectively) and noticed a significant
decrease in DNA yield with samples age. In
our case, the absence of advances in the DNA
yield is probably caused by intense thermal
treatment over the years, which smooths out
the difference between more recent and older
specimens at the LWS herbarium.
Nevertheless, some authors (Höpke &
Albach, 2018; Höpke et al., 2018; Marinček
et al., 2022) assumed that column-based DNA
isolation is preferred, resulting in relatively
better DNA yield and purity and is easier to
handle. Marinček et al. (2022) noticed that
despite the better general performance of
specialized ancient DNA isolation protocol, it
finally resulted in sequences comparable in
Figure 2. The dependence of DNA purity from the DNA yield. Calculated as Gaussian function in the
nonlinear regression module. Initial estimation of optimum and tolerance based on the weighted
average, followed by a nonlinear optimization by the Levenberg-Marquardt method in Past 4.14.
36 Plant Introduction • 103/104
Novikov & Nachychko
quality with those produced by DNA isolated
with a column-based kit.
Interestingly, the best DNA purity has been
achieved at a concentration diapason of ca.
50–90 ng/µL (Fig. 2). After that, the ratios
260/230 and 260/280 decreased, which
can be explained either by optimal DNA
concentrations in this diapason or technical
peculiarities of the spectrophotometer and
should be furtherly inspected. In most cases,
obtained 260/230 values were markedly
lower than 2.0 (mean ± standard deviation –
0.85 ± 0.36; coefficient of variation – 42.47 %;
Fig. 1 B), which indicates contamination despite
the application of the column-based DNA
isolation technology. Such 260/230 values are
close to those obtained as a result of CTAB
DNA isolation (Höpke & Albach, 2018; Höpke
et al., 2018; Kurt et al., 2022; Xie et al., 2023) and
column-based DNA isolation Marinček et al.
(2022) without additional purification. The
observed 260/280 values were below normal
level but still near 1.8 units (mean ± standard
deviation – 1.66 ± 0.36; coefficient of variation
– 17.00 %; Fig. 1 C).
In our study, the dependence of DNA yield
from the plant family of studied specimens
appeared insignificant. The samples of
Ranunculaceae specimens demonstrated the
highest mean value of DNA yield, and those
of Caprifoliaceae demonstrated the lowest.
However, the standard deviation was too
high for most analyzed families (Fig. 3). No
particular dependence on the PCR success
from the plant family or collection year was
observed.
The drying method used in the herbarium
technique was reported to significantly
affect the DNA yield and PCR success rate
(Särkinen et al., 2012; McAssey et al., 2023).
In our study, silica-dried control specimens
demonstrated 87.0–94.4 % of successful DNA
amplification. Recently collected herbarium
material (one year stored control specimen)
Figure 3. The boxplot of DNA yield from specimens of different plant families. The families represented
by single specimen and control samples are excluded from the graph.
Plant Introduction • 103/104 37
Pilot progress in DNA isolation and amplification from the LWS herbarium
Primers applied Marker
type
Successfully
amplified
(number of
included
controls)
Unsuccessfully
amplified
(number of
included
controls)
Total success
rate (with
controls), %
Success rate
(without
controls), %
trnL P6 Loop (g - h) plastid 58 (7) 5 92.1 91.1
ITS1 (ITS1 - ITS2) nuclear 24 (6) 39 (1) 38.1 32.1
ITS (ITS1+ITS2) (ITS-u1 - ITS-u4) nuclear 23 (6) 40 (1) 36.5 30.4
ITS2 (ITS2F - ITS3R) nuclear 23 (5) 40 (2) 36.5 32.1
trnH-psbA (psbA3_fv2 – trnHf_05v2) plastid 23 (5) 40 (2) 36.5 32.1
rbcLa (F - R) plastid 20 (6) 43 (1) 31.7 25.0
trnL-F (c - d) plastid 18 (5) 45 (2) 28.6 23.2
matK (3F_KIM_f – 1R_KIM_r) plastid 15 (5) 48 (2) 23.8 17.9
ITS2 (UniPlantF - UniPlantR) nuclear 14 (7) 49 22.2 12.5
Table 2. Successful DNA amplification with different markers applied.
also showed nearly identical PCR success
compared to silica-dried ones with all applied
markers. At the same time, the success of DNA
amplification for old herbarium material varied
significantly and demonstrated 12.5–91.1 % of
successful DNA amplification (Table 2). Some
of the applied markers performed better
in the sense of DNA amplification success.
The trnL P6 Loop primers demonstrated
the best performance. However, application
of this marker results in extremely short
DNA product lengths (ca. 100 bp). Such short
fragments are hard for further processing
(i.e., purification with standard protocols and
further sequencing) and, in most cases, allow
identifying the specimens only to the genus or
family level (Taberlet et al., 2007). Surprisingly,
tested nuclear DNA markers showed relatively
high amplification success. Among plastid
markers, only trnH-psbA demonstrated similar
performance. Despite the high amplification
success reported by Moorhouse-Gann et al.
(2018), UniPlant primers demonstrated the
weakest result among tested ITS primers.
Hence, trnL P6 Loop and UniPlant primers
cannot be recommended for work with the
herbarium material.
Conclusions
1. It was shown that tested column-
based DNA isolation protocol could be
successfully applied to the herbarium
material. However, the low purity of the
total DNA samples obtained should be
considered.
2. Tested primers (except for trnL P6 Loop
and UniPlant) and amplification programs
showed their reliability and can be
recommended for work with herbarium
material.
3. DNA amplification success depends on the
applied markers rather than the collection
year.
4. Nuclear markers generally outperformed
plastid ones in work with LWS herbarium
material, demonstrating better
amplification success.
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40 Plant Introduction • 103/104
Novikov & Nachychko
Nr LWS accession
Nr / field Nr
Family Species / subspecies Collection
year
Preservation
method
1 070459 Apiaceae Bupleurum tenuissimum L. 1906 pressed and dried
2 021825 Asparagaceae Muscari botryoides (L.) Mill. 1853 pressed and dried
3 114876 Asparagaceae Scilla kladnii Schur 2009 pressed and dried
4 017053 Asteraceae Achillea oxyloba (DC.) Sch.Bip. subsp.
schurii (Sch.Bip.) Heimerl
1976 pressed and dried
5 095416 Asteraceae Centaurea maramarosiensis (Jáv.) Czerep. 2002 pressed and dried
6 116027 Asteraceae Doronicum carpaticum (Griseb. & Schenk)
Nyman
1978 pressed and dried
7 117159 Asteraceae Leucanthemum rotundifolium (Waldst. &
Kit.) DC.
2012 pressed and dried
8 117383 Boraginaceae Pulmonaria rubra Schott subsp.
filarszkyana (Jáv.) Domin
2002 pressed and dried
9 077525 Boraginaceae Symphytum cordatum Waldst. & Kit. 1985 pressed and dried
10 119944 Brassicaceae Arabidopsis neglecta (Schult.) O'Kane &
Al-Shehbaz
1978 pressed and dried
11 113215 Brassicaceae Arabidopsis thaliana (L.) Heynh. 2008 pressed and dried
12 114577 Campanulaceae Campanula serrata (Kit. ex Schult.)
Hendrych
2009 pressed and dried
13 092030 Campanulaceae Phyteuma vagneri A.Kern. 1960 pressed and dried
14 118938 Caprifoliaceae Scabiosa lucida Vill. subsp. barbata Nyár. 1990 pressed and dried
15 115638 Caprifoliaceae Scabiosa lucida Vill. subsp. barbata Nyár. 2009 pressed and dried
16 116693 Caryophyllaceae Sabulina pauciflora (Kit.) A.V.Novikov 2006 pressed and dried
17 114685 Caryophyllaceae Silene nutans L. subsp. dubia (Herbich)
Zapal.
2009 pressed and dried
18 007155 Caryophyllaceae Silene zawadskii Herbich 1978 pressed and dried
19 113561 Crassulaceae Rhodiola rosea L. 2008 pressed and dried
20 113460 Crassulaceae Sempervivum carpathicum Wettst. ex
Prodan subsp. carpathicum
2008 pressed and dried
21 043251 Crassulaceae Sempervivum globiferum L. subsp.
preissianum (Domin) M.Werner
1947 pressed and dried
22 119472 Cyperaceae Carex curvula All. 1976 pressed and dried
23 116914 Gentianaceae Gentiana laciniata Kit. ex Kanitz 2012 pressed and dried
24 116676 Gentianaceae Gentiana lutea L. subsp. lutea 2005 pressed and dried
25 116786 Gentianaceae Gentiana punctata L. 2007 pressed and dried
26 116362 Gentianaceae Swertia perennis L. subsp. perennis 2011 pressed and dried
27 074413 Gentianaceae Swertia punctata Baumg. 1960 pressed and dried
28 112773 Iridaceae Crocus banaticus J.Gay 1972 pressed and dried
29 115481 Iridaceae Crocus heuffelianus Herb. 2010 pressed and dried
30 112026 Iridaceae Gladiolus imbricatus L. 1988 pressed and dried
31 110149 Iridaceae Iris graminea L. 1988 pressed and dried
32 115689 Iridaceae Iris sibirica L. 2010 pressed and dried
Appendix A. Studied herbarium material at the LWS herbarium.
Plant Introduction • 103/104 41
Pilot progress in DNA isolation and amplification from the LWS herbarium
Nr LWS accession
Nr / field Nr
Family Species / subspecies Collection
year
Preservation
method
33 116180 Juncaceae Juncus bulbosus L. 2011 pressed and dried
34 016776 Juncaceae Luzula alpinopilosa (Chaix) Breistr.
subsp. obscura S.E.Fröhner
1978 pressed and dried
35 112663 Lamiaceae Thymus alternans Klokov 1973 pressed and dried
36 120097 Lamiaceae Thymus jankae Čelak. 2014 pressed and dried
37 016733 Lamiaceae Thymus pulcherrimus Schur 1996 pressed and dried
38 116659 Linaceae Linum extraaxillare Kit. 2005 pressed and dried
39 073630 Oleaceae Syringa josikaea J.Jacq. ex Rchb. 1982 pressed and dried
40 081841 Orobanchaceae Euphrasia tatrae Wettst. 1957 pressed and dried
41 112713 Plantaginaceae Plantago atrata Hoppe subsp.
carpathica (Soó) Soó
1982 pressed and dried
42 010385 Poaceae Festuca amethystina L. subsp. orientalis
Krajina
1958 pressed and dried
43 114720 Poaceae Festuca porcii Hack. 2009 pressed and dried
44 112311 Poaceae Poa granitica Braun-Blanq. subsp.
disparillis Nyár.
1983 pressed and dried
45 012597 Poaceae Poa rehmannii (Asch. & Graebn.)
K.Richt.
1904 pressed and dried
46 110476 Poaceae Sesleria bielzii Schur 1975 pressed and dried
47 116702 Poaceae Sesleria heufleriana Schur subsp.
heufleriana
2011 pressed and dried
48 119608 Ranunculaceae Ranunculus carpaticus Herbich 1975 pressed and dried
49 104365 Ranunculaceae Ranunculus malinovskii Elenevsky &
Derv.-Sokol.
1989 pressed and dried
50 113245 Rosaceae Rosa canina L. 2008 pressed and dried
51 017272 Rubiaceae Galium transcarpaticum Stojko &
Tasenk.
1976 pressed and dried
52 017273 Rubiaceae Galium transcarpaticum Stojko &
Tasenk.
1976 pressed and dried
53 088718 Rubiaceae Galium transcarpaticum Stojko &
Tasenk.
1980 pressed and dried
54 063138 Staphyleaceae Staphylea pinnata L. 1947 pressed and dried
55 063180 Staphyleaceae Staphylea pinnata L. 1976 pressed and dried
56 107564 Staphyleaceae Staphylea pinnata L. 1998 pressed and dried
57 UA01-20dr Staphyleaceae Staphylea pinnata L. 2023 pressed and dried
58 UA01-20Si Staphyleaceae Staphylea pinnata L. 2023 silica-dried
59 UA01-12 Staphyleaceae Staphylea pinnata L. 2023 silica-dried
60 UA01-17 Staphyleaceae Staphylea pinnata L. 2023 silica-dried
61 UA01-18 Staphyleaceae Staphylea pinnata L. 2023 silica-dried
62 UA02-09 Staphyleaceae Staphylea pinnata L. 2023 silica-dried
63 UA02-14 Staphyleaceae Staphylea pinnata L. 2023 silica-dried
Appendix A. Continued.
42 Plant Introduction • 103/104
Novikov & Nachychko
Перший прогрес у виділенні та ампліфікації ДНК з матеріалу, що зберігається у
гербарії LWS
Андрій Новіков 1, Віктор Начичко 2
1 Державний природознавчий музей НАН України, вул. Театральна, 18, Львів, 79008, Україна;
novikoffav@gmail.com
2 Львівський національний університет імені Івана Франка, вул. Грушевського, 4, Львів, 79005,
Україна
Протестовано виділення ДНК із гербарних зразків, що зберігаються у гербарії LWS (Державний
природознавчий музей НАН України, Львів, Україна) за протоколом на основі силікагелевих колонок.
Виділена ДНК була ампліфікована з використанням різних ядерних і пластидних праймерів. Вихід
отриманої сумарної ДНК не показав істотної залежності від року збору та родин, до яких належали
зразки. Загалом, ДНК, отримана із зразків гербарію LWS, мала помірний вихід (середнє значення
– 56.47 нг/мкл), але відносно низьку чистоту (середнє значення співвідношення 260/230 – 0,85
і середнє значення співвідношення 260/280 – 1,66). Успіх ампліфікації ДНК старого гербарного
матеріалу коливався від 12.5 % до 91.1 % залежно від використаних праймерів. Праймери trnL P6
Loop продемонстрували найбільшу ефективність (91.1 % успішної ампліфікації), але через короткі
фрагменти отриманої ДНК не вдалося очистити продукт для подальшої обробки. Праймери UniPlant
продемонстрували найгірші результати, і лише матеріал 12.5 % досліджених зразків гербарію LWS
(за винятком контрольних), був успішно ампліфікований. Загалом, ядерні праймери, за винятком
UniPlant, продемонстрували кращу успішність ампліфікації (середнє значення – 31.5 %) при роботі
зі зразками з гербарію LWS. В той же час, пластидні праймери, за винятком trnL P6 Loop, показали
дещо нижчу успішність ампліфікації (середнє значення – 26.8 %).
Ключові слова: гербарні зразки, штрихкодування рослинної ДНК, методи екстракції ДНК, деградована ДНК, гербарій LWS
|
| id | oai:ojs2.plantintroduction.org:article-1649 |
| institution | Plant Introduction |
| keywords_txt_mv | keywords |
| language | English |
| last_indexed | 2025-07-17T12:54:24Z |
| publishDate | 2024 |
| publisher | M.M. Gryshko National Botanical Garden of the NAS of Ukraine |
| record_format | ojs |
| resource_txt_mv | wwwplantintroductionorg/3d/6aa4b1cbbf896b9755a6432273027e3d.pdf |
| spelling | oai:ojs2.plantintroduction.org:article-16492025-02-12T12:22:44Z Pilot progress in DNA isolation and amplification from the material stored at the LWS herbarium Перший прогрес у виділенні та ампліфікації ДНК з матеріалу, що зберігається у гербарії LWS Novikov, Andriy Nachychko, Viktor The isolation of DNA from the herbarium specimens deposited at the LWS herbarium (State Museum of Natural History of the NAS of Ukraine, Lviv, Ukraine) has been tested using the column-based protocol. The isolated DNA has been amplified using different nuclear and plastid primers. The yield of obtained total DNA showed no significant dependence from the year of collection and plant family of studied specimens. In general, the obtained DNA of LWS specimens had medium yield (mean – 56.47 ng/µL) but relatively low purity (mean 260/230 value – 0.85 units and mean 260/280 value – 1.66 units). The success of DNA amplification for old herbarium material varied from 12.5 % to 91.1 % depending on applied primers. The trnL P6 Loop primers demonstrated the best performance (91.1 % successful amplification), but due to short resulted DNA fragments, it was not possible to purify the product for further processing. UniPlant primers performed the worst, and only 12.5 % of samples taken from the LWS herbarium (excluding controls) were successfully amplified. In general, nuclear primers, except for UniPlant, demonstrated a better success rate (mean – 31.5 %) during the work with samples taken from the LWS herbarium. Meanwhile, the plastid primers, except for trnL P6 Loop, showed slightly lower amplification success (mean – 26.8 %). Протестовано виділення ДНК із гербарних зразків, що зберігаються у гербарії LWS (Державний природознавчий музей НАН України, Львів, Україна) за протоколом на основі силікагелевих колонок. Виділена ДНК була ампліфікована з використанням різних ядерних і пластидних праймерів. Вихід отриманої сумарної ДНК не показав істотної залежності від року збору та родин, до яких належали зразки. Загалом, ДНК, отримана із зразків гербарію LWS, мала помірний вихід (середнє значення – 56.47 нг/мкл), але відносно низьку чистоту (середнє значення співвідношення 260/230 – 0,85 і середнє значення співвідношення 260/280 – 1,66). Успіх ампліфікації ДНК старого гербарного матеріалу коливався від 12.5 % до 91.1 % залежно від використаних праймерів. Праймери trnL P6 Loop продемонстрували найбільшу ефективність (91.1 % успішної ампліфікації), але через короткі фрагменти отриманої ДНК не вдалося очистити продукт для подальшої обробки. Праймери UniPlant продемонстрували найгірші результати, і лише матеріал 12.5 % досліджених зразків гербарію LWS (за винятком контрольних), був успішно ампліфікований. Загалом, ядерні праймери, за винятком UniPlant, продемонстрували кращу успішність ампліфікації (середнє значення – 31.5 %) при роботі зі зразками з гербарію LWS. В той же час, пластидні праймери, за винятком trnL P6 Loop, показали дещо нижчу успішність ампліфікації (середнє значення – 26.8 %). M.M. Gryshko National Botanical Garden of the NAS of Ukraine 2024-12-16 Article Article application/pdf https://www.plantintroduction.org/index.php/pi/article/view/1649 10.46341/PI2024011 Plant Introduction; No 103/104 (2024); 31-42 Інтродукція Рослин; № 103/104 (2024); 31-42 2663-290X 1605-6574 10.46341/PI103-104 en https://www.plantintroduction.org/index.php/pi/article/view/1649/1557 Copyright (c) 2024 Andriy Novikov, Viktor Nachychko http://creativecommons.org/licenses/by/4.0 |
| spellingShingle | Novikov, Andriy Nachychko, Viktor Перший прогрес у виділенні та ампліфікації ДНК з матеріалу, що зберігається у гербарії LWS |
| title | Перший прогрес у виділенні та ампліфікації ДНК з матеріалу, що зберігається у гербарії LWS |
| title_alt | Pilot progress in DNA isolation and amplification from the material stored at the LWS herbarium |
| title_full | Перший прогрес у виділенні та ампліфікації ДНК з матеріалу, що зберігається у гербарії LWS |
| title_fullStr | Перший прогрес у виділенні та ампліфікації ДНК з матеріалу, що зберігається у гербарії LWS |
| title_full_unstemmed | Перший прогрес у виділенні та ампліфікації ДНК з матеріалу, що зберігається у гербарії LWS |
| title_short | Перший прогрес у виділенні та ампліфікації ДНК з матеріалу, що зберігається у гербарії LWS |
| title_sort | перший прогрес у виділенні та ампліфікації днк з матеріалу, що зберігається у гербарії lws |
| url | https://www.plantintroduction.org/index.php/pi/article/view/1649 |
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