Mycol. Res. 106 (8) : 911917 (August 2002).

# The British Mycological Society

911

DOI : 10.1017\S0953756202006275 Printed in the United Kingdom.

A genetic linkage map of Lentinula edodes (shiitake) based on AFLP markers*

Kazuhisa TERASHIMA1,2, Teruyuki MATSUMOTO2, Eiji HAYASHI3 and Yukitaka FUKUMASA-NAKAI2

" Domestic Research Fellow, Japan Science and Technology Corporation (JST ), Honmachi 4-1-8, Kawaguchi, Saitama 332-0012, Japan. # The Tottori Mycological Institute, Kokoge 211, Tottori 689-1125, Japan. $ Forest Tree Breeding Center, Ishi, Juo, Taga, Ibaraki 319-1301, Japan.
Received 31 January 2002 ; accepted 16 June 2002.

A medium-dense genetic linkage map of Lentinula edodes (shiitake) was constructed based on 203 amplied fragment length polymorphism (AFLP) markers and two mating type factors. The segregation of these markers was generated from 95 progeny of a single cross of two distantly related strains. The map consists of 11 linkage groups comprised of eight large (over 100 centimorgans (cM)) and three small (less than 100 cM) groups, and the total genetic distance of the map was 1956.7 cM. The average rate of physical size to genetic distance could be roughly estimated to be less than 18.4 kb cM", which is low compared to the values obtained for other lamentous fungi. Seventeen of the AFLP markers showed highly distorted segregation ratios (# values 6.63 ; P 0.01), and many of these were mapped in LG II (6 markers) and IV (6 markers).

INTRODUCTION Lentinula edodes, shiitake, is one of the most popular edible mushrooms in east Asia. In Japan, the production of shiitake, in terms of fresh weight and farm value, is the highest among the various cultivated mushrooms. Apart from its importance as a mushroom crop, it has also been found that L. edodes contains medicinal compounds, including lentinan, which has antitumor activity, the hypocholesterolemic eritadenine, and cortinellin, an antibacterial agent (Przybylowicz & Donoghue 1988, Matsuoka et al. 1997, Sugiyama et al. 1997). Thus, research on this fungus has increased due to its agricultural and pharmacological properties. In particular, improving the properties of the mushroom at the genetic level, so as to facilitate the cultivation of the fruit body, has been pursued in Japan for 50 years (Hashioka, Komatsu & Arita 1961, Tokimoto & Komatsu 1995). Trials on breeding for specic medicinal or dietary qualities has also recently started. Studies on shiitake include not only those investigating its uses in agriculture and pharmacology, but also its cytology, genetics, population genetics, and taxonomy (Takemaru 1961, Tanaka & Koga 1972, Tokimoto, Komatsu & Takemaru 1973, Pegler 1983,
* Contribution No. 355 of The Tottori Mycological Institute.

Nakai 1986, Fukuda et al. 1994, Hibbett et al. 1995, Hibbett, Hansen & Donoghue 1998). Genetic studies include the isolation and characterization of the various genes expressed during fruit body development (Zhao & Kwan 1999, Leung et al. 2000, Ng, Ng & Kwan 2000, Kaneko & Shishido 2001). In addition, light microscopic observations and electrophoretic karyotype analysis have suggested that this fungus contains at least eight chromosomes in the haploid genome (Tanaka & Koga 1972, Nakai 1986, Arima & Morinaga 1993). Despite these studies, however, the genomic organization of L. edodes remains poorly understood. Genetic linkage analysis using a large number of genetic markers has been conducted for various plants (Maheswaran et al. 1997, Harushima et al. 1998, Hayashi et al. 2001) and fungi (Tzeng et al. 1992, Forche et al. 2000, Larraya et al. 2000). This allows meiotic behaviour and genome organization to be claried. The resulting linkage map is also useful for ecient cross-breeding and can be used in map-based cloning of genes of interest (Farman & Leong 1998). Two linkage maps have been constructed for L. edodes, in which one comprised ve linkage groups with 21 genetic markers, including seven mutation markers for mycelial morphology, 11 auxotrophic markers, two mating type loci and one lethal factor (Hasebe 1991), and the other consisted of 3 linkage groups with 12

AFLP linkage map of Lentinula edodes allozyme markers (Bowden & Royse 1991). Thus, this linkage map does not cover the majority of the L. edodes genome and is of limited use. A high-density genetic linkage map that includes a wider variety of molecular markers is thus required for L. edodes. AFLP (amplied fragment length polymorphism) analysis was developed by Vos et al. (1995). This technique is based on the selective PCR amplication of genomic DNA restriction fragments and has been shown to reproducibly detect a large number of polymorphic loci (Vos et al. 1995). AFLP markers have also been used to estimate genetic diversity (Boucias et al. 2000, Vandemark et al. 2000, Terashima, Cha & Miura 2001, Terashima et al. 2002) and can also be employed in genetic linkage analysis (Maheswaran et al. 1997, Forche et al. 2000, Hayashi et al. 2001). In this study, we aimed to : (1) identify a large number of genetic markers using AFLP analysis ; (2) construct a linkage map that will provide a framework for future genetic studies on L. edodes ; and (3) obtain additional knowlege of genome organization of this mushroom. MATERIALS AND METHODS Establishment of the mapping population Hibbett et al. (1995, 1998) reported that the natural shiitake populations in Japan and New Zealand are phylogenetically distinct. It can thus be expected that these populations are quite dierent with regard to their genomic DNA sequences and that a cross between these populations will produce progeny with a large number of polymorphic AFLP markers. Therefore, in this study, the A567-S8 strain from Japan and the NZ 1569-S3 strain from New Zealand were selected as the parental strains used to generate the mapping population. The former strain is a basidiospore progeny from the Japanese commercial strain A567 distributed by the Akiyama Mycological Institute, Yamanashi, Japan. The latter is a basidiospore progeny from the wild strain TMIC 1569 collected in New Zealand ; they are deposited in the culture collection of the Tottori Mycological Institute (Fukuda et al. 1994). The mapping population was derived from a single cross between these two strains, and 95 basidiospore progeny generated by this cross were used to determine the segregation of AFLP markers and linkage relationships. To determine the mating types, each of the progeny was mated with four tester strains selected from the mapping population. AFLP procedure Cultures were maintained on 2 % malt-extract agar. Cultures for DNA extraction were grown at 25 mC for 4 wk on liquid MYG broth (2 % malt extract, 2 % glucose, 0.2 % yeast extract). Mycelia were harvested onto lter paper, rinsed with distilled water, and freezedried. Genomic DNA for each progeny was extracted

912 by use of the DNeasyTM Plant Mini kit (Qiagen, Hilden) and DNAs were eluted twice from the DNA binding column with 100 l AE solution. AFLP analysis was carried out by using a modication of the procedure described by Vos et al. (1995), the instruction manual of the AFLP Core Reagent Kit (Invitrogen, Carlsbad, CA), and the AFLP Microbial Fingerprinting kit (Applied Biosystems, Foster, CA). Digestion of genomic DNA and ligation with adaptors was carried out with the AFLP Core Reagent Kit. Genomic DNA (1 l of the DNA solution) was digested with endonucleases (EcoRI and MseI) using half of the reagent volumes recommended by the manufacturer. The reaction mixtures were diluted 10fold with TE (10 m TrisHCl, 0.1 m EDTA, pH 8.0). For preselective amplication, PCRs were performed in a TaKaRa PCR Thermal cycler MP (Takara Biomedicals, Shiga) with the Ej0 primer (5h-GAC TGC GTA CCA ATT C-3h) and the Mj0 primer (5hGAT GAG TCC TGA GTA A-3h) (Vos et al. 1995). Takara Ex TaqTM (Takara Biomedicals) was used as DNA Taq polymerase. Reaction conditions and the thermocycler programme used for preselective amplication were as described by Vos et al. (1995). Preselective amplication reaction mixtures were diluted 20-fold with TE before being further amplied with selective primers. Selective amplication was also performed in a TaKaRa PCR Thermal cycler MP using 0.5 pmol Ej2 primer (jAC or jAT) and 2.5 pmol Mj2 primer (jCA, jCC, jCG, or jCT) in a total reaction volume of 10 l. The Ej2 and Mj2 primer had two nucleotide extensions given in above brackets with Ej0 and Mj0 primer, respectively. The sizes of the nucleotide extensions of the selective primers were according to Majer et al. (1996) and Terashima et al. (2001, 2002). Concentrations of all reagents used except the primers were as described by Vos et al. (1995), while the thermocycling conditions employed were as described in the instruction manual of the AFLP Microbial Fingerprint Kit. Electrophoresis and detection of amplied fragments were performed by using the ABI PRIZMTM 310 Genetic Analyzer (Applied Biosystems). Linkage analysis Linkage analysis was performed using MAPMAKER version 3.0 (Lander et al. 1987) and MAPL software (Ukai et al. 1995). Linkage groups (LG) were generated by two point analysis ( group command) in MAPMAKER with a likelihood of odds (LOD) score of 3.05.0, and were compared with those formed by the nearest neighboring locus methods in MAPL with critical recombination values of 0.284 and 0.295. Subsequently, consensus groups of the markers obtained by the two methods were adopted. Ordering of markers was carried out by combining the analytical methods of the two computer programs

K. Terashima and others mentioned above. The multi-dimensional scaling (MDS) method in MAPL automatically generates order of all markers, while ordering markers using MAPMAKER is tedious. However, it is dicult to determine the local order of markers with MAPL. In this study, therefore, the ordering of the markers in the linkage groups was rst performed using the MDS method, after which the local orders were rearranged using the ripple command in MAPMAKER. Finally, the linkage map was constructed by using the Kosambi map function in the MAPMAKER program.

913 In this study, we could not determine which components comprised the B mating factor (e.g. mat B-1 from NZ1569-S3 and mat B-2 from A567-S8, or the converse pair) in the single progeny with the recombined B mating factor. Therefore, the location of mat B- and mat B- are only tentatively indicated on the linkage map.

Linkage analysis The linkage map of Lentinula edodes is shown in Fig. 1, and the features of the linkage groups are summarized in Table 1. A total of 206 markers were assigned to 11 linkage groups with LOD values that ranged from 3.7 to 4.8, as measured by MAPMAKER. The linkage groups were supported by analysis using MAPL at a critical recombination value of 0.284, in which this value was expected in organisms with an actual chromosome number of eleven (Ukai et al. 1995). The ordering of the markers in the linkage groups was rst carried out using MAPL. After this analysis, the nal order of the markers in the linkage groups was determined by MAPMAKER, after which the genetic linkage map was constructed, again by MAPMAKER. The map covers a total length of 1956.7 centimorgans (cM) with an average marker interval distance of 9.5 cM. Linkage group length varied between 22.2 cM (LG XI) and 363.5 cM (LG I), with an average of 177.9 cM per linkage group. The number of markers used to construct the linkage groups ranged from 4 (LG XI) to 37 (LG I), with an average of 18.7 markers per group. The average marker interval ranged from 7.4 cM (LG XI) to 19.6 cM (LG IX), with the minimum and maximum marker interval being 0.0 cM (between EacMcc292 and EatMcg101 in LG II) and 42.2 cM (between EacMcc450 and EatMcc374 in LG V), respectively. Forty-two AFLP markers with distorted segregation ratios were mapped and distributed into eight linkage groups (LGs I, II, III, IV, V, VII, VIII, IX). All of those in LGs II, IV, VIII and IX were inherited from parent NZ1569-S3, while those in LGs III, V and VII were inherited from parent A567-S8. Seventeen of the AFLP markers showed highly distorted segregation ratios, and many of these were mapped in LG II (6 markers) and IV (6 markers).

RESULTS AFLP markers and mating type factors AFLP analysis was performed with eight selective primer pairs and allowed the identication of 673 DNA fragments. Of these, 486 (72.2 %) represent polymorphisms between the parental strains (A567-S8 and NZ1569-S3). The total number of DNA fragments detected by individual primer pairs ranged from 67 (for the primer pair EjAC\MjCT) to 122 (EjAT\ MjCA). The number of polymorphic DNA fragments identied by each primer pair varied from 48 for EjAT\MjCG to 78 for EjAT\MjCA, with an average of 60.8 polymorphic fragments per primer pair. Considering the percentage of polymorphic DNA fragments against total number of DNA fragments, primer pairs also detected dierent level of polymorphisms, ranging from 63.2 % (EjAT\MjCG) to 83.1 % (EjAC\MjCG). Many of the polymorphic DNA fragments generated by AFLP analysis were close to each other and thus dicult to identify in the progeny making up the mapping population. Furthermore, preliminary genetic analysis suggested that some of the AFLP markers had an extremely extended map size and dramatically changed marker order. These ambiguous markers were discarded. Consequently, 203 AFLP markers were used to construct the genetic linkage map. Of these markers, 25 (12.3 %) showed signicant segregation distortion (3.84 # values 6.63 ; 0.01 P 0.05), and 17 (8.4 %) expressed highly distorted segregation ratios (# values 6.63 ; P 0.01). Of these 42 AFLP markers with distorted segregation ratios, 31 were inherited from parent NZ1569-S3, and 11 were inherited from A567S8. Mating type factors were determined by mating each of the progeny from the cross of the parental strains with tester strains. The A mating type factor (mat A) segregated with two allelic forms and the segregation rate was not signicantly distorted. For the B mating type factor (mat B), one of the progeny revealed a dierent phenotype to that of parent NZ1569-S3 or A567-S8, probably as a result of recombination between two linked subloci of the B mating factor (mat B- and mat B-). The segregation rate of the B mating type factor of the two parents was not signicantly distorted.

DISCUSSION In this study, we used AFLP to identify a large number of genetic markers in the genome of Lentinula edodes. These markers were used together with mating type factors to construct a medium-density linkage map providing a frame work of future genetic study of L. edodes. In addition, some information on the genome organization was obtained. The linkage map consists of eleven linkage groups,

AFLP linkage map of Lentinula edodes

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mat A

Fig. 1. For legend see facing page.

including eight large linkage groups (LG IVIII) over 100 cM in size and three small ones (LG IXXI) less than 100 cM long. This suggests that L. edodes has 11 chromosomes. However, previous reports, based on light microscopic observation and electrophoretic karyotype analysis, have suggested that this fungus has only eight chromosomes (Tanaka & Koga 1972, Nakai 1986, Arima & Morinaga 1993). It is possible that the three small linkage groups we identied could be homologous to partial segments of one or more of the eight large chromosomes. Alternatively, they may be true chromosomes overlooked in previous studies. It is dicult to determine the chromosome number from linkage analysis because the number of linkage groups is dependent on the analysis parameters set. However,

it is also possible in light microscopic observation to underestimate the number of chromosomes if some are small. Furthermore, in the electrophoretic karyotype analysis of L. edodes, a clear gel image could not be obtained, probably because an adequate number of protoplasts (over 10) protoplasts ml") could not be generated. Thus, whether L. edodes has eight or 11 chromosomes remains unclear. It will be necessary to use electron microscopic observations and (or) improved electrophoretic karyotype studies to resolve this question. If we assume that the eight large linkage groups identied in this study are homologous to the eight chromosomes reported previously, the average rate of physical size to genetic distance can be roughly

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mat B- mat B-

Fig. 1. Genetic linkage map of Lentinula edodes based on 203 AFLP markers and three loci of mating type factors. AFLP markers were named according to the primer designation, followed by the size of the amplied fragment in base pairs. Markers with signicant (0.01 P 0.05) and high (P 0.01) segregation distortion are respectively indicated by asterisks (*) and double asterisks (**).

estimated as less than 18.4 kb cM". This value is low if it is compared to that of the other lamentous fungi reported previously, for example, Cochliobolus heterostrophus (23 kb cM") (Tzeng et al. 1992), Bremia lactucae (25 kb cM") (Hulbert et al. 1988), Gibberella fujikuroi (32 kb cM") (Xu & Leslie 1996), Pleurotus ostreatus (35 kb cM") (Larraya et al. 2000) and Neurospora crassa (43 kb cM") (Davis 1995). If these studies are examined carefully, it appears that low ratios of physical to genetic distance in linkage maps generally occur when the linkage analyses are performed using distantly related parents. Thus, the values for C. heterostrophus and B. lactucae were generated by

distantly related crosses while the higher values obtained for the other species were generated by crosses between more closely related parents. Evidence supporting this hypothesis could be obtained by performing another linkage analysis on a cross between more closely related L. edodes parents. Of the 203 AFLP markers identied, 42 (20.7 %) showed segregation distortion (# values 3.84 ; P 0.05) and many were mapped to the almost continuous area of LG II or LG IV (Fig. 1). It has been often suggested that skewed segregation of genetic markers in fungi is caused by a biased selection of the spore isolates used as the mapping population (Kerrigan

AFLP linkage map of Lentinula edodes

Table 1. Characteristics of genetic linkage groups with AFLP markers and mating type factor in Lentinula edodes. No. skewed marker Linkage groups LG I LG II LG III LG IV LG V LG VI LG VII LG VIII LG IX LG X LG XI Total Length (cM) 363n5 277n2 229n0 219n4 199n6 175n4 174n5 150n4 97n9 47n6 22n2 1956n7 No. marker 37 34 21 24 22 18 18 15 6 7 4 206 Avg. marker interval (cM) 10n1 8n4 11n5 9n6 9n5 10n3 10n3 10n8 19n6 8n0 7n4 2 6 0 4 3 0 4 3 3 0 0 25 0n05 1 6 1 6 2 0 0 1 0 0 0 17 0n01

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et al. 1993, Larraya et al. 2000). In this study, although the germination rate of basidiospores was high (greater than 95 %), the mapping population did include spore isolates with varying growth rates. This means that sucient mycelia for DNA extraction from some spore isolates could not be obtained. The low growth rates of these isolates is probably mainly due to their unsuitable genetic composition. In L. edodes, two lethal factors have been reported, one of which was linked to mat A (Hasebe 1991). We noted that some of the skewed markers present in LG II were linked to mat A. Thus, partial expression of a lethal factor might reduce the growth rate of spore isolates and could be responsible for the segregation distortion of markers in LG II. Alternatively, it is known that gene conversion leads to non-Mendelian segregation of genetic markers in Schizosaccharomyces pombe and Saccharomyces cereviseae, and that markers near recombination hotspots frequently undergo co-conversion (Davis & Smith 2001). As the linkage analysis in this study was carried out by mating distantly related strains, it can be expected that there are many dierences in genome sequence between the parent strains, which might permit heteroduplex DNAs to be produced on a large area of homologous chromosome during meiosis. Thus, it is not unreasonable to suppose that mechanisms used to repair heteroduplex DNA, such as gene conversion, could be responsible for the segregation distortion of at least some of the markers. Linkage analysis with AFLP markers makes it possible to map the loci that control qualitative and quantitative traits of agricultural and pharmacological signicance in L. edodes, such as morphology and fruit body colour, yield of fruit bodies, fruiting seasons, resistance toward pathogens such as Trichoderma spp., and production of medicinal compounds. Furthermore, such maps will enhance our ability to eciently select strains with desired characteristics. Thus, genetic linkage analysis of L. edodes with AFLP markers will contribute to our understanding of its genome organization and will facilitate the eciency of crossbreeding programmes.

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