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Received: 9 June 2017 Accepted: 12 March 2018 DOI: 10.1111/efp.12433
ORIGINAL ARTICLE
Genomewide identification and development of microsatellite markers for Marssonina brunnea and their applications in two populations Y. Zhang1,2
| W. He1 | D.-H. Yan2
1 The Key Laboratory for Silviculture and Conservation of Ministry of Education, College of Forestry, Beijing Forestry University, Beijing, China 2
The Key Open Laboratory of Forest Protection affiliated to State Forestry Administration of China, Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, China
Summary Marssonina brunnea is an important pathogen that causes Marssonina leaf spot disease of poplar (MLDP) in various poplar species. Resistance breeding is considered as the main method for preventing this disease and requires information on genetic diversity and population structure. However, molecular markers that may be utilized in the identification of this fungus are limited. This study investigated the distribution of microsatellites in the M. brunnea genome. A total of 15,356 microsatellite markers
Correspondence Wei He, The Key Laboratory for Silviculture and Conservation of Ministry of Education, College of Forestry, Beijing Forestry University, Beijing, China. Email:
[email protected] and Dong-Hui Yan, The Key Open Laboratory of Forest Protection affiliated to State Forestry Administration of China, Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, China. Email:
[email protected]
(excluding mononucleotide repeats) were isolated from 50.1 Mb of genomic se-
Funding information The National Natural Science Foundation of China, Grant/Award Number: 31370645
lected markers could be effectively utilized in investigating the population genetic
quence. Eight M. brunnea isolates were evaluated in terms of 102 loci, followed by the selection of markers that could be utilized in investigating the population structure of 47 M. brunnea isolates from two populations. Twenty-four polymorphic microsatellite markers were developed for M. brunnea. The number of alleles per locus ranged from two to eight (average: 3.75). The polymorphism information content (PIC) of these loci ranged from 0.0408 to 0.6492. The average Shannon’s information index of these loci in the two populations was 0.3819 and 0.5351, respectively. Using these markers, M. brunnea isolates were mainly divided into two distinct clusters based on the relatedness of the sampling sites. These results indicate that the sestructure of M. brunnea. This is the first report on microsatellite markers in M. brunnea.
Editor: C. G. Fossdal
1 | I NTRO D U C TI O N
Marssonina brunnea, which belongs to the family Dermateaceae, is one of the major pathogens causing MLDP. This pathogen infects
Marssonina leaf spot disease of poplar (MLDP) is a major disease af-
poplar leaves via the conidia or ascospores in primary infection and
fecting poplar trees (Beare, Archer, & Bell, 1999; He & Yang, 1991).
extend throughout the season with the conidia (Ostry, 1987). As
MLDP generally causes black spots on the surface of the leaves and
previously reported, the sexual state of M. brunnea was not found
lesions on the petioles, resulting in premature defoliation and a sig-
in China and New Zealand, and genomic analysis showed that this
nificant reduction in photosynthesis (Spiers, 1984; Zhu et al., 2012).
pathogen might lack the capacity to perform sexual reproduction in
Furthermore, MLDP outbreaks can lead to tree death, which causes
China (He & Yang, 1991; Han, Li, & Huang, 1998; Zhu et al., 2012).
significant losses to the poplar industry, with a 30% annual de-
M. brunnea infects various poplar species (Han, Yin, Li, Huang, & Wu,
crease in average wood production having been recorded (Erickson,
2000; Spiers, 1984) and has often caused outbreaks in various coun-
Stanosz, & Kruger, 2004; Wu et al., 2012). Considering that poplar
tries in Europe (Newcombe & Callan, 1997; Spiers, 1984).
is one of the most important tree species in the world, research into the pathogenesis and control of MLDP are imperative. Forest Pathology. 2018;e12433. https://doi.org/10.1111/efp.12433
Marssonina brunnea epidemics often occur in poplar plantations in the northeast, northern and southern regions of China (Han
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et al., 2000; He & Yang, 1991). Based on its extensive area of infec-
utilized in analysing the population structure and the genetic diver-
tion, analysts have suggested that the pathogenicity of the fungal
sity of two M. brunnea populations, thereby confirming their use-
species might have changed. The resistant poplar clones I-69 and
fulness. Our method of developing SSRs may also be employed in
I-72 (Castellani, 1971) might have lost disease resistance towards
other organisms. This is the first study to report novel microsatellite
M. brunnea. Furthermore, quick-growing poplars have replaced the
markers for M. brunnea.
resistant poplar clones, including I-69 and I-72, in many forest nurseries. Novel resistant poplar clones should be cultivated for the control of MLDP. Information on the genetic diversity and population structure of M. brunnea are essential for breeding resistant poplar clones and developing other effective disease management strate-
2 | M ATE R I A L S A N D M E TH O DS 2.1 | Fungal isolates and isolation methods
gies. Our current understanding of the population genetic structure
A total of 55 M. brunnea isolates were collected in this study. Among
of M. brunnea is limited, and no useful molecular markers have been
these, eight isolates from different locations in China were used for
identified to date. Fortunately, the whole genome of M. brunnea
initial primer testing. Subsequently, 23 isolates from Beijing and 24
has been sequenced and several transcriptome analyses have been
isolates from Puyang, Henan were used in the population analyses.
completed (Chen et al., 2015; Zhu et al., 2012), thereby facilitating
All these isolates were isolated from Populus sect. Aigeiros. During
the development of molecular markers that may be employed in
isolation, leaves infected with M. brunnea were first washed with
genotyping.
sterile water, sterilized for 30 s in 75% alcohol and then kept in moist
Various polymorphic markers have been developed for diversity
conditions for 2 days at 25°C for allowing the development of fruit-
studies, and numerous molecular marker methods have been proven
ing bodies. Then, the resulting white conidia were transferred onto
to be efficient (Sollars et al., 2017; Vieira, Santini, Diniz, & Munhoz,
potato-dextrose agar (PDA) containing 50 μg/ml streptomycin sul-
2016). Microsatellites, also called simple sequence repeats (SSRs),
phate using an inoculating needle. After isolation, the isolates were
are widely used for genetic analyses of eukaryotic and prokaryotic
cultured on PDA medium at 25°C in the dark and then stored in tubes
organisms. Compared to other methods, microsatellites have several
filled with inclined PDA medium at 4°C. After sufficient fungal mycelia
advantages, such as high repeatability, high polymorphism rates and
were obtained, the genomic DNA of M. brunnea was extracted using
high abundance (Vieira et al., 2016). Specifically, microsatellites are
the cetyltrimethyl ammonium bromide (CTAB) protocol (Doyle, 1987).
simple repeats of one to six base pairs in length that occur in both coding and non-coding regions of the genome (Vieira et al., 2016). Previous studies have proven that SSRs are highly variable among
2.2 | Microsatellite survey and primer design
different populations, rendering them particularly useful for diver-
The whole genome sequence of M. brunnea was downloaded from
sity assessments (Busse et al., 2017; Yu et al., 2016). Primer devel-
the National Center for Biotechnology Information (NCBI) (URL,
opment for SSRs is easy and is commonly used between individuals
https://www.ncbi.nlm.nih.gov/genome/?term=Marssonina+bru
because the flanking regions of microsatellites are well conserved
nnea). To survey potential microsatellite loci in the M. brunnea ge-
(Vieira et al., 2016). To date, SSRs of various plant pathogens, includ-
nome, MSDB software was used (Du, Li, Zhang, & Yue, 2012) under
ing several fungal species with biphasic life cycles, have been de-
the search criteria of minimum counts of five repeat units for each
veloped for genetic studies (Della, Eyre, Danti, & Garbelotto, 2011;
mono- repeats (MNR), di- repeats (DNR), tri- repeats (TNR), tetra-
Devkota, Cornejo, Werth, Chaudhary, & Scheidegger, 2014; Gilmore
repeats (TTNR), penta-repeats (PNR) and hexa-repeats (HNR), re-
et al., 2016; Zhang, Chen, Yuan, & Meng, 2015). This study focused
spectively. Furthermore, each repeat motif class was characterized
on developing microsatellite markers based on the availability of the
by a direct repeat, the complementary counterpart and the reverse
whole genome sequence of M. brunnea.
counterpart. For example, the (AG/CT)n class of repeats consisted
A previous study indicated that the distribution of microsatellites
of (AG)n, (GA)n, (CT)n and (TC)n repeats. In addition, eight other im-
in fungi is apparently lower than in other organisms, thus potentially
portant leaf disease pathogens, namely Blumeria graminis, Erysiphe
limiting microsatellite marker development (Dutech et al., 2007).
pisi, Colletotrichum fioriniae, C. gloeosporioides, Magnaporthe grisea,
To evaluate the feasibility of microsatellite marker development
M. oryzae, Puccinia striiformis and P. triticina, were also included for
in M. brunnea, we analysed the distribution of SSRs in M. brunnea
use in the analysis of the microsatellite attributes in M. brunnea. The
relative to that of other eight important fungal pathogens that also
life cycles of all these selected fungi were biphasic (asexual and sex-
have complete genome sequences. Additionally, many studies also
ual). The genomes of these pathogens were also downloaded from
analysed microsatellite distribution before developing SSRs markers
NCBI and analysed in the same approach as that of M. brunnea. The
(Mahfooz et al., 2016; Yu et al., 2016; Zhao et al., 2015). Assessing
location of the microsatellites on the scaffolds of M. brunnea was
the distribution of microsatellites in M. brunnea might provide valu-
also visualized by MSDB.
able information for further marker development.
Primer design was conducted using the QDD program (Meglecz
The aims of this study were as follows: (i) analyse the SSR dis-
et al., 2014). To achieve higher stringency results, the following search
tribution of M. brunnea; and (ii) develop useful SSR markers for
parameters were employed: the minimum number of motif repeats was
M. brunnea. A total of 24 SSR loci were developed, which were then
five, the PCR product size ranged from 90 bp to 350 bp, the primer
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F I G U R E 1 Microsatellite frequency in the selected fungal pathogens measured by the number of SSRs per Mb
P. triticina P. striiformis M. oryzae M. grisea C. gloeosporioides C. fioriniae E. pisi B. granminis M. brunnea
0
50
100
150
200
250
300 (loci/Mb)
length ranged between 18 bp and 24 bp with an optimal length of
version 6.501 (Peakall & Smouse, 2012). The polymorphic infor-
20 bp, the annealing temperature of the primers was within the range of
mation content (PIC) of each microsatellite was obtained using the
58°C–65°C and the difference in annealing temperature in one primer
macros in Microsatellite Tools (Park, 2001). The program LOSITAN
pair was
