Bull. Natl. Mus. Nat. Sci., Ser. B, 37(3), pp. 101–111, August 22, 2011

Assessment of the DNA Quality in Mushroom Specimens:
Effect of Drying Temperature
Kentaro Hosaka* and Kunihiko Uno
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan* E-mail: khosaka@kahaku.go.jp(Received 18 May 2011; accepted 22 June 2011)

Abstract It is generally believed that drying temperature is critical for preserving high quality
DNA in biological specimens. Fruit bodies of mushrooms are therefore dried at low temperature,e.g., lower than 60°C. In this study, we assessed the DNA quality in mushroom specimens by using
different drying temperatures (35, 52, and 71°C). The quality of DNA was assessed based on gel
electrophoresis with whole genomes as well as PCR products from multiple loci. Based on the experiment using a total of seven phylogenetically distantly related species of mushrooms, we conclude that drying temperature does not significantly affect the DNA quality for subsequent studies(PCR, sequencing, etc.). Further studies are necessary to investigate presently unrepresented samples, such as big fleshy mushrooms (e.g., boletes, or Boletales) and gasteromycetes (puffballs,
stinkhorns, etc.).
Key words : Agaricomycotina, Basidiomycota, DNA quality, drying temperature, herbarium,
PCR, tissue preservation.

Introduction
It is now widely appreciated that herbarium isa great source of genetic materials (Bruns et al.,
1990; Taylor and Swann, 1994; Drábková et al.,
2002; Erkens et al., 2008; Dentinger et al.,2010). DNA data from the type specimens will
be able to solve taxonomical issues in manygroups. One particular example is by Nagy et al.
(2011) who demonstrated that a number of“unidentified” environmental DNA sequences
matched with the sequences from the type strains
and specimens. Furthermore, herbarium can potentially host a large number of presently unknown or unrepresented lineages. Brock et al.
(2008) estimated that ca. 70% of the taxonomic
diversity in herbarium is not yet represented in
the public database, i.e., GenBank.
One of difficulties concerning the DNA fromherbarium specimens is that they frequently lackhigh quality DNA. It is especially true for older,historically important specimens (e.g., types)
because DNA in specimens degrades with time
(Hummel and Herrmann, 1994; Erkens et al.,2008). The ages of specimens and degrees ofdegradation in DNA, however, are not strictly
correlated (Drábková et al., 2002; Leino et al.,2009). In fact, specimens collected more than
100 years ago often yield good quality DNA
whereas much younger specimens (e.g., less than
5 years old) often lack useful quantity and/or
quality of DNA (Dentinger et al., 2010). Therefore the quality of DNA depends more on how
specimens were collected, prepared, and maintained, than the ages (Bruns et al., 1990;
Drábková et al., 2002; Leino et al., 2009).
Because of such difficulties, a variety of DNA
extraction methods have been proposed (Bruns etal., 1990; Drábková et al., 2002; Dentinger et al.,2010). However, after DNA highly degrades,
there are only limited things we can do unless
DNA repair techniques much more efficient than

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Kentaro Hosaka and Kunihiko Uno

the ones currently available from the commercial
kits (e.g., Sigma, Qiagen, etc.) are invented. Furthermore, many herbarium specimens have been
fumigated by a variety of chemicals to prevent
insect damage (Whitten et al., 1999; Kigawa etal., 2003). Many widely available fumigants are
known to highly efficiently degrade DNA, among
which the mixture of methyl bromide and ethylene oxide is demonstrated to be one of the worst
in terms of DNA degradation (Kigawa et al.,2003). The authors’ herbarium (TNS) has beentreated by methyl bromide until relatively recently, and the specimens treated this way (ca. 10
years or older) all have highly degraded DNA.
Our initial attempt to amplify shorter fragments(300 bp or shorter) mostly failed (data notshown). For preserving valuable genetic resources, all specimens should be treated by fumigants not affecting DNA, such as sulphuryl fluoride (Whitten et al., 1999) or not fumigated at all.
Most recent attempts therefore focused onpreservation of DNA from freshly collected samples, instead of extracting DNA from old materials. A variety of preservation methods are reviewed by Nagy (2010). Among which, cryopreservation in liquid nitrogen or ultra-deep
freezer (80°C) is a single most efficient method
for virtually all types of biological materials, but
it is not feasible especially during the fieldwork(Seutin et al., 1991; Dawson et al., 1998; Nagy,2010). Several organic solvents (e.g., ethanol,
isopropanol, but especially acetone) efficiently
preserve DNA for a long term (Fukatsu, 1999;
Nagy, 2010), but air transportation of specimens
with such highly flammable substances is notrecommended.
Instead, a variety of buffers without organic
solvents have been proposed (Nagy, 2010). The
efficacy of DNA preservation using different
buffers vary among organisms. For example,
DMSO-NaCl solution was successfully applied
for marine invertebrates and avian samples(Seutin et al., 1991; Dawson et al., 1998), but
CTAB-NaCl buffer appears equally efficient forplant tissues (Rogstad, 1992). Laulier et al. (1995)
reported that guanidium thiocyanate buffer ismuch more efficient than CTAB-NaCl buffer, andworks for a variety of organisms, including virus,
fungi, and animals. It is noteworthy that acetone(though flammable) and guanidium thiocyanate
buffer can effectively preserve RNA as well as
DNA (Laulier et al., 1995; Fukatsu, 1999).
The authors’ laboratory routinely uses a modified DMSO-NaCl buffer (Hosaka and Castellano,
2008; Hosaka, 2009) for preserving field-collected mushroom samples with a good success rate.
Tissues preserved in this buffer and stored atroom temperature for several months usually
yield high quality DNA (Hosaka and Castellano,
2008; Hosaka, 2009; Hosaka et al., 2010). However, the process of preparing small tissue fragments in buffer is very tedious during the fieldwork. Since mycologists (and botanists) need toprepare dried specimens regardless of the purpose of studies, it is desirable if we can obtainhigh quality DNA from dried specimens.
Previous studies all agree that temperature andtime for dying specimens are two critical factors
for preserving quality DNA (Pyle and Adams,
1989; Bruns et al., 1990; Drábková et al., 2002;
Blanco et al., 2006; Erkens et al., 2008). It is
generally believed that quickly dried specimens
with low heat (ca. 42°C or less) are necessary for
subsequent DNA studies (Pyle and Adams, 1989;
Bruns et al., 1990; Taylor and Swann, 1994;
Johnson et al., 2003; Nagy, 2010). However,some studies reported that drying temperature
above 60°C also resulted in good quality DNA(Blanco et al., 2006; Erkens et al., 2008).
By comparing the DNA quality from phylogenetically distantly related mushroom species,
which were dried at different temperatures, wehere report that drying temperature does not significantly affect the DNA quality for subsequentstudies (PCR, sequencing, etc). Although wehave tested only seven species of mushrooms, we
believe the conclusion is applicable to manyother untested species of mushrooms. Field mycologists without storage buffers at hands therefore should dry specimens as quickly as possible(regardless of temperatures) for preserving highquality DNA.

DNA Quality in Mushroom Specimens
Table 1.

103

Mushroom species investigated in this study

SpeciesAbbreviation*

Producer

Lentinula edodes
Grifola frondosa
Flammulina velutipes
Hypsizygus marmoreus
Pholiota nameko
Agaricus bisporus
Pleurotus eryngii

L
G
F
H
Ph
A
Pl

Sakuragawa-shi, Ibaraki, JapanHOKUTO Corporation, Nagano, JapanJA Nakano, Nagano, JapanHOKUTO Corporation, Nagano, Japan
JA Tokamachi, Niigata, JapanJA Ibaraki-Kasumi, Ibaraki, JapanHOKUTO Corporation, Nagano, Japan*Abbreviations of species names are used in Figs. 1, 3 and 4.

Fig. 1. Drying procedure using a food dehydrator. a. Fresh fruit bodies for drying without paper bags. See Table
1 for abbreviations. B. Fresh fruit bodies for drying in paper bags.

Materials and Methods
Sample Preparation
A total of seven species of edible mushrooms
were purchased at the local supermarket on February 15, 2011. All of them were cultivated elsewhere in Japan, and none of the wild mushrooms
were investigated in this study. The details ofeach species are summarized in Table 1.
Fresh fruit bodies of each species were driedusing a food dehydrator (Snackmaster Express
FD-60, Nesco/American Harvest, WI, USA).
The use of food dehydrator is a desirable methodfor drying mushroom fruit bodies because it circulates warm/hot air for effective drying. The
fruit bodies were cut in half (for Agaricus,

Lentinula, and Pleurotus; Fig. 1a) prior to drying
or dried as a whole (for Flammulina, Grifola,
Hypsizygus, and Pholiota; Fig. 1a) depending on
the size. The drying temperatures were set to 35,
52, and 71°C, respectively (Table 2). For eachspecies at each temperature setting, fruit bodies
were dried either with or without paper bag (Figs.
1a, b, Table 2). Drying with enclosed paper bag
is a common practice when cross contaminationof spores from different specimens is not desirable. This, however, may inhibit effective drying
because little air circulation becomes available.
The samples were dried for 48 hours. Dried samples were photographed and stored at 20°C
until further experiments were conducted.
For each species, two additional treatments

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Kentaro Hosaka and Kunihiko Uno
Table 2.

Experimental design using different drying temperatures

Experiment No.1
1
2
3
4
5
6
7
8
1
2
3

Treatment
Dried for 48 hours at 71°C without paper bag
Dried for 48 hours at 71°C enclosed in paper bag2
Dried for 48 hours at 52°C without paper bag
Dried for 48 hours at 52°C enclosed in paper bag
Dried for 48 hours at 35°C without paper bag
Dried for 48 hours at 35°C enclosed in paper bag
Stored in DMSO buffer3 at room temperature until DNA extraction
Stored at 80°C until DNA extraction

These numbers are shown in Figs. 3 and 4.
See text and Fig. 1b for usage of paper bag.
See Materials and Methods for description of buffer.

were made without drying process (Table 2). One
treatment was to store fragments of fruit bodies
in DMSO buffer. The composition of buffer was
based on Seutin et al. (1991) with a minor modification as described by Hosaka and Castellano(2008) and Hosaka (2009). The buffer with sample fragments was stored at room temperature.
The other treatment was to freeze fruit bodies at
80°C until further experiments were conducted.
DNA Preparation, PCR and Gel Electrophoresis
DNA extraction was conducted ca. 1 monthafter the drying procedure. Dried hymenia of ca.
25 mg (dry weight) were soaked in DMSO buffer
for 1 hour prior to DNA extraction. DNA fromeach sample was extracted using a modified
CTAB extraction protocol followed by glass milk
purification methods as summarized by Hosaka(2009) and Hosaka and Castellano (2008).
Briefly, samples were ground in liquid nitrogenusing mortar and pestle, incubated in CTAB
buffer at 65°C for 1 hour, and proteins were removed using the mixture of chloroform: isoamylalcohol (24 : 1). The materials were further purified using 6 M sodium iodine buffer with glass
milk, washed with ethanol/buffer solution, and finally eluted in 100 m l of TE buffer. To assess the
DNA quality, 5 m l of genomic DNA was loaded
on 1% agarose gels stained with ethidium bromide and visualized under UV light. DNA wasinitially qualified and quantified using NanoDrop
Spectrophotometer (ND-1000, Thermo Scientif-

ic, USA), but a small amount of carry-over sodium iodine buffer prevented an accurate measurement (data not shown). The measurement fromspectrophotometer was therefore not recorded.
DNA was amplified for ribosomal DNA regions using three different combinations ofprimers. For amplifying the ITS region (ca. 600bp), the primer combination of ITS5 and ITS4(White et al., 1990) was used. For amplifying
longer fragments, the combination of ITS5 andLR7 (ca. 1600 bp; Vilgalys and Hester, 1990) or
PNS1 (Hibbett, 1996) and LR7 (ca. 3500 bp) wasused. In addition, single-copy protein coding
genes were amplified for the following loci:
RPB1, RPB2, and EF-a . The primer names andsequences were summarized in Table 3.
PCR reactions were carried out using 20 m lreaction volumes each containing: 1 m l genomic
DNA, 1 m l dNTP, (4 mM), 1 m l of each primer(8 m M), 0.5 units of Taq polymerase (TaKaRa,
Tokyo, Japan), 2 m l MgCl2 (25 mM), 2 m l Bovine
Serum Albumin (BSA). PCR products (1 m l)were electrophoresed in 1% agarose gels stainedwith ethidium bromide and visualized under UVlight.
Results
Appearance of Specimens
After 48 hours, all specimens appeared completely dried. There were no signs of incomplete
drying. Visual inspections of dried specimens,
however, revealed some differences, depending

DNA Quality in Mushroom Specimens
Table 3.

105

Primers used in this study

Sequence (5→3)

References[rDNA]
ITS5
ITS4
PNS1
LR7

GGAAGTAAAAGTCGTAACAAGG
TCCTCCGCTTATTGATATGC
CCAAGCTTGAATTCGTAGTCATATGCTTGTCTACTACCACCAAGATCTWhite et al. (1990)
White et al. (1990)
Hibbett (1996)
Vilgalys and Hester (1990)[RPB1]gRPB1-Af
fRPB1-CrGAKTGTCCKGGWCATTTTGG
CNGCDATNTCRTTRTCCATRTA

Matheny et al. (2002)
Matheny et al. (2002)[RPB2]
fRPB2-5F
bRPB2-7.1R

GAYGAYMGWGATCAYTTYGG
CCCATRGCYTGYTTMCCCATDGC

Liu et al. (1999)
Matheny (2005)[EF-1a ]EF1-526F
EF1-2218R

GTCGTYGTYATYGGHCAYGTATGACACCRACRGCRACRGTYTG

Rehner and Buckley (2005)
Rehner and Buckley (2005)

on the treatment made (Table 2). The most striking difference was discoloration of specimens,
especially the cut surface (Fig. 2). Although all
specimens with any drying temperatures showedsome degrees of discoloration, brownish discoloration was especially severe when specimens
were dried at higher temperatures (52 and 71°C
(Fig. 2).
Not all species, however, showed apparent discoloration. Dried specimens of Grifola, Pholiota,
and Pleurotus did not change their colors regardless of drying temperatures, or only slightly so at
the highest drying temperature (71°C) (Figs. 2b, d,e). On the other hand, dried specimens of Lentinula, Flammulina, Hypsizygus, and Agaricus
showed apparent discoloration (Figs. 2a, c, f, g).
The most striking was discoloration observed in
Flammulina, which became slight pale brownish(at 35°C drying temperature; Fig. 2c) to brownish to reddish brown (at 52 and 71°C drying temperatures; Figs. 2b, c). Comparing with fresh fruit
bodies with almost pure white coloration (Fig.
1a), it is apparent that dried specimens totally
lost their original color. Although the degrees of
discoloration were less severe, Lentinula (Fig.
2a), Hypsizygus (Fig. 2f), and Agaricus (Fig. 2g)
all showed apparent discoloration that was otherwise not seen in fresh fruit bodies or dried specimens treated at low (35°C) temperature.

There were generally no clear differences between specimens dried with paper bags and theones without such bags. However, one species(Agaricus) showed apparent discoloration (darkbrown) only when specimens were dried in paperbags (Fig. 2h). In addition, discoloration in
Flammulina seemed to be more severe when
dried in paper bags. This difference may be due
to slower drying when specimens were enclosed
in paper bags. Although specimens were completely dried after 48 hours, we have not checked
the status of specimens after shorter period oftime (e.g., 6, 12, or 24 hours).
Besides discoloration, differences in shapes
and sizes of fruit bodies were observed in somespecies. Fruit bodies of Flammulina have nearlystraight stalks when fresh (Fig. 1a) or dried atlower temperatures, but they became curvy or almost curly when dried at higher temperatures (52or 71°C) (Fig. 2c). At higher drying temperatures, fruit bodies show significant degrees ofshrinking (Fig. 2c), that was also observed in
Hypsizygus (Fig. 2f).
Genomic DNA
After DNA extraction and electrophoresis on
agarose gels, all samples produced good quality
DNA (Fig. 3). Although there were some differences in brightness of bands, they all showed

106

Kentaro Hosaka and Kunihiko Uno

Fig. 2. Dried fruit bodies. a–g (from left to right). Fruit bodies dried at 35, 52, and 71°C, respectively. a. Lentinula edodes, dried in a paper bag. Note slight discoloration of the cut surface as drying temperature increases.
b. Grifola frondosa dried without paper bag. c. Flammulina velutipes dried in paper bag. Note significant discoloration (brownish) of entire fruit bodies as drying temperature increases. d. Pleurotus eryngii dried in
paper bag. e. Pholiota nameko dried without paper bag. f. Hypsizygus marmoreus dried without paper bag.
Note slight discoloration and shrinking of fruit bodies as drying temperature increases. g. Agaricus bisporus
dried with paper bag. Note slight discoloration of the cut surface as drying temperature increases. h. Agaricus
bisporus dried at 71°C with (right) or without (left) paper bag. The specimen dried in paper bag has a significant discoloration. The same 10 cm scale was placed under fruit bodies in each photo.

DNA Quality in Mushroom Specimens

107Fig. 3. Comparisons of genomic DNA from mushrooms with various treatments of drying temperatures. For
abbreviations (L, G, F, H, Ph, A, and Pl), see Table 1. The numbers represent experimental design summarizedin Table 1.

clear bands of large sizes. Presence of smears
was apparent for all samples and no obvious differences were seen among different treatments.
For most samples, DNA from frozen tissues (at80°C) showed darker bands than DNA fromdried specimens. Differences in brightness ofbands appeared negligible among different drying temperatures.

PCR Amplifications
Most samples produced clear bright bands of
PCR products with any combinations of primers(Fig. 4). Amplifications of ca. 600 bp (ITS; Fig.
4a) all produced bright single bands and there
were absolutely no visible differences in brightness of bands. Amplification of the same ribosomal tandem repeat regions, but with much longer
fragments, ca. 1600 bp between ITS5-LR7 (Fig.

108

Kentaro Hosaka and Kunihiko Uno4b) and ca. 3500 bp between PNS1-LR7 (Fig.
4c), also showed absolutely no visible differences. The remaining three loci were single-copy
protein coding genes (RPB1, Fig. 4d; RPB2, Fig.
4e; EF-a , Fig. 4f). Some weak bands were observed in Hypsizygus (Fig. 4d, e; both dried at 71
or 52°C in paper bags) and Grifola (Fig. 4e;dried at 52°C without paper bag). Other differences were probably negligible.
Discussion
Despite their importance as genetic resources,
herbarium specimens have been prepared andmaintained in a variety of ways (Bruns et al.,1990). For example, there is no set standard on
how specimens should be dried. The only agreement is that specimens should generally be dried
quickly but at lower temperature (Pyle andAdams, 1989; Bruns et al., 1990; Taylor andSwann, 1994; Johnson et al., 2003; Nagy, 2010).
It is often claimed that specimens should be driedat 42°C or lower temperature (Taylor and Swann,
1994; Drábková et al., 2002; Johnson et al.,2003) or drying temperature should not exceed
60°C (Nagy, 2010). However, there seems to be
no scientific theory behind it because specimens
dried at much higher temperature often yieldhigh quality DNA (Blanco et al., 2006; Erkens etal., 2008). The present study also agrees that
DNA in specimens dried at high temperature (upto 71°C) was not significantly damaged.
The appearance of specimens varied significantly depending on drying temperatures andpresence/absence of paper bags (Fig. 2). This implies that to keep the visual appearance of thespecimens, which itself is critical for subsequent
taxonomic studies, lower drying temperatures
should be used. However, the physical conditionof specimens does not necessarily indicate thequality of DNA (Dawson et al., 1998). The sameis true for the “greenness” of leaves in plant
specimens. Although it is generally assumed thatthe greenness of leaves depends on how quickly
specimens were dried, there was no correlations
between the greenness of leaves and the qualityof DNA (Erkens et al., 2008). Our study is consistent with their findings that specimens with severe discoloration (Figs. 2c, g, h) yielded equallyhigh quality DNA (Figs. 3, 4).
Biological processes do not stop after specimens are collected. The activity of nucleases is
especially problematic because it quickly breaks
down DNA molecules to short fragments (Hummel and Herrmann, 1994; Dawson et al., 1998).
To stop such enzymatic activities, the eliminationof water from specimens should be performed as
rapidly as possible (Bruns et al., 1990; Nagy,2010). In this regard, drying temperature formushroom specimens should be set high, up to
71°C, to preserve the quality of DNA. We havenot tested higher temperatures than 71°C so noconclusions can be drawn on how high drying
temperatures can be. It can, however, easily be
assumed that very high temperatures (e.g., 100°C
or higher) simply burn the samples and probably
damage the DNA as well.
Some species of mushrooms (Grifola andHypsizygus) showed slightly weaker PCR bands
when specimens were dried at 52 or 71°C (Fig.
4). The influence on the presence of enclosing
paper bag was not apparent because such a treatment sometimes resulted in weak bands (Fig. 4d)but stronger bands were also observed (Fig. 4e).
In this study, we have not prepared replication ofeach species and we cannot conclude whether theinfrequent presence of weak PCR bands is truly
due to high drying temperatures. We argue it isprobably not the reason because the same species
dried at the highest temperature (71°C) in thisstudy showed strong bands (Fig. 4). It is also interesting to note that the frozen samples often
yielded less genomic DNA (Fig. 3). We believe it
is due to DNA condensation as shown by Johnson et al. (2003).
In this study, we have tested the quality of
DNA in freshly dried materials. Since DNA in
dried specimens degrades with time (Johnson etal., 2003; Erkens et al., 2008), the influence ofthe storage conditions also needs to be investigated. Specifically, the influence of temperature, humidity, fumigation, and UV light, which are all

DNA Quality in Mushroom Specimens

109

Fig. 4. Amplifications of different loci from mushrooms with various treatments of drying temperatures. For
abbreviations (L, G, F, H, Ph, A, and Pl), see Table 1. The numbers represent experimental design summarizedin Table 1. a. ITS (ITS5 and ITS4). b. ITS5 and LR7. c. PNS1 and LR7. d. RPB1 (gRPB1-Af and fRPB1-Cr).
e. RPB2 (fRPB2-5F and bRPB2-7.1R). f. EF-a (EFa-526F and EF1-2218R). See Table 3 for primer names
and sequences.

110

Kentaro Hosaka and Kunihiko Uno

known to cause DNA degradation, deserves rigorous investigation. In addition, this study has
tested only seven species of mushrooms. Some
mushrooms of fruit bodies with distinct morphology, such as boletes, gasteromycetes, and conks
were not included. However, we believe the conclusion is applicable to many other untested
species of mushrooms as long as specimens are
dried rapidly. To achieve this, species with big,
fleshy fruit bodies need to be cut into thin slices
prior to drying.
Our suggestions based on the results of thisstudy are following:(1) To obtain high quality DNA while maintaining the visual appearance of specimens, drying temperatures should be set low (35°C in thisstudy) as long as specimens can be dried rapidly(within 48 hours).
(2) If drying process takes longer, use higher
temperatures (up to 71°C) or cut fruit bodies into
thin slices.
(3) Use of paper bags for big, fleshy mushrooms can cause slower drying and therefore
should be avoided.
(4) Field mycologists without storage buffers
at hands should dry specimens as quickly as possible (regardless of temperatures) for preserving
high quality DNA.
(5) DNA from dried specimens should be
extracted as soon as possible or pieces of specimens should be stored in freezer until DNA
extraction.
Acknowledgments
The authors thank Yoshiaki Muramatsu,
Kyung-Ok Nam, Kazuo Nishibori, Megumi Ohtsuka and Kazuya Tsukagoshi for assisting ourcuratorial work. Thanks also go to Dr. Tamotsu
Hoshino for critically reviewing the manuscript.
This research was financed in part by a research
grant (2009–2011) of the Institute of Fermentation, Osaka, Japan, and JSPS grants-in-aid foryoung scientists (B) to KH.

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