An AFLP-markers based genetic linkage map of Heterobasidion annosum locating intersterility genes

Fungal Genetics and Biology 42 (2005) 519–527 www.elsevier.com/locate/yfgbi

An AFLP-markers based genetic linkage map of Heterobasidion annosum locating intersterility genes ˚ ke Olson, Jan Stenlid Ma˚rten Lind *, A Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, 75007 Uppsala, Sweden Received 1 October 2004; accepted 11 March 2005 Available online 19 April 2005

Abstract A genetic linkage map of the basidiomycete Heterobasidion annosum, casual agent of root rot in conifers, was constructed from a compatible mating between isolates from the North American S and P intersterility groups. In a population consisting of 102 progeny isolates, 358 AFLP markers were scored. The linkage analysis generated 19 large linkage groups, containing 6 or more markers, which covered 1468 cM. The physical size to genetic distance was approximately 11.1 kbp/cM. Segregation of three intersterility gene loci were analysed through mating of the progeny isolates with three tester strains carrying known intersterility genotypes. The loci for the two intersterility genes (S and P) were successfully located in the map. Segregation of the mating type locus was analysed by backcrossing the progeny isolates with their parental strains. The mating type locus could not be located in the map.
2005 Elsevier Inc. All rights reserved. Keywords: Heterobasidion annosum; Genetic linkage map; Intersterility genes; AFLP markers; Forest pathogen; Mating type

1. Introduction Heterobasidion annosum (Fr.) Bref. sensu lato (s.l.), casual agent of annosum root rot in conifers, is the economical most devastating pathogen in the northern hemisphere. The losses caused by devaluation of timber and growth reductions sum up to at least 500 million EUC a year for European forest owners (Bendz-Hellgren and Stenlid, 1995, 1997). Heterobasidion annosum sensu lato s.l. is a complex of different species and intersterility (IS) groups that are still partially interfertile. The groups are named after their main host preference, pine, spruce, and fir (P, S, and F); although their host ranges partially overlap (Korhonen and Stenlid, 1998). In Europe, all three intersterility groups, P, S, and F, have been identified (Capretti et al., 1990; Korhonen, 1978) and given Latin names, H. annosum, H. parviporum, and H. abietinum, *

Corresponding author. Fax: +46 18 67 35 99. E-mail address: [email protected] (M. Lind).

1087-1845/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2005.03.005

respectively (Niemela¨ and Korhonen, 1998). In North America only the P and S groups have been found (Harrington et al., 1989) but remain to be named. However, numerous reports in the literature confirms that all groups belong to different phylogenetic species (Chase and Ullrich, 1983; Garbelotto et al., 1993; Johannesson and Stenlid, 2003; Otrosina et al., 1993; Worrall et al., 1983). Individuals from the distinct groups are intersterile, i.e., generally do not mate outside the group. However, the reproduction barrier is not complete, and in laboratory mating experiments some isolates of different intersterility groups are compatible. In general, compatible mating between isolates from European IS groups P–S is rare (Stenlid and Karlsson, 1990) while successful mating among European S–F is relatively frequent (Korhonen et al., 1992, 1997). Sympatric populations including more than one IS group maintain a more strict intersterility, while allopatric populations not exposed to other groups can be more interfertile (Korhonen and Stenlid, 1998). Mating between North American S–P isolates is also readily observed in the laboratory

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(Harrington et al., 1989) although only one North American S-P hybrid with a stable natural diploid state has been isolated from spontaneous crossings in the field (Garbelotto et al., 1996, 2004). Laboratory derived S–P hybrids may form heterokaryotic mycelium with uniparental mitochondrial inheritance (Olson and Stenlid, 2001). Even though the IS genes system is present in many fungal species, there are no previous reports of these genes being mapped or characterized. Since the system has been most thoroughly evaluated in H. annosum, we have a unique opportunity to perform the first more detailed study of IS genes. Heterobasidion annosum has three distinct genetic systems which determine the outcome when two different mycelia interact; the IS system, the mating type, and the vegetative incompatibility. The IS system determines the limits of an interbreeding population, controlling the reproduction barriers by preventing mating between individuals of distinct breeding populations, making up the genetic bases to define biological species among fungi. In H. annosum the system have been postulated to consist of five genes, called S, P, V1, V2, and V3, each with a + or a
allele (Chase and Ullrich, 1990). For compatible mating, two individual homokaryotic mycelia must have a common + allele at least at one of the IS loci. All isolates belonging to the S groups are thought to possess a + allele at the SIS locus while all isolates belonging to the P groups are thought to possess a + allele at the PIS locus ensuring compatible mating within the groups. Interfertility between the two groups is thought to be mediated by the other three genes, V1, V2, and V3. Interbreeding within one group of H. annosum is limited by a bipolar (unifactorial) mating system where compatibility is controlled by one locus (Korhonen, 1978). However, the number of mating type alleles in H. annosum is large, probably more than 100 (Chase and Ullrich, 1983; Stenlid, 1985). Interaction between two homokaryotic mycelia with different mating type alleles allow for a compatible interaction leading to plasmogeny and forming of heterokaryotic mycelium. The somatic (vegetative) incompatibility system is a non-self recognition system expressed as sparse aerial mycelia and hyphal death on a macroscopic level when two incompatible heterokaryotic mycelia are confronted. Somatic incompatibility is controlled by at least three different multiallelic loci in H. annosum (Hansen et al., 1993). Multilocus marker systems such as amplified fragment length polymorphism (AFLP) are powerful approaches to create genetic linkage maps. The AFLP method is based on selective PCR amplification of restriction fragments from genomic DNA and has proved to be reliable for gathering of polymorphic DNA markers (Vos et al., 1995). Markers collected using the AFLP technique have been used to estimate genetic diversity and to create genetic linkage maps of

a number of fungal species. Such linkage maps have been made for several plant-pathogenic species, such as Blumeria graminis (Pedersen et al., 2002), Gibberella zeae (Jurgenson et al., 2002), Mycosphaerella graminicola (Kema et al., 2002), Bremia lactucae (Sicard et al., 2003), Phytophthora infestans (van der Lee et al., 1997), Leptosphariea maculans (Pongam et al., 1998), Fusarium moniliforme (Xu and Leslie, 1996), and Cochliobolus sativus (Zhong et al., 2002), but also for the human fungal pathogen Cryptococcus neoformans var. neoformans (Forche et al., 2000). Genetic linkage maps also exist for the edible mushrooms Lenintula edodes (Terashima et al., 2002), Pleurotus ostreatus (Larraya et al., 2000), Coprinus cinereus (Muraguchi et al., 2003), and Agaricus bisporus (Kerrigan et al., 1992). In this study, our objective was to identify a sufficiently large amount of AFLP markers from H. annosum to construct a genetic linkage map based on their segregation. The generation of a genetic linkage map for H. annosum will provide a framework for further genetic studies, primarily for map-based cloning of genes but also for identification of markers useful for characterisation of field populations and identification of genomic regions interesting for evolutionary studies. Our first application of the map was an attempt to locate three IS gene loci, controlling reproduction barriers of interbreeding populations.

2. Materials and methods 2.1. Acquiring the mapping population The homokaryotic strains TC 122-12 of North Amer
þ ican S (IS genotype Sþ P
V
1 V2 V3 ) and TC 32-1 of þ North American P (IS genotype S
Pþ V1 Vþ 2 V3 with the V1 allele being unknown) IS groups were selected as parental strains for the mapping population and paired on Hagem agar (Stenlid, 1985), resulting in a hybrid heterokaryon AO8 (Olson and Stenlid, 2001). The common + allele for the V3 intersterility gene allowed compatible mating while alleles of the S, P, and V2 gene will segregate in the progeny population. To produce sporocarps, the heterokaryon was inoculated onto autoclaved pieces (50 cm3) of Norway spruce wood placed in 500 ml Eflasks with 50 ml Hagem. The E-flasks were incubated for 3 months in diffuse daylight at 21 C. From spore deposits on fresh petri dishes, 105 single spore isolates were collected, using methods described by Stenlid and Rayner (1991). The 105 isolates progeny isolates were deposited in the departmentsÕ culture collection. 2.2. DNA extraction and AFLP analysis Three pieces of mycelia were inoculated in liquid Hagem medium in 9 cm petri dishes and incubated in

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darkness at 21 C for 7–10 days. Total DNA was extracted from the resulting mycelia according to established procedure (Sambrook and Russell, 2001). Analyses using the AFLP technique were preformed using the Microbial AFLP fingerprinting kit from PE Applied Biosystems as recommended by the manufacturer. However, DNA samples were digested with two distinct restriction enzymes combinations (EcoRI and MseI), or (PstI and MseI). All adapters were according to the manufacturersÕ recommendations except that the PstI-adapter had a cohesive end compatible with the PstI cleavage site. Selective AFLP-primers for the PstI cut DNA were similar to the EcoRI-primer, but had enzyme-specific parts corresponding to the recognition sequence of the PstI restriction enzyme. The reproducibility of the markers was confirmed by multiple repetitions of PCRs on a subsample of isolates and repeated scoring of the markers to make sure no human discrepancy occurred in the analysis. The AFLP-products were analysed using an ABI Prism 310 Genetic Analyser (PE Applied Biosystems). The markers obtained were designated by manual analysis using the GeneScan (PE Applied Biosystems) and Genotyper (PE Applied Biosystems) softwares. The markers were coded after their origin; either from the S parent or the P parent. Each marker was named after the nomenclature XAABBC00, where X stands for stemming from the PstI-cleaving (in which case the marker carries a P) or the EcoRI-cleaving (in which case it carries no prefix). The AA denotes the EcoRI/PstI selective primer extensions and the BB the MseI selective primer extension. The C is either an s or a p, depending on what parent it origins from. The serial number indicates the relative size of the marker compared to other markers derived from the specific parent for the primer combination in question.

V2 testers were coded as originating from the S parent, while isolates not forming clamps with the S tester were coded as originating from the P-parent in the following linkage analysis. For the mating type testing, the 102 progeny isolates were backcrossed with the parental S- and P-strains and positive mating was scored as described above. In the following linkage analysis, data for the mating type locus were coded as originating from the P-parent if clamps were formed with the S parental strain (i.e, being of a different mating type than the isolate it forms clamps with), and vice versa for isolates forming clamps with the P-parent.

2.3. Development of mapping data for the intersterility genes and the mating type genes

One hundred and two progeny isolates resulting from a cross between the two homokaryotic North American H. annosum isolates TC 32-1 and TC 122-11 from the different IS groups P and S, respectively, were analysed in this study. We found no duplicated or completely complementary isolates in the progeny set. Eleven and ten different primer combinations were scored on two sets of cleaved total DNA (21 different AFLP analyses) resulted in 358 polymorphic AFLP markers (Table 1). The number of polymorphic markers from each primer pair varied between 28 (for the AC–CA combination on the PstI-cleaved DNA) and 9 (for the AG–CT combination, also on the PstI/MseI-cleaved DNA, and the AA– CG combination, on the EcoRI/MseI-cleaved DNA), with an average yield of 20 polymorphic fragments per pair. In total, 181 of the markers originated from the S-parent, while 177 came from the P-parent (Table 1). One hundred and forty-one of the 358 markers had a segregation distortion deviating from the assumed 1:1

For the IS genotype testing, 102 progeny isolates were crossed with three tester strains; TC 111-4, Sa¨ 164, and Sa¨ 159-5 carrying the + allele for the V2, P, and S IS loci, respectively, but no other known IS + allele. Progeny and tester isolates were grown separately on Hagem for 10 days before being transferred pairwise to a new Hagem plate. After another 10 days, an agar plug from the interaction zone was transferred to a fresh Hagem plate and left to grow until the entire plate was covered. The plate was then examined for clamps under 250· magnification. For isolates forming clamps with the S-tester strain, data for the S-locus were coded as originating from the S-parent. Similarly, isolates forming clamp connections with the P or V2 testers were coded as originating from the P parent for those loci. Accordingly, isolates not forming clamps with the P or

2.4. Linkage analysis and map construction Segregation patterns for 358 AFLP markers were scored on 102 of the 105 progeny isolates and analysed with the JoinMap 3.0 program (Stam, 1993; Van Ooijen and Voorrips, 2001). The data type was coded as HAP, i.e., haploid originating from a diploid parent. Linkage groups were initially determined by results of pairwise comparisons at a minimum likelihood of odds (LOD) value of 4. The individual orders inside the groups were obtained from JoinMaps Map 3-mapping procedure (all markers, linking to at least one other marker of the same group at the pre-determined LOD-value, being used in the mapping of the group regardless of goodness-of-fit values), using a LOD lower threshold of 0.8 and recombination ratio upper threshold of 0.45 in the pairwise comparison.

3. Results 3.1. Marker development

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Table 1 Segregation and mapping data for AFLP markers Primer extensions

Markers obtained S-parent

P-parent

Total

Unlinkedc

% unlinked

Dist. seg.d

% Dist. seg.

EcoRIa AA AA AA AA AC AC AC AC AG AG AG AT

MseIb CA CC CG CT CA CC CG CT CA CG CT CG

11 12 7 6 6 11 8 11 9 6 8 9

9 12 2 14 6 12 8 12 12 10 8 6

20 24 9 20 12 23 16 23 21 16 16 15

1 0 0 2 1 2 0 3 5 4 1 0

5 0 0 10 8 9 0 13 24 25 6 0

5 8 5 6 3 9 4 7 10 9 9 1

25 33 55 30 25 39 25 30 48 56 56 0

PstIa AA AA AC AC AC AC AG AG AG

MseIb CG CT CA CC CG CT CC CG CT

6 8 16 8 10 9 5 9 6

7 6 12 5 15 3 7 8 3

13 14 28 13 25 12 12 17 9

5 2 9 2 2 2 3 5 1

38 14 31 15 8 17 25 29 11

7 5 10 9 14 2 5 9 4

54 36 36 69 56 17 42 53 44

181

177

358

50

14

141

39

a

Primer extension to primers designed to anneal to the EcoRI or PstI cleavage site. b Primer extension to primers designed to anneal to the MseI cleavage site. c Number of markers from the primer combination not linking to any other marker. d Number of markers from the primer combination with a segregation distortion deviating from the assumed 1:1 ratio at the 5% level of significance (p > 0.05, v2).

ratio at the 5% level of significance (v2 > 3.82, p > 0.05) (Table 1). Of these 141 markers with skewed segregation, 75 originated from the S-parent and 66 from the P-parent. 3.2. Linkage analysis Using the JoinMap 3.0 software, a total of 242 out of 358 AFLP markers were assigned to 19 large linkage groups containing more than 5 markers with LOD values ranging from 4.0 to 7.5 (Fig. 1). Another 70 AFLP markers were assigned to smaller linkage groups or linked to just one other marker. These markers formed another 20 linkage groups; 5 groups of 5 markers, 5 groups of 4 markers, 5 groups of 3 markers, and 5 linkage groups containing just two markers (data not shown). Still about 14% (50) of the markers remained unlinked (Table 1). The 141 markers with distorted segregation ratios were mapped and distributed into all linkage groups. The recombination frequency of each marker in the 19 large linkage groups was derived from the JoinMap software and plotted against the distal location of each mar-

ker in their respective group (Fig. 2). Recombination frequency 0.5 indicates even distribution of the marker among the progeny isolates analysed. All groups include markers with skewed segregation, although most markers in all groups are located around the 0.5 value not being heavily skewed towards any parent isolate (Fig. 2). Neither could we find any small linkage group dominated by markers with skewed segregation (data not shown). However, in linkage group 3, four markers in a row covering about 5 cM had a skewed segregation towards TC-32-1 parental isolate (P-type). The genome size calculated from the 19 large linkage groups covered a total length of 1468 cM with an average marker distance of 6.0 cM (not including linkage groups smaller than 6 markers, and including the located intersterility genes, see below). The linkage group length varied between 137 (group 3) and 39 cM (group 19), with an average of 78 cM per group, and the largest number of markers in a single group was 27 (group 1). The largest mapped distance between two markers was 36 cM (paccgs09 and paccgs02) in linkage group 3. The map size calculated on all linkage groups consisting of 2 markers or more amounted to 2252 cM in total.

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Fig. 1. The genetic linkage map of the Heterobasidion annosum genome. A total of 244 markers are included in the 19 large linkage groups. Linkage groups named in declining size in number of markers. On the left the marker position is given in centimorgans relative to the top marker.

3.3. Segregation and linkage analysis of intersterility and mating type genes The progeny isolates were paired with three tester strains Sa¨ 16-4, Sa¨ 159-5 and TC 111-4 to score the presence of the + alleles at the P, S, and V2 IS loci. Out of 101 scored isolates, 51 formed clamps with the S IS gene tester strain, while 39 out of 101 and 28 out of 96 progeny isolates formed clamps with the P IS gene tester strain and with the Vþ 2 allele carrying tester strain respectively (Table 2). The number of clamp forming progeny isolates with the P and S tester strains did not deviate from the expected 1:1 ratio, while there was a

clear under representation of recovered + alleles for the V2 gene, with a significant deviation from the expected 50% (p < 0.01, v2) (Table 2). In the mapping procedure the data were coded as if all isolates forming clamps with the particular tester strain was sharing the same genotype as the parent carrying the IS gene in question. The S IS gene was located in the middle of group 19, positioned 6 cM from the closest marker (atcgp01) on one side and 8 cM from aaccp12 on the other side, while the P gene was found in linkage group 1, only 1.4 cM from pagccp3 and 6.3 cM from acccs2 (Fig. 1). The IS gene V2 was not successfully positioned in the linkage map.

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Fig. 2. Marker segregation plot for the 19 linkage groups. The y axis describes the recombination frequency values, given in segregation skewed towards the P parent (0.5–1) or the S parent (0–0.5). The x axis describes the distance between the markers of each group in centimorgan. LG, linkage group. The spaced lines indicate the 5% level of significance (p > 0.05, v2).

Table 2 Segregation of intersterility + alleles in progeny isolates +

S P+ a Vþ 2

Tested

Found

Expected

v2

101 101 96

51 39 28

50.5 50.5 48

0.005 2.619 8.333

a Allele frequency deviating from the assumed 50% at the 5% level of significance.

Table 3 Intersterility genotypes found in the progeny isolates Intersterility genotype þ


S P V
2 S
P þ V
2

þ S P V2 S þ P þ V
2 S þ P
Vþ 2 S
P þ Vþ 2


S P V2 a S þ P þ Vþ 2

Found

Expected

v2

21 11 9 13 4 3 28 12

21.7 13.8 8.8 13.8 8.8 5.7 21.7 5.7

0.023 0.568 0.005 0.046 2.618 1.279 1.829 6.963

a

Genotype with a frequency deviating from the expected, based on observed allele frequency, at the 1% level of significance.

Table 3 shows the frequency of observed IS genotypes found among the 101 progeny isolates tested in mating experiments with the three tester strains, along with the expected frequency of genotypes given the observed allele frequency (Table 2). Genotype Sþ Pþ Vþ 2 were significantly over represented with the observed frequency of 12 compared to the expected 5.7 (p < 0.01, v2). The distribution of the other genotypes was not significantly different from expected given the allele frequencies observed for the individual loci (p > 0.05, v2). Segregation of mating type was analysed through backcrossing all progeny isolates with both parental

strains. Mating, as evident from clamp connection formation, was observed in 39 of the progeny isolates backcrossed with TC 32-1 and in 8 of the isolates backcrossed with TC 122-12. None of the backcrossed progeny isolates formed clamps with both of the parental strains, while 54 of the progeny isolates formed clamps with neither of the parental strains. In some backcrosses where no clamps were found with either of the parental strains, hyphal deaths indicative of incompatibility, was observed in one of the backcrosses. The analysis was not able to link the mating type locus to any other marker.

4. Discussion In this study, we used AFLP technique to identify 358 genetic markers in the H. annosum s.l. genome. These markers, together with three IS loci, were used to construct a genetic linkage map. This first genetic linkage map of H. annosum, built on closely spaced DNA markers, allows genome organization to be further analysed and can serve as a foundation for genome analysis and gene cloning. The potential addition of microsatellite markers to our AFLP marker based map provides the opportunity to anchor our map to information from future mapping populations. However, too few such markers have been identified for Heterobasidion spp. (Johannesson and Stenlid, 2004). Many polymorphic fragments found comparing the parental isolates (not included in the mentioned 358) had to be excluded since they were too close in size to other fragments, making it impossible to undoubtedly score them in the progeny. Out of the resulting 358 markers, 141 (39%) had a segregation distorted from

M. Lind et al. / Fungal Genetics and Biology 42 (2005) 519–527

the assumed 1:1 ratio at the 5% significance level, which is a relatively high proportion compared with some other fungal genetic linkage maps. In the analysis of Gibberella fujikuroi (Xu and Leslie, 1996), 16 out of 150 (11%) markers had skewed segregation at the same level of significance, and in P. ostreatus (Larraya et al., 2000), the number was 14%. Our results are however closer to those of L. maculans (Pongam et al., 1998), where 18 out of 62 markers (29%) were distorted, or those of A. bisporus (Kerrigan et al., 1992), with 33% distorted markers. In our study, all the markers were included in the linkage analysis since removing markers with skewed segregation did not dramatically alter the appearance of the map. Markers with distorted segregation were evenly distributed among and within the linkage groups and no clustering of skewed segregating markers could be found in our analysis. There was no significant difference between the parental strains in contribution of markers having a distorted segregation (41.4 and 37.3%, S and P parent, respectively). No linkage group consists only or mostly of markers with skewed segregation. This is in concordance with most other linkage maps of fungal genomes. However, observations from C. neoformans by Forche et al. (2000) showed markers with distorted segregation forming two separate linkage groups. Skewed segregation of genetic markers in fungi has been suggested to be caused by biased selection of spore isolation used for the mapping population (Kerrigan et al., 1992; Larraya et al., 2000). Although the germination rate of basidiospores was high and the progeny isolates used in this study showed variation in growth rate and morphology, biased selection of spores can not be excluded. The linkage analysis in this study was carried out by mating two distantly related phylogenetic species probably separated many million years ago, which may have an impact on the mechanics of sexual reproduction. One would expect many sequence differences between the genomes, which might permit heteroduplex formation on large areas of homologous chromosomes during meiosis. Thus, it is not unreasonable to suppose that the segregation distortion could have arisen due to gene conversion. Gene conversion of genetic markers in Schizosaccharomyces pombe and Saccharomyces cereviseae is known to lead to non-Mendelian segregation (Davis and Smith, 2001). The exact number of chromosomes in H. annosum is not known, but karyotyping data suggest a genome size of about 25 Mbp, spanning over approximately 10 chromosomes (Anderson et al., 1993). We observed a significant discrepancy between the number of linkage groups and the estimated number of chromosomes. Although co-migration of similar sized chromosomes is a legitimate explanation for the discrepancy between chromosome number and linkage groups we suspect a lack of saturation of the map and expect that all small as well as possibly some of the large linkage groups will be

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joined with others, once additional markers are added. In our study, 50 markers were unlinked and an additional 70 markers were found in small linkage groups suggesting that still some part of the genome contain low marker density. The genetic distance between the species could also have an impact on the distribution of markers over the genome. The suspected reduced homology between sections of chromosomes might cause fewer recombinations. Unlinked or loosely linked markers may be caused by a varying cross over frequency in different areas of the genome. This might give rise to gaps between markers, which in turn may cause fragmentation of the linkage groups. Including all small and large linkage groups the genome size expanded to a total range of 2252 cM and the physical size to genetic distance was roughly estimated to be 11.1 kbp/cM. This is a lower ratio than reported in a number of other fungi: 32 kbp/cM in G. fujikuroi (Xu and Leslie, 1996), 27.9 kbp/cM in C. cinereus (Muraguchi et al., 2003), 35.1 kbp/cM in P. ostreatus (Larraya et al., 2000), and 70 kbp/cM in the oomycete B. lactucae (Sicard et al., 2003) but well in line with C. neoformans 9.6 kbp/cM (Forche et al., 2000). Markers gathered using AFLP analyses have been suggested to cluster due to the methods likelihood to detect SNPÕs which often occur in repetitive DNA regions (Qi et al., 1998). We could not detect such clustering; our markers are relatively evenly spread over the linkage groups, spaced on average 6.0 cM apart. Non-clustering markers are similar to what have been found in C. neoformans (Forche et al., 2000), while in M. graminicola (Kema et al., 2002) and in B. lactucae (Sicard et al., 2003) such clustering have been seen. The clustering phenomenon of AFLP markers seems to be associated with high density maps; hence addition of more markers might reveal clustering in H. annosum as well. Sympatric, outcrossing species must have some barriers (genetic or ecological) to gene flow in order to maintain species cohesion. Such genetic reproduction barriers may occur through natural selection in a population when adaptation to differing ecological niches arises. In H. annosum, the IS genes system might have gradually developed during the adaptation to distinct hosts preferences. The gene flow barrier provided by the IS genes is likely to be involved in the regulation of anastomosis and plasmogeny, a critical step in the formation of dikaryotic mycelia. Intersterility group affiliation in H. annosum is strongly correlated with host preference (Korhonen, 1978). Thus, it has been suggested that pathogenicity on a host is either mediated by genes physically or genetically linked to the IS genes or actually directly mediated by the S and P IS genes themselves (Chase and Ullrich, 1990). The genetic reproduction barrier is stronger expressed in sympatric than in allopatric populations of H. annosum (Korhonen and

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Stenlid, 1998). The F IS group from southern Europe is interfertile with North American S group while intersterile with the S group from southern Europe and partially interfertile with S group from northern Europe. The analysis of the IS genes was sufficient to locate two of the loci in the map, P and S, while the data were not enough to find the third one, V2. The data for V2 deviated strongly from the assumed 1:1 segregation ratio. This is probably due to problems of irregular clamp formation in H. annosum (Korhonen and Stenlid, 1998). Especially with the V2 tester strain, few visible clamps are formed in any compatible cross as the hyphae are thinner and the clamps smaller than for other H. annosum isolates. Of the individual genotypes, only the Sþ Pþ Vþ 2 genotype had a frequency significantly deviating from that expected of a single genotype given the allele frequency found for the individual IS loci. However, the observed frequency (12) of this genotype is exactly what would have been expected if the assumed 1:1 segregation of Vþ 2 alleles had been found. This indicates that some isolates have a generally higher tendency to form clamps easier spotted with the method of choice. Another possible reason that V2 could not be located in the map could be the low density of markers in that specific region of the genome. Chase and Ullrich (1990) showed that P, S, and V2 are not linked together which is supported by our results as none of the IS genes occur on the same linkage group. The same study also showed that P and V3 are linked closely together, approximately at a distance of 4 cM. This opens the possibility to find the V3 locus while cloning the P IS gene. It is possible that the V2 gene as well as the mating type gene could be located in the map should more data be collected. To map these genes, there is also the possibility to add more AFLP markers and other markers like microsatellites to obtain a denser map. However, there is a limit to the amount of informative markers possible to derive from our population. Another prospect would be to enhance the mapping population by collecting more single spore isolates. Although a problematic and time-consuming endeavour, one possible way to overcome the mentioned difficulties with scoring visible clamp connections would be to isolate a new V2 tester strain. Although our genetic linkage map of H. annosum is not saturated and have some gaps, it can be used to locate genes and to map genetically little studied traits such as somatic (vegetative) incompatibility and quantitative traits like pathogenicity. An average relationship between genetic and physical distance of 70 kb/cM is favourable for map-based cloning (Sicard et al., 2003), indicating that our map is well suited for such a prospect. This first reported successful localisation of the IS genes S and P is the first step towards the elucidation of the almost unstudied genetic system regulating reproduction barriers in fungi. The proximity of the S IS and

P IS loci to AFLP markers gives optimism for identification and map based cloning of them. This map will form the basis for further genetic studies including the identification of markers useful for characterisation of S and P IS groups in field populations.

Acknowledgments The parental strains (TC 32-1 and TC 122-12) and tester strain (TC 111-4) were kindly provided by Dr. Thomas Chase. This project was financially supported by Swedish Research Council for Environmental, Agricultural and spatial Planning (FORMAS). Financial support from Carl Trygger Foundation is also gratefully acknowledged.

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