Phytochemistry xxx (2015) xxxxxx

Contents lists available at ScienceDirect

Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Genome analysis of medicinal Ganoderma spp. with plant-pathogenic

and saprotrophic life-styles
Ursula Kes b,1, David R. Nelson c,1, Chang Liu a,,1, Guo-Jun Yu d,e,1, Jianhui Zhang a, Jianqin Li a,
Xin-Cun Wang a, Hui Sun d,e
a

Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, 151 Malianwa North Road, Haidian District, Beijing 100193, China
University of Gttingen, Bsgen-Institute, Department for Molecular Wood Biotechnology and Technical Mycology, Bsgenweg 2, D-37077 Gttingen, Germany
Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, 858 Madison Ave., Memphis, TN 38163, USA
d
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
e
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education), Wuhan University, Wuhan, China
b
c

a r t i c l e

i n f o

Article history:
Available online xxxx
Keywords:
Ganoderma
Whole genome sequencing
Mitochondrial genome
Transcriptome
CYP450
matA
matB

a b s t r a c t
Ganoderma is a fungal genus belonging to the Ganodermataceae family and Polyporales order. Plant-pathogenic species in this genus can cause severe diseases (stem, butt, and root rot) in economically important trees and perennial crops, especially in tropical countries. Ganoderma species are white rot fungi and
have ecological importance in the breakdown of woody plants for nutrient mobilization. They possess
effective machineries of lignocellulose-decomposing enzymes useful for bioenergy production and bioremediation. In addition, the genus contains many important species that produce pharmacologically active
compounds used in health food and medicine. With the rapid adoption of next-generation DNA sequencing technologies, whole genome sequencing and systematic transcriptome analyses become affordable
approaches to identify an organisms genes. In the last few years, numerous projects have been initiated
to identify the genetic contents of several Ganoderma species, particularly in different strains of Ganoderma lucidum. In November 2013, eleven whole genome sequencing projects for Ganoderma species were
registered in international databases, three of which were already completed with genomes being assembled to high quality. In addition to the nuclear genome, two mitochondrial genomes for Ganoderma species have also been reported. Complementing genome analysis, four transcriptome studies on various
developmental stages of Ganoderma species have been performed. Information obtained from these studies has laid the foundation for the identication of genes involved in biological pathways that are critical
for understanding the biology of Ganoderma, such as the mechanism of pathogenesis, the biosynthesis of
active components, life cycle and cellular development, etc. With abundant genetic information becoming available, a few centralized resources have been established to disseminate the knowledge and integrate relevant data to support comparative genomic analyses of Ganoderma species. The current review
carries out a detailed comparison of the nuclear genomes, mitochondrial genomes and transcriptomes
from several Ganoderma species. Genes involved in biosynthetic pathways such as CYP450 genes and
in cellular development such as matA and matB genes are characterized and compared in detail, as examples to demonstrate the usefulness of comparative genomic analyses for the identication of critical
genes. Resources needed for future data integration and exploitation are also discussed.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
1.1. Importance of Ganoderma
Ganoderma P. Karst. is an important fungal genus consisting of a
morphologically diverse assemblage of mushroom taxa, all of
Corresponding author.
1

E-mail address: cliu6688@yahoo.com (C. Liu).

Contributed equally to this work.

which are characterized by unique double-wall basidiospores with

ornamented endospores. Although a mere 80 species are recognized by the Dictionary of Fungi (Kirk et al., 2011), there are 427
name records in Index Fungorum (http://www.indexfungorum.
org/). Species of Ganoderma have attracted world-wide attention
from three aspects: as therapeutic fungal bio-factories (Paterson,
2006), as plant pathogens (Hushiarian et al., 2013) and as
bio-bags of ligninolytic enzymes (Zhou et al., 2013). It is evident
from the literature that the interests in particular aspects are frequently associated with particular geographic areas and climates.

http://dx.doi.org/10.1016/j.phytochem.2014.11.019
0031-9422/ 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kes, U., et al. Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2014.11.019

U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

For example, species of Ganoderma (represented by G. lucidum

(Curtis) P. Karst. and G. sinense J.D. Zhao, L.W. Hsu & X.Q. Zhang)
are used as highly valued traditional medicines in East Asian countries including China, Japan and Korea. (A paper on an updated
nomenclature of these species is presented in this Special Issue
(in this issue) and also refer to the Editorial (in this issue).)
Scientic reports produced from these areas therefore mostly focus
on the active chemical components and therapeutic effects of
Ganoderma. In contrast, most reports on Ganoderma as plant pathogens come from South Asia including India and Pakistan, from
Indonesia and Malaysia in Southeast Asia, and North America but
there are also notable incidences in Africa. The reports regarding
the potential use for bio-energy production are mostly associated
with the US and East Asian areas. One possible reason is that
Ganoderma is a genetically highly diverse group of fungi and
Ganoderma spp. from different geographical areas might be genetically distinct and possess unique properties (Flood et al., 2000).
As therapeutic fungal biofactories, Ganoderma spp., represented
by G. lucidum and G. sinense, were found to produce a wealth of bioactive polysaccharides, oligosaccharides, triterpenoids, peptides
and proteins alcohols and phenols. Fruiting bodies and mycelium
are furthermore worthy sources of terpenoids, mineral elements,
vitamins and selections of amino acids. Health enhancing properties of Ganoderma spp. have variously been reported in prevention
and treatment of objectionable and ill-fated conditions listed as
hepatopathy, chronic hepatitis, nephritis, hypertension, hyperlipidemia, arthritis, neurasthenia, insomnia, bronchitis, asthma, gastric ulcers, atherosclerosis, leucopenia, diabetes and anorexia
(Rai et al., 2005; Xu et al., 2011). However, the anti-cancer and
immuno-modulation properties of G. lucidum are the most studied
effects, in particular the cytotoxic and apoptotic actions of b-glucans and ganoderic acids as a class of terpenoid compounds specic to Ganoderma mushrooms (Boh, 2013; Jin et al., 2012; Xu
et al., 2012, 2011). The chemical components, biological activities
and mechanisms of action are topics of intensive research and
interested readers may consult several recently published excellent reviews (Habijanic et al., 2013; Lee et al., 2012; Popovic
et al., 2013; Soares et al., 2013; Wu et al., 2013).
As plant pathogens, Ganoderma spp. have been observed to
grow on various dead or dying trees and shrubs in the Northern
Territories of Australia, including ornamental palms (such as the
golden cane palm and Carpentaria acuminata) and broadleaved
trees (amongst others Brachychiton populneus, Delonix regia, and
Casuarina equisetifolia), crop perennials (e.g. Citrus species, guava,
custard apple), and several other woody species valued for their
products (wood, seeds, gum, fragrances, bioactive compounds,
etc.) and their ecological importance (e.g. Ficus and Acacia species,
white cedar), (Hennessy and Daly, 2007). In the USA, there are
many different species of Ganoderma, of which G. applanatum (Pers.
ex Wallr.) Pat. is reported to kill aspen in the western states (US
Forest Service, 2011) and G. zonatum Murrill is considered to be a
pathogen of mature palms growing in southern regions (Elliott
and Broschat, 2000). In southeastern countries, such as Malaysia
and Indonesia, G. boninense Pat. is a major pathogen on oil palms
that eventually kills the trees. The economic loss caused by this
pathogen is estimated to be up to 500 million USD a year
(Hushiarian et al., 2013). In Mexico, G. oerstedii (Fr.) Murrill was
identied to be a tree parasite species (Mendoza et al., 2011). A
detailed evaluation of records on Ganoderma disease of perennial
crops in India concludes that members of the G. lucidum and G.
applanatum species complexes are widespread pathogens in the
subcontinent (Sankaran et al., 2005). Likewise, attacks of G. applanatum (Pers. ex Wallr.) Pat. and G. lucidum have been documented
for Pakistan on species of Pinus, Dalbergia, Artocarpus, Morus,
Cedrus, Melia, Quercus, Populus and other trees (Nasir, 2005).
Incidences of Ganoderma spp. infestations of oil palms causing

basal stem rots are also known from many tropical African countries (Miller et al., 2000). Stem, butt and root rot diseases are typical results from Ganoderma attacks (Nasir, 2005; Elliott and
Broschat, 2000). Loss of foliage of the stressed trees and die-back
of individual branches can be symptoms in stages of infections
prior to tree death (Hennessy and Daly, 2007; Paterson, 2007;
Hushiarian et al., 2013). Main routes of infections may be through
roots in the soil by vegetative spread (Irianto et al., 2006;
Hushiarian et al., 2013) but entry by spores through wounds is also
considered (Paterson, 2007; Rees et al., 2012). To manage the disease, different techniques have been attempted with varying success, such as soil mounding, surgery and removal of diseased
material, isolation trenching, ploughing and harrowing, fallowing,
chemical treatment, application of fertilizers, biological control
and selection of resistant planting materials (Hushiarian et al.,
2013). Unfortunately, measures to cure the disease or to at least
effectively stop its spread have not been found. Current disease
management relies on preventive measures reducing the frequency
of devastating infections (Paterson, 2007; Hushiarian et al., 2013).
The molecular basis of Ganoderma spp. being plant pathogens is
their ability, as white-rot fungi, to degrade lignocellulose (Paterson,
2007). In nature, Ganoderma spp. as saprotrophs have their important ecological position in nutrient mobilization from dead wood by
enzymatic decomposition (Clinton et al., 2009). The same ability
can also be employed to applications where degradation of lignocellulose is favorable, such as bio-energy production, treatment of
wastewater and bioremediation (da Coelho-Moreira et al., 2013).
The ability of Ganoderma species to degrade lignocellulose has
gained signicant attention starting from the 1980s. Several studies
have shown that Ganoderma spp. have strong abilities to enzymatically degrade lignocellulose (Adaskaveg et al., 1990; Maeda et al.,
2001; Silveira Carneiro et al., 2009; Martnez et al., 2011; Son
et al., 2010; de Andrade et al., 2012). The enzymes that participate
in lignin degradation include lignin peroxidase (LiP), manganesedependent peroxidase (MnP), laccase (Lac) and oxidases producing
hydrogen peroxide, such as glyoxal oxidase (GLOX) and aryl-alcohol
oxidases. A large number of studies have been conducted to identify
enzymes and their genes that are potentially involved in lignin degradation from Ganoderma spp. (Zhou et al., 2013). Screening for
Ganoderma spp. with stronger LME (lignin-modifying enzyme)
activities, good stability and ability to endure extreme conditions
are active areas of research.
1.2. Benets of whole genome sequencing projects
The research studies in the above mentioned areas have run
into several problems. Foremost is the confusion over the identity
of Ganoderma spp. For example, the Ganoderma spp. used as
traditional medicines are called lingzhi in China; Munnertake,
Sachitake and Reishi in Japan, and Youngzhi in Korea. It has been
difcult to determine whether or not these represent the
same species. Actually, the Chinese pharmacopeia (Chinese
Pharmacopoeia Commission, 2010 Edition) specically explains
that lingzhi refers to both G. lucidum and G. sinensis. Without
an unambiguous determination of the species identity, comparison
of chemical components and therapeutic effects obtained from various studies would be difcult. Similarly, a major problem in controlling Ganoderma caused disease is confusion over the identity of
the species causing the disease and the lack of effective tools for
early disease detection under eld conditions (Flood et al., 2000;
Paterson, 2007; Hushiarian et al., 2013; Naher et al., 2013). While
several markers such as the intergenic spacer regions (ITS) for the
ribosomal genes have been used for DNA barcoding analysis (Smith
and Sivasithamparam, 2000; Moncalvo and Buchanan, 2008), they
are not always suitable for the determination of Ganoderma spp.
(Glen et al., 2009). The inter-specic and intra-specic variations

Please cite this article in press as: Kes, U., et al. Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2014.11.019

U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

of Ganoderma are largely unknown (Pilotti et al., 2003; Pilotti,

2005; Rakib et al., 2014), thus a particular marker might not have
enough distinguishing power to discriminate closely related
species. Furthermore, it is unknown whether or not such molecular
markers are associated with particular host ranges (Nusaibah et al.,
2011) or, for example, with particular biochemical properties of
interest.
The second difculty lies in the overall lack of information
regarding the genes responsible for secondary metabolite production, ligninolytic activities and pathogenesis. Without the knowledge of the complete genetic makeup of Ganoderma spp., large
scale, brute-force screening or breeding of Ganoderma spp. or
strains with preferred characteristics would be tedious and
cost-ineffective. A metabolic engineering approach would not be
applicable for the production of active compounds and ligninolytic
enzymes. Without a gene catalog, specic drugs cannot easily be
developed to target control of the spread of pathogenic Ganoderma
spp.
There are several benets that whole genome sequencing projects can bring, such as (species specic) marker identication,
gene discovery and pathway elucidation. First and foremost, the
entire genome would provide ultimate information for the
selection of markers capable for species identication. Second,
whole genome sequencing would be the most-cost effective
method for the discovery of critical genes and the elucidation of
biosynthetic pathways for chemical compounds. Although,
approaches like transcriptome proling can also achieve the goal
of gene discovery, it might not be able to detect genes expressed
at lower levels and might not provide full-length information of
genes. Furthermore, it does not provide the information regarding
location of genes on chromosomes. Many chemical compounds in
fungi are synthesized by genes clustered together on the chromosomes. As a result, co-localization of genes would be an effective
method to discover genes involved in the biosynthesis of particular
chemical compounds (Andersen et al., 2013) and to develop strategies for the eventual concerted manipulation of their expression
(Brakhage and Schroeckh, 2011). Third, comparative analysis of
the genomes from multiple species can help elucidate the evolutionary history of critical genes (Floudas et al., 2012), such as for
the ligninolytic enzymes LiP, MnP, Lac, etc. This information can
provide guidance in screening of new species for favorable characteristics or for developing better strategies to combat pathogens.
With rapid development of DNA sequencing technologies, whole
genome sequencing of Ganoderma spp. became affordable and a
dozen research projects are already on the way. This review
intends to survey the recent progress in this area, the preliminary
results from which would usher the research and application of
Ganoderma spp. into a new era.

2. Sequencing of Ganoderma lucidum nuclear genomes

By November 2013, eleven whole genome sequencing projects
for Ganoderma species have been registered in the BioProject section of the National Center of Biotechnology Information (NCBI),
which collects biological data related to initiatives provided by
institutions or consortiums (http://www.ncbi.nlm.nih.gov/bioproject/). The details of each project and the organization involved
are shown in Table 1. Among these projects, four were conducted
by research groups from institutes in China, which include The
Institute of Medicinal Plant Development (IMPLAD), Huazhong
University of Science and Technology (HUST), and The Institute
of Edible Fungi (IEF). Two projects were conducted by the research
groups of The National Yang-Ming University (NYMU) from Taiwan. The ve remaining projects were conducted by research
groups from institutes in the United States, which include (a) The
Broad Institute of Massachusetts Institute of Technology (MIT)
and Harvard, and (b) The Joint Genome Institute (JGI). Interestingly, ve of these projects focus on the genome of Ganoderma lucidum, demonstrating the high level of interest on this
representative species for the Ganoderma genus. So far, three
assembled genomes have been published, two from strains of G.
lucidum and one from a strain of an unidentied Ganoderma species (Binder et al., 2013; Chen et al., 2012; Liu et al., 2012). One
whole genome shotgun project with unassembled sequences of a
third G. lucidum strain (GenBank BACH00000000) has also been
submitted to NCBI (Huang et al., 2013). The rest of the projects
are still in progress.
The genome assembly from IMPLAD was obtained from G. lucidum strain G.260125-1 using next-generation sequencing and
optical mapping technologies. The assembly is 43.3 Mb long and
encodes 16,113 predicted genes. Due to the better assembly of this
genome compared to those of others (see below), the richest sets of
wood degradation enzymes among all of the sequenced Basidiomycetes were identied from this assembly. Furthermore, 24 physical CYP gene clusters have been identied, which are potentially
involved in the biosynthetic pathways of various biologically active
compounds. The IMPLAD researchers claimed that the genome of
G. lucidum should be investigated as a potential model system for
secondary metabolic pathways and regulation in medicinal fungi
(Chen et al., 2012).
The HNAU assembly is from G. lucidum strain Xiangnong No. 1
using Illumina sequencing technology alone. It has a 39.9 Mb long
draft genome, which encodes 12,080 protein-coding genes.
Approximately 83% of these genes are similar to sequences found
in international databases. The rest were novel. The HNAU group
annotated their G. lucidum assembly and compared the annotated
gene models with those of other fungal genomes. The genes

Table 1
List of whole genome sequencing projects in NCBIs BioProject section.
Accession

Strain

PRJNA182009
PRJNA182007
PRJNA182006
PRJNA182005
PRJDA61381
PRJDA61379
PRJNA77007
PRJNA71455
PRJNA42807
PRJNA42873
PRJNA68313

Ganoderma
Ganoderma
Ganoderma
Ganoderma
Ganoderma
Ganoderma
Ganoderma
Ganoderma
Ganoderma
Ganoderma
Ganoderma

tornatum strain NPG1

zonatum strain POR69
miniatocinctum strain 337035
boninense strain PER71
lucidum strain BCRC 37177
lucidum strain BCRC 37180
lucidum strain Xiangnong No. 1
lucidum strain G.260125-1
sinense strain gasi0214-1
lucidum strain Hu-nongke No. 1
sp. strain 10597 SS1

Status

Organization*

References

In progress
In progress
In progress
In progress
Whole genome shotgun sequences available
In progress
High quality genome assembly available
High quality genome assembly available
In progress
In progress
High quality genome assembly available

BROAD
BROAD
BROAD
BROAD
GLRC/NYMU
GLRC/NYMU
HNAU/HUST
IMPLAD
IMPLAD
IEF
JGI

NA
NA
NA
NA
Huang et al. (2013)
NA
Liu et al. (2012)
Chen et al. (2012)
NA
NA
Binder et al. (2013)

*
BROAD: BROAD Institute of Massachusetts Institute of Technology (MIT) and Harvard, Boston, USA; IMPLAD: Institute of Medicinal Plant Development, Chinese Academy
of Medical Science, Beijing, China; IEF: Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, China; JGI: DOE Joint Genome Institute, Walnut Creek,
California USA; GLRC/NYMU: Ganoderma lucidum Research Consortium, National Yang-Ming University, Taiwan; HuNan Agriculture University (HNAU); HUST: HuaZhong
University of Science and Technology, Wuhan, China; NA: Not available.

Please cite this article in press as: Kes, U., et al. Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2014.11.019

U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

involved in the biosynthesis of the main biologically active components of G. lucidum, specically ganoderic acids, were characterized
in detail. Particularly, the HNAU group identied a fusion gene that
was only found in Basidiomycetes. Consistent with G. lucidum
being a white rot fungus with wood degradation ability, abundant
carbohydrate-active enzymes and ligninolytic enzymes were identied in the HNAU assembly (Liu et al., 2012).
The genome assembly of JGI is obtained from Ganoderma sp.
strain 19597 SS1 using a combination of ABI3730 (fosmids), 454Titanium and Illumina GAII sequencing platforms. The assembly
is 39.52 Mb long and encodes 12,910 predicted genes. The mixed
sequencing reads were assembled using a JGI specic assembly
process and Newbler (2.3-PreRelease-6/30/2009, Roche) with
default parameters. The draft assembly was then annotated using
the JGI annotation pipeline, which takes multiple inputs (scaffolds,
ESTs, known genes) and runs several analytical tools for gene prediction and annotation. Single-copy genes were identied and
compared with those from other Polyporales. Three phylogenomic
datasets, which contain 25, 71 and 356 genes respectively were
constructed to evaluate their potential for phylogenetic systematics of the Polyporales (Binder et al., 2013).
The overall comparison of the three genome assemblies is listed
in Table 2. The statistical characteristics of the three assemblies
were extracted from the corresponding papers. The sizes of the
three genome assemblies are similar, ranging from 39.5 Mb for
the HNAU assembly to 43.3 Mb for the IMPLAD assembly. The IMPLAD assembly is probably of the highest quality, in which 82 scaffolds were mapped to 13 chromosomes thanks to a physical map
constructed with the optical mapping technology. The HNAU
assembly was obtained using a typical shotgun approach and contains 634 scaffolds with a total length of 39.9 Mb. Similar to the
IMPLAD project, the JGI project used both 454 and Illumina data
to produce an assembly, which contains 156 scaffolds. The total
GC content of the genome and protein-coding genes were comparable for the three genome assemblies. The numbers of predicted
genes were also similar. 16,113, 12,080 and 12,910 gene models
were found in the IMPLAD, HNAU and JGI assemblies, respectively.
The average gene length, number of exons per gene, average exon

size, average coding sequence size, and average intron size for gene
models predicted from the three assemblies showed some level of
variation. These variations might result from different methods of
genome assembly and gene prediction used in these projects and
might not reect actual differences in the gene structures in these
genomes. Validation of these predicted models are needed to
explain the observed variations.
We compared the three genome assemblies in more detail by
plotting the number of scaffolds along with their total accumulated
length (Fig. 1a). For the HNAU assembly (dashed line), the accumulated total length increased rather slowly, suggesting that the
length of the scaffolds was similar. By contrast, the accumulated

Table 2
Characteristics of three sequenced Ganoderma genomes.
Strain name

G. lucidum
strain
G.260125-1
(Chen et al.,
2012)

G. lucidum
strain
Xiangnong
No. 1 (Liu
et al., 2012)

Ganoderma
sp. 10597
SS1 (Binder
et al., 2013)

Research group
Number of chromosomes
Length of genome assembly
(Mb)
Scaffold total number
Scaffold maximum length
(bp)
Scaffold minimum length
(bp)
GC content (%)
Number of protein-coding
genes
Average gene length (bp)
GC content of proteincoding genes (%)
Average number of exons
per gene
Average exon size (bp)
Average coding sequence
size (bp)
Average intron size (bp)
Repeat sequences (%)

IMPLAD
13
43.3

HNAU
NA
39.9

JGI
NA
39.52

82
4,834,011

634
1,953,398

156
4,606,855

2064

1004

2002

55.9
16,113

55.6
12,080

55.59
12,910

1556
59.3

1959
58.9

1541
59.0

4.7

6.3

13.8

268
1188

230
1435

148
1413

87
8.15

100
5.07

62
2.53

Fig. 1. Comparisons of the three different genome assemblies of G. lucidum strains

G.260125-1 and Xiangnong No. 1 and Ganoderma sp. strain 10597 SS1. (A)
Accumulated total assembly length vs. numbers of scaffolds; (B) dot plot comparing
the genome structures between all IMPLAD and JGI scaffolds having lengths greater
than 1 Mb.

Please cite this article in press as: Kes, U., et al. Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2014.11.019

U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

length of the IMPLAD assembly and the JGI assembly increased

remarkably; this result suggested the presence of a few very long
scaffolds along with many shorter ones. For example, the ve longest scaffolds of the IMPLAD assembly were 4,834,011, 2,306,357,
1,960,581, 1,936,600, and 1,698,588 base pairs (bp) long,
respectively. The ve longest scaffolds of the JGI assembly were
4,606,855, 3,722,621, 3,223,173, 2,972,551, and 2,861,455 bp long.
However, the JGI scaffolds were not ordered and mapped to chromosomes because of the lack of a corresponding physical map.
Since the IMPLAD and JGI assemblies appear to be of higher quality,
these two assemblies were compared further using a dot plot. All
scaffolds with a length >1 Mb from the IMPLAD and JGI assemblies
were extracted to construct the dot plot (Fig. 1b). The North-American Ganoderma sp. strain 19597 SS1 sequenced by JGI belongs to a
Ganoderma species complex (Binder et al., 2013) and consequently
it might be a species different from that sequenced by IMPLAD.
Although more work needs to be done to accurately determine
the species identity from which the materials used for sequencing
were derived, this comparison of genome assemblies suggests that,
if there are different species background, the genomes of various
Ganoderma species may share a high degree of overall similarity.
Comparing the largest scaffolds from the IMPLAD and JGI assemblies showed that the similarities between high scoring pairs ranged from 77.8% to 100%, with an average of 89.2%.
In summary, signicant variations were identied among the
reported genome assemblies. These might result from several reasons. First, the species identity of the materials used for genome
sequencing has not been determined or agreed upon. As a result,
it is unclear whether or not the materials used are of the same origin or not. Second, it suggests that the level of completion of a genome assembly affects the results signicantly. The shotgun
sequencing strategy alone is insufcient to obtain high-quality
assemblies even with high coverage produced by next generation
DNA sequencing technologies. A physical map constructed using
the optical mapping technology is invaluable to establish a chromosomal-level assembly. In spite of these variations, these assemblies allow us to appreciate the diversity and complexity of
Ganodermas genetic makeup.

3. Sequencing of Ganoderma mitochondrial genomes

The Ganoderma genus comprises approximately 80 species with
similar morphological characteristics. The lack of powerful molecular markers to determine these species has caused widespread
mislabeling and misuse of Ganoderma products, which threaten
their safe usage (Wang et al., 2012). The mitochondrion, containing
DNA referred to as the second genome, is an organelle found in
many eukaryotic cells (Henze and Martin, 2003), and its DNA
evolves faster than the corresponding nuclear DNA. Consequently,
it is more suitable to differentiate closely related organisms than
the nuclear genome (Bullerwell and Lang, 2005; Burger et al.,
2003; Ghikas et al., 2010). Hence, the analysis of mitochondrial
DNA (mtDNA) is useful for evolution and population studies on
Ganoderma species.
By mining the sequence reads generated by the Illumina platform, Li et al. (2013) successfully assembled the mitochondrial
genome (mtDNA) for G. lucidum strain G.260125-1. This genome
is a typical circular DNA molecule of 60,630 bp with a GC content
of 26.67% (EMBL Accession Number: HF570115). By contrast, the
mitochondrial genome for G. sinense has also been assembled
(GenBank Accession Number: KF673550). The total length of this
genome is 86,451 bp with a GC content of 26.8%. The alignment
of these two complete genomes is shown in Fig. 2a. A total of 15
pairs of highly similar fragments were present. Almost all of these
fragments represent the conserved regions encoding proteins, as

well as small and large ribosomal RNAs and tRNAs. The orders of
the conserved genes in the two genomes are similar.
Annotation of G. lucidum mtDNA identied genes that encode
15 conserved proteins (atp6, atp8, atp9, cob, cox1, cox2, cox3,
nad1, nad2, nad3, nad4, nad4L, nad5, nad6, and rps3), small and
large rRNAs (rns and rnl), 27 tRNAs, four homing endonucleases
(ip1, ip2, ip3, and ip4), and two hypothetical proteins (orf1 and
orf2; Fig. 2b). All genes except those encoding trnW and two hypothetical proteins are located on the positive strand. Annotation of
G. sinense mtDNA identied the same 15 conserved proteins, as
well as small and large rRNAs. In contrast to G. lucidum mtDNA,
28 tRNA genes were detected. No genes for homing endonucleases
were reported in G. sinense mtDNA. However, 33 hypothetical proteins were identied. Minor differences were observed in the species of tRNAs encoded in the two genomes. For example, trnU-UCA
is present in G. lucidum only, whereas trnE-UUC occurs in G. sinense
only. Both genomes contain three copies of trnM-CAU. G. lucidum
contains two trnR genes (trnR-UCG and trn-UCU), with each gene
containing only one copy. By contrast, G. sinense contains ve trnR
genes, including three copies of trnR-UCG and two copies of trnRUCU (Li et al., 2013).
In-depth studies have been performed for G. lucidum mtDNA,
including gene expression levels across different developmental
stages, the possible presence of long non-coding RNAs (lncRNAs),
potential transfer of DNA fragments between the mtDNA and the
nuclear genome, and genome rearrangement comparing to those
from other fungal species (Li et al., 2013). Unfortunately, no other
analyses other than the complete sequences and general annotations have been reported for G. sinense mtDNA. With the progression of the various whole genome sequencing projects (Table 1),
more mitochondrial genomes will be available and comprehensive
comparative analyses can be performed.

4. Transcriptome analysis of Ganoderma lucidum

To date, several transcriptome studies have been conducted for
various strains of G. lucidum (Table 3), although none have performed a detailed molecular species identication of the materials.
These studies differ in terms of the technologies employed and the
samples used. The samples were collected at various developmental stages, including mycelium, primordium, and fruiting body, at
various points in time. Therefore, the results cannot be easily
integrated.
One of the earliest studies was conducted in 2010, in which RNA
was extracted from 50-day-old fruiting bodies and then used to
construct a cDNA library for Sanger sequencing (Luo et al., 2010).
A total of 879 high-quality ESTs were obtained and subsequently
assembled to 600 unique sequences (Table 1). These were then
compared with those in the international nucleotide standard
database, such as Nr and Nt, to identify their possible functions.
A total of 173 unique sequences showed similarities to known
genes involved in the biosynthesis of secondary metabolites and
developmental regulation (p < 1e10). Two unique sequences
showed high similarity to genes encoding squalene epoxidase
(SE) and farnesyl pyrophosphate synthase (FPS), respectively,
which are two rate-limiting enzymes in the catalysis of triterpenoid biosynthesis in G. lucidum (Shi et al., 2010). Furthermore, a
total of 13 simple sequence repeat (SSR) motifs were identied
from these EST sequences and the candidate genes involved in
the regulation of G. lucidum development were also studied. This
data set exhibits only a limited coverage of the transcriptomic contents of G. lucidum due to the low throughput sequencing technologies used.
In the second study, next generation DNA sequencing technology was used instead of Sanger technology (Chu et al., 2012). The

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Fig. 2. Comparison of mitochondrial genomes from G. lucidum strain G.260125-1 (EMBL: HF570115) and G. sinense (GenBank: KF673550). (A) Dot plot analysis of the two
genomes. (B) Venn diagram showing the genes shared by the two genomes.

study used 16 day-old mycelia and 9 week-old fruiting bodies of G.

lucidum strain yw-1. In total, 6,439,690 and 6,416,670 high-quality
90 bp long reads were obtained from the mycelium and fruiting
body, respectively, and these reads were assembled into 18,892
unigenes for the mycelia and 27,408 unigenes for the fruiting
bodies (Table 1). The mean size of the mycelium unigenes was
498 bp and the fruiting body unigenes mean size was 514 bp, with
lengths ranging from 100 bp to over 3000 bp. Unigenes from each
sample were assembled together by the sequence clustering software TGICL, resulting in a total of 28,210 unigenes. A similarity
search was performed against the NCBI non-redundant nucleotide

database and a customized database composed of the established

genomes of ve other Basidiomycetes (Laccaria bicolor, Coprinopsis
cinerea, Schizophyllum commune, Phanerochaete chrysosporium, and
Postia placenta). A total of 11,098 and 8775 unigenes were found
similar to entries in these databases, respectively. All unigenes
were subjected to annotation by the Gene Ontology (GO), the
Eukaryotic Orthologous Group terms (KOG), and the Kyoto
Encyclopedia of Genes and Genomes (KEGG).
The unigenes were examined in detail to identify the specic
genes involved in the biosynthetic pathway for ganoderic acids,
which represent the major bioactive compounds in the medicinal

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U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

Table 3
Comparisons of the G. lucidum transcriptome contents described in four studies.

Study No./references
(Year)

Material/strain(s)

Outline

Mycelium

Primordium

Fruiting
body

Total

1. Luo et al. (2010)

50 days old fruiting bodies of a

non-specied G. lucidum strain

Numbers of ESTs
Total number of unique genes
Average unique gene length (bp)

879
600
288

879
600
288

2. Yu et al. (2012)

16-day-old mycelium and 9-week-old

fruiting body of G. lucidum strain yw-1

Number of reads
Average read length (bp)
Number of unigenes
Average unigene length (bp)
Number of genes

6,439,690
90
18,892
498
13,332

13,055

6,416,670
90
27,408
514
13,144

12,856,360
90
28,210
507
13,731

3. Huang et al. (2013)

Mono- and dikaryotic G. lucidum strains

BCRC 36123 and BCRC 37180

ESTs
ESTs
ESTs
ESTs
ESTs

1001
6,848
21,547
11059
6830

47,285

4. Ren et al. (2013)

MeJA-induced mycelium of
G. lucidum strain HG

Number of transcript-derived
fragments

3910

3910

from
from
from
from
from

5 days dikaryon
14 days dikaryon
18 days dikaryon
18 days monokaryon
30 days dikaryon

indicates that no data is available.

mushroom G. lucidum (Shi et al., 2010). A total of 13 identied

expressed unigenes were involved in the terpenoid backbone biosynthetic pathway. Among these unigenes, 7 were up-regulated in
the mycelium and six did not show signicant expression changes
in the two developmental stages (Table 4). The products of these
unigenes show high similarity to hydroxymethylglutaryl-CoA synthase (78%), mevalonate kinase (55%), phosphomevalonate kinase
(52%), mevalonate disphosphate decarboxylase (63%), farnesyl
pyrophosphate synthase (FPPS, 66%), geranylgeranyl pyrophosphate synthetase (66%), squalene synthase (SQS, 54%), squalene
monooxygenase (62%), and lanosterol synthase (LS, 61%), respectively. The expression levels of these unigenes were conrmed by
quantitative PCR (qPCR). Five genes involved in G. lucidum development were also identied: ATP synthase F1; citrate synthase;
EXG1; hydrophobin 2; and a cytochrome P450. EXG1
(AAO32563.1) is a glycosyl hydrolase. The transcriptome data
and qPCR results showed that ATP synthase F1, citrate synthase
and EXG1 might play potential roles in carbohydrate transport
and metabolism. By contrast, the cytochrome P450 was highly
expressed in the fruiting bodies. The expression of the hydrophobin gene was not conrmed by qPCR (Chu et al., 2012).
A third transcriptome proling project with the mono- and
dikaryotic G. lucidum strains BCRC 36123 and BCRC 37180 was performed to study the development of the mycelia in the rst 30 days
(Huang et al., 2013). Five mycelium samples (5 days dikaryon,
14 days dikaryon, 18 days dikaryon, 30 days dikaryon, and 18 days
monokaryon) were collected for the analyses. A total of 47,285
ESTs were obtained from these ve mycelial samples and their
lengths ranged from 50 bases to 949 bases (Table 3). Almost all
of the ESTs (47,283 out of 47,285) can be mapped to the genome
sequence of G. lucidum (Yu et al., 2012). Approximately 83% of
the EST-genome alignments (38,395) contained gaps that were
presumably introns of the multi-exon genes. Most (approximately
99.9%, 38,347/38,395) putative introns were <2 kb. ESTs aligned to
the reference genome were further clustered and merged to generate 7774 non-redundant EST genes. Alternative splice forms in G.
lucidum were also analyzed in this study. The results showed that
262 of 7774 EST genes may have alternative splice forms, including
50 and/or 30 alternative splice junctions, cassette exons, and
retained introns.
Genes that were differentially expressed after methyl jasmonate (MeJA) treatment were studied in the dikaryotic G. lucidum
strain HG to identify genes involved in the regulation of ganoderic
acid production (Table 3) (Ren et al., 2013). In that study,
cDNA-amplied fragment length polymorphism (cDNA-AFLP)

was performed to identify genes differentially expressed in

response to MeJA in G. lucidum, which can regulate production of
GA. A total of 64 primer combinations were used to identify more
than 3910 transcriptionally derived fragments (TDFs). Reliable
sequence data were obtained for 390 of 458 selected TDFs. A total
of 90 TDFs were annotated with known functions by performing a
BLASTX search of the GenBank database. Of these, 12 annotated
TDFs were assigned to secondary metabolic pathways by searching
the KEGG PATHWAY database. 25 TDFs were also selected for qRTPCR analysis to conrm the expression patterns observed with
cDNA-AFLP. The qRT-PCR results were consistent with the altered
patterns of gene expression as revealed by the cDNA-AFLP
technique. Furthermore, the transcript levels of 10 genes were
determined at the mycelium, primordium, and fruiting body developmental stages of G. lucidum. The greatest expression levels were
reached during the primordial stage for all genes except the cytochrome b2 gene, which reached the highest expression level in the
mycelium stage.
Since ganoderic acids are the major active components of G.
lucidum, enzymes related to their biosynthesis are of great interest.
Sequences found similar to those enzymes involved in the biosynthetic pathway (MVA pathway) for ganoderic acids are shown in
Table 4. In terms of their expression proles, Chen et al. (2012)
found that the genes involved in the MVA pathway were downregulated from the mycelia to the fruiting bodies (Table 4) (Yu
et al., 2012). By contrast, most genes were expressed at a higher
extent in the fruiting body stage than in the mycelial stage (Chu
et al., 2012). Huang et al. (2013) observed that the highest expression of the genes involved in the MVA pathway are observed at
18 d in the dikaryon. In addition to genes involved in the MVA
pathway, other genes likely to be involved in the biosynthesis of
ganoderic acids have also been identied. For example, 919 transcript-derived fragments exhibited altered expression patterns
after MeJA induction (Ren et al., 2013). However, since MeJA may
function as a global regulator, not all of the differentially expressed
genes after treatment are related to differential biosynthesis. Further studies should be conducted to identify factors specically
regulating the biosynthesis of ganoderic acids in G. lucidum and
other Ganoderma species. The JGI study likely used a species other
than G. lucidum; as such, their transcriptomic data were not compared in the present study.
There are two challenges facing G. lucidum transcriptome analyses. First, the materials used in various studies were usually
inconsistent. Materials from various strains were collected for
the studies at different time points in cellular growth and

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Table 4
Expression of genes involved in the biosynthetic pathway of ganoderic acids.
Gene description (product)

Chen et al. (2012)

Yu et al. (2012)

Gene_ID

Log2(M/
F)

Gene_ID

GL23502
GL26574
GL24922

1.09
0.90
1.40

3-Hydroxy-3-methyl glutaryl-CoA
reductase
Mevalonate kinase

GL24088

0.77

Unigene9161_All
Unigene27186_All
Unigene27186_All
Unigene6833_All
Unigene6227_All
Unigene2279_All

0.8
2.3
2.3
2.3
0.3
0.0

GL17879

0.58

Phosphomevalonate kinase
Mevalonate pyrophosphate
decarboxylase
Isopentenyl diphosphate isomerase
Farnesyl diphosphate synthase

GL17808
GL25304

0.05
0.69

Unigene25033_All
Unigene14395_All
Unigene6822_All
Unigene6187_All

2.1
1.2
0.5
0.6

GL29704
GL22068
GL25499
GL21690
GL23376

0.71
1.66
1.73
0.98
4.78

GL18675

GL18675

Acetyl-CoA acetyltransferase
3-Hydroxy-3-methyl glutaryl-CoA
synthase

Squalene synthase
Squalene monooxygenase
2,3-Oxidosqualenelanosterol cyclase
Geranylgeranyl pyrophosphate
synthetase
Lanosterol synthase (LS)

Huang et al. (2013)

Log2(M/
F)

Unigene9721_All
Unigene803_All

1.6
0.3
0.3
2.2
1.8

0.74

Unigene111_All
Unigene2082_All
Unigene5606_All

Unigene688_All

0.6

0.74

Unigene4718_All

1.5

Gene_ID

M_5th
daya

M_14th
daya

M_18th
daya

M_30th
daya

YMGLESTG49648

33

12

YMGLESTG52067

68

12

YMGLESTG53675

16

YMGLESTG52428

YMGLESTG50562
YMGLESTG50973

0
0

0
1

2
5

0
1

YMGLESTG47454
YMGLESTG55240
YMGLESTG52116
YMGLESTG51888

0
0
0
0

4
0
0
2

3
8
11
3

6
2
8
4

More than one gene may be found in a particular gene description.

M, mycelium; P, primordium; F, fruiting body.
: No gene was reported in the particular study.
a
The number in columns of M_5th day, M_14th day, M_18th day, and M_30th day from Huang et al. (2013) study represent the EST counts for each corresponding gene.

development, or from different cultural media. A previous study on

the production of ganoderic acids has already shown that the
above factors affected gene expression levels and bioactive compound contents (Liang et al., 2010). As a result, it is uncertain
how many of the differences observed in the aforementioned studies actually resulted from genetic diversity rather than the environmental factors. Second, the variations caused by the
technologies should also be considered. In these studies, Sanger
sequencing and next-generation sequencing technologies, such as
454 and Illumina, were employed. These technologies differ in
throughput, sequence length, and error rate, among others
(Shendure and Ji, 2008). Furthermore, added additional possible
variations to the data, make it more difcult for: (a) identication
of the complete transcriptomes of Ganoderma species and (b) comparison of the expression levels of various genes during cellular
development and production of secondary metabolites.
The use of diverse samples from different strains of the same
species at various developmental stages under different cultural
conditions is thus not helpful to address particular biological questions. Future transcriptome studies should consider the following
points from the very beginning. For example, studies should theoretically use materials from one particular, completely sequenced
strain, which has been cultured under standardized conditions,
such as medium, temperature, and length of growth, among others
(Berovic et al., 2003, 2012; Habijanic et al., 2013; Wang et al., 2014;
Wen et al., 2010). The sequencing strategy and bioinformatic data
processing steps should also be standardized. For example, the
complete genome sequences should be used as the reference to
assemble the transcriptome whenever possible.

5. Genes belonging to the cytochrome P450 family

The number of cytochrome P450 genes in fungi is highly variable. The obligate intracellular parasites Encephalitozoon cuniculi
and Nosema apis (microsporidia) have no P450s. The taphrinomycetes Schizosaccharomyces pombe, Schizosaccharomyces japonicus,

and Schizosaccharomyces octosporus each contain CYP51 and

CYP61 for ergosterol biosynthesis but no other P450s (Weete
et al., 2010). The taphrinomycetes are the earliest diverging Ascomycetes, so it is assumed that their ancestors had only these two
P450s. This is probably true for the ancestor of Dikarya and for
the Basidiomycetes descended from that common ancestor. From
here on, it is assumed that all other Basidiomycete P450s are additions to this P450 baseline.
Cytochrome P450 analysis was performed on two high-quality
nished Ganoderma genomes: G. lucidum strain G.260125-1
(Chen et al., 2012) and the Ganoderma sp. strain 10597 SS1
sequenced at JGI (Binder et al., 2013; Grigoriev et al., 2012). P450
genes are abundant in these genomes. G. lucidum has 197 P450
genes and 19 pseudogenes. Ganoderma sp. strain 10597 SS1 has
181 P450 genes and 29 pseudogenes (Syed et al., 2013). These
are among the largest fungal CYPomes, though they are smaller
than some plant CYPomes. The Ganoderma CYPs are distributed
in 42 CYP families and 14 CYP clans. Clans are clades of genes that
share a common ancestor and are the deepest reliable clusters of
P450s on phylogenetic trees. The clan system of fungi is still currently being dened, but it seems that there are approximately
25 P450 clans for fungi as a whole (from more than 5000 named
P450 sequences). Early recognized fungal clans in P. chrysosporium
(CYP51, CYP61, CYP52, CYP64, and CYP505) have been discussed
previously (Yadav et al., 2006).
A total of 377 P450 sequences from the two Ganoderma CYPomes are included in the two phylogenetic trees presented in
Fig. 3 and Fig. 4, respectively. Pseudogenes, short sequences, or
otherwise problematic sequences were omitted. Fig. 3 shows 12
of the 14 CYP clans including 194 sequences. The CYP547 clan is
the largest with 119 sequences in eight families. The CYP54 clan
has only one family, while the CYP512 family has 41 sequences.
Surprisingly, 10 of the 12 clans shown have only 1 CYP family in
Ganoderma. Orthologs are adjacent redblue pairs. The alternation
of red and blue around the tree indicates there are no species-specic gene blooms. Many of the genes have orthologs, with a few
having small expansions or contractions compared to the other

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Fig. 3. Neighbor-joining tree of 194 Ganoderma P450s from 12 CYP clans. Clans are labeled on the spokes and each clan is shown in a separate color. G. lucidum strain
G.260125-1 labels are red and Ganoderma sp. strain 10597 SS1 labels are blue. Sequence alignments were computed using CLUSTAL W and checked manually for consistent
alignment of known CYP motifs. Neighbor-joining trees were generated with the Phylip package (Ropelewski et al., 2010) using ProtDist (a program in Phylip) to compute
difference matrices. Trees were drawn and colored with FigTree ver. 1.3.1 using midpoint rooting (http://tree.bio.ed.ac.uk/software/gtree/) and labeled in Adobe Illustrator
CS ver. 11.0.0 (Adobe Systems Incorporated). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

species. The largest expansion is only 4:1 from the CYP512U subfamily in the CYP54 clan.
The remaining 183 P450 sequences in the large CYP53 and
CYP64 clans are compared in Fig. 4. Similarly, there are many
ortholog pairs and little evidence of gene blooms. In this gure,
the individual families are colored differently since there are only
2 clans in this tree. The CYP5359 family occupies half of the tree.
The CYP5035 family is also quite large. This shows the dramatic
expansion of these two families (and CYP512, 5136, 5139, and
5150 in Fig. 3). The family expansion happened before speciation
as evidenced by the large number of ortholog pairs between the
2 Ganoderma species analyzed. The large increase in intact (presumably functional) P450s in these families implies some evolutionary advantage gained by these oxidative enzymes. Some may
be involved in secondary metabolite synthesis. Others may be
involved in the oxidative degradation of the wood components,
such as lignin, once these degradation products are imported into
the fungus after pre-digestion outside the cells with peroxidases
and laccases. This seems plausible since 1063 transporters were
found in G. lucidum strain G.260125-1 (Chen et al., 2012). The fruiting bodies of these bracket fungi are hard and have cell structures
that should maintain their growth patterns. Lignin evolved in tracheophyte land plants to provide rigidity for water transporting
vessels. The woody context of bracket fungi would necessarily be
created by an independently evolved polymer analogous to lignin

in plants. Some of the Ganoderma P450s may contribute to the formation of a rigid fungal superstructure.
Chen et al. (2012) pointed out that G. lucidum strain G.260125-1
has 24 gene clusters with three or more CYP genes (suppl. Data 6 of
their paper). Some of these clusters are quite large. Three clusters
are over 200 kb in size and have more than 70 genes, larger than
many known secondary metabolite clusters. Therefore, these may
need to be broken down into 2 or more smaller clusters. The automated gene cluster nding software Antismash identied 17 gene
clusters (suppl. Data 9 of their paper), but only P450 cluster 10
overlapped with these predictions, including one of the 5 polyketide synthase (PKS) genes found in the genome. A second gene
cluster predictor, SMURF, found 5 gene clusters, but only one overlapped P450 cluster 23 (suppl. Data 10 of their paper). Comparison
of the 2 software predictors favors Antismash, since it found clusters that included all 5 PKS genes, one NRPS gene, and 11 of the 12
terpene synthase genes. SMURF found the NRPS and 2 of the PKS
genes in its clusters, but none of the terpene synthases. The
requirement for at least 3 P450s to dene a cluster may be too
stringent. There are characterized secondary metabolite gene clusters that have fewer than 3 P450s. A more successful approach may
be to look for P450s within 50 kb of a cluster anchor gene like the
NRPS, PKS, terpene synthase, and dimethylallyl tryptophan synthase (DMAT) genes. The product of DMAT is dimethylallyl tryptophan, which is the precursor of indole alkaloids in Ganoderma.

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10

U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

Fig. 4. Neighbor-joining tree of 183 Ganoderma P450s from the CYP53 and CYP64 clans. Each P450 family is shown in a separate color. G. lucidum strain G.260125-1 labels are
red and Ganoderma sp. strain 10597 SS1 labels are blue. The tree was computed and drawn in the same way as Fig. 3. (For interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.)

The P450 families in the 2 analyzed Ganoderma species and 2

other polypores P. placenta and P. chrysosporium are shown sorted
into their clans in Suppl. Table A. This set of 4 genomes has 15
clans, except for Ganoderma, which is missing CYP5153. Two of
the clans are CYP51 and CYP61 in the ergosterol pathway. These
clans frequently cluster together and may form a larger clan. The
CYP53 clan has 5 families in Ganoderma, 3 of which (CYP53,
CYP537, CYP5140) have only 1 sequence each. Singletons like these
are probably dedicated to 1 specic function. In Aspergillus niger,
CYP53A1 is encoded by the benzoate-para-hydroxylase gene
(bphA) (Faber et al., 2001). Phenolics like benzoate are toxic to
fungi and CYP53 acts as an early step in the b-ketoadipate detoxication pathway (Podobnik et al., 2008). The enzyme is highly conserved and may be a new antifungal drug target (Berne et al.,
2012). The functions of CYP537 and CYP5140 are not known.
The CYP52 clan primarily includes the CYP63 family in the
Ganoderma species. This is an expanded family for both Ganoderma
strains under comparison in this present review. CYP63 is also
moderately sized in P. chrysosporium and P. placenta. The CYP63
genes of P. chrysosporium have been studied for induction by lignin
components, with the verication that some were induced, suggesting these genes may encode lignin-degrading P450s
(Subramanian and Yadav, 2008).
The CYP54 clan has only 1 family in Ganoderma, which is
CYP512, with 41 members (Fig. 3). The CYP512A1 gene was cloned
from Trametes (Coriolus) versicolor, a lignin-degrading Basidiomycete. This gene is inducible by dibenzothiophene, and was the rst

chemical stress-induced P450 gene reported in Basidiomycetes

(Ichinose et al., 2002). The substrates for the CYP512 family have
been investigated recently (Chigu et al., 2010; da Coelho-Moreira
et al., 2013; Hirosue et al., 2011). The CYP512 members were found
to metabolize plant resins used as defense barriers to pathogens.
The CYP512 family was also expanded in wood degrading Basidiomycetes, suggesting a role in colonization of wood. Additional discussion on the roles of CYP512 family can be found elsewhere
(Syed et al., 2014).
The CYP505 clan has 7 sequences (Fig. 3), 3 ortholog pairs
CYP505D10, CYP505D12 and CYP505D13. CYP505D11 clusters
with CYP505D12 that may represent a co-ortholog. These P450s
are self-sufcient because they contain a reductase fused to the
P450 part of the sequence. The rst CYP505A1 or P450foxy was
cloned from Fusarium oxysporum (Nakayama et al., 1996), which
hydroxylates fatty acids, and forms omega-1 and omega-3 products. There are 93 named CYP505 fungal sequences obtained from
many species. Two other P450s in Ganoderma, CYP6005A1 and
CYP6005B1, are also fusion proteins and belong to the CYP6001
clan. This clan has 4 families: CYP6001, CYP6002, CYP6004, and
CYP6005, among which Ganoderma only has CYP6005. CYP6003
is in another clan by itself. These proteins are also called Psi-factor
producing oxygenases PpoA (CYP6001A1), PpoB (CYP6002A1), and
PpoC (CYP6001C1). CYP6001A1 (PpoA) from Emericella (Aspergillus)
nidulans is fused with a diooxygenase. The rst part adds 2 atoms
of oxygen, then the P450 (C-terminus) part acts like a hydroperoxide isomerase. They act in the synthesis of oxylipins (Koch et al.,

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U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

11

Fig. 5. The matA locus and its gene products. (a) The gene structure of the chromosomal regions containing the matA locus of four different Ganoderma strains. The following
genes are shown: HD1 gene a1 and HD2 gene a2 (allele numbers are indicated by the second number in the names), mip1 that encodes a mitochondrial intermediate
peptidase, b-fg that encodes an unknown conserved fungal protein and glgen that encodes a glycosyltransferase family 8 protein. The genes are indicated by the arrow-shaped
boxes (black: conserved genes; hatched: mating type genes with little conserved allele sequences; grey shaded: genes from retro-transposons; white: potential
(pseudo)genes originating possibly from transposons or retro-transposons). The arrows indicate their transcriptional direction. Numbers above the maps indicate positions in
kb on the corresponding scaffolds and contigs of the strains. The ends of the two contigs of G. lucidum strain G.260125-1 (at 0 kb and 87.4 kb, respectively) are positioned
relative to each other in their likely natural order. (b and c) Sequence alignment of HD1 and HD2 transcription factors identied from the four Ganoderma strains. N-terminal
discrimination and dimerization domains (dashed lines), stretches of K/R-rich motifs (lled boxes) as parts of potential bipartite NLSs (nuclear localization signals;
underlined), HD1- and HD2-specic peptides (open boxes) and the localization of the 60 aa-long HD2 homeodomain (underlined) with its three helices I to III (lled boxes)
and the conserved DNA-binding motif WFXNXR (WFQNRR in all Ganoderma HD2 proteins) are indicated.

2013). E. nidulans has all three. In Aspergillus fumigatus PpoA is a

linoleate 5,8-diol synthase (Jerneren and Oliw, 2012). The P450
part of these enzymes does not need molecular oxygen, since it
is supplied by the substrate of the fusion protein. This leaves the
I-helix region (traditionally the oxygen binding motif in P450s)
free to diverge, so these CYP6001 clan sequences are typically deep
branches on P450 trees. This makes them similar to the oxylipin
producing CYP74 P450s of plants and some marine animals. Some
oxylipins are developmentally signicant (Tsitsigiannis et al.,
2005). The CYP6005A and CYP6005B sequences may be making
signaling molecules in Ganoderma that are related to the products

of the Psi factor producing oxygenases. Those molecules have been

shown to be involved in fungal colonization of plants (Koch et al.,
2012).
The CYP7 clan is an evolutionary curiosity since it includes
vertebrate CYP7 and CYP8 families (not shown in Fig. 3). It is
improbable that the common ancestor of opisthokonta (fungi and
animals) had a CYP7 clan sequence. The 11 CYP clans in animals,
including CYP7, all appear to have evolved from CYP51 through
tandem duplication at 1 original locus (Nelson et al., 2013). An
alternative explanation is that they are the results of lateral transfer from animals. The functions of CYP7 clan sequences and other

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single family Ganoderma clans CYP534, CYP608, and CYP609 are

not known.
The CYP547 clan has 113 sequences out of the 194 CYP450
genes identied in G. lucidum (Fig. 3). The expansion, especially
in the CYP5150 family, suggests a function in the metabolism of
multiple related compounds. The multiplicity of closely related
sequences does not t easily with biosynthetic pathways that tend
to be relatively short linear chains of enzymes. However, it may not
be true that all members of a family work on closely related substrates. Experience from insecticide resistance shows that new
functions can be obtained from almost any P450 family (Sezutsu
et al., 2013). Such a large collection of P450s will almost certainly
have some novel functions that have developed based on opportunity or need. Similar remarks apply to CYP5359 in the CYP64 clan
and CYP5035 in the CYP53 clan (Fig. 4). One issue to consider is if 1
clan is disproportionately represented in secondary metabolite
gene clusters. A casual examination of several fungal toxins (aatoxin, trichothecene, sirodesmin, gliotoxin, and some others) for
P450s found 21 P450s in 16 families. A total of 10 were in the
CYP53 clan, 3 were in the CYP613 clan, and 2 of each were in the
CYP54 and CYP64 clans. The rest were in 4 different clans. There
may be favoritism for the CYP53 clan of P450s for use in fungal secondary metabolite biosynthesis. This has relevance for Ganoderma
as a source of medicinal compounds, as many of these will be made
in fungal gene clusters.

6. Mating type loci in Ganoderma lucidum

G. lucidum is a tetrapolar species (Adaskaveg and Gilbertson,
1986) which means that 2 distinct mating type loci (matA and
matB) control development of a dikaryotic mycelium after fusion
of 2 compatible monokaryons, as well as fruiting and sexual reproduction via formation of basidiospores on the established dikaryon.
For monokaryons to be compatible, it is essential that they differ in
alleles at both loci. Molecular analysis of the Agaricomycetes
model species, C. cinerea and S. commune, revealed that the A mating type locus in Agaricomycetes encodes 2 types of homeodomain
transcription factors (HD1 and HD2) in one or more divergently
transcribed HD1HD2 gene pairs (alphabetically named a pair, b
pair, etc.), and the B mating type locus pheromone precursors
and G-protein-coupled 7-transmembrane-domain pheromone
receptors (Ste3-type receptors). Alleles of the 2 loci can differ much
in sequence, up to a level at which one cannot easily recognize
their direct relationship. The resulting different amino acid
sequences of allelic protein products determine their distinct mating type specicities. In compatible matings, HD1 proteins from
one strain have to interact with HD2 proteins of the other strain
to form active heterodimeric transcription factor complexes for
the regulation of the expression of dikaryon-specic genes. Likewise, pheromones from one mating partner have to interact with
the pheromone receptors of the other mating partner to induce
the pheromone-response pathway required for dikaryon development and maintenance. Importantly in this system, all monokaryons can possess all types of genes. However, the HD1 and HD2
products encoded in the same allele of the A locus do not form
functional transcription factor heterodimers, and the pheromones
and pheromone receptors encoded in the same allele of the B locus
do not switch on the pheromone response pathway (Kes et al.,
2011). Below, the identication of matA and matB loci from 4 Ganoderma genomes is described in detail.
Sequences from the 3 published genome assemblies (Binder
et al., 2013; Chen et al., 2012; Liu et al., 2012) and the published
whole genome shotgun project (GenBank BACH00000000)
(Huang et al., 2013) were analyzed together for mating type loci
(James et al., 2013; this review). Candidate genes from the

G. lucidum strains BCRC 37177, G.260125-1, and Xiangnong No. 1

were identied using the BLAST module at GenBank with the program TBLASTN and the databank Whole genome shotgun contigs
(wgs). By contrast, candidate genes from Ganoderma sp. strain
10597 SS1 were identied using the TBLASTN option on the genome portal of JGI (Grigoriev et al., 2012) and data presented by
James et al. (2013). Due to poor sequence conservation, mating
type genes are generally difcult to nd based on sequence similarity. As a result, dening conserved anking genes (such as genes
mip1 and b-fg found in most Agaricomycetes next to matA) is frequently used to identify their positions within the genome (Kes
et al., 2011; James et al., 2013).
6.1. matA locus
HD1 protein Pda1 (GenBank AAS46746) and HD2 protein Pda2
(AAS46747) of Pleurotus djamor, as well as Mip1 (EKV51093), and
b-fg (EKV51091) from Agaricus bisporus var. bisporus were used
as the query sequences in database searches for matA. In the genomes of all 4 Ganoderma strains, the alleles of the A locus are about
3.6 kb in length, although they differ signicantly in sequence. In
Ganoderma sp. strain 10597 SS1, the A genes reside on scaffold 1
at position 2,506,9082,503,303 (James et al., 2013), in strain Xiangnong No. 1, the genes reside on contig_850 (AHGX01000850) at
position 241,855238,254, in BCRC37177, the genes reside on
contig03243 (BACH01002782) at position 91,03194,647, and in
strain G.260125-1, they reside on scaffold GaKu96scf_1_ctg_9
(AGAX01000044) at position 38397483, respectively.
In all 4 strains, the matA region contains only a single pair of
divergently transcribed HD1 and HD2 genes, which were subsequently specied as a1 and a2 genes (Fig. 5a). As typical for Polyporales (James et al., 2013), the HD2 gene is anked by the conserved
mip1 gene in all 4 strains. In contrast, the conserved b-fg gene is
only found to be next to the HD1 gene in G. lucidum strains
BCRC 37177 and G.260125-1. It is found to have been translocated
to another place in G. lucidum strain Xiangnong No. 1
(AHGX01000411; contig_411 at position 33,96132,989) and
Ganoderma sp. strain 10597 SS1 (scaffold 8 at position 544,298
545,271). The sites of b-fg integration in the 2 strains are identical.
As seen in the genome assembly of Ganoderma sp. strain 10597
SS1, the gene has been moved from the locus near matA to scaffold
8, which carries the matB locus (Fig. 6a). Translocation of b-fg has
brought the conserved gene glgen in G. lucidum strain Xiangnong
No. 1 directly next to matA. In other strains, there are transposon
relicts in variable composition and order located in between glgen
and matA, respectively b-fg, indicating that the anking region of
matA is a place of frequent rearrangements (Fig. 5a).
The products of the HD1 and HD2 genes for the 4 strains are
very dissimilar in sequence (Fig. 5 b and c). Therefore, they should
represent 4 different mating type specicities (Kes et al., 2011)
and allele numbers 14 have been assigned to these different matA
alleles and their respective a1 (HD1) and a2 (HD2) genes (Fig. 5a),
starting with the already published matA allele of Ganoderma sp.
strain 10597 SS1 (James et al., 2013). Sequence alignments of the
4 HD1 and the 4 HD2 proteins are shown in Fig. 5b and c, respectively. HD1 proteins of Ganoderma are about 480 amino acids (aa)
long. Strikingly, a typical HD1 homeodomain for DNA binding,
which is the most conserved region in HD1 proteins, is not recognizable in the translated sequences of Ganoderma HD1 genes
(Fig. 5b). The HD1 genes were thus dened in the alleles of the
matA locus based on their position relative to that of the divergently transcribed HD2 genes (James et al., 2013) and based on
the low level conservation of the N-terminal parts of their products
compared to those from some other wood-decay fungi. Using the
BLASTP method and the NCBI database, the rst 120140 aa of
the Ganoderma HD1 proteins were aligned with the N-termini of

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Fig. 6. The matB locus and its gene products. (a) The gene structure of the chromosomal regions containing the matB locus of four different Ganoderma strains. The likely
expansion of matB is underlined. The following genes are shown: pheromone receptor genes (prexed with ste3; the rst number afterward indicates a specic gene, a letter a
or b afterward indicates duplications of a specic gene, the second number indicates an allele of a gene) and pheromone precursor genes (prexed with ph; the rst number
afterward indicates a specic gene, the second number indicates an allele of a gene, letters a and b alleles result in the same product). The genes are indicated by the arrowshaped boxes (black: genes with highly conserved allele sequences, likely non-mating type genes; grey shaded: genes with poorly conserved allele sequences and possible B
mating-type activity; white: pheromone receptor genes inactivated by stop codon mutations and/or deletions of parts of genes; note that the ste3.1 alleles in two of the
strains cannot produce a functional protein). The arrows indicate the transcriptional direction. Blocks of matB genes closer related to each other are indicated by black, grey
and hatched boxes, respectively. Numbers above the maps indicate positions in kb on the respective scaffolds and contigs of the strains. The four contigs of G. lucidum strain
BCR 37177 have been positioned using sequence alignments of the complete contigs, so that its genes are arranged in the most similar order as those of other strains. (b and c)
Phylogenetic trees of pheromone receptors, precursors of pheromones and pheromone-like peptides (see compilation in Table 5) from four different Ganoderma strains,
calculated by the neighbor-joining method (500 bootstrap repeats). In the analysis of pheromone receptors, the sequences of seven receptors encoded in the genome of C.
cinerea strain Okayama 7 (#130) (Kes et al., 2011) were included (shaded in grey) for recognition of possible mating type and non-mating type receptors. Pheromone
precursors whose mature pheromones and pheromone-like peptides likely have identical sequences are also shaded in grey. The groupings of proteins (indicated by brackets)
base on the locations of their genes in the genomes (within matB: Mating type; outside matB: Non-mating type) and on the sequences of the 2 or 3 aa that are directly linked
to the C-terminal CAAX motif of pheromone precursors, respectively.

several HD1 mating type proteins from Dichotomus and Heterobasidion (data not shown). As shown in other species, the N-termini of
both types of mating type proteins mediate individual mating type
specicity. The N-termini are crucial for discrimination of HD1 and
HD2 proteins encoded in the same matA allele and are needed as

interfaces for heterodimerization with suitable partner proteins

encoded in other matA alleles (Kes et al., 2011). In accordance,
the N-terminal sequences of the 4 Ganoderma HD1 proteins show
only 4254% amino acid identity and 6375% similarity to each
other (Fig. 5b). Furthermore to the N-terminal dimerization and

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specicity domains, some KR-rich protein stretches are shared

with several HD1 mating type proteins of other species (data not
shown). In other species, these regions localize in sequences C-terminal to the HD1 DNA-binding motif (pfam12737: Mating_C
superfamily), whereas in Ganoderma, they follow directly the Nterminal discrimination and dimerization domains (Fig. 5b). This
further suggests that the regions encoding the HD1 homeodomain
have been deleted from the Ganoderma a1 genes. Regardless of the
internal gene deletions, the a1 genes still contain an open reading
frame for a protein consisting of all the remaining N- and C-terminal regions known from HD1 proteins in other species. Nevertheless, even in the apparent absence of the HD1 homeodomain, the
Ganoderma a1 proteins might still perform their necessary function
as partners of HD2 proteins in formation of active heterodimeric
homeodomain transcription factor complexes. This idea is supported by data from C. cinerea, which showed that HD1 proteins
can be functional even without an operational homeodomain
(Asante-Owusu et al., 1996). However, the C-terminal K/R-rich
stretches in the HD1 proteins are essential as part of bipartite NLSs
(nuclear localization signals) to transport the HD1HD2 heterodimers into the nucleus (Spit et al., 1998). From sequence alignments
made with the Ganoderma HD1 proteins and those from other species, a highly conserved 11-aa-long peptide (GPRLHAVSDSF/Y)
linked to the K/R stretches (Fig. 5b) was newly observed that is
specic to numerous Agaricomycetes HD1 proteins and whose
molecular function remains to be elucidated.
The Ganoderma HD2 genes encode proteins that are approximately 550560 aa-long. The proteins have the typical highly conserved HD2 DNA binding motif (7080% identity, 8192%
similarity), which is a classical 60 aa-long homeodomain with 3
a-helices (Kes et al., 2011). The conserved DNA-binding motif
WFQNRR can be found in the third helix (Fig. 5c). The 148
153 aa-long N-termini are less conserved, exhibiting only 3443%
identity and 4859% similarity to each other. As indicated above,
this high degree of dissimilarity is important for their functions
as recognition and dimerization domains with the N-termini of
HD1 proteins (Kes et al., 2011). Since all 4 HD2 proteins differ signicantly from each other in the N-termini, each should represent
a distinct mating type allele specicity. The regions C-terminal to
the HD2 motifs are about 350 aa-long. They are the most variable
regions in the entire protein sequences and contain only a few
short stretches of sequences with higher similarity (Fig. 5b). Notable is a newly observed 20-aa-long conserved peptide (V/
SPXHAYPXP/TYPPL/SCXY/HX,P/TFP/S) of yet unknown function
that is found in the Ganoderma HD2 proteins (Fig. 5b) and also in
several other Agaricomycetes HD2 proteins. However, data from
protein truncation experiments showed in C. cinerea that larger
parts of the poorly conserved C-termini of HD2 proteins are dispensable for function (Kes et al., 1994) and the low level of similarity between C-termini of Ganoderma HD2 proteins might
suggest this also for species of this genus.
6.2. matB locus
To identify B locus genes, pheromone receptor Rcb3 of C. cinerea
(AAO17256) was used to search the 4 genome sequences. In total, 8
to 9 different pheromone receptor genes and also parts of former
pheromone receptor genes were detected to reside in the 4 strains
within a 60100 kb long sequence region. These genes are on scaffold 8 of the genome of Ganoderma sp. strain 10597 SS1, on contig_857 (AHGX01000857) of the genome of G. lucidum strain
Xiangnong No. 1, on contig00760 (BACH01000746), contig00761
(BACH01000747), and contig00762 (BACH01000748) of the genome of G. lucidum strain BCRC 37177, and on scaffold
GaLu96scf_14_ctg_1 (AGAX010000010) of the genome of G. lucidum strain G.260125-1 (for details on positions, see (Fig. 6a)).

These regions show restricted sequence similarities among the

genomes, supporting the notion that the matB genes reside here,
and that encoded genes in all 4 strains have different B mating type
specicities. Hence, numbers 14 were assigned to the matB alleles
from the different strains following the same order as that for the
matA alleles (Fig. 6a).
Consistent with B mating type function, 5 genes for pheromone
precursors per strain were also found in these regions and 3 additional genes were found 2030 kb apart (Fig. 6a). Moreover, there
are 2 other genes elsewhere in each genome, summing up to a total
of 10 pheromone precursor genes per strain (Table 5). The rst
pheromone precursor genes were spotted in low stringency
TBLASTN searches using pheromone precursor sequences of the
polypore P. placenta (Kes et al., 2011), which detected short conserved N-terminal MDAFF motifs combined with the essential
C-terminal CAAX (C = cysteine; A = aliphatic aa, X = any aa) motifs
(compare sequences in Table 5). Additional genes were then
obtained by cross-checking the TBLASTN search results with the
identied Ganoderma pheromone precursors. The marking of
genomic positions by PF08015 (pfam08015: fungal mating-type
pheromone) on the JGI portal helped additionally in some
instances for Ganoderma sp. strain 10597 SS1. In general however,
due to their short length and the high sequence variation of the
encoded proteins, pheromone precursor genes are difcult to nd
through the TBLASTN method alone, even under conditions of lowest stringency and with inactivated lters for low complexity
regions. All 6 frame-translations of this entire DNA region were
manually inspected to search for N-terminal MDAFF-like motifs
and C-terminal CAAX-motifs, which are indicative of pheromone
protein sequences. No new pheromone precursor genes were
found.
From other species, it is observed that matB pheromone receptor genes and matB pheromone precursor genes localize together.
Other genes for pheromone receptors and for pheromone-like peptides can be present in addition but apparently have no function in
mating. Such non-mating-type genes do not necessarily come
together in the genome (Kes et al., 2011). Some of pheromone
receptor genes in Ganoderma have neighboring pheromone precursor genes (Fig. 6a), hinting to B mating type function. Starting from
a pheromone receptor gene with a neighboring pheromone precursor gene, the receptor genes in Ganoderma sp. strain 10597 SS1
were named ste3.1ste3.8 and the linked pheromone precursor
genes ph1ph8. All were given the allele number 1 (Fig. 6a). Next,
a phylogenetic analysis was done with receptor sequences of all
4 Ganoderma strains and in addition the sequences for B-matingtype and non-mating-type receptors of C. cinerea (Fig. 6b), using
ClustalX and the neighbor-joining function in the program MEGA
(Tamura et al., 2011). In the C. cinerea matB locus, there are 3 paralogous groups of pheromone receptor and pheromone precursor
genes, all of which have mating type function but act independently from each other (Kes et al., 2011; Riquelme et al., 2005).
In the phylogenetic tree of pheromone receptors, the matingtype-active receptors of C. cinerea can be found in clusters with
Ganoderma receptors coming from genes that have neighboring
pheromone precursor genes (compare Fig. 6a and b). It is thus
likely that at least 3 paralogous groups of matB genes and products
exist in Ganoderma, similar to the Subgroups 13 seen in C. cinerea
and other Agaricales (Kes et al., 2011; Riquelme et al., 2005).
Other receptors in Ganoderma appear to have diverged from those
with putative mating type function (Fig. 6b) and might be nonmating type receptors similar to extra receptors found in other
Agaricomycetes (Kes et al., 2011). Analysis with the TMpred program at the ExPASy portal (Swiss Institute of Bioinformatics, Lausanne, Switzerland) predicted that all of the Ganoderma receptors
have strong 7-transmembrane helices as to be expected for functional receptors.

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15

Table 5
Sequences of pheromone precursors as deduced from the Ganoderma genomes.
Strain/position in genome

Namea

Sequence of pheromone precursor

Ganoderma sp. strain 10597 SS1 (JGI scaffold 8)

1,522,2241,522,346
Ph1-1
1,520,5571,520,388
Ph2-1
1,509,5861,509,425
Ph3-1
1,507,9281,508,116
Ph4-1
1,506,4151,506,549
Ph5-1
1,454,9251,455,062
Ph6-1
1,448,2451,448,126
Ph7-1a
1,443,9421,444,061
Ph8-1

MDEFENLFGSIETKWDAPMTLEVPVNADDPSHAGVYCVIA
MDAFSFTLDELLAPQHPPAADPLPSPSSSDDEPKGMPVDFEYINNNYSHSWCTVA
MDYFESLHASFLKSETSSAQSLDIVDISLPADIPVPVDEEHQPSTSHFWCVIV
MDEFATITVSLSEPTTSFSPSVPPLGLQLPAHERAAVLARLSSSLPVNEDNNDQYTAYCIII
MDSFFVISEPLYDSPGQAVELSDDLPNDSEHYGGGQTSFYCVIA
MDAFFVIASPIQADEPTSSSVEEIEEIFVDYENLNNSWHSGCVIA
MDAFFTISSPIPVDEPAVEEILIDSEGSSTSEHSGCIIA
MDAFFTIAPAVPVEEPAADIVFINCDSGTGDNHSGCIIA

Ganoderma sp. strain 10597 SS1 (JGI scaffold 16)

200,939201,082
Phl1-1
202,641202,781
Phl2-1

MDAFFVISAPIVASPESATSSSSELDETFVDQDSFGYVGWHAGCVVA
MDAFFHLAAPIPSQEPACSSSSEGTEVFVDEDTFGYVGVHSGCIIA

G. lucidum strain Xiangnong No.1 contig_857 (GenBank Accession No. AHGX01000857)

90,21790,035
Ph2-2
MDSFDTLSLPLVTLEDTHTLSSSSAFGETCAPSGVPSLLDPPVNQEVIDGNGTYSWCTIA
92,10992,231
Ph1-2
MDEFDIFDISLGISLQEPLSGDIPVNEDDPSRAGVYCIIA
100,547100,753
Ph4-2
MDEFSIITTLPSDHSETHLNPISPPYGPSRPFANISPHQRRALLAHIASSVTVDEDSNDQYQAYCIIA
103,674103,823
Ph5-2
MDIFEPIFIAPPIPKLPSPPEPSDIDESDVLADFEHSNPASPKFYCVIS
b
108,751108,554
Ph3-2
MDDFAFTVIAPPIPDAGVAVDVGDIVLIDEDRPWHFASACTIA
151,870151,733
Ph6-1
MDAFFVIASPIQADEPTSSSVEEIEEIFVDYENLNNSWHSGCVIA
158,563158,682
Ph7-1b
MDAFFTISSPIPVDEPAVEEILIDSEGSSTSEHSGCIIA
162,882162,763
Ph8-1
MDAFFTIAPAVPVEEPAADIVFINCDSGTGDNHSGCIIA
G. lucidum strain Xiangnong No. 1 contig_841 (GenBank Accession No. AHGX01000841)
14,30414,438
Phl1-2
MDAFFVISAPIVASPESATSSSSEYDETFVDQDSFGYVGWHAGCVVA
16,00316,143
Phl2-2
MDAFFHLAAPIPSPEPASSSSSEGTEVFLDEDTFGYVGVHSGCIIA
G. lucidum strain BCRC 37177 contig00761 (GenBank Accession No. BACH01000747)
40534175
Ph1-3
MDAFTTIVSIATTGETTAEITNDQREVTYGSSSYSWCTVA
297467
Ph2-3
MDDFTTFSAFLETEASLKDRPSVDGPPAIMSPTDGVPVDMEYITGNSSSYSWCVVA
G. lucidum strain BCRC 37177 contig00760 (GenBank Accession No. BACH01000746)
37,32337,117
Ph4-3
MDEFCIIASLSSEHSETHLNSIDSRYSPSRSFGGISSQERRALLAHIASSVAIDEDSNDQYQAYCVIA
33,99533,852
Ph5-3
MDTFELLFIAPPIPELPSSLESSDAAESDVLADYEHNNPASPKFYCVIS
30,68030,874b
Ph3-3
MDEFIFTVIAAPVPDTGATADVEEIVLIDEDRPWHFGMGCTIA
G. lucidum strain BCRC 37177 contig00759 (GenBank Accession No. BACH01000745)
22,40322,266
Ph6-2
MDAFFVIASPIQAEEPANSSVEEIEETFVDHDSLNNNWHSGCVIA
29,26029,379
Ph7-2
MDAFFTISSPVPVDEPAVEEILVGSDSSSTSEHSGCVIA
33,62633,507
Ph8-2
MDAFFTIAPPVPVDEPVADVEFINYDDGTSDNHSGCTIA
G. lucidum strain BCRC 37177 contig00934 (GenBank Accession No. BACH01000906)
26,50526,362
Phl1-3
MDAFFVISAPVIASPECAPSSFSEVDETFVDQETFGYVGWHAGCVVA
24,84124,698
Phl2-3
MDAFFYIADPIPSQEPAPSSSSPAAPETFFDHDTFGYIGVHSGCIVA
G. lucidum strain G.260125-1 GaLu96scf_14_ctg_1 (GenBank Accession No. AGAX01000010)
141,651141,529
Ph1-4
MDAFVTIVSIVTTGETTAEITNDQREVTYGSSSYSWCTIA
145,527145,357
Ph2-4
MDDFATFSAFLETEASLKDQPSVDGPPAIMPPTDGVPVDMEYITGNSSSYSWCVVA
163,198163,052
Ph5-4
MDEFFVLAEPIPVDQPSDTGSSDVPAIDYPSEFEQYGSGQNTFFCVVA
164,566164,396
Ph3-4
MDLFELTHDLFLEPEAEVAVSSSPLDTEDPSLVIDLPVPVDSEHQPSSSHFWCIVA
168,957169,169
Ph4-4
MDTFDITFAISDGSPQGAPSAALSPVSSPTGSPVAPVVMDAADTPDLDFEQVPADFDHRSSGFGAYCVIA
204,460204,579
Ph8-3
MDAFFTIAPPVPVDEPTADVEFINYDDGSGDNHSGCTIA
209,131209,012
Ph7-3
MDAFFTISSPVPVDEPAVEEILVSSDSSNSSEHSGCVIA
215,985216,122
Ph6-3
MDAFFVIASPIQTDEPASSSVEEIEEVFVDQDSLSHNYHSGCVIA
G. lucidum strain G.260125-1 GaLu96scf_22_ctg_3 (GenBank Accession No. AGAX01000061)
122,680122,826
Phl1-4
MDAFFVISAPLVTSSESAATSSSSEVDEPFVDQDSFGYVGWHAGCVIA
124,338124,484
Phl2-4
MDAFFYLAAPIPSQEPASEPSSPSENPEVFVDHDTFGYIGVHSGCIIA
a
Ph stands for pheromones encoded by genes within the chromosomal region containing the matB locus, Phl for pheromone-like peptides encoded by genes unlinked to
matB.
b
Intron between last sense codon and stop codon.

The alleles of the likely mating-type pheromone receptor genes

within the matB locus of the 4 strains and of likely non-mating type
pheromone receptor genes positioned in the anking chromosomal
regions (Fig. 6a) were dened by the clustering of receptors in the
phylogenetic tree (Fig. 6b), as well as protein length (size ranges
are 414436, 438477, 476494, 591601 aa) and general gene
structure. Among the strains, the genes do not simply line up in
the same order. Inversions and relocations of genes and group of
genes are observed. This is true for pheromone receptor genes, as
well as for pheromone precursor genes (Fig. 6a). Particularly many
rearrangements occurred directly within the closer conned
regions of matB (covering about 1930 kb; Fig. 6a), together with
apparent duplications of ste3.4 genes in strains BCRC 37177 and
G.260125-1, and ste3.2 genes in strain Xiangnong No. 1. Blocks of

closer related genes are recognized between the matB alleles of

the different strains, which include both pheromone precursor
and pheromone receptor genes. In summary, each combination of
2 distinct matB alleles shares one closer related block of genes with
each other, and differs stronger in a second block of genes. In such
a manner, discrete mating type specicities should be granted for
all 4 strains in all possible breeding combinations.
Table 5 summarizes the sequences of all pheromone precursors
identied from the 4 Ganoderma genomes, including those whose
genes are unlinked to matB. All sequences start with an MDXF
motif, contain an internal dipeptide of charged aa for N-terminal
pheromone processing (such as EH, ED, RE, DR, DD) and end with
an CAAX motif required for isoprenylation and carboxymethylation
of the conserved cysteine after C-terminal proteolytic processing

Please cite this article in press as: Kes, U., et al. Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2014.11.019

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U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

acids at these positions contribute to the interaction of pheromones with compatible pheromone receptors (Riquelme et al.,
2005; Szabo et al., 2002). Pheromones should not interact with
receptors encoded by the same matB allele (Kes et al., 2011;
Riquelme et al., 2005). Guesses could be made as to which of the
pheromones might interact with which foreign receptors. However, due to the variable positions of alleles of pheromone precursor and pheromone receptor genes within the alleles of the matB
locus, a clear answer of whether products of alleles of a specic
pheromone precursor gene will interact with only allelic products
of a dened receptor gene can only be given by experimental work.

7. Resources for mining Ganoderma genomes

Fig. 7. Screen shot showing the web resources available for Ganoderma. (a) JGIs
web portal (http://genome.JGI.doe.gov/Gansp1/Gansp1.home.html); permission to
use the screen shot of the web site has been granted by Dimitrios Floudas from
Clark University, Worcester, MA; (b) GaluDB (http://www.medfungi.org/galu).
Permission to use the screen shot of the web site has been granted by Chang Liu
from Institute of Medicinal Plant Development, Chinese Academy of Medical
Science, Beijing, China.

(Kes et al., 2011). Some pheromone precursor genes are highly

conserved between 2 strains (such as for Ph6-1 and Ph8-1;
Fig. 6a) and others encode identical precursor sequences from distinct DNA sequences (such as for Ph7-1a and Ph7-1b, Table 5). In
addition, a number of distinct precursors will result in identical
mature pheromones upon processing (marked in Fig. 6c). Mature
pheromones of Ganoderma should consist of 1013 aa. The phylogenetic tree of all pheromone precursors shown in Fig. 6b may not
support well the grouping by allele origin, because the underlying
ClustalX alignment based mainly on the relatively conserved short
N- and C-terminal sequences of the 3970 aa long pheromone precursors of overall highly variable sequences (see Table 5). However,
the clustering revealed a possibly meaningful grouping by the 2 or
3 aa that are directly neighbored to the CAAX motif (Fig. 6c). Amino

Although the INSD (International Nucleotide Sequence Database), such as NCBI and EMBL, are the most reliable sources of public information, databases for particular taxonomic groups are also
needed to help to provide rich information related to nonsequence information of the species. Furthermore, the complete
genome sequences of various Ganoderma species and strains, along
with the transcriptomes of various developmental stages from
Ganoderma species or strains, are becoming more available. There
is a need for a centralized resource that can be used to integrate
and compare various genome and transcriptome data sets. Several
efforts have been taken to meet this challenge.
Two web resources are available for Ganoderma. One has been
set up by JGI (Grigoriev et al., 2012) and is shown in Fig. 7a. The
JGI portal provides various data for the JGI sequencing projects. It
provides: (1) a genome browser, so that users can browse the
genes of the sequenced genome; (2) gene annotations based on
GO, KEGG, KOG, CLUSTERS, and SYNTENY; and (3) a download page
for the sequences of scaffolds, coding sequences, transcripts, proteins, EST clusters, and gene models. The portal is well designed,
user friendly, and aesthetically pleasing. The data are well-organized and can be easily searched and downloaded.
The second web resource is set up and maintained by IMPLAD
(Chen et al., 2012) and is shown in Fig. 7b. The web server is associated with the whole genome sequencing project led by IMPLAD.
Similar to the JGIs web portal, it provides a page allowing users to
download various types of data related to IMPLADs G. lucidum genome sequencing projects. Two categories of data are provided. One
is the primary experimental data, which includes the genome
assemblies, other genome-sequencing data, 454 transcriptomic
data, and RNA-Seq data. These can be downloaded from GenBank
under the project accession PRJNA71455. The other category is
the secondary analysis data, which includes gene models, gene
annotations, compound structures, and gene clusters. Tools can
be divided further into 4 sub-categories and support functions,
such as: (1) database searches based on sequence similarity; (2)
the retrieval of annotations and sequences of a list of genes, and
the IDs of genes around a particular gene of interest; (3) the curation of gene models; and (4) the identication of gene clusters and
the mapping of genes to chromosomes.
One of the limitations for the above web resources is that they
focused on particular sequencing projects. As a result, these web
resources lack integration with other sources of Ganoderma information. To provide an integrated information hub, we are currently
constructing another web resource Gano portal at IMPLAD (http://
www.medfungi.org/gano). The Gano portal aims to provide a centralized resource for these datasets, because of the rapid generation of genome and transcriptome data sets. The Gano portal
contains the following pages: (1) a Ganoderma diversity page,
which list the reported Ganoderma species and their related information; (2) a Gano identication page, which allows users to
upload a DNA barcode sequence for the Gano sample under their

Please cite this article in press as: Kes, U., et al. Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2014.11.019

U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

study. The sequences will then be compared to the backend database for species determination; (3) a Gano genome page, which
lists all the whole genome sequencing projects on Gano, their afliated publications, and if there are any, the afliated datasets; (4) a
Gano transcriptome page, which lists all Gano transcriptome projects, their afliated publications, and the afliated data sets; (5)
a gene search page, which provides users a BLAST searchable database that combines all coding sequences, transcripts, and protein
sequences from various whole genome sequencing and transcriptome projects; (6) a download page, which keeps a reference set
of genomes, coding sequences transcripts, and proteins for download; and (7) a page listing the Ganoderma type strains and organizations maintaining their culture collections. We hope that
through community effort, this portal could support the retrieval
and comparison of genomic and transcriptomic data related to
Ganoderma.
8. Discussion
8.1. Major discoveries
One of the key problems cited in numerous reports is the difculty in dening the species identity of the samples involved. Comparisons of the genome sequences of 2 of the G. lucidum strains and
transcript sequences identied from the 4 transcriptomic studies
have shown high degree of discrepancies. Although, these differences might be attributed to different developmental stages, different growth conditions and different sequencing technologies, the
presence of such large degree of discrepancies cast doubt on the
identical species origin of the samples claimed in these studies. Fortunately, the wealth of information provided by the nuclear and
mitochondrial genomes will provide a comparative resource for
the discovery of novel molecular markers for species determination.
Similar to all other genome sequencing projects, the most valuable output of these studies is the identication of genes that are
involved in various biological processes. For example, 24 gene clusters have been discovered in the genome of G. lucidum strain
G.260125-1, which are potentially responsible for the biosynthesis
of biologically active compounds. Particularly, the analyses of the
CYP450 genes and genes at the mating type loci can serve as good
examples for exploiting genomic information to discover genes
that are responsible for important biochemical and developmental
processes. Additional studies can be conducted to compare genes
that are involved in lignin degradation and plant pathogenesis,
which might result in the identication of genes that are responsible for these processes. Potential drug targets can also be identied
by comparing the genetic makeup of infective and non-infective
Ganoderma spp. This information on genes and pathways also lays
the foundation for marker-guided breeding, as well as using metabolic engineering technologies to overproduce active chemical
compounds or ligninolytic enzymes of interests.
8.2. Limitations
There are several limitations in these current studies, such as
unconrmed species identity, biased species sampling, limiting
sample size and less than ideal sequencing quality. First, most of
the studies described above focused on G. lucidum, as it is the
best-studied species of Ganoderma for its pharmacological activities. The research groups conducting these studies are mostly from
China, where G. lucidum is of particular economic importance.
However, the species identity in the G. lucidum complex lack consensus. The medicinal species has been assumed to be G. lucidum,
but evidence has emerged that this is a different species
(Moncalvo et al., 1995). Wang et al. (2009) divided Asian speci-

17

mens classied as G. lucidum into two clades; both were separated

from the European G. lucidum. One clade, composed of tropical collections, represented G. multipileum; while the identity of the other
clade was unknown. Later, Wang et al. (2012) recognized the
unknown clade as G. sichuanense. Another paper by Cao et al.
(2012) found that the holotype of G. sichuanense was not conspecic with the unknown clade and proposed the unknown clade
as a new species G. lingzhi, representing the widely cultivated species in China. Second, only 2 dened strains of G. lucidum have been
sequenced so far with genomes being assembled. Considering the
large degree of sequence variations observed between the 2 genomes, a larger sample size is needed to paint a complete picture
of the sequence variations at the genome level for G. lucidum,
which are critical for breeding. Lastly, the quality of the available
assemblies varies signicantly, as the scaffolds can be placed to
chromosomes in only one of the three completed assemblies. The
less than perfect assembly would hinder the discovery of gene
clusters as well as the understanding of the history of genome evolution, such as gene duplication, rearrangement, etc. (A paper on an
updated nomenclature of these species is presented in this Special
Issue (in this issue) and also refer to the Editorial (in this issue).)
8.3. Future directions
The availability of genome sequences have ushered in a new era
of research on Ganoderma spp. Future work can be divided into
efforts focused on (1) genome sequencing projects alone; (2) utilization of genome information for functional genome studies; and
(3) application of Ganoderma spp. in the areas of healthcare, bioenergy production and plant protections. For additional genome
sequencing projects, better planning would be benecial. For
example, the list of species to be sequenced should be prioritized
based on their economic importance and/or social impact. The
sample size for the same species should be increased to understand
better the intra-specic and inter-specic variations. The priority
for sequencing either the nuclear genome or the mitochondrial
genome should be considered. For example, if the goal is to develop
new markers for species determination, sequencing of the organelle genome probably should take precedence over the sequencing of the nuclear genome, as the size of organelle genome is
about 1/10001/500 of that of the nuclear genome. Assemblies of
higher quality are preferred and additional technologies such as
the construction of genetic or physical maps should be taken into
consideration during the planning phase of any projects attempting to obtain high-quality assemblies.
With the availability of sequences of more genomes, in-depth
genome information mining and the development of functional
genomics tools in order to validate the functions of predicted genes
and gene clusters will become the crucial tasks. The interest in
Ganoderma spp. rose from their potential applications in health
care, bio-energy production and plant protection. As a result, genome studies have to be conducted with these potential applications
in mind. Species or strains with particular characteristics, such as
the ability to produce a larger variety of chemical compounds or ligninolytic enzymes with high activities should be studied rst and
genomic information should be integrated with those data on
chemical or enzymatic activities. A centralized resource, which will
be maintained for long term, will allow researchers working on
particular aspects of Ganoderma to access and exploit the rich information generated from these genome sequencing projects.
9. Author contributions
C.L. designed and organized the writing of the paper; C.L. and
J.L. wrote Sections 13, 7 and 8; G.Y. and H.S. wrote Section 4;

Please cite this article in press as: Kes, U., et al. Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2014.11.019

18

U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

D.N. wrote Section 5; U.K. wrote Section 6; J.H.Z. critically reviewed

the manuscript. X.C.W. created and maintains the Gano portal. All
authors have read and agreed to the contents of this paper.
Acknowledgements
We thank Mr. Kai Wu for helping with comparing the mitochondrial genomes and an anonymous reviewer for constructive
advice on the manuscript. This study was supported by a research
grant for Returned Overseas Chinese Scholars, the Ministry of
Human Resources of China (No. 431207) granted to C. Liu, Program
for Changjiang Scholars and Innovative Research Teams in Universities of Ministry of Education of China (Grant No. IRT1150) and
grants from the National Science Foundation of China (Grant No.
81373912 to C. Liu). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the
manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
11.019.
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Please cite this article in press as: Kes, U., et al. Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2014.11.019

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U. Kes et al. / Phytochemistry xxx (2015) xxxxxx

Ursula Kes studied biology at the Ruhruniversity

Bochum (Germany) and graduated with focus in botany
in 1983. After a PhD study in microbiology at the
Technical University of Berlin and postdoctoral periods
at the Free University Berlin, Queen Mary and Westeld
College at the University of London and the Department
of Plant Sciences in Oxford, she was awarded a Violette
and Samuel Glasstone Fellowship of the University of
Oxford. In 1994, she became a group-leader at the ETH
Zurich in Switzerland, where she did her habilitation as
post-doctoral degree and obtained a venia legendi in
molecular microbiology and genetics. Since 2001, she is
Full Professor at the Georg-August-University in Gttingen where she chairs the
Department of Molecular Wood Biotechnology and Technical Mycology. Her
research eld covers litter and wood decay fungi, developmental processes of
mushrooms, and molecular genetics, genomics and proteomics of basidiomycetes.

David R. Nelson graduated from the University of Texas

Health Science Centre at San Antonio with a PhD degree
in 1985. After post-doctoral periods at the University of
Texas Medical School at Houston, and the University of
North Carolina at Chapel Hill, he became Assistant
Professor at University of Tennessee (Memphis) in 1994,
where he was promoted to Full Professor in 2014. Prof.
Nelson is a world-renowned expert on P450 classication and evolution. He has named more than 26,000
P450 sequences and has participated in many genome
projects in plants, fungi and animals.

Chang Liu graduated from the University of Minnesota

in 2001, Twin Cities Campus, obtaining his PhD degree.
After graduation, he worked in GlaxoSmithKline Corp
(Research Triangle Park, USA), Yale University and The
University of Hong Kong. He has been a Professor in the
Institute of Medicinal Plant Development, Chinese
Academy of Medical Science in Beijing since 2010. He
has published in journals such as Nature Communications, Science and PNAS. Recently, his research has
focused on the genomic aspects of medicinal fungi and
he has been a primary contributor for the sequencing
and analyses of the nuclear and mitochondrial genomes

Jianhui Zhang joined Professor Lius lab in 2012 as a

postdoctoral fellow. For the two last years, he was
engaged in computational identication of gene families
from medicinal plants (Salvia miltiorrhiza) and mitochondrial genome analyses of medicinal fungi (Ganoderma lucidum). His research interest is using genomic
information, molecular biology, bioinformatics and
biochemistry to understand problems of fundamental
importance in plant and fungal biology.

Jianqin Li graduated from the China Agricultural University in Beijing, obtaining her PhD degree in year
2010. After graduation, she worked in the Institute of
Medicinal Plant Development, Chinese Academy of
Medical Science as a postdoctoral fellow. She had participated in Ganoderma lucidum genome sequencing
projects and published in journals such as Nature
Communications and PLOS ONE. Her works focused on
the mitochondrial genome and non-coding RNAs of
Ganoderma lucidum. Recently, she took on a position as a
scientic editor of the Journal of Agricultural Biotechnology.

Xin-Cun Wang has worked on taxonomy and phylogeny of Ganodermataceae Donk since 2005 in the Institute of Microbiology, Chinese Academy of Sciences,
Beijing and received his PhD degree in 2013 from the
University of Chinese Academy of Sciences. In the same
year, he became a postdoctoral fellow in the Institute of
Medicinal Plant Development, Chinese Academy of
Medical Sciences, Bejing, focusing on the development
and application of DNA barcoding technologies and the
understanding of organelle genome evolution in fungi
and plants. He had published several articles about
species identity and identication in journals such as
Molecular Ecology Resources, Taxon, PLOS ONE etc., and identied the widely
cultivated Ganoderma species G. lucidum in China as G. sichuanense J.D. Zhao & X.Q.
Zhang with his colleagues based on microscopic and molecular evidence.

of Ganoderma lucidum.

Guo-Jun Yu graduated in 2010 from the Henan Agricultural University at Zhengzhou, Henan Province,
obtaining his Bachelor degree. Now he is working for his
PhD degree at the Wuhan University. He is interested in
lectins, transcriptomics, proteomics and glycoproteomics of medicinal fungi.

Hui Sun obtained her PhD degree in 2000 under the

supervision of academician Lianhui Xie and Professor
Qiyin Lin, in Fujian Agricultural University. After graduation, she has focused on research of active proteins/
peptides in medicinal fungi. She has nished a series of
works on transcriptome, proteome, and active peptides
and proteins in medicinal fungi in the past years. Currently, she is interested in lectins from medicinal fungi
which may be developed to serve as antitumor medicines and useful glycan identication tools in the future.

Please cite this article in press as: Kes, U., et al. Genome analysis of medicinal Ganoderma spp. with plant-pathogenic and saprotrophic life-styles. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2014.11.019