Molecular mapping of a new gene Rrs4CI11549 for resistance to barleyscald (Rhynchosporium secalis)

Vishwanath Patil1, Åsmund Bjørnstad1,* and James Mackey2

1Department of Horticulture and Crop Sciences, Agricultural University of Norway, P. O. Box 5022, Ås,N-1432, Norway; 2Department of Plant Breeding Research, Swedish University of Agricultural Sciences,Uppsala, S-75007, Sweden; *Author for correspondence (e-mail: asmund.bjornstad@ipf.nlh.no; fax: +4764947802)

Received 18 October 2002; accepted in revised form 24 March 2003

Key words: Hordeum vulgare L., Molecular markers, QTL, Resistance genes, Rhynchosporium secalis

Abstract

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar‘Ingrid’ and the resistant accession ‘CI 11549’ (‘Nigrinudum’) was evaluated for resistance in the pathogenRhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance ‘CI11549’ against isolate ‘4004’. One is an allele at the complex Rrs1 locus on chromosome 3H close to the cen-tromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC3-linesinto ‘Ingrid’ from ‘CI 2222’ (another ‘Nigrinudum’) resistance seems governed by one locus close to the telo-meric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessivegene rh8 reported to be present in ‘Nigrinudum’. Various resistance donors of Ethiopian origin designated as‘Nigrinudum’, ‘Jet’ or ‘Abyssinian’ were identical to a great extent with respect to markers, but differed in re-sistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications fortheir use as differentials in scald tests and screening of germplasm collections are discussed.

Introduction

Scald, a foliar disease caused by Rhynchosporiumsecalis (Oud.) J.J. Davis, is a major disease of barley(Hordeum vulgare L.) and occurs in all major barleygrowing regions of the world. The pathogen is char-acterized by an extensive genetic variability and fre-quent changes of the population (Tekauz 1991), ap-parently the result of a high mutation rate (Goodwinet al. 1994), or possibly sexual recombination (Sala-mati and Tronsmo 1997; Salamati et al. 2000). Con-ventional resistance breeding based on field-testingcan further be made more difficult by considerableenvironmental variation, even when testing in care-fully controlled environments (Grønnerød et al.2002). In addition, individual R. secalis isolates mustbe passed through the host regularly to maintain viru-lence, and repeated in vitro culturing can lead to phys-iological changes (Graner and Tekauz 1996).

In previous studies various major genes conferringrace-specific resistance to R. secalis have been deter-mined (Dyck and Schaller 1961a, 1961b; Baker andLarter 1963; Wells and Skorporad 1963; Habgood andHayes 1971). At least 14 different genes or loci, mostbased on seedling resistance, have been described(Søgaard and von Wettstein-Knowles 1987; Barua etal. 1993; Abbott et al. 1995; Graner and Tekauz 1996;Garvin et al. 2000). However, until the advent of de-tailed genetic maps, it was possible to assign only afew of these to specific chromosomes provisionally(either 3H or 4H) (Bockelmann et al. 1977). With themore recent molecular markers, scald resistancegenes have been mapped to barley chromosomes 1H,3H, 4H, 6H and 7H (Abbott et al. 1992; Barua et al.1993; Schweizer et al. 1995; Graner and Tekauz1996; Garvin et al. 1997, 2000). Recently, locus des-ignations have been revised (Bjørnstad et al. 2002),

169© 2003 Kluwer Academic Publishers. Printed in the Netherlands.Molecular Breeding 12: 169–183, 2003.

and one locus called Rrs4CI 11549, is described in thispaper.

A total of 11 alleles have been listed at the Rrs1locus on chromsome 3H (Bjørnstad et al. 2002). Anumber of molecular markers are available for thislocus (Graner and Tekauz 1996; Penner et al. 1996;Russell et al. 1997; Grønnerød et al. 2002). In addi-tion, Barua et al. (1993) localized a resistance genedesignated Rhy on chromosome 3H using bulked seg-regant analysis, but the exact position of this locushas remained obscure.

Wells and Skorporad (1963) for the first time de-scribed the scald resistance of barley cultivar ‘CI2222’, also referred to as ‘Nigrinudum’ (literallymeans ‘black and naked seeds’), to be conferred by arecessive gene, rh8. Baker and Larter (1963) foundthat two complementary recessive genes rh6 and rh7confer resistance in the cultivars ‘Jet’ and ‘Steudelli’.All three accessions, which share a common Ethio-pian origin, are characterized by black seeds, and withthe exception of ‘Steudelli’, are naked. The three re-sistance genes rh6, rh7 and rh8 were, however, foundto be distinct based on the susceptibility of the F1 hy-brids between ‘Nigrinudum’ (‘CI 2222’) and either‘Jet’ or ‘Steudelli’. Later Habgood and Hayes (1971)confirmed these findings, but concluded that rh6 wasan allele at the Rrs1 locus, and therefore renamed itto rh5. In addition, they described the same gene rh8in another ‘Nigrinudum’ (‘CI 11797’), and the un-linked rh11 in ‘CI 4364’ and ‘CI 4368’.

The name ‘Nigrinudum’ has thus been used syn-onymously for more than one genotype of differentorigin. The source of origin for the cultivars with CInumbers can be obtained from the USDA (UnitedStates Department of Agriculture), GRIN (Germ-plasm Resources Information Network) online data-base (http://www.ars-grin.gov/npgs/searchgrin.html).‘CI 2222’ (Wells and Skorporad 1963) from Ethiopia,‘CI 11797’ (Habgood and Hayes 1971) from theNetherlands, and ‘CI 11549’ (Bockelmann et al.1980) from Japan have all been referred to as ‘Nigri-nudum’ in the literature. Similarly ‘CI 2222’ is la-beled ‘Jet’ in an USDA accession (USDA, http://www.ars-grin.gov/cgi-bin/npgs/html/acchtml.pl?1018985), a name usually given to ‘CI 967’. This createsa great confusion for the geneticist/plant breeder,when including either ‘Jet’ or ‘Nigrinudum’ as differ-ential varieties. Both ‘CI 2222’ and ‘CI 11549’ wereused as donors in the near-isogenic line developmentprogram in ‘Ingrid’ (Patil et al. 2002). In the GRINdatabase the cultivar ‘CI 11549’ is listed as of Japa-

nese origin. However, we failed to trace this cultivarin the ‘Barley Germplasm Center’, Okayama Univer-sity, Kurashika, Japan. Further there are no records ofblack seeded and naked barley grown in Japan (K.Sato, personal communication). Therefore it is possi-ble that this cultivar is of Ethiopian origin, but wasintroduced from Japan to the USDA.

The three aims of the present study were: (i) tomap the scald resistance in ‘CI 11549’ using an ‘In-grid’ × ‘CI 11549’ doubled haploid (DH) population,F2 population and back cross (BC3) lines between‘Ingrid’ × ‘CI 11549’; (ii) to compare this resistanceto that in BC lines from ‘Ingrid’ × ‘CI 2222’, to as-sess their identity; and (iii) to compare the responsesof 11 different Ethiopian resistance donors, to differ-ential isolates and molecular marker patterns.

Materials and methods

Plant materials

Doubled haploid linesA population of 79 F1 DH lines of a cross betweenthe susceptible barley genotype cv. ‘Ingrid’ and theresistant ‘CI 11549’, was used for the genetic analy-sis. The first 56 were generated with the H. bulbosum(Jensen 1976) method and the remaining by antherculture (Bjørnstad et al. 1993).

Backcross linesThe BC lines were made at the Swedish Universityof Agricultural Sciences, Uppsala, Sweden. The do-nor parents ‘CI 11549’ and ‘CI 2222’ were bothcrossed with ‘Ingrid’ (recurrent parent), and the F1

hybrids and the resistant offspring (BC1F2) back-crossed to ‘Ingrid’. Further backcrosses and selectioncycles produced lines for the present analysis thatwere in the BC3-F2 and BC3-F4 generations in caseof ‘CI 11549’; BC6-F2 and BC7-F6 with respect to‘CI 2222’.

F2 linesThe F2 generation arising from the cross ‘Ingrid’ בCI 11549’ was tested thrice to analyze the phenotypicreaction to scald.

DonorsTo investigate possible synonyms or duplicates anddifferences between various Ethiopian donors the fol-lowing accessions were chosen and analyzed. Black

170

and naked seeds: ‘Jet’ (‘CI 967’), ‘Jet’/‘Nigrinudum’(‘CI 2222’), ‘Nigrinudum’ (‘CI 11549’); black andcovered seeds: ‘Steudelli’ (‘CI 2226’), ‘CI 2230’;white and covered seeds: ‘CI 1227’, ‘Abyssinian’ (‘CI1233’), ‘Abyssinian’ (‘CI 1237’), ‘Abyssinian’ (‘CI668’), ‘CI 4364’ and ‘Benton’ (‘CI 6168’).

Inoculation tests

DHs‘Ingrid’, ‘CI 11549’ and 79 DHs were tested for re-action to R. secalis (isolate ‘4004’) in two indepen-dent experiments (Fall 1996 and Spring 1999), eachreplicated twice, with a fully randomized design andparents repeated thrice each per replicate.

BC linesThe BC lines between ‘Ingrid’ × ‘CI 11549’ and ‘In-grid’ × ‘CI 2222’ were used in inoculation tests. Theselines were tested in spring 1999 with isolate ‘4004’.

F2 linesThere were three independent tests involving the ‘In-grid’ × ‘CI 11549’ F2 generation (Fall 1996, Spring1999 and Spring 2001). In all tests the F2 lines werechallenged with isolate ‘4004’.

DonorsThe donors were challenged with six different isolatesof R. secalis during April 1998. Salamati and Tron-smo (1997) have described five Norwegian isolates(‘2’, ‘16–6’, ‘17’, ‘22’ and ‘28–3’), which are differ-entially virulent against all donors and have been usedin screening breeding lines (Reitan et al. 2002). Thenull isolate ‘4004’ (Race DK 23) was from Jørgensenand Smedegaard-Petersen (1995).

Growth conditions and scoring of symptoms

Growth conditions, inoculation, scoring scale and dis-ease rating were as described by Grønnerød et al.(2002) and Reitan et al. (2002). Briefly, a 0 to 5 scalewas used, where disease rating less than 2 was clas-sified as resistant reactions (R), 2.0–3.0 as moderatelyresistant (MR), 3.1–4.0 as moderately susceptible(MS) and 4.1–5.0 as susceptible (S). All scores aremeans of 3–4 plants, averaged over replicates, ifavailable. Inocula with an approximate spore titer ofca 2.0 × 105 ml−1 were made according to Salamatiand Tronsmo (1997). The disease scoring and scalewas as described by Grønnerød et al. (2002). ‘Ingrid’

was used as susceptible control in all tests. Means ofthe 4–5 plants per unit, means of replicates and meansof tests were analyzed for DH lines and donors. In F2

tests individual plant scores were used.

DNA extraction

Genomic DNA from the DH lines, parents and donorswas extracted from two-week-old seedlings accordingto standard CTAB procedures described by Kleinhofset al. (1993). The seedlings were incubated in the darkovernight to improve the quality of extracted DNA.In case of the BC and F2 lines, individual plants fromeach of the classes 0, 1–2 and 5 scores were selectedat random after they were scored for the scald reac-tion.

Bulked segregant analysis

Two pools of bulked DNAs were created based on thescald resistant or susceptible phenotypes. The bulkscomprised of equal amounts of pooled DNAs fromfive susceptible and five resistant individual DH gen-otypes, respectively.

Molecular markers

Molecular markers, viz., restriction fragment lengthpolymorphism (RFLP), simple sequence repeat(SSR), sequenced tagged sites (STS) and amplifiedfragment length polymorphism (AFLP) were used.Southern blots and RFLP analyses were performedusing 32P-dCTP as described by Kleinhofs et al.(1993). The RFLP probes, SSRs and STS used alongwith references, are listed in Table 1. Segregation ofthe SSR alleles was analyzed by denaturing polyacry-lamide gel (5%) electrophoresis (Saghai Maroof et al.1994) followed by silver staining. The polymerasechain reaction (PCR) amplification for the differentSSR and STS markers was performed as described bythe various authors. The polymorphic primers werelater used with all the 79 DH lines. The BC lines werescreened with the five STS and 45 SSRs listed inTable 1. A sample of the F2 population from the 2001test was screened with two STS (agtc-17 and STS/MWG680) and four SSRs (HVM 27, HVM 36, HVM60, Bmac 209 and Bmag 225). AFLP analysis wasperformed as described by Vos et al. (1995) on thetwo parents and 79 DH lines with modifications andselective primers as described by Grønnerød et al.(2002).

171

Tabl

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234*

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0806

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Bm

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Bm

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Bm

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ac06

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5

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1996

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172

The donors were screened with markers in twochromosomal regions around Rrs1 (chromosome3HL) and Rrs2 (7HS) where previous mapping workhad identified major loci giving resistance against R.secalis (Bjørnstad et al. 2002). They were also testedwith the STS ‘Ylp’, diagnostic for the Yd2 resistancelocus against barley yellow dwarf virus (Ford et al.1998), located close to Rrs1 (Grønnerød et al. 2002).

Linkage and QTL analysis

Genetic linkage analysis was performed using Join-Map™ version 2.0 (Stam and van Ooijen 1995).Drawmap version 1.1 (Ooijen van 1994) was used todraw the genetic linkage maps. The programs con-sider Kosambi’s Mapping functions for calculatingmap distances (Kosambi 1944). QTL analysis wasdone using the PLABQTL-software (Utz and Melch-inger 2000), with both the simple interval mapping(SIM) and composite interval mapping (CIM). Em-pirical LOD thresholds were determined by 1000 per-mutations according to Churchill and Doerge (1994).For SIM the 5% significance level was approximately1.8 and for CIM approximately 2.0. Various markersflanking the significant peaks from SIM were used ascofactors for CIM analysis, but only ‘agtc-17’, repre-senting the well-known Rrs1 locus is reported to con-firm the presence of linked QTL. The presence of theinteraction between the detected QTL and environ-ment (QTL × E) was tested using SIM on the basis ofgenotype means of 4–5 plants per unit. Alternatively,the means across the replicates within a test, or acrossthe tests were used. All reported R2 values are vali-dated averages from cross validation of 5 sub-seg-ments.

Results

Segregation of resistance in DH lines and F2 lines(‘Ingrid’ × ‘CI 11549’)

DH linesThe results of the inoculation tests are summarized inFigure 1. ‘CI 11549’ displayed an intermediate infec-tion type 2 (R) in both the tests, showing a high de-gree of consistency among tests and replicates. In test1, three DHs did not germinate. The 76 DHs tested,segregated 18R: 10MR: 7MS: 41S, i.e., 28R: 48S, aslightly skewed ratio. However, for the first 56 DHsgenerated by the H. bulbosum technique, the ratio was

25R: 30S. The DHs originating from anther cultureshowed a strongly skewed ratio of 3R: 18S. In test 2,the severity of infection was lower. The 78 DHs (oneDH did not germinate) segregated 10R: 24MR:12MS: 32S, i.e., 34R: 44S. In the lines generated bythe H. bulbosum techniques the ratio was 28R: 26S.The DHs made by anther culture segregated 6R: 18S.Thus the segregation ratios were relatively consistentacross the tests.

F2 linesThere were three different inoculation tests of F2 pop-ulation, summarized in Figure 2. The results werefairly consistent across the tests, indicating incom-plete dominance of the resistance. The inoculationtests showed a considerable range of symptoms in‘Ingrid’ (2–5) as well as ‘CI 11549’ (1–3). The paren-tal distributions were not overlapping in test 1, butslightly so in test 2 and test 3. The results were notconsistent with recessive resistance, but rather withincomplete dominance or additivity.

Mapping of resistance trait

A total of 59 SSRs, 5 STS, and 5 RFLPs (Table 1)were found to be polymorphic for the present crossafter screening the two parents and the bulks (suscep-tible and resistant). Most of the polymorphisms wereclustered around the centromeric region on chromo-some 3H. A linkage group with all the STS markers,13 SSRs and two RFLPs mapping a distance of 98.2cM was assigned to chromosome 3H (Figure 3). In-terestingly, HVM36b was mapped to chromosome3H, contrary to Liu et al. (1996). A map based on the56 DHs prepared by the H. bulbosum method did notdiffer much from one based on all DHs (including theanther culture lines), except for reshuffling of someof the closely linked markers. There were other link-age groups, but they did not show any QTL (resultsnot shown). The results of the screening of the par-ents and bulks clearly indicated that the resistancewas localized on chromosome 3H. Hence the chro-mosome 3H map was used for QTL analysis.

QTL analysis

There were no QTL × test (QTL × E) interactions, andhence analyses were performed on the basis of over-all DH means across tests. The QTL detected by SIMand CIM are listed in Table 2. Two QTL with LODthresholds far exceeding the significance levels found

173

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by permutations were detected. The first QTL(Rrs1CI11549) was located at 71 cM between SSRmarkers STS/MWG680 and agtc-17, with a supportinterval of 4 cM. The second QTL (Rrs4 CI 11549) waspositioned at 93 cM between HVM36b and HVM60,with a support interval of 4 cM. The two QTL ex-plained 65% of the cross validated phenotypic vari-ance (Table 2a). In the less severe test 2, there werethree QTL peaks. The first two were the same as inthe previous test and the third QTL was detected at37 cM between HVM15 and HvLTPPB (support in-terval 14 cM). They explained 49% of the cross vali-dated phenotypic variance (Table 2b). When the av-erage of the two inoculations was considered, onlythe two QTL common to both tests were detected.

The final two-locus model explained 56% of the phe-notypic variation in SIM among DH means (Table2c).

These results were basically confirmed by CIManalyses, with even higher R2 values (Table 2). Thethird QTL detected in SIM analysis of test 2 disap-peared in CIM (Table 2b), indicating an artifact. Thetwo QTL explained 70% of the phenotypic variationin the CIM analysis (Table 2c). These results indicatethat there are two QTL, 22 cM apart, and that theyrecombine in certain of the DH plants and act addi-tively. This two-locus model was also confirmed bySIM and CIM analysis of only the H. bulbosum-de-rived lines (Table 2). No significant associations weredetected at other chromosomes. Importantly, the telo-meric region of chromosome 7H did not show anyassociation between the markers and scald resistancewhen inspected and checked for QTL.

Confirmation of QTL in the F2 generation

The two-locus model was subjected to an independentF2 population test 3. It was assumed that among theplants with intermediate resistance type (2–3), cross-over genotypes with one locus should occur as ob-served in the DH lines. Given the map distance be-tween the two QTL (22 cM), the gametes rrs1, Rrs4or Rrs1, rrs4 should occur at a frequency of approxi-mately 0.1 and their homozygotes about 0.01. In ourpopulation of 150 F2 genotypes, about 75 plants withresistance reaction scores 2–3 were investigated withmarkers (Figure 2) flanking the two QTL. Two appar-ent recombinant homozygotes rrs1/rrs1, Rrs4/Rrs4and two heterozygotes Rrs1rrs1, Rrs4rrs4 were de-tected. Both showed intermediate resistance reac-tions. These resistant plants had retained the alleleson chromosome 3H. Escapes were also detected, asingle plant with all ‘Ingrid’ alleles, but with a scoreof 2.0, as well as a plant with ‘CI 11549’ alleles anda score of 3.0. Both could be expected from the over-lapping parental distributions. We see the resistancein a few recombinant genotypes as confirmation ofour model.

Marker-based comparison of BC lines

‘CI 11549’The results of BC lines are presented in Table 3. Av-erage scores are used in all cases. Of various SSR andSTS primers listed in Table 1, only 16 SSRs and allfive STS were found to be informative. The resistant

Figure 3. Genetic map of barley chromosome 3H. QTL were de-tected between STS/MWG680 – agtc-17 (Rrs1) and HVM36b –HVM60 (Rrs 4). Linkage analysis was performed on 79 DH plantsfrom a cross between ‘Ingrid’ × ‘CI 11549’ with RFLPs, SSRs andSTS. Distances (in cM) between loci are listed on the left side ofthe diagram.

176

BC3 F2 lines, A98 (score 2–3) and A100 (score 1) re-tained the ‘CI 11549’ allele at agtc-17, but otherwisewere identical to the susceptible line A99 (score 4–5)in all markers on chromosome 3H. These lines werehowever differentiated by the ‘CI 11549’ alleles at 4loci on chromosome 7H (Bmag021, Bmag0206,Bmag007 and HVM04). This indicated that the BClines had loci for R. secalis resistance distal on chro-mosome 7H. A possible reason for this discrepancybetween the DH mapping results and BC-lines maybe that the ‘CI 11549’ used for BC and DH line de-velopment had different genotypes.

‘CI 2222’The BC lines from ‘CI 2222’ also retained only thedonor agtc-17 allele, irrespective of their scores.However, the BC line A135 (score 1) also retainedYLM, and another BC line A128 (score 1–2) ampli-fied Bmag021 located at the tip of chromosome 7H.Possibly they may represent different NILs from thesame donor. This, however, needs confirmation ofprogeny from intercrossing the NILs, like in the studyby Patil et al. (2002).

Table 2. Genetic positions for R. secalis isolate 4004 resistance, QTL detected by Simple Interval Mapping (SIM) and tested by CompositeInterval Mapping (CIM) of mean disease severity data with ‘Ingrid’ × ‘CI 11549’ population

QTL Position (cM) Flanking

markers for

the QTL

detected

+ Pos LOD add R2% Present in

CIM

SIM & CIM

in H. bulbo-

sum lines

a. List of QTL detected on chromosome 3H in the fall 1996 test. (agtc-17 was cofactor for CIM)

1 71 STS/

MWG680 –

agtc-17

0 15.77 −1.25 69.6 yes yes

2 93 HVM36b –

HVM60

1 15.92 −1.32 69.9 yes yes

Cross validated R2% = 65.42 75.67

b. List of QTL detected on chromosome 3H in the spring 1999 test. (agtc-17 was cofactor for CIM).

1 71 STS/

MWG680 –

agtc-17

0 10.64 −0.79 43.1 yes yes

2 93 HVM36b –

HVM60

1 7.71 −0.86 54.1 yes yes

3 54 HVM15 –

HvLTPPB

4 4.54 −0.64 28.20 no no

Cross validated R2% = 49.41 49.89

c. List of QTL detected on chromosome 3H with the average of Fall 97 and Spring 99 tests. (agtc-17 was cofactor for CIM).

1 71 STS/

MWG680 –

agtc-17

0 14.60 −1.01 65.0 yes yes

2 93 HVM36b –

HVM60

2 12.23 −1.10 58.5 yes yes

Cross validated R2% = 56.51 70.29

+Pos Position (cM) of the QTL relative to the flanking marker.add Additive QTL effect at the location of scanning, the negative sign indicates the contribution of ‘CI 11549’ QTL alleles that lower thescald score.R2% Coefficient of determination, percentage of the phenotypic variance which is explained by the putative QTL. All R2 values are fromcross validating of five segments, the detected QTL and their estimated positions used for a simultaneous multiple regressions to obtain finalestimates of the additive and dominance effects.

177

Tabl

e3.

Res

ults

ofth

epa

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Response of ethiopian barley accessions

Marker similarityTable 3 shows a complete marker identity in ‘CI11549’ and ‘CI 2222’. The overall marker similaritybetween the 11 donors of Ethiopian origin was alsohigh. ‘Jet’ (‘CI 967’) was identical with the previoustwo in the 3HL and 7HS regions analyzed, but clearlydifferent from ‘Steudelli’ (‘CI 2226’). ‘Abyssinian’(‘CI 668’), however, was completely identical withthe ‘Nigrinudums’, except for the presence of Ylp, asdid the donors ‘CI 2230’, ‘CI 1227’, ‘CI 1237’. Ylpmaps close to Rrs1 at the centomeric region of 3H(Grønnerød et al. 2002). The donors ‘CI 1227’, ‘CI1233’, ‘CI 1237’ and ‘CI 4364’ carried the same dis-tinct alleles on chromosome 7HL. The YLM locuswas present in all donors. Thus, in this sample of ac-cessions there are strong similarities in these chromo-somal regions.

Resistance reactions to the six different isolates ofR. secalisThe resistance reactions of Ethiopian barley acces-sions were clearly differential (Figure 4). ‘CI 2222’and ‘CI 11549’ were nearly identical, but ‘Jet’ (‘CI967’) was moderately susceptible to all isolates ex-cept ‘4004’. ‘Steudelli’ varied from moderately sus-ceptible to moderately resistant, but was weaker than‘Jet’ (‘CI 967’) in response to inoculation by ‘4004’,conforming to previous experience (S. Grønnerødpersonal communication). ‘CI 2230’ was susceptibleto all isolates except for ‘17’. In the covered/white-seeded group ‘CI 1233’ was susceptible to isolate‘16–6’, apart from that ‘CI 1233’ and ‘Abyssinian’(‘CI 668’) were moderately resistant to all isolates.‘CI 1227’ and ‘CI 4364’ exhibited variable reactions,while ‘CI 1237’ and ‘CI 6168’ were susceptible to allexcept isolate ‘2’. Clearly, resistance to R. secalisbore little relationship to the presence of Ylp, a diag-nostic of the gene Yd2 conditioning barley yellowdwarf virus (BYDV) resistance in spite of its closelinkage to Rrs1 (Ford et al. 1998; Grønnerød et al.2002).

Discussion

Mapping of scald resistance in ‘CI 11549’

DH linesMost of the resistant plants were of intermediate types(scores 2–3) corresponding to the resistant parent ir-respective of the tests. The reduced disease severityin the test 2 could be attributed to the slightly lowhumidity, a fact strongly affecting scald severity(Tekauz 1991; Salamati and Tronsmo 1997; Grøn-nerød et al. 2002).

The segregation ratios of the DHs were slightlyskewed. This differed, however, between DHs gener-ated by the H. bulbosum method and by anther cul-ture, the latter showing strongly skewed segregationwith ‘Ingrid’ alleles prevailing, which could be attrib-uted to superior response of this cultivar to in vitroregeneration. Our results confirm previous studies inthis regard (Bjørnstad et al. 1993; Devaux et al.1995). Skewed segregation may also be strong inDHs made by the H. bulbosum method (Grønnerød etal. 2002). The lines from H. bulbosum technique con-formed to 1:1 ratio suggesting a single segregatinglocus involved in conferring resistance against R.secalis race ‘4004’.

QTL (SIM and CIM) analysis, however, identifiedat least two linked and additive loci (ca. 71 and 93cM) on chromosome 3H. This was further confirmedby inspecting the raw data from molecular markersand disease scores, clearly identifying resistant re-combinants. The proximal QTL at 71 cM betweenSTS/MWG680 and agtc-17 coincides with the wellknown Rrs1 locus. The distal locus between HVM36band HVM60 may be identical to the resistant locusthat Schaller et al. (1963) located 27.5 cM apart fromthe Yd2 gene, including two linked and one indepen-dent locus (C. Qualset, personal communication).This locus seems to have been confounded with Rrs1in much of the subsequent literature, as in the 3H mapof Wettstein-Knowless (1992). It may however beidentical with Rhy (Barua et al. 1993). Our initialdesignation rrsx, in accordance with Rhy, was up-dated to Rrs4CI 11549 by Bjørnstad et al. (2002). To-gether these two genes (Rrs4 and Rrs1) account for60% of the cross-validated phenotypic variance (Ta-ble 2c), which we consider satisfactory given the un-certainty associated with such tests.

The segregation ratio of the F2 population indi-cated an incomplete dominance of the two genes(Rrs1 and Rrs4). The F2 segregation confirmed the

179

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above model, in spite of the slightly overlapping pa-rental distributions.

Are ‘CI 11549’ and ‘CI 2222’ identical?

The deviations between the BC-lines and DHs fromthe cross ‘Ingrid’ × ‘CI 11549’ observed by themarker analysis was ascribed to heterogeneity in theseed source. Alemayehu and Parlevliet (1997) re-ported a strong variation in scald resistance betweenand within the Ethiopian landraces. Brown (1985) re-ported that different resistance genes might exist incultivars with the same name when a common seedsource has not been used. Ceoloni (1980) reportedthat ‘Nigrinudum’ (‘CI 11549’) was resistant to 71%of R. secalis isolates, while Salamati and Tronsmo(1997) found that ‘NGB 10681’/‘CI 11549’/‘Nigrinu-dum’ was fully susceptible to 93% of the isolates and‘CI 2222’ was susceptible to only 23%. They did nottest these two with the mostly avirulent isolate ‘4004’used in the present study. In our tests ‘CI 2222’ wassimilar to ‘CI 11549’ both in its phenotypic responsesto R. secalis isolates and in the molecular marker pat-terns, confirming their common Ethiopian origin. Inspite of this, both between DH and BC lines from ‘CI11549’ and between BC lines from ‘CI 2222’ diver-gent marker genotypes at Rrs1, Rrs4 and Rrs2 wereobserved. This leads us to conclude that there are atleast three loci conferring resistance in ‘CI 11549’ and‘CI 2222’: Rrs1 close to YLM, Rrs4 close to HVM60and Rrs2 close to Bmag0021, possibly the same al-lele as Rrs2Steudelli (Bjørnstad et al. 2002). There wasno trace of the recessive gene rh8 (Wells and Skor-porad 1963) in either of the Nigrinudums. Thus, thetwo Nigrinudums are identical in phenotypic reactionto scald and molecular marker patterns.

Should genotyping replace phenotyping ingermplasm collections?

The results in Figure 4 clearly show differential reac-tions. Although none of the donors were completelyresistant against all the six isolates, they showed avaried range of phenotypic reactions. Also, a diver-sity of scald reactions exist among the BYDV-resis-tant lines, although Yd2 and Rrs1 are closely linkedand resistance to both diseases tend to be associatedwith increasing altitude in the Ethiopian highlands(Engels 1994; Demissie and Bjørnstad 1996). Fur-thermore, while all donors amplified YLM, only thedonors ‘CI 2230’, ‘CI 1227’, ‘CI 1237’ and ‘Abys-

sinian’ amplified Ylp. Those that amplified Ylp alsohad minimal phenotypic symptoms and ELISA scoresafter virus inoculation (Bjørnstad, Munthe and Pal-tridge, unpubl. results). Ford et al. (1998) suggestedthat both markers might be used in marker-assistedselection for Yd2. However, our results show thatYLM is rather a marker for Ethiopian origin, not forYd2. When we scored the two STS markers in 89Ethiopian lines used in an RFLP study (Bjørnstad etal. 1997), only 10 amplified Ylp, but all amplifiedYLM (Paltridge and Bjørnstad, unpubl. results). Also,non-Ethiopian donors of resistance to R. secalis like‘Brier’ and ‘Hudson’ contain the allele at YLM(Bjørnstad et al. 2002).

Except for Ylp, which was developed from a pro-tein in NILs with Yd2, most groupings of lines basedon resistance seem to bear little relation to markers,be they morphological or molecular. This relates tothe suggestion to replace phenotypic screening of ger-mplasm collections by genetic markers at or close tothe loci of interest bypassing the need for extensivecrossing work. Given linkage disequilibrium a uniquemarker allele might hold promise of a unique alleleof interest or, in general, that unique marker diversityindicates uniqueness in other traits (Gilbert et al.1999; Asíns 2002). Conversely, identical marker alle-les might indicate allelic identity, or at least allelism.

Our data do not support such an approach. TheRrs1, Rrs2 and Rrs4 markers available could not dis-tinguish between the donors, in spite of differentialresistance reactions at one or more of the loci in-volved. In the Ethiopian germplasm studied, linkagedisequilibrium is simply not high enough. In screen-ing such a gene pool, unless the average mutation rateof a marker locus has followed suit with evolution ofnew resistance specificities, phenotyping should notbe replaced. On the other hand, in the deliberately in-trogressed breeding gene pool studied by Reitan et al.(2002), linkage disequilibrium was high enough, sothat the marker-assisted approach indicated allelismand identity more precisely than the phenotyping.

Conclusions

Overall, there was no trace of the rh8 gene in the twoNigrinudums tested (‘CI 11549’ and ‘CI 2222’). In-stead, we found two linked and incompletely domi-nant scald resistances in ‘CI 11549’ on chromosome3H (Rrs1 and Rrs4) and a locus close to the telomericregion of chromosome 7H (probably allelic to Rrs2)

181

in ‘CI 2222’. The two Nigrinudums are identical inphenotypic reaction to scald and molecular genotype.The other conclusion that can be inferred from theseresults is that the heterogeneity in landraces and CInumbers leads to increased confusion in the differen-tial cultivars for scald resistance. Therefore it ishighly recommended to replace them with nearisogenic lines. A set of such lines has recently beenpresented (Patil et al. 2002) and have been furtheranalysed in other papers (Bjørnstad et al. 2002; Patilet al. 2002).

Acknowledgements

We thank Anne Guri Marøy for technical assistance.The collaboration with Dr. N. Paltridge (Adelaide) onthe initial screening of Ethiopian donors with the Ylpand YLM amplicons is kindly acknowledged, as is thecollaborations of Dr. T. Munthe (Norwegian Crop Re-search Institute) for BYDV inoculation and ELISAtests of the same genotypes. We thank the anonymousreferees for their valuable comments. This researchwas mainly funded by a grant from the Nordic GeneBank, project 1997:1.

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