Lenti-AFLP.pdf

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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 amplified 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 filamentous fungi. Seventeen of the AFLP

markers showed highly distorted segregation ratios (# values 6.63; P0.01), and many of these were mapped in

LG II (6 markers) and IV (6 markers).

I N T R O D U C T I O N

Lentinula edodes, shiitake, is one of the most popularedible mushroomsin east Asia. In Japan, the production

of shiitake, in terms of fresh weight and farm value, isthe highest among the various cultivated mushrooms.

Apart from its importance as a mushroom crop, it hasalso been found that L. edodes contains medicinalcompounds, including lentinan, which has antitumor

activity, the hypocholesterolemic eritadenine, andcortinellin, 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. Inparticular, improving the properties of the mushroom

at the genetic level, so as to facilitate the cultivation ofthe fruit body, has been pursued in Japan for 50 years(Hashioka, Komatsu & Arita 1961, Tokimoto &

Komatsu 1995). Trials on breeding for specific med-icinal or dietary qualities has also recently started.

Studies on shiitake include not only those investi-gating its uses in agriculture and pharmacology, but

also its cytology, genetics, population genetics, andtaxonomy (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 studiesinclude 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 micro-

scopic observations and electrophoretic karyotypeanalysis have suggested that this fungus contains at

least eight chromosomes in thehaploid genome(Tanaka& Koga 1972, Nakai 1986, Arima & Morinaga 1993).

Despite these studies, however, the genomicorganization 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 beclarified. The resulting linkage map is also useful forefficient 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 five linkage groups with 21genetic markers, including seven mutation markers for

mycelial morphology, 11 auxotrophic markers, twomating type loci and one lethal factor (Hasebe 1991),and the other consisted of 3 linkage groups with 12

Mycol. Res. 106 (8): 911917 (August 2002). # The British Mycological Society

DOI: 10.1017\S0953756202006275 Printed in the United Kingdom.

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


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AFLP linkage map of Lentinula edodes 912

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 ofmolecular markers is thus required for L. edodes.

AFLP (amplified fragment length polymorphism)analysis was developed by Vos et al. (1995). Thistechnique is based on the selective PCR amplification of

genomic DNA restriction fragments and has beenshown to reproducibly detect a large number of

polymorphic loci (Vos et al. 1995). AFLP markers havealso 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 beemployed in genetic linkage analysis (Maheswaran etal. 1997, Forche et al. 2000, Hayashi et al. 2001). In thisstudy, we aimed to : (1) identify a large number of

genetic markers using AFLP analysis; (2) construct alinkage map that will provide a framework for future

genetic studies on L. edodes ; and (3) obtain additionalknowlege of genome organization of this mushroom.

M A T E R I A L S A N D M E T H O D S

Establishment of the mapping population

Hibbett et al. (1995, 1998) reported that the naturalshiitake populations in Japan and New Zealand arephylogenetically distinct. It can thus be expected that

these populations are quite different with regard totheir genomic DNA sequences and that a cross between

these populations will produce progeny with a largenumber 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 theparental strains used to generate the mapping popu-

lation. The former strain is a basidiospore progenyfrom 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; theyare deposited in the culture collection of the TottoriMycological Institute (Fukuda et al . 1994). The

mapping population was derived from a single crossbetween these two strains, and 95 basidiospore progeny

generated by this cross were used to determine thesegregation of AFLP markers and linkage relationships.

To determine the mating types, each of the progeny wasmated with four tester strains selected from the mappingpopulation.

AFLP procedure

Cultures were maintained on 2 % malt-extract agar.

Cultures for DNA extraction were grown at 25 mC for4 wk on liquid MYG broth (2% malt extract, 2%

glucose, 0.2% yeast extract). Mycelia were harvestedonto filter paper, rinsed with distilled water, and freeze-dried. Genomic DNA for each progeny was extracted

by use of the DNeasyTM Plant Mini kit (Qiagen, Hilden)

and DNAs were eluted twice from the DNA bindingcolumn with 100 l AE solution.

AFLP analysis was carried out by using a modi-fication of the procedure described by Vos et al. (1995),

the instruction manual of the AFLP Core Reagent Kit(Invitrogen, Carlsbad, CA), and the AFLP MicrobialFingerprinting kit (Applied Biosystems, Foster, CA).

Digestion of genomic DNA and ligation withadaptors was carried out with the AFLP Core Reagent

Kit. Genomic DNA (1 l of the DNA solution) wasdigested with endonucleases (EcoRI and MseI) using

half of the reagent volumes recommended by themanufacturer. The reaction mixtures were diluted 10-fold with TE (10 mTrisHCl, 0.1 mEDTA, pH 8.0).

For preselective amplification, PCRs were performedin a TaKaRa PCR Thermal cycler MP (Takara

Biomedicals, Shiga) with the Ej0 primer (5h-GACTGC GTA CCA ATT C-3h) and the Mj0 primer (5h-

GAT GAG TCC TGA GTA A-3h) (Vos et al. 1995).Takara Ex TaqTM (Takara Biomedicals) was used as

DNA Taq polymerase. Reaction conditions and thethermocycler programme used for preselective ampli-fication were as described by Vos et al . (1995).

Preselective amplification reaction mixtures werediluted 20-fold with TE before being further amplified

with selective primers.Selective amplification was also performed in a

TaKaRa PCR Thermal cycler MP using 0.5 pmol Ej2primer (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 twonucleotide extensions given in above brackets with

Ej0 and Mj0 primer, respectively. The sizes of thenucleotide extensions of the selective primers were

according to Majer et al. (1996) and Terashima et al.(2001, 2002). Concentrations of all reagents used exceptthe primers were as described by Vos et al. (1995), while

the thermocycling conditions employed were as de-scribed in the instruction manual of the AFLP

Microbial Fingerprint Kit.Electrophoresis and detection of amplified fragments

were performed by using the ABI PRIZMTM 310Genetic Analyzer (Applied Biosystems).

Linkage analysis

Linkage analysis was performed using MAPMAKERversion 3.0 (Lander et al. 1987) and MAPL software

(Ukai et al. 1995). Linkage groups (LG) were generatedby two point analysis (group command) inMAPMAKER with a likelihood of odds (LOD) score

of 3.05.0, and were compared with those formed bythe 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


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K. Terashima and others 913

mentioned above. The multi-dimensional scaling

(MDS) method in MAPL automatically generates orderof all markers, while ordering markers using

MAPMAKER is tedious. However, it is difficult todetermine the local order of markers with MAPL. In

this study, therefore, the ordering of the markers in thelinkage groups was first performed using the MDSmethod, after which the local orders were rearranged

using theripple

command in MAPMAKER. Finally,

the linkage map was constructed by using the Kosambi

map function in the MAPMAKER program.

R E S U L T S

AFLP markers and mating type factors

AFLP analysis was performed with eight selective

primer pairs and allowed the identification of 673 DNAfragments. Of these, 486 (72.2%) represent poly-

morphisms between the parental strains (A567-S8 andNZ1569-S3). The total number of DNA fragments

detected by individual primer pairs ranged from 67 (forthe primer pair EjAC\MjCT) to 122 (EjAT\MjCA). The number of polymorphic DNA fragments

identified by each primer pair varied from 48 forEjAT\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 different level of poly-morphisms, ranging from 63.2% (EjAT\MjCG) to

83.1% (EjAC\MjCG). Many of the polymorphicDNA fragments generated by AFLP analysis were

close to each other and thus difficult to identify in theprogeny making up the mapping population. Fur-

thermore, preliminary genetic analysis suggested thatsome of the AFLP markers had an extremely extendedmap size and dramatically changed marker order.

These ambiguous markers were discarded. Conse-quently, 203 AFLP markers were used to construct the

genetic linkage map. Of these markers, 25 (12.3%)showed significant segregation distortion (3.84#

values 6.63; 0.01P0.05), and 17 (8.4%)expressed highly distorted segregation ratios (# values

6.63; P0.01). Of these 42 AFLP markers with

distorted segregation ratios, 31 were inherited from

parent NZ1569-S3, and 11 were inherited from A567-S8.Mating type factors were determined by mating each

of the progeny from the cross of the parental strainswith tester strains. The A mating type factor (mat A)segregated with two allelic forms and the segregation

rate was not significantly distorted. For the B matingtype factor (mat B), one of the progeny revealed a

different phenotype to that of parent NZ1569-S3 orA567-S8, probably as a result of recombination between

two linked subloci of the B mating factor (mat B-andmat B-). The segregation rate of the B mating typefactor of the two parents was not significantly distorted.

In this study, we could not determine which components

comprised the B mating factor (e.g. mat B-1 fromNZ1569-S3and matB-2 from A567-S8, or theconverse

pair) in the single progeny with the recombined Bmating factor. Therefore, the location of mat B- andmat B- are only tentatively indicated on the linkagemap.

Linkage analysis

The linkage map ofLentinula 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, asmeasured by MAPMAKER. The linkage groups were

supported by analysis using MAPL at a criticalrecombination value of 0.284, in which this value wasexpected in organisms with an actual chromosome

number of eleven (Ukai et al. 1995).

The ordering of the markers in the linkage groupswas first carried out using MAPL. After this analysis,the final order of the markers in the linkage groups was

determined by MAPMAKER, after which the geneticlinkage map was constructed, again by MAPMAKER.The map covers a total length of 1956.7 centimorgans

(cM) with an average marker interval distance of9.5 cM. Linkage group length varied between 22.2 cM

(LG XI) and 363.5 cM (LG I), with an average of177.9 cM per linkage group. The number of markers

used to construct the linkage groups ranged from 4 (LGXI) to 37 (LG I), with an average of 18.7 markers pergroup. The average marker interval ranged from 7.4 cM

(LG XI) to 19.6 cM (LG IX), with the minimum andmaximum marker interval being 0.0 cM (betweenEacMcc292 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 thosein LGs II, IV, VIII and IX were inherited from parent

NZ1569-S3, while those in LGs III, V and VII wereinherited 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).

D I S C U S S I O N

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 mapproviding a frame work of future genetic study of L.edodes. In addition, some information on the genomeorganization was obtained.

The linkage map consists of eleven linkage groups,


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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 11chromosomes. However, previous reports, based onlight microscopic observation and electrophoretic

karyotype analysis, have suggested that this fungus hasonly eight chromosomes (Tanaka & Koga 1972, Nakai1986, Arima & Morinaga 1993). It is possible that the

three small linkage groups we identified could behomologous to partial segments of one or more of the

eight large chromosomes. Alternatively, they may betrue chromosomes overlooked in previous studies. It is

difficult to determine the chromosome number fromlinkage analysis because the number of linkage groupsis 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 karyotypeanalysis of L. edodes, a clear gel image could not beobtained, probably because an adequate number of

protoplasts (over 10) protoplasts ml") could not begenerated. Thus, whether L. edodes has eight or 11chromosomes remains unclear. It will be necessary to

use electron microscopic observations and (or) im-proved electrophoretic karyotype studies to resolve this

question.If we assume that the eight large linkage groups

identified in this study are homologous to the eightchromosomes reported previously, the average rate ofphysical 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 amplified fragment in base pairs.

Markers with significant (0.01P 0.05) and high (P0.01) segregation distortion are respectively indicated by

asterisks (*) and double asterisks (**).

estimated as less than 18.4 kb cM"

. This value is low ifit is compared to that of the other filamentous fungireported previously, for example, Cochliobolus hetero-strophus (23 kb cM") (Tzeng et al . 1992), Bremialactucae (25 kb cM") (Hulbert et al. 1988), Gibberella

fujikuroi (32 kb cM") (Xu & Leslie 1996), Pleurotusostreatus (35 kb cM") (Larraya et al . 2000) andNeurospora crassa (43 kb cM") (Davis 1995). If these

studies are examined carefully, it appears that lowratios of physical to genetic distance in linkage maps

generallyoccur when the linkage analyses are performedusing distantly related parents. Thus, the values for C.heterostrophus and B. lactucae were generated by

distantlyrelated crosses while the higher values obtainedfor the other species were generated by crosses betweenmore closely related parents. Evidence supporting this

hypothesis could be obtained by performing anotherlinkage analysis on a cross between more closely relatedL. edodes parents.

Of the 203 AFLP markers identified, 42 (20.7%)showed segregation distortion (# values 3.84;P0.05) and many were mapped to the almostcontinuous area of LG II or LG IV (Fig. 1). It has been

often suggested that skewed segregation of geneticmarkers in fungi is caused by a biased selection of thespore isolates used as themapping population (Kerrigan


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Table 1. Characteristics of genetic linkage groups with AFLP markers and mating type factor in Lentinula edodes.

Linkage

groups

Length

(cM)

No.

marker

Avg. marker

interval (cM)

No. skewed marker

0n05 0n01

LG I 363n5 37 10n1 2 1

LG II 277n2 34 8n4 6 6

LG III 229n0 21 11n5 0 1

LG IV 219n4 24 9n6 4 6

LG V 199n6 22 9n5 3 2LG VI 175n4 18 10n3 0 0

LG VII 174n5 18 10n3 4 0

LG VIII 150n4 15 10n8 3 1

LG IX 97n9 6 19n6 3 0

LG X 47n6 7 8n0 0 0

LG XI 22n2 4 7n4 0 0

Total 1956n7 206 25 17

et al. 1993, Larraya et al. 2000). In this study, althoughthe germination rate of basidiospores was high (greater

than 95%), the mapping population did include sporeisolates with varying growth rates. This means that

sufficient mycelia for DNA extraction from some sporeisolates could not be obtained. The low growth rates ofthese isolates is probably mainly due to their unsuitable

genetic composition. In L. edodes, two lethal factorshave been reported, one of which was linked to mat A

(Hasebe 1991). We noted that some of the skewedmarkers present in LG II were linked to mat A. Thus,

partial expression of a lethal factor might reduce thegrowth rate of spore isolates and could be responsiblefor the segregation distortion of markers in LG II.

Alternatively, it is known that gene conversion leadsto non-Mendelian segregation of genetic markers

in Schizosaccharomyces pombe and Saccharomycescereviseae, and that markers near recombination hot-

spots frequently undergo co-conversion (Davis & Smith2001). As the linkage analysis in this study was carriedout by mating distantly related strains, it can be

expected that there are many differences in genomesequence between the parent strains, which might

permit heteroduplex DNAs to be produced on a largearea of homologous chromosome during meiosis. Thus,

it is not unreasonable to suppose that mechanisms usedto 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 itpossible to map the loci that control qualitative andquantitative traits of agricultural and pharmacological

significance in L. edodes, such as morphology and fruitbody 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 efficiently select

strains with desired characteristics. Thus, geneticlinkage analysis of L. edodes with AFLP markers will

contribute to our understanding of its genomeorganization and will facilitate the efficiency of cross-breeding programmes.

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Corresponding Editor: J. I. Lelley

Documents

Lenti-AFLP.pdf