Bull. Natl. Mus. Nat. Sci., Ser. B, 39(2), pp. 53–60, May 22, 2013

Assessment of the DNA Quality in Mushroom Specimens:a Recovery of the Whole ITS Sequence from
Fragmented DNA of the Type Specimen1
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 22 February 2013; accepted 25 March 2013)

Abstract DNA from four type specimens of mushrooms (Agaricales, Basidiomycota, Fungi)
stored in the herbarium of National Museum of Nature and Science since 1950ʼs were extracted
and PCR ampliﬁcation was attempted. Because the whole ITS region (ca. 650 bp) could not be
ampliﬁed, DNA were assumed to be highly degraded to less than 600 bp. To assess the degree ofdegradation, we used a microﬂuidics-based platform. This revealed that the most frequently present fragments were less than 230 bp long. Based on this result, we have designed six new primers
to speciﬁcally amplify approximately 200–400 bp products of Amanita imazekii. A total of 8 contigs were assembled to successfully reconstruct the whole ITS region of the species, and thesequence recovered by this approach had a 100% match with the reference sequence.
Key words : Amanita, DNA barcoding, fumigation, holotype, old specimens.

Introduction
As pointed out by many researchers, numerous
specimens stored in herbaria are great source ofgenetic materials (Bruns et al., 1990; Taylor andSwann, 1994; Drábková et al., 2002; Erkens etal., 2008; Dentinger et al., 2010). Undoubtedly,type specimens, especially holotypes, are themost important specimens for solving taxonomic
issues. If DNA data from the type specimens
become available, we can have a direct link
between species names and sequence data, as
shown by Nagy et al. (2011). Furthermore, herbarium can potentially host a large number ofpresently unknown or unrepresented lineages.
Brock et al. (2008) estimated that ca. 70% of thetaxonomic diversity in herbarium is not yet represented in the public database.
1

Both authors contributed equally to this work.

When we deal with the type specimens, some
difﬁculties include the ages of specimens andpoor quality of DNA. Although the ages of specimens and degrees of degradation in DNA are notstrictly correlated (Drábková et al., 2002; Leinoet al., 2009), DNA in specimens degrades withtime (Hummel and Herrmann, 1994; Erkens etal., 2008). This process is unavoidable because
DNA molecules degrade as a result of hydrolysis,alkylation, and oxidation even under an ideal
condition, i.e., constant low temperature (Willerslev and Cooper, 2005). Therefore to rescue highquality DNA in type specimens, DNA should be
extracted as soon as possible or small pieces of
specimens should be stored under more ideal
conditions for future DNA studies.
It is also claimed that the quality of DNA
depends on how specimens were collected, prepared, and maintained (Bruns et al., 1990; Drábková et al., 2002; Leino et al., 2009). For exam-

54

Kentaro Hosaka and Kunihiko Unople, previous studies generally agree thattemperature and time 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). Although drying by low
heat (ca. 42°C or less) is often preferred to preserve high quality DNA (Pyle and Adams, 1989;
Bruns et al., 1990; Taylor and Swann, 1994;
Johnson et al., 2003; Blanco et al., 2006; Nagy,2010), we have reported, by comparing the DNA
quality from phylogenetically distantly related
mushroom species, that drying temperature does
not signiﬁcantly affect the DNA quality for subsequent studies (Hosaka and Uno, 2011).
More importantly, many herbarium specimens
have been fumigated by a variety of chemicals toprevent insect and fungal damage (Bridson andForman, 1998; 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 andethylene oxide is demonstrated to be one of theworst in terms of DNA degradation (Kigawa etal., 2003). Ideally, all specimens should betreated by fumigants not affecting DNA, such assulphuryl ﬂuoride (Whitten et al., 1999) or not
fumigated at all, but many historically important
specimens, especially the type specimens, havealready been suffered from such fumigants for along time.
Our research group has initiated a project of
DNA barcoding all type specimens in our fungal
herbarium. To achieve this goal, however, we atleast need to obtain sequences of the whole ITS
region (ca. 600–700 bp), which is now considered an ofﬁcial DNA barcode region for fungi(Schoch et al., 2012). Since we have experienced
difﬁculties amplifying the regions longer than
600 bp from old (ca. 10 year or older) specimens
stored in our herbarium (Hosaka and Uno, 2011),
we assumed that DNA in such specimens arehighly degraded due to the ages and fumigation.
However, we have not assessed the sizes of DNA
fragments so far.
Because repeated destructions of specimens

for molecular studies should be avoided, we haveinitiated DNA extraction from taxa with large
and ﬂeshy fruit bodies. Among them, a genus
Amanita contains more than 900 species worldwide (Tulloss, 2005) with great diversity in East
Asia (Zhang et al., 2004). Furthermore, thegenus has been a subject of molecular phylogenetic studies (Oda et al., 1999, 2004; Zhang etal., 2004). Our herbarium possesses ca. 20 typespecimens of the genus from various ages
(1950ʼs to 2000ʼs) and therefore we have selected
the specimen collected relatively recently(1990ʼs) as a case study to assess the DNA
quality.
In addition to a standard PCR using generalprimers, we also used the Agilent Bioanalyzer(Palo Alto, CA, USA) to visualize the quantity of
DNA in each fragment size. This technology uses
a microﬂuidics-based platform for sizing, quantiﬁcation and quality control of DNA (http://www.
home.agilent.com) and it has a potential of further application, such as evaluating quality andquantity of total RNA (Miller et al., 2004). By
using this method, we can assess degrees ofDNA fragmentation more objectively than using
a standard gel electrophoresis. We here report theresults of Bioanalyzer assay and a recovery of
the whole ITS sequence from fragmented
genomic DNA of the type specimen.
Materials and Methods
Type specimen used in this study
The holotype of Amanita imazekii T. Oda, C.
Tanaka & Tsuda (TNS-F-101519) was collected
in 1996 and has been stored in the fungal herbarium of National Museum of Nature and Science(Fig. 1). It was described as a new species in2001 (Oda et al., 2001) but the whole ITS region
had already been sequenced and been depositedin GenBank under the accession number
AB038764 (submitted on February 23, 2000).
The sequence was obtained before the specimen
was ofﬁcially registered at the TNS herbarium.
To compare the quality of genomic DNA, three
additional type specimens collected and stored inAssessment of the DNA Quality in Mushroom Specimens

55

USA). Samples (1 μl) of genomic DNA were
loaded into each well of High Sensitivity DNA
LabChip for analysis. The obtained data wereanalyzed using the Expert Software 2100 (Agilent Technologies, Palo Alto, CA). The Bioanalyzer assay was processed and analyzed following the manufacturerʼs instructions.

Fig. 1. Label of the holotype of Amanita imazekii
housed at the TNS herbarium.

the TNS herbarium from earlier times (1950ʼs to1980ʼs) were used as well. These specimens
include Amanita pseudoporphyria (TNS-F237281, collected in 1956), Lyophyllum sykosporum (TNS-F-237381, collected in 1986), andRussula subnigricans (TNS-F-237524, collected
in 1954).
DNA extraction
DNA was extracted from the gill tissues stored
in DMSO buffer (Seutin et al., 1991; Hosaka,2009) overnight. Tissues were ﬁrst ground underliquid nitrogen using a mortar and pestle. Groundtissues were then transferred to new 1.5 ml tubes
using clean spatulas, and CTAB buffer wasadded. DNA was extracted using the modiﬁed
CTAB extraction protocol (Doyle and Doyle,1987) followed by glass milk puriﬁcation methods as summarized by Hosaka (2009) andHosaka and Castellano (2008). Brieﬂy, groundsamples were 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 6M
sodium iodine buffer with glass milk, washedwith ethanol/buffer solution, and ﬁnally eluted in
100 μl of TE buffer.
Assessment of DNA fragmentation using the
Bioanalyzer
The sizes of DNA were determined by microcapillary electrophoresis using the Agilent Bioanalyzer (Agilent Technology, Palo Alto, CA,

PCR and sequencing
DNA sequence data were obtained from theinternal transcribed spacer regions (ITS) of thenuclear ribosomal DNA. For amplifying thewhole ITS region, the primer combination of
ITS5 and ITS4 (White et al., 1990) was used.
Because the ampliﬁcation of the whole ITS
region was difﬁcult for this sample, we havedesigned six new primers located internally
between ITS5 and ITS4 (Fig. 4, Table 1). As
mentioned above, the ITS sequence of the same
specimen has already been deposited in GenBank(accession number ＝ AB038764), which wasused as a reference sequence for primer design.
Primers were designed using the Primer 3 ver.
0.4.0. (http://primer3.sourceforge.net/) to amplify
approximately 200–400 bp products.
PCR reactions were carried out using 20 μl
reaction volumes each containing: 1 μl genomic
DNA, 1 μl dNTP, (4 mM), 1 μl of each primer(8 μM), 0.5 units of Taq polymerase (TaKaRa,
Tokyo, Japan), 2 μl MgCl2 (25 mM), 2 μl Bovine
Serum Albumin (BSA). Cycling parameters were
1 cycle of 94°C for 3 min, 30 cycles of 94°C for
1 min, 51°C for 30 s and 72°C for 1 min, with aﬁnal extension at 72°C for 15 min. PCR products
were electrophoresed in 1% agarose gels stainedwith ethidium bromide and visualized under UVlight. When ampliﬁcation bands were conﬁrmed,
PCR products were then puriﬁed using the ExoSap-IT (Millipore, Molsheim, France) anddirectly sequenced using the Big Dye Terminator
Cycle Sequencing Kit (Applied Biosystems Inc.,
Norwalk, CT, USA), following the manufacturerʼs instructions. The obtained sequences wereedited and assembled along with the reference
sequence from the type specimen (accession
number AB038764) using ATGC Ver. 7.0.2

56

Kentaro Hosaka and Kunihiko Uno(Genetyx Corporation, Tokyo, Japan).
Results
Type specimen used in this study
Visual inspection revealed that the specimen
had no severe insect damage, except the one
caused prior to collection (Fig. 2). No fungal
growth, which can make DNA extraction problematic, was observed, either (Fig. 2). Major
microscopic features such as basidiospores,
basidia, and hyphal structures were well preserved.
DNA extraction and initial PCR attempt
By using general primers for the ITS region(ITS5 and ITS4), no visible bands were obtained.
The results were the same after several attemptsof DNA extraction. We have puriﬁed the PCR
products for direct sequencing, but no informative sequences, including the ones from contaminants, were obtained.
Assessment of DNA fragmentation using the
Bioanalyzer
Electropherogram of four DNA samples fromthe holotype specimens (Fig. 3) revealed thatDNA from Amanita imazekii was fragmented,

Fig. 2. Type specimen of Amanita imazekii. The
scale at the bottom represents 15 cm.

but the quality (in terms of degrees of fragmentation) and quantity of its DNA was higher than theother three DNA samples, which were from thespecimens collected in earlier times (1954 to1986). The analysis using the Expert Software
2100 (Agilent Technologies, Palo Alto, CA) indicated that the peak of the DNA from Amanita
imazekii was located at 229 bp. On the other
hand, the other three DNA samples had peaks at
ca. 100 bp. The average size of DNA fragmentsfrom Amanita imazekii, when measured between
50 bp to 1000 bp, was ca. 363 bp.
PCR using newly designed primers andsequencing
Our attempt to amplify approximately 200–
400 bp products using the combination of existing (ITS5 and ITS4) and newly designed primers(Amaima-F1, F2, F3, R2, R3, R4) (Fig. 4, Table1) was successful. The ampliﬁed bands were typically bright and clear, and direct sequencing
after puriﬁcation was mostly successful. A totalof 8 contigs were assembled to reconstruct thewhole ITS region. The sequence recovered bythis approach had a 100% match with the reference sequence from the same specimen (acces-

Fig. 3. The result output of the Bioanalyzer assay.
Taxon names of each DNA samples are followed by the year of collection in parentheses.
The numbers of the X axis indicate the relative
length of DNA fragments, not the actual basepairs. The numbers of the Y axis indicate therelative quantity of DNA fragments. The sharp
peak at the left side is a size marker (35 bp).

Assessment of the DNA Quality in Mushroom Specimens

sion number＝AB038764).
Discussion
Despite the fact that macro- and microscopic
characters are well maintained (Fig. 2), no PCR
bands were obtained using general primers (ITS5
and ITS4) to amplify ca. 650 bp. This is probably
not due to insect and/or fungal infestation
because no bands or sequences from contaminants were obtained, and the PCR using newly
designed internal primers (Fig. 4, Table 1) successfully ampliﬁed the target regions. The longest PCR products we obtained were ca. 400 bp
and the ampliﬁcation of fragments shorter than
400 bp had a 100% success rate. This is consistent with the result of the Bioanalyzer assay (Fig.
3), which indicated that the most frequently present fragments were ca. 230 bp. The other three
type specimens we have tested all possessed
highly fragmented DNA because the peaks ofelectropherogram were located at ca. 100 bp. The
older ages and therefore more repeated dose of
fumigations must have contributed to this phenomenon.
The DNA of the holotype of Amanita imazekii,
however, was not free from fragmentation, either.
Although we do not have a detailed lab record,
the ampliﬁcation and sequencing of the whole57

ITS region was probably not a problem when
the reference sequence (accession number＝
AB038764) was submitted to GenBank in 2000.
This strongly indicates that fragmentation of
DNA occurred after the specimen was relocatedto the TNS herbarium.
Our herbarium (TNS) had been treated bymethyl bromide until relatively recently. The
chemical had been applied twice a year until
2003 or so. The mixture of methyl bromide andethylene oxide or other chemicals, such as
methyl iodide, can more severely degrade DNA
than methyl bromide alone (Kigawa et al., 2003),
but the moderate degradation observed in DNA
of Amanita imazekii indicates that a few years
doses of methyl bromide have apparently negative impacts. The TNS herbarium has beentreated by sulphuryl ﬂuoride (Vikane®) since2004, and we assume that DNA degradation by
fumigation after 2004 has little effect because
this chemical is claimed not to affect the DNA
molecules (Whitten et al., 1999; Kigawa et al.,2003).
We tested using only one DNA extraction protocol, and this may bias our results. A variety of
DNA extraction methods have been proposed(Bruns et al., 1990; Drábková et al., 2002;
Dentinger et al., 2010; Izumitsu et al., 2012), andsome methods may be more efﬁcient for extract-

Fig. 4. PCR primer map of the ITS region. The PCR primers designed speciﬁcally for this study are depicted asgray arrows.
Table 1.

Primers used in this study.

Primer5′-sequence-3′

SourceAmaima-F1
Amaima-F2
Amaima-F3
Amaima-R2
Amaima-R3
Amaima-R4
ITS5
ITS4

ATTGGAATGAAAACTCTGGTTGAATGTTATTGGCAAGGT
TTATTGCTGGCCTTTTTGGTTGACCTTGCCAATAACATTCAGCAATAATTCCCAATATCCGTCAATTTGTCAACACGGCTGGAAGTAAAAGTCGTAACAAGG
TCCTCCGCTTATTGATATGC

This study
This study
This study
This study
This study
This study
White et al. (1990)
White et al. (1990)

Kentaro Hosaka and Kunihiko Uno

Fig. 5. A portion of the ITS region reconstructed from contigs shorter than 400 bp.

58

ing highly degraded DNA because DNA fragments shorter than 200 bp do not efﬁciently bindto silica particles in glass milk. Nevertheless, afailure of PCR ampliﬁcation of 600 bp products
and the results of the Bioanalyzer assay (Fig. 3)
are consistent with our hypothesis that DNA
from four type specimens we tested are alldegraded.
A failure of PCR ampliﬁcation can also be
caused by interstrand crosslinks by alkylation orintermolecular crosslinks by Maillard reaction(Willerslev and Cooper, 2005). Crosslinks are
not the direct cause of DNA degradation, butsuch crosslinks may decrease the efﬁciency of
DNA extraction, or cause fragmentation of DNA
during the extraction procedure. Oxidative andhydrolytic modiﬁcation of nucleotides, especially
CG → TA transitions, may also cause the PCR
failure (Willerslev and Cooper, 2005), but our
results of contig assembly (Fig. 5) indicate such
substitutions did not occur at the ITS region.
The whole ITS region of Amanita imazekii
was successfully recovered because the degree ofdegradation was not as severe and the reference
sequence was available. This is probably not thecase for many other type specimens, which werecollected in earlier times and therefore possess
highly degraded DNA. To recover long fragments of DNA from such specimens, we have todeal with even shorter fragments by using“primer walking” (Fraser and Fleischmann,
1997) or a next-generation sequencing approach.
A problem of DNA degradation in historicallyimportant specimens is common to the herbaria
around the world. For example, PCR ampliﬁcation of some specimens stored in Chinese herbaria from the late 1980ʼs was not possible(Hosaka et al., 2010). Because DNA in specimens degrades with time (Hummel and Herrmann, 1994; Erkens et al., 2008), we strongly
suggest that DNA from historically important
specimens should be extracted as soon as possible, or small pieces of specimens should be
stored under more ideal conditions for future
DNA studies. One way of preventing further degradation of DNA is a cryopreservation in liquidAssessment of the DNA Quality in Mushroom Specimens

nitrogen or ultra-deep freezer at −80°C (Nagy,2010). If such facilities are not available, use of
several organic solvents, such as acetone(Fukatsu, 1999), or DNA preservation buffers(Rogstad, 1992; Laulier et al., 1995; Dawson etal., 1998; Johnson et al., 2003; Nagy, 2010)
should be considered.
Acknowledgments
The authors thank Kazuo Nishibori and
Megumi Ohtsuka for assisting our curatorial
work. Thanks also go to Kyung-ok Nam forassisting our molecular work. This research was
ﬁnanced in part by the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid forYoung Scientists A (24680085), and the Center
for Molecular Biodiversity Research at the
National Museum of Nature and Science to KH.
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