a genetic map of blumeria graminis based on functional genes, avirulence genes, and molecular...

Fungal Genetics and Biology 35, 235–246 (2002)doi:10.1006/fgbi.2001.1326, available online at http://www.idealibrary.com on

Functional Genes, Avirulence Genes,and Molecular Markers

Carsten Pedersen,* ,1 Søren W. Rasmussen,† and Henriette Giese* ,2

*Plant Research Department, Risø National Laboratory, P.O. Box 49, DK-4000 Roskilde, Denmark; and†Department of Yeast Genetics, Carlsberg Laboratory, Gammel Carlsbergvej 10, DK-2500 Valby, Denmark

Accepted for publication November 21, 2001

A genetic map of Blumeria graminis based on func-tional genes, avirulence genes, and molecular mark-ers. Fungal Genetics and Biology 35, 235–246. Agenetic map of the powdery mildew fungus, Blumeriagraminis f. sp. hordei, an obligate biotrophic pathogenof barley, is presented. The linkage analysis was con-ducted on 81 segregating haploid progeny isolatesfrom a cross between 2 isolates differing in sevenavirulence genes. A total of 359 loci were mapped,comprising 182 amplified fragment length polymor-phism markers, 168 restriction fragment length poly-morphism markers including 42 LTR-retrotransposonloci and 99 expressed sequence tags (ESTs), all theseven avirulence genes, and a marker closely linked tothe mating type gene. The markers are distributed over34 linkage groups covering a total of 2114 cM. Fiveavirulence genes were found to be linked and mappedin clusters of three and two, and two were unlinked.The Avra6 gene was found to be closely linked to mark-ers suitable for a map-based cloning approach. A link-age between ESTs allowed us to demonstrate exam-ples of synteny between genes in B. graminis andNeurospora crassa. © 2002 Elsevier Science (USA)

1 To whom correspondence should be addressed. Fax: �45 46774282.E-mail: [email protected].

2 Present address: Department of Ecology, The Royal Veterinary andAgricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg,Denmark.

All rights reserved. 235

AFLP; RFLP; synteny; map-based cloning.

The ascomycete fungus, Blumeria graminis f. sp. hor-dei, causing the disease powdery mildew of barley, is anobligate biotroph. The pathogen–host interaction is con-trolled by specific resistance genes in the plant and match-ing fungal avirulence genes according to the gene-for-genehypothesis. A large number of powdery mildew resistancegenes have been identified in barley (Jahoor and Fisch-beck, 1993; Jørgensen, 1994; Schonfeld et al., 1996) andtwo cloned alleles of the Mla locus have recently beenshown to belong to the type of resistance genes containinga coiled-coil domain, a nucleotide binding site, and aleucine-rich repeat domain (Halterman et al., 2001; Zhouet al., 2001). A corresponding large number of avirulencegenes are predicted to be present in the fungus and theirinheritance has been studied by linkage analyses (Chris-tiansen and Giese, 1990; Jensen et al., 1995; Brown andSimpson, 1994). These studies have shown some of theavirulence genes to be linked. Relatively few fungal aviru-lence genes have been characterized to date. They bear noresemblance to each other and in some cases it has beenpossible to assign a function in pathogenicity to the en-coded proteins, other than their role as elicitors in race-specific resistance (Lauge and De Wit, 1998; White et al.,2000).

Direct physical interaction between the products of aresistance gene and those of an avirulence gene accordingto the ligand-receptor model has so far been demonstrated

A Genetic Map of Blumeria g

Pedersen, C., Rasmussen, S. W., and Giese, H. 2002.

inis Based on

Index Descriptors: powdery mildew fungus; ESTs;

ram

1087-1845/02 $35.00© 2002 Elsevier Science (USA)

in only a few cases. These include two bacterial plantdiseases controlled by the resistance gene–avirulence genepairs, Pto-AvrPto of tomato (Scofield et al., 1996; Tang etal., 1996) and RPS2-AvrRpt2 of Arabidopsis (Leister andKatagiri, 2000). Recently, Jia et al., (2000) showed a directinteraction between the products of the rice resistancegene Pi-ta, conferring resistance toward the fungal patho-gen Magnaporthe grisea, and those of the correspondingavirulence gene Avr-Pita (Bryan et al., 2000; Orbach et al.,2000). These genes and the avirulence genes, PWL1 andPWL2, from Magnaporthe grisea were isolated by map-based cloning (Kang et al., 1995; Sweigard et al., 1995). Atpresent, the map-based cloning approach is consideredmore robust and reliable than techniques relying on directinteraction such as the two-hybrid system or phage displaysystem using resistance gene products as the bait.

The powdery mildew fungus is well-suited to map-based cloning as it is possible to carry out controlledcrossings, and all molecular marker types can be useddirectly for linkage analysis, as it is haploid throughout itsasexual life cycle. Two preliminary genetic maps based onthe segregation of restriction fragment length polymor-phism (RFLP) markers and avirulence genes have beenpublished (Christiansen and Giese, 1990; Brown andSimpson, 1994) and the chromosome complement hasbeen studied by light microscopy and pulsed-field gelelectrophoresis (Borbye et al., 1992). The genome of B.graminis has a high content of repetitive DNA, part ofwhich consists of a SINE-like repeat (Rasmussen et al.,1993), a LINE-like repeat (Wei et al., 1996), and LTR-retrotransposons (C. Pedersen, unpublished results).Many isolates of B. graminis also contain a linear self-replicating plasmid (Giese et al., 1990).

Map-based cloning requires tight genetic linkage be-tween the molecular markers and the genes of interest andgenomic libraries of large-insert clones. The aim of thisstudy was to develop a genetic map of B. graminis basedon avirulence genes and expressed sequence tag (EST)markers (i) as the basis for positional cloning of avirulencegenes, (ii) to provide a framework for a genomic sequenc-ing project, and (iii) to provide the background for studiesof possible synteny with other fungi. In addition, the studyallows us to analyze the inheritance of avirulence genesand genomic distribution of functional genes and LTR-retrotransposable elements. Different marker systems areavailable for genetic analyses and we have combined am-plified fragment length polymorphism (AFLP) markers,which are relatively fast to obtain, with RFLP markers,using mainly single-copy cDNA clones. The mappedcDNA clones facilitate screening of genomic libraries andcontig assembly. The use of expressed sequence tags as

markers (Thomas et al., 2001) makes it possible to relatefunction to a large proportion of the mapped genes.

MATERIALS AND METHODS

Fungal and Plant Material

The B. graminis isolates C15, sampled in 1964 in Den-mark (Wiberg, 1974), and JEH31, sampled in 1972 inDenmark by J. E. Hermansen (pers. comm.), were crossedas described in Giese et al. (1990). The progeny isolateswere obtained from nonsporulating single colonies as de-scribed by Brown et al. (1992). The map was developedusing 81 progeny isolates. The isolates were maintainedand propagated on the susceptible barley cultivar ‘Pallas.’

Tests for Virulence

The virulence spectra of the parent isolates, C15 andJEH31, were tested on barley near-isogenic lines of thevariety ‘Pallas’ with known resistance genes (Kølster et al.,1986) and found to differ in seven avirulence loci (Table1). The virulence spectra of the progeny isolates weredetermined on ‘Pallas’ isogenic lines P02, P03, P04B, P09,P12, and P17 and on ‘Black Russian’ (Table 1). Virulencetests were initially carried out on leaf segments placed onbenzimidazol–agar and later confirmed by repeated testson whole plants grown in multihole plastic containers. Theinfection types were determined 7–8 days after inocula-tion using the disease rating scale developed by Torp et al.(1978).

DNA Extraction

Pure powdery mildew DNA for AFLP analysis wasisolated from conidia as described by Justesen et al.(1996). The conidia were collected by shaking infectedplants over a settling tower placed on a glass plate. ForRFLP analysis a mixture of barley and B. graminis DNAwas isolated from heavily infected barley leaves by a CTABprocedure (Poulsen et al., 1993).

AFLP Analysis

The AFLP proceedure was as described by Vos et al.(1995) with the following modifications: The PCR prod-ucts were labeled directly by incorporating [�-32P]dCTPduring the selective amplification and the restriction en-zyme concentration was reduced from 5 to 1 U per 0.5 �g

236 Pedersen, Rasmussen, and Giese

© 2002 Elsevier Science (USA)All rights reserved.

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genomic DNA. For the preamplification we used P-, E-,and M-primers corresponding to the sequence of the P-,E-, and M-adapters. The preamplification consisted of 35cycles: a 30-s denaturation step at 94°C, a 30-s annealingstep at 50°C, and a 1-min extension step at 72°C. For theselective amplification two selective nucleotides wereadded to each primer. All 16 possible MseI primers wereused in combinations with 14 different PstI primers. Onlythe PstI primers with CC and GG as selective nucleotideswere not used. Also EcoRI primers with the selectivenucleotides AT and AA were used. The 20-�l selectiveamplification mixture contained 1 �l of 10-times-dilutedpreamplification PCR product, 50 ng of each primer, 10mM Tris–HCl (pH 9), 50 mM KCl, 1.5 mM MgCl, 0.01%gelatine, 0.1% Triton X-100, 50 �M the four nucleotides,1 �Ci [�-32P]dCTP, and 0.4 U Taq polymerase (Promega).The selective amplification cycle conditions were as for thepreamplification, apart from the annealing temperature,which was reduced from 65 to 56°C with 1°C per cycleover 10 cycles followed by 27 cycles with annealing at56°C. After amplification, 8 �l loading buffer (98% form-amide, 10 mM EDTA, 0.5 mg/ml bromophenol blue, and0.5 mg/ml zylene cyanol) was added and the samples weredenatured. The samples were loaded onto a 4.5% dena-turing polyacrylamide gel, which had been prerun for 30min at 80 W. The pBR322 plasmid, digested with MspIand labeled with [�-32P]dCTP using Klenow polymerase,served as marker. The samples were electrophoresed at 80W until the xylene cyanol front reached the bottom of thegel. Gels were fixed in 10% acetic acid, dried at 80°C, andexposed to X-ray films for about 4 days.

The AFLP markers were designated by the primercombination used in the PCR and the size of the fragment.Hence, PTCMAA-350 indicates the marker obtained byamplification with a PstI primer with TC as selective

nucleotides combined with a MseI primer with AA asspecific nucleotides and a polymorphic fragment size ofapproximately 350 bp.

RFLP Analysis

RFLP analysis was carried out using (i) genomic multi-copy sequences, known to produce informative fingerprintpatterns (Christiansen and Giese, 1990), (ii) end fragmentsfrom BAC clones, and (iii) cDNA clones (Table 2). Thegenomic clones were for simplicity named with the letterprefix M instead of GEE used by Christiansen and Giese(1990). As an example, clone M365 is identical toGEE365. The cDNA clones were from a library con-structed from germinating conidia (Justesen et al., 1996)and used in an EST project (Thomas et al., 2001; http://www.crc.dk/phys/blumeria/) and from cDNA librariesmade from epidermal strips of infected barley leaves10–12 h after infection (the penetration stage) and 14–16h after infection (the haustorium formation stage) (unpub-lished results) (Table 2). The mating-type locus wasmapped using a 1050-bp fragment obtained by MspI di-gestion of a 2.1-kb PCR fragment produced by the primerset V2R2-V2L, a gift from Drs. Braendle and McDermott(Zurich, Switzerland). They have shown that the primersproduce a polymorphism closely linked to the mating-typelocus (Braendle, et al., 1997).

RFLPs were detected by standard Southern analysis(Sambrook et al., 1989) of EcoRI, BamHI, HindIII, BglII,and EcoRV restriction-digested DNA from the parentisolates, C15 and JEH 31. Eight to 10 �g of DNA wereused in each analysis and EcoRI-digested barley DNA wasused as control.

The EST markers are named according to the designa-tion of the clones analyzed by Thomas et al. (2001), omit-

TABLE 1

Scoring of Infection Types of 81 Progeny Isolates of a C15 � JEH31 Cross

Differential barley line Resistance genes

Parental infectiontypea

Parental typesamong progeny

Probability of a 1:1segregation P(X � �2)C15 JEH31 C15 JEH31

Pallas 02 Mla3 4 0–1 45 36 0.317Pallas 03 Mla6, Mla14 0 4 44 37 0.436Pallas 04B Mla7 0 1–2 48 31 0.055Pallas 09 Mla10, Mla(Du2) 0 4 41 40 0.911Pallas 12 Mla22 4 0 42 39 0.738Black Russian Mla2, Mla(BR2) 4 1–2 40 38 0.911Pallas 17 Mlk1 1–2 4 41 40 0.820

a Infection types according to Torp et al. (1978): “0” full resistance; “4” full susceptibility.

237A Genetic Map of Blumeria graminis


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ting the zeros between the letter “C” and the (counting)numbers.

Single-Nucleotide Polymorphism (SNP)Search and Scoring

Single-nucleotide polymorphism markers were devel-oped to map selected BAC and YAC ends that could notbe mapped in the cross using RFLP analyses. The BACends were sequenced using vector primers for the Be-loBAC vector (Wang et al., 1997) and BigDye chemistry.The YAC ends were TAIL-PCR amplified according toLiu and Whittier (1995) and the products were sequenced.Based on the BAC and YAC end sequences, primer pairswere developed to amplify 200- to 400-bp fragments ofgenomic DNA from both parents. These fragments werethen sequenced and screened for SNPs using the softwarepackage Sequencher (GeneCode Corp., MI, U.S.A.). Toscore the SNP in the progenies, fragments were PCR-amplified and the products digested with an appropriaterestriction enzyme to allow identification of the two geno-types.

Data Analysis and Map Construction

AFLP fingerprints were compared visually and themarkers were scored according to presence or absence ofpolymorphic DNA fragments. The multicopy RFLP mark-ers were likewise scored by the presence or absence ofbands, while the single-copy RFLP markers could bescored as shifts in the fragment sizes. A few progenyisolates were shown to contain more than one band usingsingle-copy probes, indicating the presence of more thanone isolate, and were discarded. A �2 test was used tocalculate the probability of the expected 1:1 segregation.Linkage analysis was conducted using Joinmap, version 2(Stam, 1993) with the population type code HAP, whichdescribes a population structure of haploid progenies de-rived from one heterozygous diploid individual. The link-age groups were based on a LOD threshold value of 3.5and the mapping distances were calculated using the Ko-sambi mapping function. Only markers causing a jumpthreshold value of 4 or less are positioned on the map. Themap was drawn from JoinMap output files using DrawMap(Ooijen, 1994) and PowerPoint (Microsoft).

TABLE 2

RFLP Markers Except Those Derived from the Clones from the Blumeria EST Project (Thomas et al., 2001) That Are Listed in Table 4

Name Clone type Sequence type/homologya Number of copiesb Number of mapped loci

M160 gDNA LTR retrotransposon, pol polyprotein 15 7M161 gDNA No homology 5 2M365 gDNA LTR retrotransposon, pol polyprotein 20 10M367 gDNA LTR retrotransposon, pol polyprotein 20 7M373 gDNA LTR retrotransposon, gag-like protein 30 8M472 gDNA LTR retrotransposon, pol polyprotein 8 4MP53 gDNA No homology 15 3PAM4 gDNA LTR retrotransposon, pol polyprotein 20 6D24-1 cDNA No homology 40 8C411 cDNA No homology 25 7E9-1 cDNA Ribosomal protein 1 1E11-1 cDNA Actin related protein, ARP 2/3 complex 1 1cEgh2 cDNA Glycerol-6-phosphate 1 1cEgh16 cDNA No homology, cEgh16 family 5 1cEgh27 cDNA No homology 1 1P2910-12hai cDNA No homology 1 1P1514-16hai cDNA No homology 2 1P2314-16hai cDNA No homology 1 1GR20 cDNA cEgh16-related family 7 1GR21 cDNA No homology 2 1P7D9 cDNA No homology 1 1B86D5 BAC end No homology 10 4B37D2 BAC end — 20 1YAC389F YAC end No homology — 1C15-MAT PCR prod. No homology, unlinked to MAT locus 15 1

a The sequence type/homology column shows the results of BLAST searches.b Estimated by Southern hybridizations.



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Search for Synteny

EST sequences, which mapped to the same locus, weresearched against the Neurospora database (data release2, September 2001) at the Whitehead Institute, Centerfor Genome Research (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/) using the BLAST similaritysearch for translated nucleotides (tBLASTn). The se-quences were first translated to amino acid sequence andthe correct reading frame was identified when possible.Otherwise, all possible reading frames were used for thesearch. The criteria for proper identification of N. crassahomologues were set at a P(N) value of the BLAST searchat 1e-08 or below.

RESULTS

Virulence Analysis

The cross segregated for seven avirulence loci as shownin Table 1. The segregation data fit a 1:1 segregation withthe exception of Avra7, which shows a slightly distortedsegregation (48:31). Scoring for segregation was consistenton differential lines producing infection types 0 or 4 (Ta-ble 1). However, with the resistance genes Mla7 and Mla2(Pallas 04B and Black Russian, respectively), the differ-ences between the high- and low-infection types were lesspronounced and it is possible that some progeny weremisscored (Table 1). The presence of additional resistancegenes in Pallas 03, Pallas 09, Black Russian (Mla14,Mla(Du2), and Mla(BR2), respectively) did not result insegregation of additional avirulence genes, showing thatboth the parental isolates are avirulent toward these resis-tance genes.

AFLP Analysis

The modified AFLP procedure gave stronger but lessdistinct bands than those seen using the traditional proto-col with labeled primers. This is probably due to a higher

background level and the occurrence of “double bands”produced by direct labeling of both DNA strands as alsoseen with silver staining (Briard et al., 2000).

About 200 primer combinations were applied to theparental isolates and those giving the most informativebanding patterns were used for mapping. The primercombinations selected for mapping produced on averageabout five markers per combination.

RFLP Analysis

Four hundred and twenty sequenced cDNA clones(EST clones) were tested for their ability to identifyRFLPs. One hundred and five (25%) were polymorphicand 99 were mapped (Tables 3 and 4). Ten clones previ-ously found to produce multiple banding patterns weresequenced and tested. Eight were genomic clones, ofwhich most contain LTR retrotransposons, and 2 werecDNA clones, D24-1 and C411, which also contain repet-itive DNAs (Table 2). These 10 clones produced 62 RFLPmarkers (Tables 2 and 3). Mapping of BAC ends by RFLPwas successful in two cases, both of which resulted inmultiple banding patterns. One BAC end fragment,B86D5-R, produced 4 unlinked markers.

SNP Analysis

Attempts to develop SNP markers for mapping of spe-cific BAC and YAC ends failed in most cases due to a verylow level of SNPs. More than 2300 bp were sequencedoriginating from 10 different loci, but only two SNPs weredetected. The one mapped in this strategy was derivedfrom a YAC end (YAC389F). There is a BseRI site in thePCR-amplified fragment of the JEH31 isolate, but not inC15, which was used in the SNP scoring.

Linkage Analysis, Map Construction, andMarker Distribution

A total of 381 markers were included in the linkageanalysis at the selected LOD of 3.5 (Fig. 1). Five markers

TABLE 3

Summary of the 359 Mapped Genes and Anonymous B. graminis Markers

Total markers AFLP Avr loci SNP RFLP

Subcategories of RFLP markers

“MAT” locus ESTs Repeats BAC ends

358 182 7 1 168 1 99 63 5

Note. The RFLP markers are subdivided according to clone type (the four right columns).



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could not be placed on any linkage group and two groupscontained only 2 markers and are not presented in Fig. 1.Thirteen markers were excluded as they caused a consid-erable “jump” in goodness-of-fit (Stam, 1993). This may bedue to “false” linkages, as the number of markers is highrelative to the number of progeny isolates. It brings the

total number of mapped markers and genes down to 359,of which about half are AFLP markers and the other halfmainly RFLPs (Tables 2 and 3). When we raised the LODscore to 4, four linkage groups split into two and a fewterminal markers became separated from their groups but,in general, the map remained unchanged.

TABLE 4

B. graminis EST Clone-Derived Markers

Clone GenBank Accession No. Copiesa Mapped to linkage groupb Function or homologyc

C007 AW787872-3 1 3 Ribosomal protein L22C013 AW787982-3 1 27 Mitochondrial porin, outer membraneC024 AW787998-9 1 3 Libase 1 precursorC082 AW787924-5 1 11 ATP synthase gamma chainC120 AW787971-2 1 24 DNA-directed RNA polymerase IIIC127 AW788026-7 1 19 Hypothetical 110.0-kDa protein YM84C174 AW788072-3 1 4 20S proteasome alpha 5 subunitC225 AW788134-5 1 14 Trisephosphate isomerase gene, TIMC245 AW788169-70 1 6 Putative mitochondrial ATP-dependent proteaseC290 AW788227-8 1 2 Iron transport multicopper oxidase, FET3C293 AW788233-4 1 31 40S ribosomal protein S19C323 AW788277-8 1 1 ChitinaseC341 � AF189366 1 15 Chitin synthase Chs2, Blumeria graminisC342 AW788304-5 1 1 Hypot., mitochon. carrier prot. Ygr096wpC350 AW788314-5 1 9 60S Ribosomal protein L37C387 AW788361-2 1 18 60S ribosomal protein L34C412 AW788389-90 1 6 Palmitoyl-protein thioesterase precursorC447 AW788438-9 2–3 31 Heat shock protein, HSP70C457 AW788452 1 12 Protein–tyrosine–phosphataseC458 AW788453-4 1 1 Pyruvate dehydrogenaseC474 AW788472-3 1 15 T-complex protein 1, alpha subunit homologC484 AW788480-1 1 5 Acid proteinase eapc precursorC502 AW788508-9 1 11 Similarity to protein kinaseC513 AW788520-1 1 2 40 S ribosomal protein, S15C541 AW788557-8 1 33 CalmodulinC549 AW788569-70 1 27 Aspargine synthetaseC562 AW788591-2 1 32 Sorbitol utilization protein Sou2C622 AW788670-1 1 12 Aldehyde dehydrogenaseC640 AW788696-7 1 14 Myosin 2, MYO2C642 AW788700-1 1 7 Peptidylprolyl isomeraseC711 AW788789-90 1 15 Vacuolar ATP synthase subunit DC759 AW788857-8 1 11 Phe-RNA synthetaseC765 AW788867-8 1 11 Proteosome subunit Pre2pC804 AW788912-3 1 6 Nucleoside diphosphate kinase I, NDK IC806 AW788915-6 1 or 2 32 Isotrichodermin C-15 hydroxylaseC824 AW788942-3 1 2 NADH dehydrogenase 78-kDa chain precursorC907 AW789077-8 1 9 Acidic profilinC945 AW789140-1 ca. 8 27 Heat shock protein, Hsp 30C1084 AW789373-4 1 25 Superoxide dismutase precursorC1181 AW789532-3 1 3 Glutamine synthetaseC1190 AW789545-6 1 5 Possible apospory-associated protein CC1192 AW789549-50 1 2 Extragenic suppressor of the bimD6 mutationC1244 AW789627-8 1 1 Transporter, MSF (S. pombe), PHO87 (yeast)C1256 AW789651-2 1 13 60S ribosomal protein L14D746 AW791691 Multicopy 2 18S RNA gene

a Estimated by Southern hybridizations.b According to Fig. 1.c According to http://www.crc.dk/phys/blumeria/.



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The expected 1:1 segregation was verified using �2 tests.Seventeen markers (5%) showed distorted segregation at the5% level. These markers were confined to four regions on themap. Three of these regions also contained linked markerswith slightly distorted segregation below the 5% significancelevel. The regions are located between the markers C1244and EATMGT-182 on group 1, M160-2 and M367-2 ongroup 12, and C427 and PAAMTT-230 on group 13.

Three of the seven avirulence genes, Avra10, Avra22 (Syn.Avrc), and Avrk1, are closely linked and make up linkagegroup 34 which contains no other markers (Fig. 1). Avra3

and Avra2 are linked and belong to linkage group 29 andthe last two avirulence genes, Avra6 and Avra7, are locatedon groups 15 and 25, respectively. The closest linkagebetween a molecular marker and an avirulence gene is ongroup 15 where Avra6 is cosegregating with two AFLPmarkers and closely linked to the EST marker C711.

The AFLP markers are distributed with varying densi-ties throughout the map. EST markers are found on alllinkage groups with the exceptions of groups 8, 10, 20, 22,and 34. There are clusters of several linked EST markerson linkage groups 1, 2, 4, 12, 14, 27, and 31. Some of theLTR retrotransposons, M367, M365, M161, M373, andPAM4, are also linked to one another.

Synteny Studies

Sixteen EST sequences were searched against the Neu-rospora database and significant homologies were found to12 sequences (69%) (Table 5). Two of the genes representgene families (C622 encoding an aldehyde dehydrogenaseand C447 encoding an HSP-70) and have several homo-logues in N. crassa which weaken their utility for syntenystudies. This allows for synteny analyses of three pairs ofcosegregating EST markers (groups 4, 11, 27). One coseg-regating pair of ESTs on linkage group 11, C502 withhomology to protein kinases and C082 with homology toATP synthases, was found to have two close homologues inN. crassa residing on the same supercontig (68). Closerinspection showed these to be located on the same contig(2.648) separated by approximately about 3000 bp. An-other pair, C549 and C013, on linkage group 27, werelikewise found to have homologues in N. crassa on thesame contig (2.246) separated by about 2000 bp.

DISCUSSION

Genetic Mapping

The present genetic map represents the most detailedanalysis of the B. graminis genome to date and for the first

time includes the map positions of a range of functionalgenes. The AFLP markers and RFLP markers derivedfrom ESTs and LTR retrotransposon elements are dis-persed throughout the genome and the 359 markers givedense marker coverage on the linkage groups identified.The largest distance between markers is 26 cM. Thirty-four linkage groups were identified and the map size isestimated to be in the region of 2100 cM. Filamentousascomycetes are reported to have relatively small genomesof around 35 Mb or smaller (Kupfer et al., 1997); however,we have indications that the genome of B. graminis isbigger (unpublished results). The genetic maps of M.grisea, another plant pathogenic ascomycete, have been inthe range of 600–900 cM (Romao and Hamer, 1992;Sweigard et al., 1993; Farman and Leong, 1995) and thegenetic map of N. crassa has a length of about 1000 mapunits (Radford and Parish, 1997). However, a linkage mapof the human fungal pathogen Cryptococcus neoformanshas a size of 1355 cM and the total size was estimated tobe about 1900 cM, showing that large linkage maps are notexceptional in fungi (Forche et al., 2000). The large mapsize estimated for the B. graminis genome in the presentstudy is ascribed to a high level of recombination. Ourunpublished results from linkage analyses of two othercrosses involving different B. graminis parents also re-sulted in large linkage maps, indicating that this is notexclusively a genotypic effect. Cytological data have sug-gested the presence of seven to eight chromosomes in B.graminis (Borbye et al., 1992) and genetic mapping car-ried out by J. Brown (John Innes Centre, UK, pers.comm.) suggests a maximum of 12 major linkage groupsand a smaller map size. It was not possible in our presentanalysis to merge any of the 34 identified linkage groups.Analyses of more progeny isolates would probably permitthe construction of fewer and larger linkage groups, butthe genetic distances would still be large. It is possible thatenvironmental factors influence recombination frequency.Our crosses were carried out in a green house withouttemperature control in the spring. Such a period with hightemperatures could possibly affect the recombination fre-quency. A high level of recombination is an advantage forhigh-resolution mapping in connection with map-basedcloning, but it requires a higher number of progeny toconstruct a cohesive genetic map.

Distorted segregation was observed for 5% of the mark-ers belonging to three different regions of the map.Linked markers showed a lower degree of distortion andthe distortion was not associated with marker type. Dis-torted segregation is a common phenomenon in linkageanalyses and can be explained by linkage between markersand genes that favor or act against the survival of the



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FIG. 1. A genetic map of Blumeria graminis f. sp. hordei composed of 34 linkage groups covering 2114 cM. The map contains seven avirulence genes(Avrxy) located on groups 15, 25, and 34, a marker (MAT) linked to the mating-type locus, 182 AFLP markers, and 169 RFLP markers of which 99 areEST markers, 40 are LTR-retrotransposable element markers, and 6 are mapped BAC or YAC ends. RFLP markers are in boldface and the EST markersare underlined. The numbering of linkage groups is according to length (in cM) and is not related to chromosomes.


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individual progeny. It is expected to have a larger effect ina haploid organism as the selection that causes the distor-tion can act virtually during the whole haploid life cycle.This does not appear to be a problem in our case, and amuch larger degree of distortion (18% of markers) wasobserved in the fungus C. neoformans (Forche et al.,2000).

Synteny

The inclusion of functional genes in the B. graminismap not only improves its usefulness in generating land-marks for map merging and assembly of BAC and YACcontigs but also facilitates synteny studies. Most of thecDNA clones are only partially sequenced (Thomas et al.,2001) but a putative function has been assigned to 48 of

the 99 mapped genes (Table 4). This is not sufficient tocarry out a detailed synteny search of the N. crassa ge-nome, but nevertheless eight pairs of cosegregating ESTmarkers were used for BLAST searches against the N.crassa database. We only analyzed cosegregating markersbecause the genomic sequence of N. crassa is made up ofunordered supercontigs and only very closely linked mark-ers can be expected to be found located on the samesupercontig. Only five pairs of B. graminis ESTs hadsignificant homology to sequences in N. crassa and couldbe used for synteny analyses. However, two of the ESTsinvolved, C622 and C447, represent multigene familieswith several members both in B. graminis and N. crassa(Tables 4 and 5), which makes synteny comparisons diffi-cult. Of the last three EST pairs examined synteny wasfound for two of them. The N. crassa homologues of the

FIG. 1—Continued



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EST clones C502 and C082 on group 11 are very closelylinked with only about 3000 bp separating the 2 genes, andthe N. crassa homologues of C549 and C013 are likewiselocated next to each other. Two more cases of microsyn-teny between B. graminis and N. crassa have recentlybeen detected using EST clones physically mapped to thesame BAC clone of B. graminis (unpublished results),indicating that microsynteny might be a general tendency.These are the first indication of synteny between thegenomes of B. graminis and another filamentous fungus.Synteny between M. grisea and N. crassa has recentlybeen described (Hamer et al., 2001) and synteny has beenfound among different yeast species (Ozier-Kalogeropou-los et al., 1998; Llorente et al., 2000). When the genomicsequences of N. crassa and Aspergillus nidulans are fullyassembled this will facilitate further comparisons betweenfungal genomes and if extensive synteny is detected it willhelp exploiting knowledge gained in the model species.The rice blast fungus, M. grisea, is a well-studied plantpathogenic ascomycete, and a number of pathogenicitygenes and several avirulence genes have been character-

ized from this species (Kang et al., 1995; Sweigard et al.,1995; Orbach et al., 2000). Systematic EST and genomicsequencing has also been initiated (Zhu et al., 1999).Synteny between the genomes of Blumeria and Magna-porthe will, therefore, be particularly helpful for identify-ing interesting genes in B. graminis. Genetic analyses of B.graminis genes that are related to pathogenicity will showwhether they cosegregate with avirulence genes.

Mapping of Avirulence Genes

It is striking that the mapped avirulence genes, Avra10,Avra22, and Avrk1, are clustered tightly in one group of only3 cM that is unlinked to any of the other linkage groups.Brown and Jessop (1995) determined a distance of 4.1 cMbetween Avra10 and Avrk1, which is in agreement with ourresults. Jensen et al. (1995) found a distance of 2.4 cMbetween Avra10 and Avrk and a weak linkage of 46.3 cM toAvra6, which could not be confirmed in the present study.Christiansen and Giese (1990) found 9.3 cM betweenAvra10 and Avra22 and linkage to a marker of the LTR-

TABLE 5

Results of BLAST Searches of Cosegregating EST Marker Pairs against the Neurospora Database (Release 2, September 2001) Containing37,869,317 bp (About 90%) of the Genomic Sequence

Linkage group Marker Homology of EST clone in Blumeria Neurospora homologues BLASTXa Contigb/supercontig Linkage groupc

2 C1192 Extragenic suppressor Found 3e-20 2.806/101 III2 C491 No homology Not found — — —4 C269 No homology Found 2e-16 2.333/18 VIII4 cEgh2 Glucose-6-P-isomerase Found 7e-31 2.519/36 IV

11 C502 Similarity to protein kinase Found 1e-42 2.684/68 —11 C082 ATP synthase � chain Found 6e-30 2.684/68 —12 C457 Protein–tyrosine–phosphatase Found 2e-14 2.230/12 VII12 C622 Aldehyde dehydrogenase 5 found 5e-26 2.43/2 III, VI

1e-18 2.158/8 II, V5e-11 2.514/35 VI5e-10 2.155/8 II, V2e-09 2.8/1 I

17 D361 No homology Found 5e-10 2.203/10 V17 C423 No homology Not found — — —26 C119 No homology Not found — — —26 C493 No homology Not found — — —27 C549 Asparigine synthase Found 9e-40 2.246/14 V27 C013 Mitochondrial porin Found 3e-37 2.246/14 V31 C293 40S ribosomal protein S19 Found 2e-40 2.320/17 III31 C447 HSP70 (multigene family) 4 found 4e-53 2.742/83 II

9e-42 2.237/13 I, VI3e-36 2.86/4 I2e-30 2.637/56 IV

a The results of the tBLASTx searches are indicated with the P(N) values.b The contig/supercontig numbers refer to the designation of the second release (September 2001) of the assembly of the Neurospora sequence at

Whitehead Institute.c The linkage group numbers refer to the genetic map of Perkins et al. (2000).



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retroposon sequence M365. This indicates that this islandof closely linked Avr genes belongs to a larger linkagegroup as suggested by the map of Jensen et al. (1995). Inthis map, one linkage group was suggested to contain up to11 different Avr genes with distances ranging between 2.4and 46.3 cM between individual genes. The close linkagebetween Avra3 and Avra2 has been reported previously byJørgensen (1988). Our unpublished linkage maps from twocrosses between the B. graminis isolates HL and Race1,segregating for the Avra1 and Avra31, and the isolates C15and TY4 showed close linkage between the MAT locus andthree RFLP markers in both crosses. By combining theseresults, it is clear that Avra1 and Avra31 belong to linkagegroup 9 in the current map. The M365 clone produces anRFLP marker linked to the MAT gene in all three crosses,further confirming the location of these two Avr genes.

One avirulence gene, Avra6, cosegregates with twoAFLP markers and is closely linked to the single-copy ESTclone, C711, encoding an ATP synthase subunit. This geneprovides an excellent landmark for the isolation of YACand BAC clones and the 1-cM distance is considered avery tight linkage in this population with high recombina-tion frequencies. The characterization of different Avrgenes from B. graminis will provide important insight intothe function and evolution of these genes and will be animportant tool for studies of resistance in barley.

ACKNOWLEDGMENTS

This work was supported by a grant from The Danish ResearchCouncil under the Biotech III Programme. We thank Torben Nielsen,Henrik Kristensen, and especially Rikke Sillesen for technical assistanceand Solveig K. Christiansen for critical reading of the manuscript.

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a genetic map of blumeria graminis based on functional genes, avirulence genes, and molecular...

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