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Restriction Fragment Length Polymorphism-Mediated Targeting of the ml-o Resistance Locus in Barley (Hordeum vulgare

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Restriction Fragment Length Polymorphism-Mediated Targeting of the ml-o Resistance Locus in Barley (Hordeum vulgare
  Proc. Nati. Acad. Sci. USA Vol. 88, pp. 3691-3695, May 1991 Genetics Restriction fragment length polymorphism-mediated targeting of theml-o resistancelocus in barley (Hordeum vulgare) (backcross lines/Erysiphe graminis f.sp. hordei/intravarietalcrosses/linkage drag/plant resistance gene) KARIN HINZE, RICHARD D. THOMPSON, ENRIQUE RITTER, FRANCESCO SALAMINI, AND PAUL SCHULZE-LEFERT Max-Planck-Institut fur Zuchtungsforschung, Abteilung fur Zuchtungsforschung und Ertragsphysiologie, D-5000 Cologne 30, Federal Republic of Germany Communicated by J. Schell, December 5, 1990 ABSTRACT The ml-o locus in barley confers resistanceto all known races of the fungus Erysiphegraminis f.sp. hordei. Since the molecular mechanisms underlying ml.o-mediated resistance are currentlyundefined, experiments havebeen initiated toisolate the gene by means of its map position. A collection of backcross lines containing ml-o alleles derived from six barley genotypes allowedus to identify a set of DNA markers very tightly linked to theresistance locus. These markers span an unexpectedly small segment of 8.6 centimor- gans on chromosome 4 that includesthe Ml-o locus. Two of the markers cosegregate with theresistance locus on the basis of 44 homozygous resistant plants identified within a segregating F2 population derived from an intravarietal cross. Colinearit of theresistance-linked markers was confirmed in anF2 mapping population derived from a wide cross between Hordeum vulgare subsp.vulgare and Hordeum vulgaresubsp. spontaneum. The twomarkers cosegregating with theresistance locus in the former cross define in the latter cross an interval of 2.4 centimorgans within which Ml-o is most probably situated. The set of linked markers opens up the possibility of carrying out a bidirectional chromosomal walk or jump to the gene. Erysiphe graminis f.sp. hordei is the causal agent of powdery mildew disease in barley. Several loci conferring resistance against powdery mildew infection havebeen identified and mapped through classical linkage analysis (1-3). Among these mi-o has two outstanding features: resistance alleles are recessive (most probably through the loss of function of the gene and they mediate worldwide resistance to all known races of the fungus  4 . A largecollection of spontaneous andinduced mi-o mutants in different barley genotypes have been described phenotypically andhavebeen used in linkage studies (4,5). The locus maps to the long arm of chromosome 4  6 . Despite an extensive genetic and physiological char- acterization of mi-o mutants (7-9) the molecular mechanisms underlying ml-o-mediated resistance are not yet understood. In anattempt to identifythe corresponding gene product(s) by a reverse genetics (10) approach, a strategy basedon a search for very tightly linked DNA markers has been adopted. The identification of such markers usuallyrequires a preexisting restriction fragment length polymorphism (RFLP) map built up from avery large number of probes. To bypass this time-consuming process, we used a collection of mi-o near-isogenic lines and the method of colabeling andsimultaneousprobingof sets of anonymous clones (11). In this study we analyzed eight mi-o backcross lines (BC lines), produced through seven backcrossesof different mio mutantswith cultivarIngrid, which served astherecurrent parent. Importantly, the donor parents represent six different genetic backgrounds. The tight linkage among RFLP markers identified in the mi-o BC line survey was assessed in the F2 population of the wide cross Hordeum vulgare subsp. spon- taneum x Hordeum vuigaresubsp. vulgare cv. Aramir. Finally we measured distances of the markers relativeto Ml-o through analysis of the homozygous mi-o plants in an F2 population derived from the intravarietal cross of Hordeum vulgare cv. Carlsberg II Mi-o x Hordeum vulgare cv. G.Zweiz. ml-oll. MATERIALS AND METHODS PlantMaterial. The intravarietal cross was carried out between lines Hordeum vulgare subsp. vulgare cv. Carlsberg II Mi-o and Hordeum vulgare subsp. vulgare cv. G.Zweiz.mi-oil (HOR 23937),the wide cross between lines Hordeum vulgare subsp. spontaneumand Hordeum vulgare subsp.vulgare cv. Aramir. Allmi-o BC lines were a gift from James MacKey. They were generatedthroughseven backcrosses with H. vulgare subsp.vulgare cv. Ingrid followed by at leastsix selfings. Table 1 summarizes appropriate information from all mi-o mutants that wereused as donor parents in the backcross procedure. Genomic Library. The source of probes utilized in this study is a genomic DNA library from Hordeum vulgare cv. Carlsberg II. In brief, genomic DNA was digested to com- pletion withPst I and size fractionated through ultracentrif- ugation on sucrose gradients. Size-selected DNA (0.5-3.0kilobases) was inserted into plasmid vector pUC18 followed by transformation into Escherichia coli strain TG-2. Library Screening. A primary screening procedure elimi- nated clones coding for repetitive or organellar sequences; plasmid DNA preparation (12) was followed by digestion withPst I, separation of DNA fragments on 1 agarose gels, and transfer to nylon membranes (HybondN; Amersham). Those clones that gave rise to hybridization signals with labeled total genomic, or mitochondrial, or chloroplastbarley DNA were discarded. The mi-o BC-survey filters were prepared essentially as described by Gebhardt et al. (14). DNA restriction enzymes used for RFLP analysis wereAlu I, Hae III, Rsa I, and Taq I, all of them recognizing a different 4-base pair(bp) target sequence. Probe Labelings and Hybridizations. DNA inserts from barley plasmid clones wererecovered and stored in agarose plugs after separation from vector DNA on 1 low-melting- point agarose gels. After melting of the insert containing agarose at 70 C,generally four tosix insert DNAs (2-5 ng each) were mixed, labeled with 20 ACi of [a-32P]dCTP (3000 Ci/mmol; 1 Ci = 37 GBq) (15), and hybridized simultane- ously to mi-o BC-linesurvey filters. All hybridizations were Abbreviations: BC line, backcross line; RFLP, restriction fragment length polymorphism; cM, centimorgan.3691 The publication costs of this article were defrayed in part by page charge payment. This article must therefore behereby marked  advertisement in accordance with 18 U.S.C.§1734 solelyto indicate thisfact.  Proc. Natl. Acad. Sci. USA 88 (1991) Table 1. ml-o mutant alleles Allele MutantMother variety Mutagen mi-ol M66 HaisaX-ray mlo3 M.C.20 Maltesia Heda r-Ray mlo4 SR 1 Foma X-rayml-o5 Ris0 5678 Carlsberg II ENS mi-o8Ris0 7372 Carlsberg II EMS mi-o9 SZ 5139bDiamant EMS mi-olO SR 7 Foma T-Raymi-oil G. Zweiz. G. Zweiz. Spontaneous EMS, ethylmethane sulfonate. G. Zweiz., GrannenloseZwei- zeilige. carried out in 10 dextran sulfate/i M NaCl/1 sodium dodecyl sulfate/80 ug ofdenatured salmonsperm DNA per ml at 650C overnight. Linkage Analysis. In the wide cross, maximum likelihood estimates of recombinationfrequency of all pairs of poly- morphic loci defined by single restriction fragments were calculated as described in Ritter et al. (16). This calculation includes multipointlinkage and the EM algorithm for han- dling missing values. Since in the intravarietal cross only homozygous resistant ml-o plants were analyzed, maximum likelihood estimates ofrecombination frequencies were cal- culatedwithintherecessive class of a 1:2:1 segregating F2 populationaccording to table 6, formula 16, of Allard (17): p= (h + 2b)/2n, where p = recombination frequency, n = number of homozygous recessive individuals in F2, h = number of heterozygousrecombinant individuals in F2, andb = number of homozygous recombinant individuals in F2. The standard error withintherecessive class was calculated according to Allard (17): SD =   Test for Resistance. Powdery mildew infections were car- ried out in the greenhouse with a random field populationof Erysiphe graminis f.sp. hordei isolated and multiplied on Hordeum vulgare cv. Golden Promise. Four-hundred thirty- nine F2 seedlings from the intravarietal cross were infected with powdery mildew conidia 10 days after sowing. Plants were grown at 18'C and scored 10 days after infection. RESULTS Identification of RFLPs Through ml-o BC Lines. Approxi-mately 1100 anonymous genomic Pst I clones from barley detecting single/low-copy DNA sequenceswere tested by hybridization to ml-oBC-linesurvey filters (see Materials and Methods). Only five clones were identifiedthat display RFLPs between DNA from the recurrent parent (cv. Ingrid) and DNAs from at least two of the eight tested ml-o BC lines. As a representative example, the results obtained with oneof the clones, bAN61, are shown in Fig. 1. The data produced when this probewas hybridized to DNA fromeach BC lineare assigned to three groups:  i BC lines mi-ol,mi-oS, andml-o8 display a differentpattern compared to the recurrent parent cultivar Ingrid (Fig. 1A, lanes 1, 4,5, and 9 . The RFLP patterns obtained with each of these BC lines arealso present in the DNAs from the respective donor parents (Fig. lb, lanes 1 and 4 . It is therefore concluded that in BC lines ml-ol, mi-oS, and ml-o8, clone bAN61 defines a locus of donor parental srcin that is retained throughout the back- cross procedure while selecting for mi-o resistance.  ii DNAs from BC lines mi-o3, ml-o4, ml-o9, andml-olO and from therecurrent parent produce a homomorphic fragment pattern (Fig. LA, lanes 2, 3, 6,7, and 9 . A polymorphism, however, differentiates the recurrent anddonor parental DNAs from these four BC lines (Fig. lb, lanes 2,3,6,7, and 9 . Therefore the donor parental pattern was replaced by the one from therecurrent parent during the backcross proce- _  n  t ui : 00 00 0o 0 E EE E EE m m m m m m 0 t0 E m L-- 0 E ua M: 0. a _ AWL ANN 1 2 3 4 5 6 7 8 9 FIG. 1. Genomic Southern analysis of mi-o BC lines. The upper panel shows for each mio BC line analyzed and therecurrentparent (cultivarIngrid) a graphical representation of barley chromosome 4. Introgressed fragments, including mi-o alleles derived from six different donor parentalgenetic backgrounds, areindicated for each BC line. BC lines ml-o4/mi-olO and ml-oS/ml-o8 are derived from identical donor parentalgenetic backgrounds and aretherefore denoted by an identical graphicalunderlay. The lower partdisplaysthe RFLP detected by probe bAN61 ongenomic hybridization filters containing DNAs from each mio BC line (lanes 1-8 ina) and the respective donor parents(lanes 1-8 in b). Lane 9 representsthepattern detected with genomic DNA from therecurrent parent (RP). dure, indicating that locus bAN61 in these four BC lines is outsidethe introgressed fragment containingmi-o.  iii DNA from BC line ml-oll is homomorphic in comparison to therecurrent parent DNA (, lanes8 and 9). Since, however,probe bAN61 detects an identical fragment pattern in the DNA from the donor parent (Fig. lb,lane 8), bAN61 is judged to be noninformative for BC line mi-oil   A summary of the informationobtained from the mio BC- line survey filters for each of the identified RFLP markers is given in Table 2. Additional characterization of each mio BC line was basedonmarkers known to map distalto mi-oon chromosome 4 (K.H., unpublished results). With the exception of the cDNA coding forthe /3-amylase gene (18), none of the probes is informative for all BC lines. Genetic Mapping of RFLP Markers Identified Through ml-o BC Lines. Location of thefive RFLP markers identified in the mi-o BC-line surveyon barley chromosome 4 was verified through a barley chromosome 4 addition line in wheat  ref. 19; data not shown). Furthermore, an F2 populationof 147 individuals ofa wide cross between Hordeum vulgaresubsp. spontaneum and Hordeum vulgare subsp. vulgare cv. Aramir was used to measure geneticdistances between the respec- tive loci (Fig. 2 . Four of the five probes (bAP91, bAL88/2,bAO11, and bAN61) were found to be informative. They identifyfour polymorphic loci distributedwithin a total distance of only 8.6 cM.The fifth probe, bBE54, defines two loci (a and b) on the same linkage group (see also below). Locus bBE54b, alsoidentified through the mi-o BC-line survey, was not informative inthis wide cross-derived map- ping population. The second locus, bBES4a, maps at a distance of 24.5 cM in a centromeric position with respect to bAP91 and is not a marker ornot informative for the BC lines tested(seealso Table 2 . Additional information with respect to map position within the linkage groupwas gained through theavailability of a 3692 Genetics: Hinze et al.  Proc. Natl. Acad. Sci. USA 88 (1991) 3693 Table 2. mi-oBC-line survey usingbarley chromosome 4 RFLP markers RFLP probe mi-c bBE54 f3-Amylase BC line bAN61 a b bAP91 bAL88/2 bA11 cDNA bAL57 b4-104ami-cl + NI ++ + NI - NI m-o3 - NI ++ ++ - NI m-o4 - NININI NI+ -NI mi-oS + NI ++ + NI - NINI ml-o8 + NI + ++ NI - NINI mi-c9 - NINI ++ NI - NI mi-dO - NININI NI +- NI mi-c11 NI - NI +++ - +, The donor parental polymorphism is still present in the BC line; -, the donor parental polymorphism is replaced through the recurrent parent polymorphism in the BC line; NI,noninformativeprobe: the donorand recurrent parent have an identical restriction fragment pattern. cDNA coding forthe f3-amylase gene: this gene (BmyJ locus) is known to map in a telomeric position with respect to Mi-o (20). Linkage was found between a polymorphism detected through the f8-amylase cDNA probeand all RFLP loci identified in the mi-o BC-line analysis (Fig. 2 . With respect to theposition of thislateral marker, the chromosomal segment bordered by bAOJJ and bAN61 can be located at a position on the long arm of chromosome 4, which approxi- mates to the Mi-o position based on multipoint tests using classical phenotypicmarkers  6, 21). Genetic Distancesof RFLP Markers to Ml-o. We analyzed anF2 population segregating forresistance to mildew con- ferred by an mi-o allele to assess the genetic distances between Mi-oand the RFLP markers identified in the mi-o BC lines. A total of 439 F2 plants from an intravarietal cross between Hordeum vulgare subsp. vulgare cv. Carlsberg II Mi-o x cv. G. were infected withErysiphe graminis f.sp. hordei spores. Resistant and susceptible plants L b4-104/1 I   bBE54a -bAN61 -bAP91   bAL88/2 -bAO11 -Bmyl FIG. 2. Linkage map of RFLP markerson barley chromosome 4. Estimated genetic distancesbe- tween RFLP markers are indi- cated by numbers in centimorgans (cM) based on multipointlinkageanalysis (16). The data were ob- tained from 147 individuals in a segregating F2 population from the wide cross Hordeum vulgare subsp.vulgare x Hordeum vul- gare subsp. spontaneum. segregated according to a ratio of 1:3.7 (expected, 1:3). This deviation from the expected ratio was probably due to misclassifications of resistant and susceptible plants: Plants that allowed the growth of very few fungalcolonies were scored as susceptible in our bioassay. It has been reported, however, that mio resistance may frequently allowa very low level ofcolony formation on theinfectedleaves (22). DNA from 44 clearly resistant plants (homozygous mi-o mio) was analyzed (Fig. 3 . Loci bAL88/2 and bAOil cosegregate with the allele mi-o since in all 44 plants only the respective RFLP fragments derived from the resistant parent G. Zweiz. ml-oll arepresent. Probes bAP91 and bBE54 display only in plant 35 a state of heterozygosity of the RFLP alleles derived from the two parents: these twomarkers therefore cosegregate and map at a distance of 1.1 cM relativeto mi-o. Probe bAN61 detects four plants (nos. 8, 15, 30, and 35) that are heterozygous for bAN6M. Since in the wide cross bAN61 maps 5.4 cM centromeric from bAP91 (Fig. 2), it was expected that plant 35-with a heterozygosity for bAP91- should also beheterozygous for bAN61.The results from this intravarietal cross are therefore consistent with the linear order and distances among loci bAL88/2,bAP91, and bAN61 established by the wide cross summarized in Fig. 2.  Linkage-Drag Around Ml-o. Basedon the genetic dis- tances amongchromosome 4 loci defined by RFLP markers we estimatethe maximum size of theintrogressed fragment for each mi-o BC7 line (Fig. 4). BC lines mi-o3, ml-o4,ml-o9, and ml-oO contain the smallestdetectableintrogressed frag- ment size bracketed by bAN61 and Bmyl. Due to a lack of informative probes we cannot assess a centromericborder for BC lines ml-oSand mi-o8. It should be noted, however, that none of the remaining BC lines contains donor DNA extend- ing intothe other arm of chromosome 4 since marker b4-104a-according to map position-is still diagnostic forthe ml-o-containing chromosome arm. Toward the telomeric end of the chromosome none of the BC lines retains the donor parental allele of the Bmyl locus. Therefore the size of the introgressed fragment telomeric from mi-o in all cases must be smaller than 17 cM (assuming mi-c to be located between bAOIl and bAL88/2). In a BC7 line according to Stam and Zeven (23) areplacementofa donor parental marker positioned at this distance to the selected marker locus is, however,expected to occur only witha probability of p = 0.59. If the same calculation is done forthe marker couple ml-c-bAN61, which defines an interval centromeric from mi-o, replacementof the donor parental marker bAN61 is expected to occur with p = 0.39. Since we find that at least in four of eight mi-o BC lines donor parental alleles of bAN61 are replaced through therecurrent parent alleles, these data indicate that thelinkage drag around Mi-o is at least not larger than predicted from the theoretical estimates of Stam and Zeven. 7.6 19.1 5.4 0.8 2.4 (t 1.2) 15.6  I-l Genetics: Hinze et al.  Proc. Natl. Acad. Sci. USA 88 (1991) 43.5 -- bBE54a 5.6 -1 1.1 ( 2.2) bAN61 - bBE54b F2: ml-o ml-o individuals e Ps A b 4gE Za we Ofw o 0   A _sm ~ ~.  ~u~W,, .,,U@.Wm _ PON i m of U bAP mI-o 0 |- bAL88 2 1cM   O q~o -. ....w mine VW~ R* L-bAO11 1 ^ *- £ FIG. 3. Genetic distances of chromo- some 4 RFLP markers to Mi-o. The right half shows the results of Southern hy- bridizations obtained by the indicated marker probes with genomic DNAs from 44 homozygous ml-o-resistant F2 plants. Hybridization patterns obtainedwith DNAs from the resistant (Pr) and the susceptible (Ps) parent are shown in ad- dition. The arrowheads mark patterns obtainedwith plant 35 indicating het- erozygosity for bAP9J, bBE54b,and bAN61. On the left, geneticdistances of RFLP markers relative to mi-o are indi- cated bynumbers in cM based on maxi- mum likelihood estimates of recombina- tion frequencies calculated within the re- cessive class of a 1:2:1 segregating F2 population (see text and ref. 17). DISCUSSION Cloning by map positionrequires the identification of RFLP markers very tightly linked to the target gene. This demands a screening procedure for avery high number of genomic clones. The effectiveness of analyzing BC lines by simulta- neous probing with sets of genomic clones has been demon- strated here and in a previous report (11). If such an approach is taken, our results strongly suggest the screening of a collection of BC lines producedfrom as many different genetic backgrounds as possible for the following reasons:  i We frequently identified genomic clones detecting polymor- phismsbetween the DNA from a single BC line and the recurrentparent. All of these clones, however, define loci not linked to Mi-o (by F2 segregation criteria of the wide cross) and therefore identify Mi-o unlinked DNA segment(s) of donor parental srcin retained during the backcross proce- dure.  ii An analysisincluding 48 barley varieties revealed that in the barley genome restriction polymorphisms are detected only with anaverage frequency of 43 based on restriction enzymes identifying 6-bp target sequences (24). If only two barley lines are considered this predicts that in a pool of anonymous probes only half of the probes will be informative. Since many resistance genes in barley and other cereals were introgressed by intravarietal crosses,* this ne- cessitates theinclusion of several genetic backgrounds in a BC-line survey to maximize the percentage of informative probes. Each of the mio BC lines used in this study has been backcrossed seven times. According to Stam and Zeven (23), the size of the introgressed fragment that is retained in the marker chromosome should be 30 cM with a SD of20 cM (assuming for chromosome 4 a total length of 150 cM). We therefore expected to identify 25-35 Mi-o linked markers withinthe poolof 1100 analyzed barley clones (calculated on the basis of a Poisson distribution and assuming a total length of 1.050 cM forthe barley genome map). Since random *Vortrage fur Pflanzenzuchtung, Proceedings of the Eucarpia Cereal Section Meeting, 1984, Weihenstephan, F.R.G. probes may detect more than one locus (e.g., bBE54), this number is more likelyto be underestimation. There is, however, a clear discrepancy between the expected value and the number of Mi-o linked markers actuallyidentified (atotal of five). Also, the size of the introgressed fragment that is defined by these markers (8.6 cM) does not correspond to the prediction. We speculate that the low frequency of Mi-o linked markers and their confinement to a small chromosomal fragmentaround MI-o are due to a nonrandom srcin of cloned genomic Pst I fragments in thevicinity of this locus. Since all tested Pst I clones detect nonrepetitive low- or single-copy-number DNA sequences, the underrepresenta- tion ofsuchsequencescould reflect an overrepresentationof repetitive DNA in thevicinity of the locus. There are several reports describing colinearity of markers if wide (including interspecific) and intravarietal-based link- age data are compared. Different distances between single marker couples have, however, been found frequently (25, 26). A comparison of our linkage data based on the wide- and intravarietal crosses (Figs.2 and 3) indicates colinearity of Mi-o linked markers. In addition, we also find a good correspondence of marker-based distances in both crosses. The exception, however, is the marker couple bAOI- bAL88/2, forming a 2.4-cM interval only in the wide cross, whereas both markers cosegregate with mi-o in the intrava- rietal cross. The simplestexplanation for thisresult is an inadequate sample size of homozygous mi-o plants in the intravarietal cross. Increasing the sample size should identify resistant plants in which a recombination has occurred be- tween ml-o-bAL88/2 and/or mi--bAOIL. Young andTanksley (27) describe that backcross breeding was largelyineffective in reducing the size of linked DNA around the Tm-2 gene, a resistance gene introgressed from Lycopersicon peruvianum into Lycopersicon esculentum (28). They describe a Tm-2 BC line that retained, even after 11 backcrosses, one complete chromosome arm plus a por- tion of theother arm from the donor parent, indicating a strong linkage drag aroundTm-2. Our results suggest that the linkage drag around mio may be lower than expected from thetheoretical estimates described by Stami andZeven (22). 3694 Genetics: Hinze et al.  Proc. Natl. Acad. Sci. USA 88 (1991) 3695 BC7 -ml -o BC7 -_mi-3 BC7 -mi-a4 BC7-mI-o5 BC7 -mI-o8 BC7 -mi-a9 BC7 -ml-a BC7 -ml-a 0 .0 I L-I- m U, O .0 0   I I- CO: I- Z X . O .0 .0 .0 1 E m /L-   f ~~~~~~~~~~~~~ 7.6 19.1 5.4 0.82.415.6 4, 4::::::::---------------............ 4/ _ ... I4 ha ----- Bat~~i FIG. 4. Graphical chromosome 4genotypes of barley lines showing the estimated sizes of the introgressed segments flanking Mi-o. The top line denotes the relative position of RFLP markerson barley chromosome 4. Numbers indicate distances in cM. The mi-o locus (dotted line) is assumed to be situated in the 2.4-cM interval bracketed bymarkers bAL88/2 and bA011.Locus Bmyl maps proximal to the telomeric end of the chromosome, whereas markers b4-104a and bBE54a are oriented toward the centromere. The lower part shows a graphical representation of thatpart of chromosome 4 that flanksthe Ml-o locus in eachanalyzed BC7 line. White regions denote recurrent parentsegments, black regions denote donor parental-derived segments, and darkly stippled regions indicate segments in which crossovers occurred. A possible explanation for these two divergent findings could be the factthat recombination tends to besuppressed in genotypesheterozygous for a chromosomalsegment srci- nating from different species (29), an effectthat is even amplified at centromere-proximal positions (30). Since Tm-2 was introgressed through interspecific crosses and is locatedclose to the centromere (31) whereas mi-o was introgressed through intravarietal crosses and maps distal from the cen- tromere, we predict a significantly smaller linkage drag for mio introgression. Although we cannot completely exclude a location of Mi-o neither centromeric from bAL88/2 nortelomeric from bAOII, theavailable data indicate that thegene(s) is most likely to reside in the interval bracketed by markers bAOII and bAL88/2. This forms a rationale to start from the twomentionedmarkers a bidirectional walk or jump (32, 33) toward the Mi-o gene. The set of available x-ray-induced mio mutants will provide a powerful tool to assist long-range physical mapping on thebasis of the two markers cosegre- gating with theresistance.In addition, a search for additional RFLP markers withinthe bAO1J-bAL88/2 interval and the molecular analysis of a larger population of resistant plants to find recombinants to the neighboring markers should parallel the long-rangecloning approach. We are grateful to Prof. James MacKey, Dr. S. Ceccarelli, and Dr. Mike D. Gale for theirgifts of mi-o BC lines, F1 kernels derived from the wide barley cross, and the 0-amylase cDNA clone. Ingrid Herzer is gratefully acknowledged for her excellent technicalassistance. We thank members of the Jeffery L. Dangl laboratory for commentsand suggestions and J. L. Dangl and Thomas Debener for critical reading of the manuscript. 1. Jensen, J. P.   Jorgensen, J. H. (1981) Tidsskr. Planteavl 85, 303. 2. Schwarzbach, E.   Fischbeck, G. (1981) Z. Pflanzenzacht. 87, 309-318. 3. 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We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

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