Springer-VerlagTokyohttp://www.springer.de102650918-94401618-0860Journal of Plant ResearchJ Plant ResLife Sciences19610.

1007/s10265-005-0196-4

J Plant Res (2005) 118:57–60 © The Botanical Society of Japan and Springer-Verlag Tokyo 2005
Digital Object Identifier (DOI) 10.1007/s10265-005-0196-4

TECHNICAL NOTE

Hirokazu Tsukaya • Yu Iokawa • Makiko Kondo •

Hideaki Ohba

Large-scale general collection of wild-plant DNA in Mustang, Nepal

Received: August 27, 2004 / Accepted: December 28, 2004 / Published online: February 3, 2005

Abstract The deposit of DNA samples of wild plants that

correspond to voucher specimens is highly informative and Introduction
greatly enhances the value of the herbarium specimens. The
Society of Himalayan Botany (SHB), Tokyo, has assembled Some of the most vital information used in phylogenic stud-
general collections of flowering plants of the Sino- ies is that obtained from DNA sequences (e.g., review by
Himalayan region for more than 40 years. In a trial of the Mishler 2000); most herbarium specimens, however, cannot
collection of these types of bioresources for use in basic be used for DNA extraction. The inclusion of corresponding
research, we adopted FTA cards, which have recently been DNA samples with herbarium specimens is therefore highly
used for large-scale collection of DNA of humans, micro- informative, and greatly enhances the value of the specimen
organisms and viruses, for the general collection of DNA (Prance 2001). Accordingly, large-scale collection of the
samples of wild plants during a botanical expedition in DNA of cultivated plants is ongoing in some research
Mustang, Nepal, in 2003. Three hundred and fifty-five plant institutes (e.g., more than 13,000 DNA specimens have been
specimens from Mustang, Nepal, were collected along with accumulated at the Royal Botanic Gardens, Kew, UK;
the corresponding DNA samples. Examination of the qual- http://www.kew.org/data/dnaBank/homepage.html).
ity of the DNA samples by PCR demonstrated the utility of However, no organized trials of the general collection of
the collection system. The identification of all of the speci- DNA during field research have been carried out. In gen-
mens collected, as well as data from the specimens, will be eral, plant researchers bring plant material from the field to
presented on the Flora of Nepal Database website (http:// their laboratories as silica-gel-dried leaves, from which
ti.um.u-tokyo.ac.jp/default.htm), which is open to the pub- DNA is extracted (Prance 2001). However, this method of
lic. The DNA resources will be identified on the website and DNA sampling is not suitable for long-term (over several
distributed openly by the SHB to researchers worldwide for weeks or longer), large-scale, general collections in remote
basic research. areas, because large amounts of silica gel are required to
preserve the leaves of many specimens. Due to this incon-
Key words Bioresource · FTA card · Himalayan botany · venience, researchers do not usually perform mass DNA
General collection of DNA · Nepal (Mustang) collections and, instead, choose to sample the DNA of par-
ticular taxa, with the result that only limited types of DNA
samples are collected, even during long-term botanical
expeditions. This practice is wasteful, given that DNA sam-
H. Tsukaya (*) · M. Kondo ples collected with the corresponding herbarium specimens
National Institute for Basic Biology/Okazaki Institute for Integrated are highly useful for plant studies, and that plants that
Bioscience, Okazaki, Aichi 444-8585, Japan
Tel. +81-564-55-7512; Fax +81-564-55-7512 inhabit remote areas like the Sino-Himalaya are not easily
e-mail: tsukaya@nibb.ac.jp accessible. Therefore, general collection should include not
H. Tsukaya
only herbarium specimens but also DNA samples.
School of Life Sciences & School of Advanced Sciences, The Graduate Simple kits for DNA isolation have recently become
University for Advanced Studies Shonan Village, Hayama, Japan available. We previously evaluated the utility of these kits
H. Tsukaya under field conditions in remote areas, and found that FTA
Graduate School of Science, Kyoto University, Kyoto, Japan cards (Whatman) are useful for the collection of plant
Y. Iokawa DNA, without the need for special equipment (Tsukaya
Department of Biology, Joetsu University of Education, Niigata, Japan 2003) as reported earlier for genomes of rice and tobacco
H. Ohba (Li et al. 2000). FTA cards are paper cards that immobilize
University Museum, University of Tokyo, Tokyo, Japan nucleic acids. Collection of DNA from leaf samples on these
58
cards is straightforward, requiring personnel only to squash
young leaves on the cards and then allow them to dry at
room temperature. The dried cards can be stored at room
temperature for long periods without special care. DNA
samples immobilized on FTA cards can only be used for
PCR reactions, and cannot be used for experiments that
require large amounts of free DNA, such as the construc-
tion of genomic libraries. However, the ease of collection of
DNA samples in the field by means of these cards is of great
value. In fact, FTA cards have recently been extensively
used for large-scale DNA collection of humans (e.g., Forrest
et al. 2004), microorganisms (e.g., Subrungruang et al. 2004;
Lampel et al. 2004), and the HIV virus (e.g., Li et al. 2004).
However, the use of FTA cards for large-scale collection of
wild-plant DNA has not been reported so far. In our pre-
liminary sampling with FTA cards during field research in
Java, Indonesia, application of FTA cards for the general
collection of plant DNA successfully revealed traits of nat-
ural hybridization between two Impatiens species (Tsukaya
2004). Based on this idea, the first trial for the preparation
of a large number of sets of dried specimens and DNA
samples of wild plants was carried out in the Himalayas by
a team from the Society of Himalayan Botany, Tokyo,
Japan, in 2003.
Materials and methods
A botanical expedition was carried out in upper Mustang,
Nepal, from 25 June to 9 July 2003, as a cooperative project
Fig. 1. A storage box (right) containing 355
DNA specimens from Mustang immobilized
on FTA cards, and an FTA card (left) with
spots of DNA samples from 20 plant
specimens. The unit of scale is 1 cm. Note
the compactness of the DNA samples
between the Department of Plant Resources, Nepal, and
the University of Tokyo, Japan. Details of the route of the
expedition and the itinerary have been described elsewhere
(Iokawa and Yonekura 2004).
To avoid cross-contamination in the DNA collection,
each DNA sample was spotted in a square of ca. 1.8 ¥
1.8 cm; the squares had been marked with pencil on the
FTA cards (Whatman Japan, Tokyo, Japan). A leaf segment
from each individual was applied to the FTA card, covered
with a small piece of parafilm (Pechiney Plastic Packaging,
WI, USA), and then squashed with a pen (Tsukaya 2003).
Next, each spot was marked with a specific number indicat-
ing the collection number. The cards were then dried in a
small plastic box with a small amount of silica gel (Fig. 1).
All of the procedures were carried out at a campsite on the
mountain. The cards were kept in a plastic box at room
temperature in the field before being brought to the labo-
ratory in Japan. PCR reactions can be performed after
washing the FTA cards three times for 5 min each with a
buffer supplied with the cards, and three times for 5 min
with Tris-EDTA (pH 8.0) buffer, and then drying the cards
in an oven at 50°C. For PCR amplification, 1-mm2 pieces
were used as templates in 50-ml reactions containing
Pyrobest DNA polymerase (TaKaRa Biomedicals, Tokyo,
Japan). To amplify the ITS and trnL-F loci, PCR was per-
formed as described previously (Tsukaya 2004), using prim-
ers described elsewhere (White et al. 1990; Douzery et al.
1999; Tsukaya et al. 2003). Following amplification, 3 ml
of each product was loaded on 1% agarose gels for
electrophoresis.

Results and discussion
Extensive floristic studies of the Himalayas have been car-
ried out for more than 40 years by the late Prof. H. Hara of
the University of Tokyo and his successors in the Society of
Himalayan Botany (SHB), Tokyo, Japan (Kanai 2002; Ohba
2002). The Flora of Nepal project commenced in 1993 as an
international cooperative project between Nepal, the UK,
and Japan, and a database has been constructed of the flora
and the field research (Akiyama and Ohba 2004). The goal
of the present study was to examine the feasibility of per-
forming combined collection of DNA and voucher herbar-
ium specimens, in order to maximize the value of general
collections of plant specimens carried out by the SHB in the
Sino-Himalayas. DNA samples were collected by one of us
(Y.I.) during a botanical expedition in Mustang, Nepal, with
the help of members of the expedition team. DNA spots for
355 specimens were collected on FTA cards (Fig. 1) with
voucher herbarium specimens. To evaluate the quality of
the DNA samples, PCR was performed on 40 randomly
selected DNA samples to amplify the nuclear ITS region
and the plastid trnL-F region, under standard PCR reaction
conditions. These PCR conditions successfully amplified
fragments of the nuclear ITS region in 28 of the 40 speci-
mens (70%; Fig. 2a, b), and in 34 of the 40 specimens the
trnL-F region of the plastid DNA was successfully amplified
(85%; Fig. 2c, d). Some of samples that were not amplified
still might be, using different PCR conditions. These results
demonstrate the utility of the FTA cards for large-scale
general collection of plant DNA during a long-term field
expedition in a remote area.
The use of FTA cards for general collection of plant
DNA at remote sites requires special care in the following
areas. For some plants, in particular the Gramineae, DNA
collection on FTA cards was difficult due to the small
amounts of tissue extract obtained from leaves of these
plants; for 144 specimens of this type, in the present trial,
silica-gel-dried samples were collected for later DNA
Fig. 2a–d. PCR examination of
the quality of the DNA templates a b
collected in Mustang. a, b PCR
products of the ITS region from
40 independent DNA samples
immobilized on FTA cards. c, d
PCR products for the trnL-F
region from 40 independent
DNA samples immobilized on
FTA cards. Twenty samples were
electrophoresed on 2% agarose
gels alongside size markers
c d
59
extraction. But this does not necessarily mean that FTA
cards cannot be used for Gramineae. This problem can be
overcome by choosing folded young leaves as sources of
DNA. In fact, DNA from Oryza sativa was amplified with-
out problem (Lin et al. 2000; our unpublished results).
More importantly, precise application of the voucher
specimen on the card spot is necessary. In the present trial
before the start of the expedition, we drew lines on the FTA
cards with pencil to prepare squares of 1.8 ¥ 1.8 cm for
mounting a single DNA sample (Fig. 1, left). Each square
was named with the combination of the number of the card
and the square number seen in the Fig. 1 (e.g., 3–12 for
square number 12 on card #3). This design enabled us to
spot DNA of various samples at high density. Moreover, this
design appeared to be effective at preventing cross-
contamination among samples, because all the amplified
bands were stable and single (Fig. 2), suggesting only one
DNA species was amplified for each sample. Our on-going
sequencing also shows that cross-contamination is quite
rare among the samples (data not shown). However, if one’s
budget is sufficient to allow the use of more FTA cards,
lesser density of DNA spotting is, of course, better for pre-
venting contamination and for collecting sufficient DNA for
each specimen (although the 1.8 ¥ 1.8 cm spot can supply
more than 200 templates for PCR).
In our present trial, one person (Y.I.) was assigned to
deal with the spotting of DNA on the FTA cards, while the
other members participating in the expedition supplied him
with segments of a leaf that corresponded to each herbar-
ium specimen with a collection number. The collection
number is used for all processes of administration of the
specimen collected in the Flora of Nepal Database (http://
ti.um.u-tokyo.ac.jp/default.htm). To administer both spot
numbers and the corresponding collection numbers with-
out mistake, one additional person was needed, because
spots on the FTA card have no special characteristics or
features except for their color (Fig. 1). Preparation of
herbarium specimens was carried out by the collectors
themselves.
60
Using this method, one botanical expedition was able to
accumulate about 500 DNA specimens, and at least 70% of
the specimens are expected to be easily used in PCR anal-
ysis (Fig. 2). Based on the results, we judged that this project
should be continued. This bioresource will aid in PCR-
based research of Himalayan plants that are not easily avail-
able to the vast majority of plant researchers. We would also
like to invite the worldwide research community of plant
taxonomists to join in our trials.
Establishment of a stock of DNA resources for
Himalayan plants
The general collection of DNA samples, together with
voucher specimens, requires a high level of organization,
not only for the initial collection of samples and specimens,
but also for the effective usage of the collection, which
includes the distribution of DNA samples to researchers
worldwide. Therefore, in collaboration with members of the
Society of Himalayan Botany, Tokyo, we have established a
stock of DNA resources for Sino-Himalayan plants. The
general collection of DNA samples will be carried out dur-
ing every botanical expedition conducted by the SHB. All
voucher specimens will be deposited in the herbarium of
the University of Tokyo (TI). Electronic information on the
corresponding specimens, including scientific name, locality,
date, and other information, has been released on the Flora
of Nepal Database website (http://ti.um.u-tokyo.ac.jp/
default.htm). In the near future, the website will be updated
to indicate whether the DNA sample corresponding to each
specimen is available. At the same time, we will distribute
the collected DNA materials when requested. Although
each spot of DNA in our trial can supply more than 200
templates for PCR reaction, and the template can be used
in at least 20–30 PCR reactions, DNA samples are limited
research resources. Thus, rules for the distribution of mate-
rials will soon be determined. This trial represents a great
advance in the understanding of the diversity of plants in
the Himalayas.
Acknowledgements The authors wish to give special thanks to the
following colleagues who assisted in the botanical expedition in the
field: Dr. T. Miyazaki (University of Tokyo, Japan), Dr. K. Yonekura
(Tohoku University, Japan), Dr. Y. Ibaragi (Tokushima Prefectural
Museum, Japan), and Dr. R.K. Uprety (Ministry of Forest and Soil
Conservation, Nepal). The Mustang botanical expedition was carried
out as a cooperative project between the Department of Plant
Resources, Nepal, and the Department of Botany, University Museum,
University of Tokyo, Japan. This study was supported by a grant from
the Midori-ikusei-Zaidan, the SOKENDAI group research project
from the Graduate University for Advanced Studies, and a Grant-in-
Aid from the Ministry of Education, Science, and Culture, Japan and
the Japan Society for the Promotion of Science.
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