Constructing a new integrated genetic linkage map
and mapping quantitative trait loci for vegetative
mycelium growth rate in Lentinula edodes
Wen-Bing GONG
a,b, Wei LIU
b,c, Ying-Ying LU
b,d, Yin-Bing BIAN
a,b,
Yan ZHOU
a,b, Hoi Shan KWAN
e, Man Kit CHEUNG
e, Yang XIAO
a,b,*
aKey Laboratory of Agro-Microbial Resource and Development (Ministry of Agriculture),Huazhong Agricultural University, Wuhan 430070, Hubei Province, PR China
bInstitute of Applied Mycology, Huazhong Agricultural University, Hubei Province 430070, PR China cInstitute of Hydrobiology, Chinese Academy of Sciences, Hubei Province 430072, PR China
dInstitute of Crop Genetic Resource, Guizhou Academy of Agricultural Sciences, Guiyang 550006, Guizhou Province, PR China
e
School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, PR China
a r t i c l e i n f o
Article history:Received 27 October 2013 Received in revised form 7 January 2014
Accepted 8 January 2014 Available online 21 January 2014 Corresponding Editor:
Mike Lorenz
Keywords:
Genetic linkage map Quantitative trait loci Shiitake mushroom
Vegetative mycelium growth rate
a b s t r a c t
The most saturated linkage map for Lentinula edodes to date was constructed based on a mono-karyotic population of 146 single spore isolates (SSIs) using sequence-related amplified poly-morphism (SRAP), target region amplification polypoly-morphism (TRAP), insertionedeletion (InDel) markers, and the mating-type loci. Five hundred and twenty-four markers were located on 13 linkage groups (LGs). The map spanned a total length of 1006.1 cM, with an average marker spacing of 2.0 cM. Quantitative trait loci (QTLs) mapping was utilized to uncover the loci regu-lating and controlling the vegetative mycelium growth rate on various synthetic media, and complex medium for commercial cultivation of L. edodes. Two and 13 putative QTLs, identified respectively in the monokaryotic population and two testcross dikaryotic populations, were mapped on seven different LGs. Several vegetative mycelium growth rate-related QTLs uncov-ered here were clustuncov-ered on LG4 (Qmgr1, Qdgr1, Qdgr2 and Qdgr9) and LG6 (Qdgr3, Qdgr4 and Qdgr5), implying the presence of main genomic areas responsible for growth rate regulation and control. The QTL hotspot region on LG4 was found to be in close proximity to the region con-taining the mating-type A (MAT-A) locus. Moreover, Qdgr2 on LG4 was detected on different me-dia, contributing 8.07 %e23.71 % of the phenotypic variation. The present study provides essential information for QTL mapping and marker-assisted selection (MAS) in L. edodes.
ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction
Lentinula edodes (shiitake), an edible mushroom native to East Asia, is one of the most widely cultivated edible mushrooms
in the world (Bruhn et al. 2009). Despite being an important part of Asian cuisine culture, L. edodes has been increasingly adopted in Western cooking (Tur1o et al. 2008). Pharmacologi-cally, L. edodes is renowned for its antitumor and antiviral
* Corresponding author. Key Laboratory of Agro-Microbial Resource and Development (Ministry of Agriculture), Huazhong Agricultural University, Wuhan 430070, Hubei Province, PR China. Tel.:þ86 27 87282221; fax: þ86 27 87287442.
E-mail addresses:xysubmission@gmail.com,xiaoyang@mail.hzau.edu.cn(Y. Xiao).
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f u n b i o
1878-6146/$e see front matter ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2014.01.001
activities, and it is the first medicinal macrofungus to enter the realm of modern biotechnology (Bisen et al. 2010). As a lig-nin-decomposing fungus, L. edodes possesses a powerful ligni-nolytic enzyme system (Kwan et al. 2012) and a huge potential for the bioconversion of lignocellulosic wastes (Gait an-Hernandez et al. 2006; Philippoussis et al. 2007).
New and fine strains are crucial for a sustainable develop-ment of L. edodes production and marketing. L. edodes is a tetra-polar heterothallic basidiomycete. Two monokaryons of L. edodes with different mating alleles at both the A and B mat-ing-type loci are able to fuse and generate a dikaryon which is distinguished by the presence of clamp connections. In tra-ditional hybridizations, crosses between compatible mono-karyons are required to generate a series of hybrid dikaryons, and genotypes could only be indirectly selected based on the phenotypes of those hybrids in subsequent sys-tematic selections (Foulongne-Oriol et al. 2012). However, ap-propriate molecular markers could be used to select genotypes directly based on the linkage relationships between markers and target genes in marker-assisted selection (MAS). This approach could greatly increase the genetic improve-ment efficiency of L. edodes strains.
Most of the important economic and agronomic traits of L. edodes, such as yield and mycelium growth rate, are quantita-tive traits controlled by multiple genes or quantitaquantita-tive trait loci (QTLs). As a result, it is difficult to improve these traits ge-netically by traditional breeding methods. By contrast, the uti-lization of molecular markers, genetic map and subsequent QTL mapping is more powerful in dissecting the genetic archi-tecture of quantitative traits. It is also useful for carrying out MAS, which could increase the precision and efficiency of sub-sequent screening in breeding programs (Foulongne-Oriol et al. 2011).
Genetic maps of several other edible mushrooms, includ-ing Agaricus bisporus (Kerrigan et al. 1993; Callac et al. 1997; Moquet et al. 1999; Foulongne-Oriol et al. 2010, 2011), Pleurotus ostreatus (Larraya et al. 2000; Park et al. 2006; Sivolapova et al. 2012), Pleurotus pulmonarius (Okuda et al. 2009) and Pleurotus eryngii (Okuda et al. 2012), have been reported previously. For L. edodes, several molecular genetic maps are currently avail-able. Using 32 single spore isolates (SSIs) of the dikaryotic strain L-54,Kwan & Xu (2002)constructed the first molecular genetic map of L. edodes. The map was based on random am-plified polymorphic DNA (RAPD) markers and spanned a dis-tance of 622.4 cM. Based on 95 SSIs, high-efficiency amplified fragment length polymorphism (AFLP) markers were also used to construct linkage maps of L. edodes (Terashima et al. 2002, 2006). Moreover, a genetic map spanning 908.8 cM was generated by tetrad analysis using 92 basidiosporic strains (Miyazaki et al. 2008). Just recently, low-coverage resequencing was used to rapidly genotype 20 SSIs and construct a genetic linkage map of L. edodes (Au et al. 2013). However, all these ge-netic maps were constructed with a small mapping popula-tion (less than 100 SSIs) and a low marker density (less than 300 markers). Population size and marker density are two main factors affecting the number and effects of QTLs detected, as well as the accuracy and precision of QTL esti-mates (Yu et al. 2011). Therefore, it is necessary to construct a high-quality genetic map of L. edodes based on more markers and a larger mapping population for better QTL mapping.
Several types of molecular markers are usually utilized in genetic mapping of edible mushrooms. PCR-based AFLP markers have been widely used for their relatively high repro-ducibility and efficiency (Foulongne-Oriol 2012). Sequence-related amplified polymorphism (SRAP) (Li & Quiros 2001) gen-erates PCR-based markers targeting open reading frames (ORFs). Improved from SRAP, target region amplification poly-morphism (TRAP) uses known partial sequence of a candidate gene as a fixed primer (Hu & Vick 2003). Because of their sim-plicity, reliability, moderate throughput, and facile sequenc-ing of selected bands (Lin et al. 2003), SRAP and TRAP have been widely used in genetic mapping of plants (Gao et al. 2007; Yu et al. 2007; Chu et al. 2008). However, they were used scarcely in edible mushrooms.
As a quantitative trait, mycelium growth rate has attracted tremendous interest from mycologists due to its correlation with yield, inhibitory ability against competitors, and sexual rec-ognition system (Larraya et al. 2001, 2002; van der Nest et al. 2009; Sivolapova et al. 2012). In P. ostreatus, the relationship between mycelium growth rate and the mating-type A (MAT-A) gene has been reported in monokaryons, with statistical analysis re-vealing a higher growth rate in monokaryons bearing the A2 mating allele than those bearing the A1allele (Larraya et al. 2001). The result was supported by the linkage found between a QTL controlling dikaryotic mycelium growth rate and the MAT-A locus (Sivolapova et al. 2012). For L. edodes, only one puta-tive QTL for vegetaputa-tive mycelium growth rate of dikaryons on the potato dextrose agar medium was detected previously. This QTL and the MAT-A locus were not mapped on the same linkage group (LG) (Miyazaki et al. 2008). Research on vegetative mycelium growth is still in its infant stage, and currently, related systematic studies are very limited on monokaryons and dikary-ons growing on the synthetic and complex media. Promising loci for genetic improvement of vegetative mycelium growth rate re-main elusive, and thus it is necessary to dissect the QTLs con-trolling the vegetative mycelium growth rate of L. edodes.
In this study, we employed a monokaryotic population of 146 SSIs and constructed a new molecular linkage map con-taining 524 loci of L. edodes. QTL analyses of vegetative growth rate on different media were also carried out using one mono-karyotic population and two testcross dimono-karyotic populations. The objectives of our study were to: (1) construct a high-quality linkage map for further genetic research; and (2) detect QTLs controlling the vegetative growth rate in L. edodes.
Materials and methods
Fungal strains and populations
Monokaryotic strains L205-6 (mating-type: A1B1) and W1-26 (A2B2) of L. edodes were selected respectively from germinating single spore cultures (monokaryons) of the cultivated strains L205 and WX1. The hybrid strain L6-26 was generated from the pairing between L205-6 and W1-26. The monokaryotic pop-ulation consisted of 146 SSIs from mature fruiting bodies of strain L6-26. Mating-types of the monokaryotic strains were determined as previously reported (Darmono & Burdsall 1992; Li et al. 2007). Two tester monokaryotic strains, 741-15 (A3B3) and 741-64 (A4B4), were obtained from protoclones of
the wild dikaryotic strain LeQC741S from Sichuan Province as previously described by Kawasumi et al. (1987). All the 146 SSIs in the monokaryotic mapping population were paired with the two tester strains 741-15 and 741-64 to produce test-cross dikaryotic populations LQ-15 and LQ-64, respectively. Both LQ-15 and LQ-64 contained 146 dikaryotic strains. L. edo-des strains L205 (CCTCC AF 2013008) and LeQC741S (CCTCC AF 2013009) were deposited in the China Center for Type Cul-ture Collection whereas strain WX1 (ACCC 50926) was depos-ited in the Agricultural Culture Collection of China.
In the construction of the linkage map, only the monokary-otic segregating population including 146 SSIs was used, while in QTL mapping, all the 146 SSIs were used for the monokary-otic growth rate analysis, and all the dikarymonokary-otic strains in LQ-15 and LQ-64 were used for dikaryotic growth rate analysis.
DNA extraction
Mycelia of the 146 SSIs were incubated in liquid medium of malt extract, yeast extract and glucose (MYG) (2 % malt ex-tract, 2 % glucose, 0.1 % peptone, and 0.1 % yeast extract) at 25 C and collected by filtering. Total genomic DNA was extracted from the freeze-dried mycelia using the cetyltrime-thylammonium bromide (CTAB) procedure (Murray & Thompson 1980). DNA was then detected as previously de-scribed (Xiao et al. 2010) and was diluted to 50 ng/mL.
Marker development SRAP and TRAP markers
SRAP analysis was performed with 56 (7 8) primer pairs (Supplemental Table S1). PCR reactions were performed in a total volume of 20mL consisting of 2 mL 10 PCR buffer, 50 ng template DNA, 3 mM dNTP mix, 8mM forward primers, 8mM reverse primers, 40 mM MgCl2, and 1.75 U Taq polymer-ase (TaKaRa, Japan). PCR amplifications were carried out in a MyCycler Thermal Cycler (Bio-Rad, USA) under the following conditions: 94C for 5 min, 5 cycles (at 94C for 1 min, 35C for 1 min, and 72C for 1 min), then 35 cycles (at 94C for 1 min, 50C for 1 min, and 72C for 1 min), and finally 72C for 7 min. The 43 TRAP primer pairs used in this study are listed in Supplemental Table S2. In the TRAP analysis, the PCR reaction mixture and the amplification program used were the same as those for the SRAP analysis except that there were 12.5mM for-ward primers (fixed primers) and 3mM reverse primers (ran-dom primers) in the 20mL mixture.
Insertionedeletion (InDel) markers
A total of 15 polymorphic primer pairs were chosen via an ini-tial screening of 25 InDel test primers (Supplemental Table S3) using DNA samples of L205-6 and W1-26 as templates. All PCR reactions were performed in 20mL mixture containing 2 mL 10 PCR buffer, 50 ng template DNA, 5 mM dNTP mix, 10 mM forward primers, 10 mM reverse primers, 40 mM MgCl2, and 1 U Taq polymerase. PCR amplifications were per-formed at 94C for 3 min, 35 cycles (at 94C for 30 s, 60C for 45 s, 72C for 1 min), and finally at 72C for 10 min.
Amplified fragments of SRAP, TRAP and InDel markers were all detected by electrophoresis on 6 % denaturing
acrylamide gels stained with silver nitrate solution. SRAP markers were named by the forward and reverse primers used, followed by the approximate size of bands in base pairs (e.g. ME1-EM4-250); TRAP markers by the forward primer and reverse primer numbers, followed by the size of the bands (e.g. baw28-2-80); and InDel markers by the InDel primer name (e.g. S502R/F).
Linkage analysis and mapping
At each locus, a genotype identical to L205-6 was marked as “a”, while that to W1-26 was marked as “b”. All markers were evaluated by the Chi-square test for deviation from the expected 1:1 ratio among the strains in the monokaryotic mapping population. JoinMap V3.0 (Van Ooijen & Voorrips 2001) was used for the linkage analysis. Data of the monokary-otic mapping population were coded by the HAP (haploid) model. Markers were classified into LGs based on the mini-mum likelihood of odds (LOD) score of 3.0. Kosambi’s mapping function (Kosambi 1943) was used for calculating the linkage relationships between markers, recombination rates and ge-netic distances. The gege-netic map was graphically exhibited using Mapchart (Voorrips 2002).
Estimation of genome length, map coverage and marker distribution
The expected genome length (Le) was estimated using two dif-ferent methods. In the first method, 2s (s is the average marker spacing over the entire linkage map) were added to the length of each LG to account for terminal chromosome regions (Fishman et al. 2001). In the second method, the length of each LG was multiplied by (mþ1)/(m1), where m is the num-ber of markers on each LG (Chakravarti et al. 1991). The two es-timates were then averaged and used as the expected genome length. The theoretical map coverage was estimated using the equation c¼ 1e2dn/Le, where c is the proportion of the ge-nome within d cM of a marker, n is the number of markers in the map and Le is the estimated genome length (Remington et al. 1999; van der Nest et al. 2009).
The marker distribution between different LGs was evalu-ated by comparing the actual marker density with expected values under a Poisson distribution. Assuming an identical marker density across all LGs, the expected number of markers in the i-th LG (LGi) was calculated by the equation li¼ m$Gi/Pi$Gi, where m is the total number of markers and Giis the length of LGi after adding 2s (Remington et al. 1999; Foulongne-Oriol et al. 2010). The marker distribution within each LG was tested by whether the observed genetic positions of the markers are under a uniform distribution or not, using the KolmogoroveSmirnov test (Foulongne-Oriol et al. 2010).
Phenotype determination and QTL mapping
Monokaryotic growth rates of the 146 SSIs in the mapping pop-ulation were determined on the MYG medium as previously described (Gong et al. 2013). For the LQ-15 and LQ-64 popula-tions, growth rates of all dikaryotic strains in both the MYG and the complete yeast medium (CYM: 2 % glucose, 2 % agar, 0.2 % peptone, 0.2 % yeast extract, 0.1 % K2HPO4, 0.05 %
MgSO4$7H2O, 0.046 % KH2PO4) were determined. The final growth rate was calculated as the radial extension of each my-celial colony per day (Olson 2006). To evaluate the dikaryotic growth rates on the medium for commercial cultivation of L. edodes, glass tubes (18 mm in diameter, 18 cm in length) con-taining 18 g of mixed sawdust (SD) medium (79 % hardwood sawdust, 20 % wheat bran, and 1 % gypsum; 60 % humidity) were utilized for both testcross dikaryotic populations. The in-oculum was placed at the top of each tube, and the growth rate was determined as the ratio of the distance colonized by the mycelium (in millimeters) to the growth time (20 d after inocu-lation). Three repetitions were performed for all the above-mentioned samples. Both the monokaryotic and dikaryotic samples were incubated at 25C in the dark.
For each medium, the effects of genotype on vegetative mycelium growth rate were assessed by one-way analysis of variance (ANOVA). For the synthetic media (MYG and CYM), the effects of tester strains and media on dikaryotic growth rates were analyzed by two-way ANOVA. For the mixed saw-dust medium, the effects of the tester strains on dikaryotic growth rates were evaluated by the paired sample t-test. The Pearson procedure was used for correlation analyses between all types of mycelium growth rates in the three different pop-ulations and media. SPSS (Statistical Package for the Social Sciences) version 17 (SPSS Inc., Chicago, IL, USA) was used for all the data analyses.
For QTL mapping, each phenotypic data set was input to WinQTLCart 2.5 (Zeng 1994; Wang et al. 2012), and composite in-terval mapping (CIM) was used for QTL analysis with model 6 at a walking speed of 1 cM (number of control markers¼ 5; window size¼ 10 cM). For each trait, the threshold for significant QTLs (P< 0.05) was calculated by a 1000 permutation test. A threshold of 2.5 was used to declare the presence of a QTL. According to Larraya et al. (2002), QTLs were named as Q(m/d)grx, where m is the monokaryotic state, d is the dikaryotic state, gr is the growth rate, and x is a consecutive QTL number. QTLs detected in different media or populations were assumed to be the same when their confidence intervals (CIs) overlapped and the addi-tive values had the same sign (Foulongne-Oriol et al. 2012).
Results
Marker development
A total of 716 polymorphic markers (including MAT-A and MAT-B) were detected in the monokaryotic population. For SRAP, 25 out of 56 primer pairs produced a total of 225 clear polymorphic bands. The number of DNA bands for each SRAP primer pair ranged from four to 17, with an average of nine. For TRAP, 474 polymorphic markers were obtained from 43 primer pairs, and three to 20 markers were amplified by each primer pair, with an average of 11. Fifteen out of 25 InDel markers were confirmed to be polymorphic in the monokaryotic population.
Linkage analysis and mapping
In the segregation analysis of the 716 polymorphic markers in the monokaryotic population, 134 markers (37 SRAP markers
and 97 TRAP markers) (18.7 %) showed highly distorted segre-gation ratios (P< 0.01) and 27 markers (10 SRAP markers and 17 TRAP markers) (3.8 %) showed significant segregation distor-tion (0.01 P < 0.05). The 134 markers that displayed high dis-tortion were excluded in the downstream linkage analysis, due to the failure to group all the markers into different LGs in a pre-liminary analysis if they were included. Among the remaining 582 markers, 32 were unable to be grouped into any LG and another 26 markers were identical to others (similarity value ¼ 1.000). Thus, these 58 markers were also excluded from the 582 markers in the final linkage map. Finally, a total of 524 markers (172 SRAP markers, 343 TRAP markers, 7 InDel markers, and the MAT-A and MAT-B loci) were mapped into 13 LGs, covering a total length of 1006.1 cM, with an average distance of 2.0 cM between markers (Table 1,Fig 1). The num-ber of markers per group varied from six to 110, with an average of 40. The LG length ranged from 21.6 cM to 134.2 cM, with an average of 77.4 cM. The average marker spacing per LG ranged from 1.0 cM to 4.6 cM. The largest interval between two adja-cent markers was found to be 21.6 cM on LG13, and no interval larger than 20 cM was found on the other LGs. A total of 25 (4.8 %) skewed markers (0.01 P < 0.05) were distributed on nine different LGs, ranging from zero to six for each group. Three segregation distortion regions, defined as regions with three or more closely linked markers exhibiting significant seg-regation distortion (Paillard et al. 2003), were found on LG3, LG6 and LG7. MAT-A and MAT-B were mapped, respectively, on LG4 and LG11.
Based on the current genetic linkage map, the estimated genome length (Le) of L. edodes was found to be 1066.2 cM (an average between 1058.1 cM as calculated by method one and 1074.2 cM by method two; refer to Materials and Methods for details). Based on this value, the theoretical map coverage was roughly estimated to be nearly 100 % (located within 10 cM of one marker), 99.3 % (located within 5 cM of one marker) and 86.0 % (located within 2 cM of one marker) of the genome. Two-tailed cumulative Poisson distribution test revealed a disproportionate distribution of markers across the 13 LGs (Table 1). Compared to the estimated values, LG1 and LG9 had significantly more markers, while LG8 and LG11 had fewer. Markers showing a significant deviation from the uniform distribution were detected in LG2, LG4, LG9, and LG10 (Table 1).
QTL mapping for vegetative mycelium growth
The estimated growth rates (averages of three repetitions) of different populations of L. edodes and on different media were displayed in Table 2. Compared to the monokaryons, the dikaryons grew faster and had smaller coefficients of var-iation. One-way ANOVA indicated a significant effect of geno-type on vegetative mycelium growth rate in the three populations and media ( p< 0.01). Significant effects of tester strains ( p< 0.01) and media ( p < 0.01) on dikaryotic growth rates were also revealed by two-way ANOVA. Moreover, paired sample t-test suggested significant effects of tester strains on dikaryotic growth rate on the SD ( p< 0.01) medium. Correlation analyses between mycelium growth rates in dif-ferent populations and media were performed using the Pear-son procedure (Table 3). In the LQ-15 population, positive
correlations between dikaryotic growth rates on all the three media were revealed. Dikaryotic growth rates on MYG and CYM showed highly positive correlations (r¼ 0.646, p < 0.01), while the correlations of MYG and CYM with SD were rela-tively low (MYG-SD: r¼ 0.338, p < 0.01; CYM-SD: r ¼ 0.275, p< 0.01). Similar results were also found in the LQ-64 popula-tion. However, no significant correlations were found between the dikaryotic growth rates of LQ-15 and LQ-64 on CYM and SD media except for the MYG medium. In addition, monokaryotic growth rates correlated positively, but only to a low extent, with dikaryotic growth rates on both MYG (r ¼ 0.246, p< 0.01) and CYM (r ¼ 0.171, p < 0.05) in LQ-15.
QTLs detected by the CIM method in different media and populations were listed inTable 4. For monokaryotic growth rate, putative QTLs Qmgr1 and Qmgr2 were detected, respec-tively, on LG4 and LG9 of the genetic map. The two QTLs to-gether explained 13.28 % of the phenotypic variation. The lengths of the CI (LOD-1 confidence interval) for Qmgr1 and Qmgr2 were 9.5 cM and 6.7 cM, respectively. Both favorable al-leles at the two loci came from the parental strain W1-26. Qmgr1 was mapped in the region from 68.1 cM to 77.6 cM of LG4. It was linked to the InDel marker S278R/F, and was adja-cent to MAT-A (70.9 cM on LG4). On the other hand, Qmgr2 was located on LG9 (from 43.0 cM to 49.7 cM) and linked to the SRAP marker ME1-EM4-850.
For dikaryotic growth rate, a total of 13 QTLs were detected in the testcross populations LQ-15 and LQ-64, explaining 6.17 %e23.71 % of the phenotypic variation. In LQ-15, two QTLs were located on LG4, and three others were mapped on LG6. The lengths of CI for these five QTLs ranged from 1.1 cM to 6.6 cM. Among these QTLs for dikaryotic growth rate, Qdgr2 was detected across all three media, with high per-centages of phenotypic variation explained (PVE). Qdgr4 and Qdgr5 were found in both the MYG and CYM media. Qdgr1, Qdgr2 and Qdgr3 showed negative additive effects, while
Qdgr4 and Qdgr5 showed positive additive effects. Qdgr1 was most likely located in a 6.3 cM region (from 64.3 cM to 70.6 cM) on LG4, adjacent to MAT-A. The CI of Qdgr2 partially overlapped with that of Qmgr1 as detected in the monokary-otic population. Both QTLs showed negative additive effects with favorable alleles from W1-26. In LQ-64, a total of eight pu-tative dikaryotic mycelium growth rate-related QTLs were identified, contributing 6.17 %e11.15 % of the phenotypic var-iation. Qdgr6 and Qdgr7 were located on LG13 and showed neg-ative additive effects. Qdgr8, Qdgr9 and Qdgr11 were scattered on LG1, LG4 and LG2, respectively. Moreover, the CI of Qdgr9 partially overlapped with that of Qdgr2 as detected in LQ-15. Qdgr10, Qdgr12 and Qdgr13 were located on LG7 (Table 4,Fig 1).
Discussion
Molecular markers used in this study
Efficient and user-friendly molecular markers are highly valuable tools for genotyping of mapping populations. In this study, SRAP and TRAP, two rapid and powerful PCR-based marker techniques, were used to develop markers for genotyping. Compared to anonymous markers, these gene-targeted markers provide ample opportunities to inte-grate gene information and phenotypic trait variation, and are important for QTL mapping (Shokeen et al. 2011). SRAP and TRAP target potentially functional genes, and may pro-vide an additional avenue to further genetic studies involved in markeretrait association (Li & Quiros 2001; Hu & Vick 2003; Yu et al. 2007). Unlike AFLP, these two techniques re-quire no extensive pre-PCR processing of templates (Liu et al. 2005). They are also highly efficient in generating a large number of markers (Liu et al. 2005; Gao et al. 2007), which is also the case in the current study on L. edodes. On average,
Table 1e Characteristics of linkage groups in the current genetic linkage map of L. edodes.
Linkage group No. of markers No. of skewed markersa Observed length (cM) Average marker spacing (cM) Largest interval (cM) Poisson test P-valueb KS test P-valuec LG1 110 2 134.2 1.2 12.7 1.42E-06 0.110 LG2 50 1 97.2 2.0 16.1 0.531 0.007 LG3 19 6 54.7 3.0 11.1 0.032 0.125 LG4 64 3 118.7 1.9 12.3 0.310 0.005 LG5 18 0 51.9 3.1 8.8 0.034 0.457 LG6 56 6 86.1 1.6 12.5 0.042 0.214 LG7 38 3 98.0 2.6 17.8 0.041 0.133 LG8 12 0 50.7 4.6 13.7 0.001 0.246 LG9 62 1 63.0 1.0 9.6 2.62E-06 1.11E-05 LG10 39 0 93.1 2.5 15.7 0.105 3.03E-08 LG11 18 0 68.1 4.0 18.1 0.001 0.05 LG12 6 2 21.6 4.3 12.2 0.031 0.293 LG13 32 1 68.8 2.2 21.6 0.283 0.348 Total 524 25 1006.1 Average 40 2 77.4 2.0d 14.0 a 0.01 P < 0.05.
b The marker distribution between different LGs was detected using the two-tailed cumulative Poisson distribution test; a P-value of 0.025 cor-responds to a significance level of 0.05.
c Uniform distribution of markers along each LGs was detected using the KolmogoroveSmirnov test.
d The average marker spacing over the whole map was calculated by dividing the total length of all LGs by the number of marker intervals (the number of mapped markers minus the number of LGs).
S502R/F 0.0 flp1-4-90 10.2 zin2-4-710 12.1 chd1-2-450 14.0 ME4-EM3-140 16.2 ME3-EM2-410 17.2 pep1-3-160 18.0 ste3-2-310 19.6 ME4-EM3-175 20.8 ME4-EM3-400 21.1 ME5-EM7-360 22.1 ME3-EM2-350 23.4 ME3-EM2-300 24.1 ME7-EM8-480 24.5 zip1-4-1100 25.0 ME5-EM7-660 25.2 rab-4-650 25.6 chd1-2-1100 25.7 ste3-2-500 25.9 ftf2-3-630 26.3 man1-4-800 26.9 man1-4-390 27.2 cho-2-500 27.7 ME6-EM8-400 28.6 ME6-EM1-560 30.3 tel-3-720 32.2 mlg1-2-470 44.9 cut-4-345 46.5 mip-3-800 48.0 exg1-4-660 49.0 ftf3-3-600 49.8 zin2-4-900 50.7 ftf3-3-40 51.6 ste3-3-200 52.1 zin1-3-212 53.2 ftf3-3-380 53.8 mlg1-2-335 54.3 ME3-EM2-450 54.6 ME3-EM2-460 54.8 ME7-EM8-320 55.0 ME5-EM5-200 55.2 zin2-4-100 55.4 mlg1-2-320 55.5 mip-4-750 55.6 dmc1-4-450 55.7 ME4-EM7-260 56.0 mdh1-4-150 56.1 dmc1-4-480 56.3 dmc1-4-1100 56.4 ME5-EM8-480 56.7 ME5-EM5-480 56.9 dmc1-4-245 57.4 lup23-3-900 58.1 ME6-EM1-760 58.9 zin1-3-380 59.8 exg1-4-510 61.0 LG1[1] exg1-4-500 61.4 ME7-EM6-180 61.8 ME3-EM8-85 63.5 lup23-3-380 65.9 ME4-EM2-170 66.0 dmc1-4-175 68.5 eln3-4-600 72.5 mip-2-460 73.0 ME3-EM8-280 76.6 ftf2-3-600 * 79.6 tlg1-3-630 82.1 ME5-EM7-600 82.7 ME3-EM3-580 * 85.4 mlg1-2-310 87.7 ME6-EM8-75 89.5 ME5-EM7-620 92.7 icb-2-450 94.8 ME3-EM3-390 102.2 icb-2-400 105.7 zin2-4-480 106.8 ME6-EM8-100 107.8 dmc1-4-600 108.9 ghf30-2-175 110.4 mip-3-430 110.9 hlh-4-520 111.5 ME4-EM3-380 111.8 ME6-EM8-245 ME4-EM8-420 112.3 cut-4-500 112.5 ME5-EM5-260 112.8 ME6-EM8-210 113.1 ME1-EM4-250 113.6 ME4-EM8-430 114.2 cho-2-950 114.7 chi1-4-340 115.5 mdh1-4-235 116.2 zin2-4-380 117.1 ME5-EM8-400 117.9 mip-3-300 118.2 ME6-EM3-430 118.9 ghf30-2-370 120.6 ME6-EM1-280 121.2 ME5-EM5-460 122.7 ME7-EM5-545 124.2 ME7-EM5-540 124.3 chi2-3-355 125.9 ME3-EM5-205 126.7 chi2-3-370 126.9 ME6-EM3-600 127.4 ME6-EM3-240 127.6 ME7-EM5-980 128.3 icb-2-50 129.4 mip-2-400 132.1 ME3-EM5-220 134.2 Qdgr 8 7. 09% LG1[2] chi2-3-450 0.0 pep1-3-910 16.1 icb-2-335 19.1 tel-3-470 21.0 tel-2-430 21.4 tel-2-210 22.3 mip-3-230 23.4 exg1-4-300 25.3 zip1-4-225 * 37.9 ME4-EM8-245 39.6 tyr-3-80 39.9 ME2-EM6-60 40.7 ME2-EM6-80 41.4 ME4-EM3-450 43.4 gpd-4-390 50.2 ME5-EM7-500 50.8 exg1-4-255 52.1 icb-2-260 52.9 zip1-4-480 54.0 ME6-EM1-610 55.1 ME5-EM5-135 55.4 ME5-EM7-375 55.5 tel-4-310 55.8 ME1-EM2-240 56.1 gb1-4-600 56.3 mip-4-240 56.4 ME4-EM3-500 56.8 ME1-EM4-380 57.1 chi1-4-360 57.6 zip1-4-200 57.8 zin1-3-450 57.9 tlg1-3-320 58.2 cut-4-290 58.4 cut-4-690 58.6 zin2-4-850 59.3 pro1-4-780 59.7 mip-2-160 60.0 ME1-EM7-240 60.5 ftf2-3-180 61.4 man1-4-350 62.0 pro1-4-800 62.7 dmc1-4-495 63.4 ftf2-3-160 64.6 tel-2-480 69.2 ME5-EM7-480 74.8 eln3-4-280 76.3 icb-2-470 78.8 S413R/F 80.3 zip1-4-800 83.1 pro1-4-700 97.2 Qdgr 11 6. 4 4 % LG2 tel-2-460 0.0 chi2-3-290 9.9 ftf3-3-410 18.3 mip-2-360 19.6 ste3-3-320 * 20.8 flp1-4-750 * 21.6 mip-3-700 22.2 ste3-4-950 * 22.6 cut-4-240 * 22.8 exg2-3-350 * 23.3 exg2-3-230 23.7 zin1-3-170 24.2 ME5-EM8-185 25.3 exg2-3-640 27.0 ste3-4-130 28.4 rab-4-150 * 35.6 ftf1-2-800 40.0 mip-2-420 51.0 chi2-3-600 54.7 LG3
nine SRAP and 11 TRAP markers were produced per primer pair. These are comparable to an average of 8.5 AFLP markers reported in a previous study (Terashima et al. 2006). The application of SRAP and TRAP markers greatly im-proved both the marker density and the total coverage of the genetic map in L. edodes.
Apart from SRAP and TRAP markers, InDel markers were also used in this study. These markers were derived from the L. edodes draft genome sequence (unpublished data in Prof. Kwan’s lab), which was not available in previous studies. Compared with AFLP, these sequence-based markers could fa-cilitate the integration of genetic map and genome sequence. However, only seven InDel markers were available in the cur-rent genetic map, making it difficult to establish a correlation between the genetic map and the genome sequence. More InDel markers are thus desired to improve the present genetic map. To our knowledge, this is the first report of using SRAP, TRAP and InDel markers for genetic linkage mapping in edible mushrooms.
In the monokaryotic population, 22.5 % of the markers were found to be skewed. This is similar to a value (20.7 %) re-ported previously (Terashima et al. 2002). Several hypotheses have been proposed to explain distorted segregation, such as biased selection of SSIs, expression of lethal factors and unbalancing selection of mating-types (Larraya et al. 2000; Foulongne-Oriol et al. 2010). The mapped skewed loci appeared to locate together as segregation distortion regions in the genetic map, as previously reported in L. edodes (Terashima et al. 2002), P. pulmonarius (Okuda et al. 2009) and P. eryngii (Okuda et al. 2012). The cluster distribution of skewed loci seemed to indicate that the segregation distortion of markers is most likely caused by genetic factors rather than statistical bias (Li et al. 2010).
Features of the current genetic map
A genetic map with the highest marker density in L. edodes to date was constructed here using SRAP, TRAP and InDel molec-ular markers. Compared to previous studies (Table 5), the cur-rent study involved the largest mapping population (146 SSIs), which could ensure the accuracy of genetic mapping (Foulongne-Oriol et al. 2010). Additionally, the present genetic map contained 13 LGs with 524 markers, and spanned 1006.1 cM, with an average marker distance of 2.0 cM. In terms
of total length, the current map was close to that constructed by tetrad analysis (908.8 cM) (Miyazaki et al. 2008), shorter than that of the AFLP-based map (1398.4 cM) (Terashima et al. 2006), and longer than those of the other two maps (Kwan & Xu 2002; Au et al. 2013).
Thirteen LGs were revealed in this study. The LGs outnum-bered the chromosomes of L. edodes as revealed using contour-clamped homogeneous electric field (CHEF) gel electrophore-sis, in which only eight chromosomes were found in aw33 Mb genome (Arima & Morinaga 1993). Recently, the ge-nome of L. edodes has been sequenced and the total length of the assembly was estimated to be 40.2 Mb (Kwan et al. 2012). Therefore, there may be more than eight chromosomes in L. edodes. Indeed, the number of chromosomes in L. edodes has remained unclear (Terashima et al. 2002). As demonstrated in A. bisporus and Flammulina velutipes, the assignment of DNA probes designed from genetic linkage maps to CHEF-separated chromosomes may be a more effective karyotyping approach (Sonnenberg et al. 1996; Tanesaka et al. 2012). Addi-tional experiments on hybridization of genetically mapped markers to CHEF blots are thus needed to reveal the actual number of chromosomes in L. edodes. In this study, a larger LG number could be attributed to an absence of suitable markers to link LGs belonging to the same chromosome, as well as high LOD values (5.0e8.0 in some cases) used for grouping in linkage mapping. The number of markers mapped in previous genetic maps of L. edodes varied from 69 to 289 (Table 5) whereas 524 markers were mapped in the current map. The average marker distance reported here (2.0 cM) is shorter than those in any previously reported genetic maps (Table 5). There was only one gap (>20 cM) reported in the cur-rent map.
The MAT-A locus was located on a relatively large LG in the current map. The same finding has been reported previously (Terashima et al. 2006; Miyazaki et al. 2008; Au et al. 2013). MAT-A and the InDel marker S278R/F, derived from scaffold S278 of the shiitake draft genome (unpublished data in Prof. Kwan’s lab), were located on the same LG, which was in con-gruence with previous findings (Au et al. 2013). Both the InDel marker DTF1R/F derived from scaffold S214 (Supplemental Table S3) and MAT-B also belonged to the same LG as previ-ously reported (Au et al. 2013). LG4 and LG11 in our map corre-sponded respectively to LG2 and LG11 in the map constructed byAu et al. (2013).
Fig 1e Genetic linkage map of L. edodes and QTL locations for mycelium growth rate detected in the monokaryon, LQ-15 and LQ-64 populations. The name of each linkage group is denoted by “LG”, followed by the group number on the top; LG1 is divided into LG1 [1] and LG1 [2] for better presentation. Marker names are provided on the right side of each LG, and genetic positions of the markers are shown on the left side. SRAP markers are named by the forward and reverse primers used, followed by the approximate size of bands in base pairs (e.g. ME1-EM4-250); TRAP markers by the forward primer and re-verse primer numbers, followed by the band size” (e.g. baw28-2-80); and InDel markers by the primer name (e.g. S502R/F). InDel markers are underlined in the figure. Markers showing segregation distortion (0.01£ P < 0.05) are indicated with *. Using this map, two QTLs controlling the mycelium growth rate of monokaryon (Qmgr1 and Qmgr2) and 13 QTLs controlling the mycelium growth rate of dikaryon (Qdgr1 to Qdgr13) were respectively detected in the monokaryotic population and the testcross populations (LQ-15 and LQ-64). These QTLs are shown on the right side of the LGs. The percentage value following each QTL represents the R2value (percentage of explained phenotypic variation). The length of each QTL bar represents the LOD-1 confidence interval. Solid lines represent the LOD-2 confidence intervals for QTLs with LOD scores of estimated threshold based on a 1000 permutation test (P< 0.05), and dashed lines for QTLs with LOD scores between 2.5 and the es-timated threshold. *, ** and *** after the QTL names indicate QTLs detected on MYG, CYM and SD media, respectively.
hsf-3-160 0.0 icb-2-1100 7.9 hsf-3-155 8.9 ME5-EM8-60 19.2 cut-4-630 20.4 rab-4-255 * 21.4 ste3-3-1100 23.5 ME5-EM8-780 25.0 chi1-4-1100 27.1 ftf3-3-350 27.7 ME3-EM2-230 28.5 ME1-EM2-280 * 30.4 ME4-EM7-230 30.8 ME4-EM2-280 31.0 ME4-EM8-415 * 31.2 ME4-EM8-410 31.6 hsf-3-980 31.8 ME1-EM6-680 32.0 ME5-EM7-160 33.2 ME3-EM8-170 34.9 ME7-EM8-960 36.7 mip-3-480 37.1 cut-4-810 37.7 baw28-2-100 38.0 ME6-EM8-230 38.2 cut-4-790 38.7 ME6-EM8-550 39.2 icb-2-1000 39.5 ghf30-2-345 40.4 zin1-3-214 40.9 pep1-3-630 41.2 tel-2-620 42.1 ME2-EM6-120 42.4 ME6-EM8-170 43.7 baw28-2-800 44.4 ME6-EM3-80 45.7 ME3-EM1-295 46.3 cho-2-370 48.1 mip-2-350 48.9 ME7-EM5-640 51.1 hlh-4-230 51.7 baw28-2-80 52.4 tel-3-400 56.1 rab-4-640 58.3 exg2-3-180 60.6 MAT-A 70.9 S278R/F 71.2 ME2-EM6-470 83.5 ME2-EM6-460 84.1 ME6-EM3-225 88.7 hsf-3-1000 89.5 ghf30-2-160 90.7 tyr-3-70 91.0 ME6-EM3-230 91.1 mip-3-380 93.6 zin2-4-430 94.2 dmc1-4-950 95.8 zin2-4-280 97.1 ghf30-2-400 100.4 mip-4-330 105.7 gb1-4-490 107.3 icb-2-210 109.6 zip1-4-295 111.2 gb1-4-140 118.7 Qm gr 1 6. 8 9 % Qdg r1 16 .79% Qdgr 2* 23 .7 1 % Qdg r2* * 1 8 .5 1% Q d gr 2 *** 8 .07 % Qdg r9 9. 54% LG4 exg1-4-320 0.0 ftf2-3-590 8.8 exp1-4-170 15.4 ME1-EM4-550 16.2 ME5-EM5-240 18.8 ste3-3-255 19.7 baw28-2-320 20.5 ste3-3-480 21.6 lac4-4-950 21.8 lac4-4-110 22.0 exg2-3-240 23.5 exg2-3-410 24.1 ftf1-2-550 27.3 ME4-EM2-160 29.9 icb-2-150 35.7 cut-4-370 37.1 ME1-EM6-390 43.7 ftf2-3-560 51.9 LG5 ghf30-2-580 0.0 tyr-3-750 6.7 tlg1-3-650 9.5 ME3-EM8-580 12.9 exp1-4-660 14.7 ME7-EM6-195 16.2 tyr-3-450 17.0 ste3-3-210 18.4 tyr-3-380 18.9 ste3-2-265 19.6 zin2-4-110 19.7 gpd-4-500 20.5 exp1-4-600 21.1 zin2-4-400 21.6 ME4-EM8-220 22.3 ME3-EM3-160 22.9 ME3-EM8-950 25.2 ME3-EM1-420 33.3 lup23-3-950 34.7 ME4-EM3-900 36.2 rab-4-500 36.7 ME4-EM8-540 37.5 zin1-3-218 38.0 ME4-EM3-440 38.7 ftf2-3-310 39.0 zin1-3-216 39.5 ME2-EM5-710 mip-4-550 39.8 ME6-EM8-490 40.0 icb-2-460 40.4 mip-2-465 40.7 ME6-EM1-450 41.3 hsf-3-60 42.1 zin2-4-150 42.4 ME4-EM3-438 43.1 chi2-3-350 44.3 zip1-4-500 45.2 ME7-EM8-220 47.5 ME6-EM1-350 48.1 exg2-3-380 49.6 ME5-EM8-290 50.9 cho-2-350 63.4 ME4-EM8-490 * 64.8 exg2-3-600 65.7 icb-2-420 * 67.6 ME6-EM8-370 * 68.0 ME6-EM1-360 * 68.5 ME7-EM5-400 69.2 ME2-EM5-306 * 69.9 zin1-3-275 70.7 zin1-3-420 71.6 gpd-4-350 * 72.9 ME3-EM5-360 74.2 rab-4-520 77.2 zip1-4-300 79.5 lup23-3-980 86.1 Qdgr 3 6. 5 8 % Q dgr 4 * 9. 0 9 % Qdg r4 ** 16. 8 3% Qdgr 5* 6. 67% Qd gr 5 ** 12 .5 8% LG6 Fig 1e (continued).
mlg1-2-490 0.0 ME3-EM5-380 2.4 mip-3-1090 5.6 ME6-EM1-570 10.4 ME7-EM5-645 13.5 lac4-4-540 14.9 mlg1-2-420 15.8 mlg1-2-160 16.5 ME5-EM7-780 17.3 ME1-EM5-580 18.8 ME3-EM5-680 19.5 tel-3-160 20.4 ME4-EM3-70 21.3 icb-2-330 22.9 chd1-2-480 23.8 chi1-4-200 25.1 chi1-4-190 25.6 ftf1-2-150 28.4 cho-2-800 30.7 cut-4-340 33.2 icb-2-800 35.7 mip-4-1100 53.5 mip-4-350 59.8 ME2-EM6-510 62.0 hsf-3-900 * 63.5 mip-2-170 * 64.7 ME5-EM7-340 * 65.5 mip-3-550 66.5 ME3-EM3-280 68.2 ME1-EM2-500 69.7 rab-4-295 72.5 zin2-4-460 73.4 chd1-2-800 76.2 ME3-EM5-980 79.0 zin2-4-360 92.9 ME6-EM8-920 93.8 ME6-EM3-650 95.9 dmc1-4-520 98.0 Qd gr 1 0 7. 0 5 % Qdg r1 2 7 .14 % Qdgr 13 6. 17 % LG7 exp1-4-760 0.0 exg1-4-250 13.7 hlh-4-220 15.4 ME3-EM3-80 17.6 tel-2-420 19.2 ste3-4-380 19.9 ME1-EM2-900 20.8 lac4-4-230 22.3 ste3-2-600 24.6 ste3-3-510 27.3 flp1-4-710 38.6 S234R/F 50.7 LG8 zin2-4-250 0.0 ste3-3-530 5.3 tlg1-3-810 9.5 ME6-EM3-440 10.4 zip1-4-50 13.2 gpd-4-400 14.1 ftf2-3-340 16.1 icb-2-405 17.2 ME6-EM1-380 18.2 zin1-3-418 19.0 zin2-4-650 19.8 lac4-4-250 20.4 ste3-3-640 21.0 ME5-EM7-330 21.4 ftf3-3-240 22.6 ME4-EM2-610 23.4 ghf30-2-350 23.7 ghf30-2-310 24.2 mlg1-2-360 24.8 ME7-EM5-280 25.3 ste3-2-650 25.5 ME3-EM8-410 25.7 gb1-4-380 25.8 cut-4-90 26.1 mlg1-2-140 26.2 ME5-EM5-280 ghf30-2-550 26.4 ME4-EM3-310 26.7 hlh-4-1000 26.8 ME2-EM5-420 27.0 pro1-4-120 27.1 zin1-3-650 27.2 ME2-EM6-440 27.3 ME5-EM5-790 27.7 ME1-EM6-410 ME3-EM5-560 27.8 ME2-EM6-70 28.0 ME5-EM7-260 ME5-EM8-840 28.1 pep1-3-245 pro1-4-50 28.2 ME6-EM3-365 28.7 chd1-2-200 28.9 ME4-EM2-500 29.1 ghf30-2-40 29.8 ftf1-2-650 30.3 ME7-EM5-820 30.6 exg1-4-620 30.9 baw28-2-500 31.3 mdh1-4-210 31.5 gb1-4-550 32.2 zin2-4-240 33.5 gb1-4-500 35.0 tyr-3-420 35.7 exg2-3-750 37.6 ste3-3-500 38.5 pep1-3-680 39.9 ME6-EM1-340 40.8 chi2-3-380 * 42.0 ME1-EM4-850 44.3 ftf2-3-540 53.9 flp1-4-260 63.0 Qm gr 2 6. 3 9 % LG9 Fig 1e (continued).
zip1-4-190 0.0 lac4-4-1200 8.8 ME4-EM3-480 15.9 ME2-EM6-310 18.4 S11R/F 27.4 mlg1-2-340 36.7 ftf3-3-95 40.9 ME4-EM7-500 56.6 pep1-3-720 60.2 exp1-4-740 66.1 gpd-4-340 67.3 ghf30-2-395 71.0 ME4-EM2-700 71.9 hsf-3-180 72.9 ftf3-3-190 75.0 ME7-EM5-530 76.3 ME3-EM8-720 78.4 mip-4-880 79.7 ME6-EM8-300 80.0 ME5-EM8-250 80.6 ftf2-3-320 81.1 cut-4-380 81.5 baw28-2-880 81.9 zin2-4-700 82.3 ste3-2-410 82.4 mip-3-1100 82.5 tel-3-280 83.2 tel-2-270 83.3 ME5-EM8-154 83.7 ME6-EM1-780 84.0 pro1-4-1000 84.3 tel-4-275 84.7 cut-4-605 85.0 gpd-4-250 85.4 ME4-EM2-60 86.1 ME5-EM7-320 86.7 ME6-EM3-800 88.2 mip-2-455 90.6 mip-2-500 93.1 LG10 BBL1R/F 0.0 DTF1R/F 9.9 MAT-B 28.1 exp1-4-190 30.1 exg1-4-680 36.9 exg1-4-610 38.6 mip-4-230 40.8 baw28-2-950 42.0 gb1-4-330 43.1 cut-4-382 43.7 exp1-4-220 43.9 zin1-3-270 44.1 ste3-4-180 44.8 ftf3-3-850 46.4 mip-4-250 48.3 zip1-4-390 49.7 ME4-EM2-750 57.6 exp1-4-180 68.1 LG11 mlg1-2-400 * 0.0 exg2-3-550 12.2 ME6-EM8-160 14.1 mip-3-150 14.4 tel-3-650 16.6 ME7-EM6-290 * 21.6 LG12 gpd-4-120 * 0.0 gpd-4-125 9.5 mip-2-155 11.8 ME7-EM6-320 13.6 mip-4-900 15.2 icb-2-430 17.1 zin1-3-285 17.5 ME4-EM7-390 17.9 ME2-EM6-580 18.4 rab-4-270 18.5 ME2-EM6-570 18.7 exg2-3-500 19.0 ME5-EM7-385 19.4 ftf2-3-220 20.0 mip-4-560 21.4 flp1-4-100 23.6 flp1-4-150 25.2 mip-4-225 46.8 pep1-3-360 48.5 gpd-4-600 50.3 ftf3-3-320 51.9 ste3-2-1100 52.4 mdh1-4-390 53.0 ME1-EM6-730 53.7 ftf3-3-950 54.5 pro1-4-360 55.2 ME6-EM3-320 56.2 zin2-4-225 57.1 cho-2-410 58.8 ME4-EM2-295 60.3 chd1-2-650 66.2 ME7-EM5-250 68.8 Qdg r6 8. 21% Qdgr 7 11. 1 5% LG13 Fig 1e (continued).
The physical distance per unit of recombination for L. edo-des was roughly estimated to be 40.0 kbp/cM in our map, which was close to that of P. ostreatus (35.1 kbp/cM) (Larraya et al. 2000) and A. bisporus (33.1 kbp/cM) (Foulongne-Oriol et al. 2010).
QTLs controlling the vegetative mycelium growth rate
For fundamental and applied interest, the dissection of ge-netic architecture of mycelium growth has been carried out in edible mushrooms using QTL mapping or other methodol-ogies (Larraya et al. 2001, 2002; Foulongne-Oriol 2012; Sivola-pova et al. 2012). In this study, we used three different segregation populations to uncover the loci regulating and controlling the vegetative mycelium growth rate of L. edodes. Two QTLs accounting for the monokaryotic mycelium growth rate were detected using SSIs, whereas 13 dikaryotic myce-lium growth rate-related QTLs were identified based on two testcross dikaryotic populations across three different media. Notably, several vegetative growth rate-related QTLs in L. edo-des uncovered here were clustered on LG4 (Qmgr1, Qdgr1, Qdgr2, and Qdgr9) and LG6 (Qdgr3, Qdgr4 and Qdgr5). Among them, the robust QTL Qdgr2 was found in three media in LQ-15, contributing 8.07 %e23.71 % of the phenotypic variation. These results suggested the predominant role of a few princi-pal genomic regions in regulating the vegetative mycelium growth rate.
Qmgr1, Qdgr2 and Qdgr9 were all linked with the InDel marker S278R/F, which was 0.3 cM away from MAT-A. Qmgr1
and Qdgr2 probably corresponded to the same gene since they were mapped on consistent locations with the same negative additive effect, while Qdgr9 was probably on a different locus with a different sign in the additive value. The linkage between MAT-A and Qmgr1, Qdgr1, Qdgr2, and Qdgr9 revealed an associa-tion of vegetative mycelium growth with sexual recogniassocia-tion in L. edodes. Indeed, similar associations have been observed in P. ostreatus (Larraya et al. 2001; Sivolapova et al. 2012) and Amylos-tereum areolatum (van der Nest et al. 2009), in which the MAT-A locus was positioned in the same region as the putative signifi-cant QTLs associated with mycelium growth rate. A possible ex-planation for the association is that the recognition loci are subject to evolutionary forces (balancing selection and sup-pressed recombination) that are markedly different from those acting on the rest of the genome (van der Nest et al. 2009). It is likely that mycelium growth is influenced by balancing selec-tion acting on its recogniselec-tion loci, or the genomic regions flank-ing these loci (van der Nest et al. 2009).
QTLs for dikaryotic mycelium growth rates detected in LQ-15 and LQ-64 showed no consistency in location, indicating strong effects of the two compatible tester strains on dikary-otic mycelium growth rate. The localization of QTLs was dif-ferent on difdif-ferent media in the same population, implying a considerable effect of the substrate on phenotypic variation. SD is a complex medium used to produce shiitake mushroom, therefore QTLs detected in it would provide more instructive clues for breeding schemes.
In our study, some TRAP markers, generated from several potentially functional genes, were closely linked to QTLs
Table 2e Statistical characterization of vegetative mycelium growth rates of L. edodes in different populations and media.
Trait Range Minimum
value Maximum value Mean standard error Standard deviation Variance Coefficient of variation (%) MGR (mm d1) 3.000 0.267 3.267 1.219 0.046 0.551 0.303 45.2 DGR-15myg (mm d1) 2.778 2.778 5.556 4.630 0.046 0.549 0.301 11.9 DGR-15cym (mm d1) 2.666 2.667 5.333 4.135 0.051 0.610 0.372 14.8 DGR-15sd (mm d1) 2.350 2.100 4.450 3.598 0.037 0.441 0.195 12.3 DGR-64myg (mm d1) 2.611 2.722 5.333 4.539 0.032 0.384 0.148 8.5 DGR-64cym (mm d1) 2.444 2.778 5.222 4.559 0.036 0.432 0.187 9.5 DGR-64sd (mm d1) 2.408 2.567 4.975 4.065 0.035 0.423 0.179 10.4
Notes: MGR (monokaryotic growth rate) means growth rates of the 146 SSIs in the mapping population.
DGR-15myg, DGR-15cym and DGR-15sd (DGR: dikaryotic growth rate) indicate growth rates of the 146 dikaryons in LQ-15 on the MYG, CYM and SD media, respectively.
DGR-64myg, DGR-64cym and DGR-64sd indicate growth rates of the 146 dikaryons in LQ-64 on the MYG, CYM and SD media, respectively.
Table 3e Correlation coefficients between vegetative mycelium growth rates of L. edodes in different populations and media.
Trait DGR-15myg DGR-15cym DGR-15sd DGR-64myg DGR-64cym DGR-64sd
MGR 0.246 (0.003) 0.171 (0.041) 0.155 (0.065) 0.152 (0.068) 0.058 (0.495) 0.064 (0.447) DGR-15myg 0.646 (<0.001) 0.338 (<0.001) 0.174 (0.038) 0.086 (0.309) 0.052 (0.541) DGR-15cym 0.275 (0.001) 0.101 (0.229) 0.018 (0.834) 0.021 (0.805) DGR-15sd 0.092 (0.275) 0.021 (0.802) 0.123 (0.142) DGR-64myg 0.591 (<0.001) 0.305 (<0.001) DGR-64cym 0.252 (0.002)
Notes: The abbreviations in this table follow those inTable 2. Numbers in brackets represent P-values. The correlations are significant with
controlling the vegetative mycelium growth rate (Table 4, Fig 1). These genes included cell wall degradation-related en-zyme-encoding genes (mlg1), putative fungal-specific tran-scription factor ( ftf2) and the glyceraldehyde-3-phosphate dehydrogenase gene ( gpd ). The mlg1 gene encodes a gluca-nase of the GH16 family (Sakamoto et al. 2009), which could break down glucans. The TRAP marker, mlg1-2-420, was found to be linked to Qdgr12, suggesting an involvement of mlg1 in carbohydrate degradation of L. edodes in the mixed SD medium. In L. edodes, gpd is expressed constitutively and strongly during each stage of fruiting-body development (Hirano et al. 1999). This gene encodes a key enzyme in both glycolysis and gluconeogenesis, and may serve to supply en-ergy and substrates for mycelium growth. The fungal-specific transcription factor ftf2 encodes proteins that contain a Zn(2)-C6 fungal-type DNA-binding domain in the
N-terminal, as well as a fungal-specific transcription factor do-main at the center (Sakamoto et al. 2009). In Candida albicans, some transcription factors containing the Zn(2)-C6 motif
were found to be involved in filamentous growth
(MacPherson et al. 2006). TRAP markers could target poten-tially functional genes and may provide additional clues for the conversion of mapped QTLs to candidate genes.
Conclusions
Molecular breeding is a powerful tool for genetic improvement of crops and animals. However, there is still a long way to uti-lize this method in genetic improvement of edible mushrooms due to the deficiency of genetic research on their important economic and agronomic traits. In the present study, a high-quality genetic map of L. edodes was constructed, and a total
Table 5e Comparison of different genetic linkage maps of L. edodes.
Description Kwan &
Xu (2002)
Terashima et al. (2006)
Miyazaki et al. (2008)
Au et al. (2013) Current study
Population size 34 95 92 20 146
Main genetic markers RAPD AFLP RAPD, SCAR,
Genes
Sequence-based markers
SRAP, TRAP, InDel
Map total length (cM) 622.4 1398.4 908.8 637.1 1006.1
Number of mapped markers 69 166 289 200 524
Number of linkage groups 14 11 11 13 13
Average marker spacing (cM)* 11.3 9.0 3.3 3.4 2.0
Number of gaps (>20 cM) 6 9 2 0 1
Note: * The average marker spacing (cM) over the whole maps was calculated by dividing the total length of all LGs by the number of marker intervals.
Table 4e QTLs controlling the mycelium growth rate of L. edodes detected in different populations and media.
Population Medium QTL LG Position (cM)a Markerb LOD Additive effectc CI (cM)d R2(%)
Monokaryon MYG Qmgr1e 4 71.21 S278R/F 2.77 0.14 68.1e77.6 6.89
Qmgr2e 9 44.31 ME1-EM4-850 2.58 0.14 43.0e49.7 6.39
LQ-15 MYG Qdgr1 4 67.61 MAT-A 4.85 0.25 64.3e70.6 16.79
Qdgr2 4 73.21 S278R/F 9.60 0.27 71.2e77.4 23.71 Qdgr3 6 16.21 ME7-EM6-195 3.22 0.17 15.4e18.2 6.58 Qdgr4 6 34.71 lup23-3-950 4.38 0.20 34.1e35.6 9.09 Qdgr5 6 41.31 ME6-EM1-450 3.15 0.18 40.7e42.1 6.67 CYM Qdgr2 4 72.21 S278R/F 7.89 0.27 71.1e77.7 18.51 Qdgr4 6 34.71 lup23-3-950 7.57 0.30 34.3e35.4 16.83 Qdgr5 6 41.31 ME6-EM1-450 5.47 0.27 40.7e42.6 12.58 SD Qdgr2 4 71.21 S278R/F 3.21 0.13 70.9e76.4 8.07 LQ-64 MYG Qdgr6e 13 8.01 gpd-4-125 2.76 0.11 0e11.3 8.21 Qdgr7 13 21.01 ftf2-3-220 3.40 0.13 20.1e21.4 11.15
CYM Qdgr8e 1 99.91 ME3-EM3-390 2.61 0.12 95.0e105.1 7.09
Qdgr9e 4 77.21 S278R/F 2.75 0.13 71.6e83.8 9.54
Qdgr10 7 33.21 cut-4-340 3.01 0.12 33.2e34.5 7.05
SD Qdgr11e 2 80.31 S413R/F 2.67 0.11 79.8e81.9 6.44
Qdgr12 7 15.81 mlg1-2-420 2.96 0.12 15.2e16.6 7.14
Qdgr13e 7 21.31 ME4-EM3-70 2.54 0.11 19.5e22.9 6.17
a Position of the LOD score peak in cM. b Nearest marker to the LOD score peak.
c The additive effect of QTLs; a minus sign means that the favorable allele of the QTL came from the parental strain W1-26, while a plus sign means that the favorable allele of the QTL came from the parental strain L205-6.
d Confidence intervals supported by LOD-1.
of 15 putative QTLs for the vegetative mycelium growth rate were detected in the subsequent QTL mapping. A QTL hotspot region on LG4 of the current genetic map, which was found to be associated with both the monokaryotic and dikaryotic my-celium growth rates, was reported here. The present study demonstrates the potential of using the current linkage map to identify QTLs for traits that are more difficult to measure in L. edodes, such as yield and yield-related traits. In summary, this study lays a solid foundation for establishing efficient ge-netic and breeding programs in L. edodes. With the completion of genome sequencing of L. edodes (Kwan et al. 2012), more and more sequence tag site markers, such as simple sequence re-peat (SSR) and single nucleotide polymorphism (SNP) markers, are expected to be developed for studies on genetic map construction and QTL mapping of L. edodes.
Disclosure statement
None of the authors has any conflict of interest to disclose.
Acknowledgments
This work was financially supported by National Natural Sci-ence Foundation of China (No. 31000929). We thank Prof. Bo Wang (Sichuan Academy of Agricultural Sciences) for provid-ing the wild strain of L. edodes.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.funbio.2014.01.001
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