effects of introgression and recombination on haplotype ......resistance to both soil-borne...

Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.106.063800

Effects of Introgression and Recombination on Haplotype Structure andLinkage Disequilibrium Surrounding a Locus Encoding

Bymovirus Resistance in Barley

Silke Stracke,* Thomas Presterl,†,1 Nils Stein,* Dragan Perovic,*,‡

Frank Ordon‡ and Andreas Graner*,2

*Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK), D-06466 Gatersleben, Germany, †University of Hohenheim, Institute ofPlant Breeding, Seed Science and Population Genetics, D-70593 Stuttgart, Germany and ‡Institute of Epidemiology and

Resistance Resources, Federal Center for Breeding Research on Cultivated Plants, D-06449 Aschersleben, Germany

Manuscript received July 20, 2006Accepted for publication November 16, 2006

ABSTRACT

We present a detailed analysis of linkage disequilibrium (LD) in the physical and genetic context of thebarley gene Hv-eIF4E, which confers resistance to the barley yellow mosaic virus (BYMV) complex. Eighty-three SNPs distributed over 132 kb of Hv-eIF4E and six additional fragments genetically mapped to itsflanking region were used to derive haplotypes from 131 accessions. Three haplogroups were recognized,discriminating between the alleles rym4 and rym5, which each encode for a spectrum of resistance toBYMV. With increasing map distance, haplotypes of susceptible genotypes displayed diverse patternsdriven mainly by recombination, whereas haplotype diversity within the subgroups of resistant genotypeswas limited. We conclude that the breakdown of LD within 1 cM of the resistance gene was generatedmainly by susceptible genotypes. Despite the LD decay, a significant association between haplotype andresistance to BYMV was detected up to a distance of 5.5 cM from the resistance gene. The LD pattern andthe haplotype structure of the target chromosomal region are the result of interplay between low re-combination and recent breeding history.

THE ever-increasing availability of nucleotide se-quence, and the concomitant improvement thatthis brings to our understanding of the organization ofcomplex crop plant genomes, has created the oppor-tunity to identify trait-related genes and to analyze theirallelic diversity. The association between phenotype andgenotype in diverse populations represents a power-ful approach, but the applicability and design of suchanalyses depend critically on the extent and pattern oflinkage disequilibrium (LD) present in the study popu-lation. Cultivated barley (Hordeum vulgare ssp. vulgare)has many of the hallmarks known to be associated witha high level of LD. Its effective recombination rate isdramatically reduced by its predominantly inbreed-ing habit, with an estimated outcrossing rate of �5% inwinter barley and ,0.5% in spring barley (Giles et al.1974; Doll 1987; Abdel-Ghani et al. 2005). Domesti-cation and intensive selection have introduced majorbottlenecks in genetic variation, and these are thoughtto be largely responsible for the perceived narrownessof the modern gene pool (Badr et al. 2000; Russellet al. 2000; Matus and Hayes 2002). LD in modern

spring barley, as estimated from a whole-genome survey,extends over distances of at least 10 cM, indicative of anextensive conservation of the genetic identity of barleychromosomes (Kraakman et al. 2004). However, esti-mates of genomewide LD conceal localized variation,which, as has been shown for a number of species, canbe substantial and independent of the mating sys-tem (Gupta et al. 2005). In the self-pollinating speciesArabidopsis thaliana LD varies from ,10 kb in global(Tian et al. 2002) to 50–250 kb in local populations(Nordborg et al. 2002, 2005; Aranzana et al. 2005).Within a given set of maize (an outbreeding species)accessions, the extent of LD has been documented tovary widely around different genes and chromosomalregions (Remington et al. 2001). On chromosome1, for example, it has been shown to decline within0.1–0.2 kb (Tenaillon et al. 2001), while in a regionunder high selection pressure, it can extend up to600 kb (Palaisa et al. 2004).

Little is known of the structure of LD in the physicaland genetical vicinity of genes in cultivated barley (re-viewed in Gupta et al. 2005). As the success of associa-tion studies in this major crop species depends criticallyon this knowledge, we have set out to evaluate LD in awell-characterized region of chromosome 3H, whichharbors Hv-eIF4E, a gene encoding an important virusresistance. Two recessive alleles, rym4 and rym5, confer

1Present address: KWS SAAT AG, D-37574 Einbeck, Germany.2Corresponding author: Leibniz-Institute of Plant Genetics and Crop

Plant Research (IPK), Corrensstrasse 3, D-06466 Gatersleben, Germany.E-mail: [email protected]

Genetics 175: 805–817 (February 2007)

resistance to both soil-borne Bymoviruses, barley yellowmosaic virus (BaYMV) and barley mild mosaic virus(BaMMV) (Kanyuka et al. 2005; Stein et al. 2005). Thiscombination of pathogens is commonly referred to asthe barley yellow mosaic virus complex (BYMV). Car-riers of rym4 are resistant to BaMMV, BaMMV-Sil, andBaYMV-1, but are susceptible to BaYMV-2, while thosecarrying rym5 are resistant to BaYMV-1, BaYMV-2, andBaMMV, but not to BaMMV-Sil (Kanyuka et al. 2004).Although at least seven independent loci conferringresistance to BYMV have been identified in barley todate (Ordon et al. 2005), European breeding has re-lied heavily on rym4 and rym5, both of which originatefrom single germplasm accessions. The Dalmatian land-race Ragusa is the source of rym4 (Huth 1985), whilerym5 derives from the Chinese landrace Mokusekko 3(Konishi et al. 1997; Graner et al. 1999; Friedt et al.2000).

The cloning of Hv-eIF4E (Stein et al. 2005; Wickeret al. 2005) provides a focus for an LD analysis. Takinginto consideration that breeding for resistance in Europedid not start until the 1980s, and that the Hv-eIF4E locushas been subjected to a high selection pressure over ashort timescale, this case study reveals much of howgenome structure and dynamics can be shaped by plantbreeding.

MATERIALS AND METHODS

Plant material: Included in the study were 127 cultivatedbarley cultivars and landraces originating from Europe, Asia,and America, along with four accessions of wild barley (ssp.spontaneum) collected in either Turkey or Israel. Geographicalorigins, pedigrees (where available), and phenotype withrespect to reaction to BYMV are listed in supplemental TableS1 at http://www.genetics.org/supplemental/. Accessions car-rying rym4 included Ragusa, 19 European cultivars, and fourAsian landraces. With respect to rym5, five European and sixAsian accessions, including the donor Mokusekko 3, wereanalyzed. All these accessions were tested in the field and bymechanical inoculation to verify their rym4/rym5 status (Götzand Friedt 1993; Ordon et al. 1993) (supplemental Table S1at http://www.genetics.org/supplemental/). The remainingresistant accessions have not been tested for allelism torym4 and rym5 (rym?; Table S1 at http://www.genetics.org/supplemental/). Nevertheless, their resistance is likely due tothe presence of genes other than rym4/rym5, since theirhaplotypes differed markedly from those of the verified rym4and rym5 accessions. In the context of this study, thesegenotypes are referred to as noncarriers of rym4/rym5 andwere merged with the group of susceptible cultivars. GenomicDNA was extracted from a single plant per accession asdescribed elsewhere (Graner et al. 1991).

Population structure: Sixteen barley expressed-sequence-tag (EST)-derived simple sequence repeat markers (SSRs)(EST–SSRs) were selected to characterize population struc-ture (supplemental Table S2 at http://www.genetics.org/supplemental/) (Thiel et al. 2003). The selection of appro-priate markers was based both on map position (to obtain aneven distribution across the genome) and on informativeness(high polymorphic information content value) (Weber 1990;Anderson et al. 1993). The markers were organized into mul-

tiplex sets comprising between three and six different primerpairs. Amplicons were generated with the Multiplex PCR kit(QIAGEN, Chatsworth, CA), using an amplification programof 95�/15 min, followed by 40 cycles of 94�/30 sec, 60�/30 sec,and 72�/15 sec, with a final extension step of 72�/10 min. PCRproducts were separated on 6% polyacrylamide gels usingthe ABI377 system (Applied Biosystems/Applera, Darmstadt,Germany), and profiles were analyzed with the software pack-ages GeneScan 3.7.1 und Genotyper 3.7. The assessment ofgenetic population structure was performed within a Bayesianframework using a Markov chain Monte Carlo algorithm tosample from the joint posterior distribution of the subpopu-lation allele frequencies, and assignment of individuals toparticular subgroups was effected with STRUCTURE version2.1 (Pritchard et al. 2000a; http://pritch.bsd.uchicago.edu/structure.html). For a K setting of between 2 and 6, 10 inde-pendent simulations were performed, using the admixturemodel and a burn-in of 500,000 followed by 1,000,000 iterations.

To assess the applicability of an association analysis withinmodern breeding material, association tests were performedin 51 European winter barley cultivars, comprising 20 rym4carriers and 31 susceptible accessions. To infer the populationstructure of this sample, the estimated individual member-ship coefficient in each subgroup was accounted for with astructured population association test (STRAT) (Pritchardet al. 2000b). The test statistic is constructed by computing thelikelihood ratio to test the null hypothesis that allele frequen-cies of subpopulations at the candidate locus are independentof phenotype. Phenotypic association was tested by treatingsingle haplotypes as multiallelic marker loci, including onlysingle polymorphic sites at which the minor allele occurred ata frequency of at least 0.05. Empirical P-values of the observedtest statistic per marker locus and per polymorphic site werecalculated using 100,000 permutations.

The variation in phenotype due to population structure wasassessed by a multiple linear regression model (PLABSTATversion 2H; Utz 1993). The estimated membership fractionsfor each genotype in K clusters of K ¼ 3 to K ¼ 6 in the entireset and K ¼ 2 for the subset of European winter cultivars wereconsidered. The coefficient of determination from the re-gression (R 2) was applied to quantify the effect on the trait ofthe genetic background structure.

SNP discovery and genotyping: A number of mutations inHv-eIF4E are known to confer resistance to BYMV. Thus thescan for polymorphic nucleotides within the set of 131 ac-cessions was focused on an assembly of 12 genomic fragmentswithin and surrounding Hv-eIF4E (Figure 1). The size of eachfragment was between 175 and 1039 bp, encompassing in total6.9 kb. This represents a genetic interval of �5.5 cM proximalto, and 0.9 cM distal to, Hv-eIF4E (Table 1). The LD structurein relation to physical distance was analyzed on the basis of sixfragments spanning the �132-kb BAC contig AY661558 es-tablished during the cloning of Hv-eIF4E (Stein et al. 2005;Wicker et al. 2005). This included three genic fragments,covering all five exons (645 bp) and flanking intron regions(1840 bp), one fragment from the adjacent distally mappinggene Hv-MLL (marker No519; 1048 bp upstream), and onenoncoding fragment mapping both distally (No969) andproximally (No1134) to the target. The proximal fragmentsGBR1843, GBS0526, and GBS0419 have been previouslymapped in the Oregon Wolfe mapping population (OWB,Dom 3 Rec) (Costa et al. 2001), which consists of 94 F1-derived doubled haploid progeny (Stein et al. 2007). Thedistal fragments GBR1845, GBR1851, and GBS1020 weremapped in 115 segmental recombinant inbred lines ofAlraune 3 W122.37, a population genetically equivalent to.4800 F2 progeny (AW) (Pellio et al. 2005). Because of a lackof polymorphism between the parents of both these mapping

806 S. Stracke et al.

populations, it was not possible to combine all the markersonto a single map. Therefore, for the LD analysis, genetic mapdistances were considered separately for the OWB and AWpopulations.

In preparation for sequencing, DNA fragments were ampli-fied using fragment-specific PCR profiles, as given in supple-mental Table S3 at http://www.genetics.org/supplemental/.PCR products were purified (MinElute 96 UF PCR puri-fication, QIAGEN), and 50–100 ng of product was used astemplate for cycle sequencing with DYEnamic ET dye termi-nator chemistry (Amersham Bioscience, Freiburg, Germany)on a capillary automatic sequencing device (MegaBace 1000,Amersham). Alignments were compiled and analyzed usingSequencher v4.1 (Gene Codes, Ann Arbor, MI) and Bioeditv4.7.8 (Tom Hall, North Carolina State University; http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The data set de-scribing all genotyped polymorphic sites is available on request.

Statistical analyses: For the purpose of statistical analyses,indels were regarded as single sites. Estimates for LD wereobtained via the software package TASSEL (version 1.0.9,http://www.maizegenetics.net/bioinformatics/tasselindex.htm), applying the measurement r 2 (squared correlationcoefficient) (Hill and Robertson 1968). To estimate thestrength of LD between haplotypes, we used the measure D9(Lewontin 1964) modified for multiple alleles by calculatinga weighted average of D9 value where the weights are theproducts of the corresponding allele frequencies (Hedrick1987; Farnir et al. 2000). Significance of LD is determined by atwo-sided Fisher’s exact test for biallelic sites and by permu-tations (setting number 1000) for multiallelic sites. To precludebias of low-frequency alleles on the LD calculation, poly-morphic sites featuring allele(s) with a frequency of #0.05were excluded. Values of r 2 and D9 were either plotted as afunction of the pairwise distance between the polymorphicsites or displayed as an LD matrix as generated by TASSEL.

For the clustering of haplotypes across the 132-kb contigand the assignment to haplogroups, neighbor-joining trees(Saitoh et al. 2004) were constructed on the basis of Kimuratwo-parameter distances and pairwise deletions of gaps,applying MEGA version 2.1 (Kumar et al. 1994). To test therobustness of derived tree topologies, 1000 bootstraps wereperformed. To estimate gene genealogies, a haplotype networkof Hv-eIF4E was obtained by statistical parsimony (Templetonet al. 1992), using the program TCS version 1.18 (Clement et al.2000). This program calculates the probability of parsimonyfor all pairwise differences until the probability exceeds 0.95.

To assess genomic variability across the entire target regionand to measure and compare the diversity within and acrossthe designated haplogroups, both nucleotide (pi) and haplo-type (Hd) diversities were estimated using DnaSP (Version 3.51;

Rozas and Rozas 1999; http://www.ub.es/dnasp/) software.For this purpose, indels were treated as single sites. Overall,12 of the 127 H. vulgare genotypes were excluded from theanalysis because of missing data.

RESULTS

Population structure: The 16 EST–SSR loci revealed77 alleles, with 2–9 alleles/locus. The distribution ofthese alleles was in close accordance with the taxo-nomic and geographic subgroups defined in the germ-plasm collection, as illustrated by a Bayesian approach(Figure 2). However, it was difficult to fully determinethe optimal number of subgroups, since the poste-rior probabilities for the number of clusters increasedsteadily. On the assumption of two subgroups (K ¼ 2),the population was divided into winter and springtypes. A stepwise increase to K ¼ 6 led to the gradualseparation of the Asian from the European accessions,of the two-row from the six-row spike types, and of thecultivated (vulgare) from the wild (spontaneum) types.Importantly, all rym4 carriers and all European rym5carriers displayed allele frequencies similar to thoseof susceptible accessions belonging to different sub-groups. Thus population structure could account foronly 21% (K ¼ 3) to 29% (K ¼ 6) of the variation inBYMV resistance.

Structure of LD: The 12 genomic fragments encom-passing the target region were resequenced across thecollection of 131 accessions. A total of 83 polymorphicsites, comprising 78 SNPs and five indels, were identi-fied. Of the 83 sites, 5 were triallelic, and 4 of these,in addition to 14 of the biallelic sites, had an allelefrequency of #0.05 and were excluded from the sub-sequent analysis. Across the entire collection, a consid-erable degree of LD was evident within the physicalcontig (Figure 3A), while at the genetic level, ther 2 value dropped sharply to ,0.3 within 1 cM of thetarget (Figure 3B). Despite the diminishing r 2 value,a repeated increase of LD (r 2 . 0.3) was observed ata larger distance. This was not an artifact of popula-tion stratification, as the allele distribution of the

Figure 1.—Combinedgenetic and physical mapof the region surroundingHv-eIF4E on chromosome3H. Genetic distances incentimorgans are indicatedfor markers flanking the lo-cus in the OWB and thehigh-resolution AW maps.The arrow indicates the cen-tromere (C). The schema-tic arrangement of markerfragments is depicted (notto scale) on the physical BACcontig (AY661558). Hv-MLL,barley MCT-1-like protein.

Haplotype Structure Surrounding Resistance Locus 807

corresponding pairs of polymorphic sites did notassociate with population structure.

To compare the LD structure between rym4/rym5-resistant and -susceptible genotypes (including non-carriers of rym4/rym5), r 2 was determined separately forthese two subgroups (Figure 4). The extended structureof LD observed for the 132-kb contig was confirmed forall subgroups, but with varying strength. In the sub-group represented by the rym4 (n ¼ 24) and the rym5(n¼ 11) carriers, most pairs of polymorphic sites withinthe interval displayed complete LD (r 2 ¼ 1), while thesusceptible subgroup (n ¼ 96) showed fewer high r 2values. LD within the susceptible group fell, as it did forthe entire set, within 1 cM of the target. On the otherhand, the rym4/rym5 subgroup was characterized by

a significantly inflated LD across the entire geneticregion, demonstrating a high degree of conservationwithin resistant genotypes.

Haplotype structure: Haplotypes were generated onthe basis of the polymorphic sites, and their structureacross the entire genetic interval was analyzed. Con-sidering each individual fragment, between three andseven haplotypes achieved a frequency .2% (Table1). LD strength between haplotypes of a single DNAfragment within subgroups of resistant (rym4 andrym5 carriers) and susceptible genotypes revealedremarkable differences (supplemental Figure S1 athttp://www.genetics.org/supplemental/). Compared tothe susceptible group, resistant accessions had higherr 2 values across the entire region.

Figure 2.—Estimated population structure based on 16 EST–SSRs. The population can be partitioned into K subpopulations,which are color labeled. A bar, whose colored segments represent the individual’s estimated contribution to the individual sub-populations, represents each accession. Black vertical lines separate accessions of different resistance phenotype, origin, andgrowth habit. U, America; Hs, ssp. spontaneum.


For the 132-kb contig, 22 haploytpes were identifiedon the basis of 54 polymorphic sites (Figure 5). The rym4carriers included two haplotypes (haplotype 1 rym4-Aand haplotype 2 rym4-E), which matched the two broadgeographical origins of Asia (A) and Europe (E). Allrym5 carriers shared the same haplotype (haplotype 4rym5). Noncarriers of rym4/rym5 were represented by19 haplotypes. More than half of them (10/19) weresingletons while .67% of those genotypes belonged toone of the three major haplotypes 3, 5a, and 17. Using aneighbor-joining method, the haplotypes clustered intothree major clades, hereafter referred to as haplogroupsI–III (Figure 6). The formation of these haplogroupsappeared to be independent of BYMV resistance (Fig-ure 5). Thus, haplogroup I included three haplotypescomprising the European and Asian rym4 carriers (hap-lotypes 1 and 2) and a group of highly conserved sus-ceptible two-row winter cultivars (haplotype 3). Only 6of the 54 scored sites varied within this haplogroup.Haplogroup II contained rym5 carriers (haplotype 4)and geographically diverse accessions (haplotypes 5–12),which included both no, or as yet undetermined, re-sistance alleles/genes. A clear dimorphism betweenhaplogroups I and II was obtained for half of the poly-morphic sites of the contig. Dimorphic sites were lo-cated mainly in noncoding sequences, while 15 of the16 polymorphic exon sites were shared between thetwo haplogroups. Only one cultivar (Posaune, haplo-type 13) was intermediate between these groups, con-taining a signature of recombination both up- anddownstream of the Hv-eIF4E exon 1. Haplogroup III(haplotypes 14–20) had a composite structure with atleast three origins. The sequence data of No1134 areclosely related to haplogroup I, with only two poly-morphic sites present (Figure 5, position 29084n and29201n). Moreover, variation within Hv-eIF4E fragment1 resulted in a sequence, which is clearly distinct fromthat present in haplogroups I and II. The pattern ofpolymorphic sites downstream of Hv-eIF4E fragment1, however, resembled haplogroup II. This apparentpatchwork on either side of fragment 1 may indicate the

occurrence of several historical recombination eventswithin Hv-eIF4E. The overwhelming reduction in hap-lotype and nucleotide diversity within the haplogroups,as compared to what is present across the entire col-lection, validates the grouping (supplemental Table S4at http://www.genetics.org/supplemental/). A separateanalysis indicates that, within a given haplogroup, lessdiversity is present in the noncoding than in the codingsequence. Importantly, all sites within the coding se-quence of Hv-eIF4E generate an amino acid exchange(s)in the protein sequence.

A haplotype network for Hv-eIF4E (Figure 7), con-structed with a statistical parsimony algorithm, illustratesthe relationships between the haplotypes between andwithin the haplogroups and emphasizes the genetic dis-tance between the haplogroups I and II/III, with thepresumed recombinant haplotype 13 representing theonly link between them. Haplotypes 5 and 17 are centralwithin the network and have generated a number ofdescendant haplotypes. Both are present in a signifi-cant number of accessions and are broadly distributedwith respect to growth habit and origin. Thus they areprobably the most ancient in the germplasm set. Incontrast, on the basis of the termini of the network, thetwo resistant haplotypes, rym4-E and rym5, must haveemerged rather recently.

The genetic diversity within the European (n ¼ 80)and Asian (n ¼ 32) subgroups was compared across thephysical contig. Despite the 2.5-fold excess of Europeanmaterial within the collection, the Asian accessions in-cluded a larger number of haplotypes. The more fre-quent occurrence of singletons in the latter set (sevenvs. four) is largely responsible for its apparent wider di-versity. With respect to common haplotypes (frequencywithin a subgroup $0.05), six were represented in theEuropean set (95.5% of the total diversity) and sevenin the Asian set (80.5% of the total diversity). Four ofthese common haplotypes were shared between thesubgroups (haplotypes 4, 5a, 17, and 20; Figure 5). As aresult, Hd and p-values were comparable between theEuropean (Hd¼ 0.806 6 0.022; pi¼ 0.00484 6 0.00020)

Figure 3.—LD structure at Hv-eIF4E. Plots show the pairwise LD measurement r 2 related to (A) physical, and (B) genetic dis-tances. The data consist of 67 polymorphic sites with a minor allele frequency .0.05 in the set of 131 accessions. Nonsignificant r 2

values (P . 0.05) are indicated by open dots. Mean values of r 2 are given by asterisks for (A) windows of 1 and 10 kb distance and(B) identical genetic distances.


and Asian (Hd¼ 0.910 6 0.025; pi¼ 0.00441 6 0.00049)sets.

Association analysis: Since BYMV resistance was mostfrequent among the European winter barleys, a test forassociation between the candidate locus and resistance

was carried out for the subgroup of 51 European win-ter cultivars, of which 20 are rym4 carriers and theremainder are susceptible accessions. To ascertain thepopulation structure within this group, a model-basedclustering algorithm was applied. The average-likelihood

Figure 4.—Strength and extent of LD in different germplasm sets. Polymorphic sites in the investigated interval with a minorallele frequency .5% were considered for a pairwise calculation of LD across (A) the entire collection of 131 accessions, (B) thenon-rym4/rym5-resistant subpopulation of 96 accessions, and (C) the set of 35 rym4 or rym5 carriers. Each point in the LD matrixrepresents a comparison between a pair of polymorphic sites, with the r 2 values displayed above the diagonal and the P-values forFisher’s exact test below. Points on the diagonal correspond to comparisons of each site with itself. Polymorphic sites locatedwithin the fragments of the physical contig are indicated. Color codes for r 2 and P-values are given.


values from 10 runs reached a maximum at K ¼ 2 andfell for higher values (data not shown). The populationexplained 6% of the variation in BYMV resistance.Structured population association tests were carriedout between BYMV resistance and haplotype at both the12 marker fragments and at 55 of the 83 polymorphic sites(excluding those where the minimum allele frequencywas ,0.05). The association was significant (P , 0.01)for all nine loci mapping between No1134 and GBS1020,comprising the physical contig across Hv-eIF4E and thedistal 1-cM region (Table 2). At six loci in this interval(No1134, Hv-eIF4E 1–3, No519, GBR1845M), rym4 car-riers possessed haplotypes that were not observed in anysusceptible accessions. Locus GBR1843, located 6.5 cMproximally to the physical contig, showed only a weakassociation (P , 0.05) with resistance. Over 56% of theSNPs were significantly associated with the resistantphenotype (P , 0.01), confirming the outcome of thehaplotype test (supplemental Table S5 at http://www.genetics.org/supplemental/).

As a control, association tests were carried out be-tween BYMV resistance and alleles of the evenly distrib-uted SSR markers across the genome (supplementalTable S5 at http://www.genetics.org/supplemental/).

Of 11 polymorphic SSR markers in the European winterbarleys set, 10 were not associated with the phenotypeand only one marker located on 6H showed a weakassociation (GBM1021; P ¼ 0.035).

DISCUSSION

We have described a detailed evaluation of LD andhaplotype patterns surrounding the gene Hv-eIF4E, whichencodes a heavily utilized virus resistance in Europeanbarley.

Population structure: Population structure has amajor impact on patterns of LD and, consequently, onthe outcome of association studies (Pritchard et al.2000b). The diverse collection of cultivars and landracesfrom Europe, Asia, and America selected in this studycould be clustered on the basis of growth habit, earmorphology, geographical origin, and subspecies and issimilar in genetic breadth to collections used in othergenomewide marker analyses in barley (Melchinger et al.1994; Ordon et al. 1997; Thiel et al. 2003). Importantly,however, the population structure did not disturb theassociation between haplotype and BYMV resistance.

TABLE 1

Summary of all sampled marker fragments

MarkerDistancein cMa

Position oncontig in bpb

Fragmentsize in bp

Polymorphicsites Haplotypesc Best BLASTx hit Score

GBR1843 5.5 OWB — 347 4 4 Unknown proteinP0458E05.14(Oryza sativa BAC05614)

233 1.0E-60

GBS0526 4.5 OWB — 930 9 4 Putative 60S ribosomal proteinL38 (O. sativa BAC79676)

132 1.0E-30

GBS0419 2.2 OWB — 371 4 3 Putative b-fructofuranosidase(O. sativa BAC05626)

354 9.0E-97

No1134 0.02 AW 28,97329,373

401 11 6 None AY661558

Hv-eIF4E 0 — 101,606 879 12 6 Eukaryotic initiation factor oftranslation 4E (Hv-eIF4E)

AY661558fragment 1 102,484

Hv-eIF4E 0 — 103,313 1039 15 7 Eukaryotic initiation factorof translation 4E (Hv-eIF4E)

AY661558fragment 2 104,351

Hv-eIF4E 0 — 106,146 565 12 6 Eukaryotic initiation factor oftranslation 4E (Hv-eIF4E)

AY661558fragment 3 106,710

No519 0 — 107,786 499 2 3 Monocarboxylic acidtransporter-like (Hv-MLL)

AY661558108,284

No969 0 — 161,182 175 2 3 None AY661558161,356

GBR1845 0.4 AW — 265 5 4 Putative cytochrome B5(O. sativa BAB63673)

110 8.0E-31

GBR1851 0.7 AW — 424 5 4 Unknown protein P0518C01.5(O. sativa BAB63668)

75 8.0E-13

GBS1020 1.0 AW — 1011 2 3 Putative cell cycle switchprotein (O. sativaBAB63690)

312 4.0E-89

a Relative to ATG of Hv-eIF4E.b Start and end position of the corresponding fragment based on contig AY661558.c No. of haplotypes with a frequency .0.02 in the entire collection.


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Linkage disequilibrium and genomic pattern: Inself-pollinating species such as A. thaliana and rice, LDhas been observed to extend several kilobases be-yond a target gene (Hagenblad and Nordborg 2002;Nordborg et al. 2002; Garris et al. 2003; Hagenbladet al. 2004; Olsen et al. 2004), and can even reach thecentimorgan range (Nordborg et al. 2002; Zhu et al.2003; Aranzana et al. 2005). Corresponding resultsapply at the Hv-eIF4E locus. The structure of the threeconserved haplogroups in the physical vicinity of Hv-eIF4E reflected a high level of LD across the 132-kbinterval. Similarly, a sustained level of LD has beenrevealed over a 212-kb stretch flanking the hardnesslocus in European elite barley cultivars (Caldwell et al.2006). With respect to genetic distance, LD around Hv-eIF4E fell below the critical threshold of r 2 ¼ 0.3 within,1 cM. This result is inconsistent with a genomewideestimate for LD of up to 10 cM, as reported amongmodern spring barley cultivars (Kraakman et al. 2004).However, it has been conclusively established for bothplants and humans that LD is highly variable, reflect-ing the combined influences of population structure,genomic region under consideration, and the numberof polymorphic sites available (Akey et al. 2003; Keet al. 2004).

The drastic decay of LD at the genetic level, as it wasobserved in this study, was mainly attributable to thesusceptible accessions. While in both rym4- and rym5-resistant groups considerable haplotype conservationpersisted, the haplotypes of susceptible accessions re-vealed a high level of recombination in the regionsflanking the 132-kb fragment. Various forces can con-tribute to haplotype conservation, including (a) priorgenetic bottlenecks resulting in a low effective population

size, (b) introgression of rym4 and rym5 from a restrictednumber of sources, (c) a very recent history of intensiveselection for resistance against BYMV, (d) a lack of re-combination in the target region, and (e) gene function.While it is difficult to demonstrate a specific bottleneckaffecting the accessions investigated in this study, theeffect of severe domestication-related bottlenecks onhaploytpe diversity between modern barley cultivarsand wild ssp. spontaneum has been repeatedly described(Badr et al. 2000; Matus and Hayes 2002; Piffanelliet al. 2004). A comparison between the diversity at Hv-eIF4E within wild and cultivated barley should providemore information regarding the history of this locus.Generally, modern breeding has narrowed the geneticbase by altering allele frequencies. With the increaseduse of ssp. spontaneum as a donor for resistance genes,novel alleles have been introgressed into the gene poolof cultivated barley. The typical outcome of such intro-gressions has been analyzed elsewhere (Ivandic et al.1998; Pillen et al. 2004). While it initially generates aspike in genetic diversity, strong selection for the exoticallele will gradually erode the frequencies of the ‘‘old’’alleles, even leading to their complete loss (Russellet al. 2000; Collins et al. 2001; Bundock and Henry2004). Such a scenario is likely to have occurred duringbreeding for resistance to BYMV. The analysis of pedi-gree and molecular marker data provides evidence thatthere was only a single source each of rym4 and rym5in European elite germ plasm (Huth 1985; Granerand Bauer 1993; Friedt et al. 2000). Thus, strong se-lection for resistance resulted in an enrichment ofthe corresponding alleles in European winter barley.A similar situation applies to the selection of yellowendosperm in maize, which started in the early 20th

Figure 6.—Haplotype relationships within132-kb interval. The midpoint-rooted neighbor-joining tree is based on the sequence ofNo1134, Hv-eIF4E fragments 1–3, No519, andNo969 of the 22 haplotypes designated in Fig-ure 5. The numbers on the branches indicatethe frequency (%) with which a clade appearedin 1000 bootstrap samples. Branch lengths areproportional to the probable number of substitu-tions per site using Kimura two-parameter distan-ces. Haplogroups I, II, and III are indicated.


century, and has been traced to two independent intro-gression events. As a result, genetic variation in the vi-cinity of the target locus Y1 is very low (Palaisa et al.2003, 2004).

The short timescale over which intensive selection forBYMV resistance has operated has provided as yet onlylimited opportunities for recombination around theresistance gene. However, targeted selection for rym4/rym5 is probably not the only reason for the observedLD, as the pedigree triplets (parent1, parent2, off-spring) formed by the susceptible cultivars Ursel, Ultra,Villa, and Volla, which are all represented in the col-lection, also showed no recombination across the entire

region genotyped. Since susceptible alleles of Hv-eIF4Eare not likely to be subjected to selection pressure, theobserved pattern is more probably an outcome of re-lated pedigrees and a limited number of meiotic eventsduring the breeding process.

A low recombination rate in the target region haspossibly also governed the size of the introgressionsegment and the LD pattern. In this regard, a compar-ison of genetic and physical distances in the region ofHv-eIF4E revealed marked differences in recombina-tional activity and, in particular, a reduction proximalto the gene and an increase distal to it (Stein et al.2005). Except for marker fragment No1134, which

Figure 7.—The Hv-eIF4Ehaplotypenetwork.Numberscorrespond to the haplotypedesignations in Figure 5.Lines represent mutationalchanges and solid circles in-dicate intermediate haplo-types. The 95% confidenceinterval is 13 steps. Haplo-types shared between morethan one genotype are indi-cated by squares. Shadedsquares correspond to rym4-E (1), rym4-A (2), and rym5(4) haplotypes, respectively.Haplotypes showing strongindependence from growthhabit and origin are markedwith a hatched pattern. Hap-logroups I, II, and III areindicated.

TABLE 2

Structured population association tests between haplotype frequency at single-marker loci and rym4-encoded BYMV resistance

K ¼ 1 K ¼ 2Marker locus Distance in cMa x2 d.f. TSb P-value TSb P-value

GBR1834 5.5 11.40 4 6.41 0.0134* 7.39 0.0427*GBS0526 4.5 1.89 1 0.95 0.1670 1.55 0.2288GBS0419 2.2 7.39 1 3.99 0.0051** 2.66 0.0812No1134 0.02 31.18 3 16.29 ,0.0001*** 17.00 ,0.0001***Hv-eIF4E -1 0 31.34 3 16.44 ,0.0001*** 16.92 ,0.0001***Hv-eIF4E -2 0 31.04 3 16.19 ,0.0001*** 16.94 ,0.0001***Hv-eIF4E -3 0 31.04 3 16.19 ,0.0001*** 16.94 ,0.0001***No519 0 31.00 2 16.00 ,0.0001*** 17.27 ,0.0001***No969 0 7.64 2 4.04 0.0209* 6.97 0.0066**GBR1845 0.4 30.58 1 15.80 ,0.0001*** 16.78 ,0.0001***GBR1851 0.7 20.93 2 10.84 ,0.0001*** 11.79 ,0.0001***GBR1020 1.0 16.20 1 8.35 ,0.0001*** 9.27 ,0.0001***

*P # 0.05, **P # 0.01, ***P # 0.001.a Relative to ATG of Hv-eIF4E.b Test statistic of STRAT (TS).


borders an interval, characterized by a low ratio ofphysical-to-genetic distance (0.8–2.3 Mb/cM), the con-tig is located in a region with a high ratio (30–50 Mb/cM). The low recombination frequencies are consistentwith the large proportion of transposable elementsequence present in the 439.7-kb BAC contig, whichcontains only one genic island of 10 kb harboring Hv-eIF4E and Hv-MLL (Wicker et al. 2005). Meiotic re-combination in eukaryotes is confined mainly to genes(Thuriaux 1977; Civardi et al. 1994; Dooner andMartinezFerez 1997), and studies in maize have con-firmed that genes located close to retrotransposonshave less recombinational activity than those presentwithin gene clusters (Fu et al. 2002). A possible addi-tional factor contributing to LD relates to selectionpressure on genes located on either side of the BACcontig. The maintenance of LD across several genes hasbeen demonstrated in maize, although it was largelyrestricted to gene-rich regions (Palaisa et al. 2004).

Further investigations are clearly needed to deter-mine whether the genomic patterns observed can beattributed to a single factor such as limited sample size,limited effective population size, the small number ofgenerations since introgression, or low recombination,or whether it is the result of a combination of some or allof these forces.

Haplotype network for the Hv-eIF4E locus: On thebasis of the derived network model, it can be suggestedthat the resistant and susceptible alleles have descendedrelatively recently from a common ancestor. The sepa-ration between haplogroups I and II lends strength tothe notion that both rym4 and rym5 have an indepen-dent evolutionary history. Evidence for a geographicpattern has been provided for several loci in wild barley(Morrell et al. 2003, 2005), but the strong dimorphismassociated with rym4 and rym5 did not correspond to anygeographic subdivision. The fact that the germplasmhas been subjected to breeding, material exchange, andsubsequent introgression events of course may obscuresuch a division. Thus, to gain a better understanding ofthe evolution of the haplogroups, comprehensive allelegenotyping within a set of geographically widely distrib-uted landraces and wild barleys will be required.

Surprisingly, the strong clustering of haplotypes iden-tified across Hv-eIF4E was exclusively attributable topolymorphic sites in noncoding sequence, while thegenetic variation within each group was due mainly toamino acid replacement mutations in the exons. Whythe dimorphism among the haplogroups is due largelyto noncoding polymorphic sites remains unexplained.In particular, the haplotype diversity within haplo-group II is mostly attributable to rare polymorphic sitespresent in single Asian genotypes (haplotypes 8–12).Ongoing tests for allelism will show whether thesegenotypes carry new resistance alleles at the Hv-eIF4Elocus, or whether the resistant phenotype is conferredby an independent locus.

Consequences for association studies: The mainte-nance of the resistant haplotype blocks, resulting fromrecent, nonrecombined introgression event(s), has re-sulted in the formation of significant associations be-tween resistance and haplotypes up to a distance of atleast 1 cM from the resistance gene. Such a coarse levelof resolution is inadequate for map-based gene cloning.On the other hand, it does imply that a fairly low markerdensity is sufficient to detect associations between atarget region and resistance. If this is typical for othergenes within breeding germplasm, the prospects aregood for identifying chromosomal segments associatedwith traits of interest. Further resolution may be achiev-able by using populations that have been intermatedover many generations, thereby promoting the break-down of linkage blocks. This situation is not commonin breeding material and is more likely to be found inlandraces and wild populations. For spp. spontaneum,levels of LD comparable to that of the outbreeding spe-cies maize have been reported (Lin et al. 2002; Morrellet al. 2005; Caldwell et al. 2006), and these should besufficient to provide the genetic resolution necessary toidentify the functional polymorphism associated withthe trait variation.

We thank M. Koornneef for his critical review of the manuscript, S.Wiedemann and S. Jakob for helpful discussions, and K. Trnka fortechnical assistance. We also acknowledge the linguistic advice ofhttp://www.smartenglish.co.uk in preparing this article.

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