The Mla (Powdery Mildew) Resistance Cluster Is Associated With Three NBS-LRR Gene Families and Suppressed Recombination Within a 240-kb DNA Interval on Chromosome 5S (1HS) of Barley

Corresponding author: Roger P. Wise, USDA-ARS-Corn Insects and Crop Genetics Research Unit, Department of Plant Pathology, 409 Bessey Hall, Iowa State University, Ames, IA 50011-1020., rpwise{at}iastate.edu (E-mail)

Communicating editor: B. S. GILL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster.

GENES in plants that confer resistance to fungal pathogens frequently display characteristic gene-for-gene specificity as originally described by FLOR 1956 . In nature, there are many resistance (R) genes in the host, each with unique specificities to particular pathogen isolates. These R genes are often tightly linked or represented by many alleles. The specificities among host-resistance determinants and their corresponding pathogen isolates have been useful for the genetic analyses of several resistance-gene clusters (SHEPHERD and MAYO 1972 ; PARAN et al. 1991 ; DICKINSON et al. 1993 ; JONES et al. 1993 ; SUDUPAK et al. 1993 ; KESSELI et al. 1994 ; LAWRENCE et al. 1995 ; RICHTER et al. 1995 ; HU et al. 1996 ; reviewed by ANDERSON et al. 1997 ).

A large number of Ml specificities, which confer resistance to the powdery mildew fungus, Erysiphe graminis f. sp. hordei, have been identified in barley, Hordeum vulgare L. These variants are distributed among 11 groups: Mlat, Mla, Mlk, Mlnn, Mlra, MlGa, and Mlp on chromosome 5 (1H; reviewed by JORGENSEN 1994 ), Mlg and mlo on chromosome 4 (4H; GORG et al. 1993 ; BUSCHGES et al. 1997 ), MlLa on chromosome 2 (2H; GIESE et al. 1993 ), and Mlh on chromosome 6 (6H; JORGENSEN 1994 ). Thirty-two specificities at the Mla locus have been differentiated by their specific reaction to unique isolates of E. graminis (GIESE 1981 ; GIESE et al. 1981 ; WISE and ELLINGBOE 1983 , WISE and ELLINGBOE 1985 ; JAHOOR and FISCHBECK 1993 ; reviewed by JORGENSEN 1994 ; KINTZIOS et al. 1995 ). Hence, due to its highly variable nature, the Mla-resistance cluster is an excellent model for the investigation of specific recognition in gene-for-gene interactions among small grains and obligate fungal pathogens (KEEN 1990 ; THOMPSON and BURDON 1992 ; CRUTE and PINK 1996 ). In our earlier studies, we developed a high-resolution recombinant population (selected from 3600 gametes) that makes possible the simultaneous analysis of a number of specificities of the Mla cluster (MAHADEVAPPA et al. 1994 ). Of the 32 Mla specificities, the Mla6, Mla14, Mla13, and Ml-Ru3 variants present in this recombinant population are all flanked by the Xbcd249.1 and Xmwg036 RFLP loci (DESCENZO et al. 1994 ; DESCENZO and WISE 1996 ).

In preparation for positional-cloning of the Mla locus, we used random amplified polymorphic DNA (RAPD; WILLIAMS et al. 1990 ), amplified fragment length polymorphism (AFLP; VOS et al. 1995 ), restriction fragment length polymorphism (RFLP; BOTSTEIN et al. 1980 ), and sequence-tagged site (STS) methods to saturate the Mla region with molecular markers. We used these markers to identify yeast artificial chromosomes (YACs) from the cultivar Franka, and bacterial artificial chromosomes (BACs) from the cultivar Morex, that are tightly linked to and spanning the Mla cluster. At least 11 copies of nucleotide-binding site/leucine-rich repeat (NBS-LRR) resistance-gene homologues (RGHs) were identified from the Mla-spanning, Morex BAC contig. The 11 RGHs are present in three distinct families and are dispersed throughout the 240-kb, Mla-spanning region.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Overview of the high-resolution mapping population:
The barley lines used to set up the original cross for the mapping population are nearly isogenic, differing by one or more unique Mla specificities in the introgressed region (MOSEMAN 1972 ). Each of the lines was characterized quantitatively for its respective infection kinetics and resistance specificity (WISE and ELLINGBOE 1983 ). Crosses were constructed between the Franger- [cereal introduction (C.I.) 16151] and Rupee-derived (C.I. 16155) isogenic lines. C.I. 16151 contains the Mla6 and Mla14 specificities for resistance to E. graminis, whereas C.I. 16155 contains Mla13 and Ml-Ru3 (JORGENSEN 1994 ). The flanking endosperm storage-protein-encoding genes, Hor1 and Hor2, were used to select for genetic recombinants in the Mla region. These polypeptides are distinctly polymorphic between the lines containing different Mla alleles, and recombinant phenotypes can be readily visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of endosperm tip extracts (DOLL and ANDERSEN 1981 ). A total of 1800 F₂ seeds (representing 3600 F₁ gametes) were screened by this method. The final population presently consists of 286 individual F₄ homozygous lines, each representing an independent recombination event between the Hor1 and Hor2 loci, which are 8.1 cM apart and bracket the Mla cluster (DESCENZO et al. 1994 ; MAHADEVAPPA et al. 1994 ).

Powdery mildew resistance screening:
Infection types (IT) were scored as described in MAHADEVAPPA et al. 1994 . The infection types 0, 1, or 2 are considered resistant reactions while the infection types 3 or 4 are considered susceptible (WISE and ELLINGBOE 1983 ). The Franger- (C.I. 16151), Rupee- (C.I. 16155), Kwan (C.I. 16143, containing Mlk)-derived lines, in addition to Manchuria (C.I. 2330), were used as controls (MOSEMAN 1972 ). Families that segregated with any isolate were retested with at least 16 individuals per line. Sixteen individuals were used to ensure 99% probability of observing at least one homozygous recessive individual (MATHER 1951 ).

Bulk design:
A 3-cM window bracketing the Mla cluster was defined via the recombination breakpoints in our high-resolution, recombinant population (DESCENZO et al. 1994 ). We used bulk segregant, RAPD, and AFLP analyses (GIOVANNONI et al. 1991 ; MICHELMORE et al. 1991 ; CHURCHILL et al. 1993 ; VOS et al. 1995 ) to compare pools of 14 (for RAPD) or 16 (for AFLP) DNAs that were homogeneous within the window for either the Mla6 and Mla14 or the Mla13 and Ml-Ru3 resistance specificities.

RAPD and STS analysis:
RAPD analysis was carried out using 10-base oligonucleotide primers synthesized from both Operon Technologies (Alameda, CA) and Oligonucleotide Synthesis Laboratory (University of British Columbia, Vancouver, Canada). A total of 40 Operon (Operon Technologies) and 699 University of British Columbia (Carlson) arbitrary nucleotide sequences were used in this analysis. Map positions of RAPD (and subsequently AFLP) polymorphisms were initially positioned via a low-resolution interval-mapping population, followed by all the recombinants between Xbcd249.1 and Xmwg036 in the high-resolution mapping population.

PCR amplification was performed in a 25-µl reaction volume with a 1x reaction buffer supplied by the manufacturer [20 mM Tris-HCl (pH 8.4), 50 mM KCl], 1.5 mM MgCl₂, 0.001% gelatin, 0.1 mM each of dNTP, either 5 µM decamer RAPD- or 20 µM STS-primer, 50 ng of genomic DNA, and 0.625 units of Taq DNA polymerase (GIBCO BRL, Rockville, MD). The following programs were used for amplification: for RAPD, one cycle for 1 min at 94°; 44 cycles for 5 sec at 94°, 30 sec at 36°, 1 min at 72°, with a final extension of 9 min at 72°; for the STS analysis, one cycle for 3 min at 94°; 29 cycles for 30 sec at 94°, 1 min at 60°, 1 min at 72°, with a final extension of 4 min at 72°. All PCR amplifications were performed in a PTC-100 programmable thermocycler (MJ Research Inc., Watertown, MA). Amplification products were resolved by electrophoresis at 80 V for 4 hr on a 2% thin (3-mm) agarose gel containing 1x TBE buffer (0.089 M Tris, 0.089 M Borate, 0.002 M Na₂EDTA; SAMBROOK et al. 1989 ) and 1 µg/ml ethidium bromide.

Cloning of polymorphic RAPD fragments:
DNA fragments were isolated by extracting an agarose plug with the small end of a pasteur pipette followed by placement in 100 µl sterile double-distilled water (ddH₂O) to elute overnight at 4°. One microliter of eluted DNA/ddH₂O solution was used as a template for reamplification with the original 10-base oligonucleotide primer. DNA inserts were purified via a modified NA45 membrane (Schleicher & Schuell, Keene, NH) extraction, ligated into pGEM-T (Promega, Madison, WI), and transformed into the Escherichia coli TB-1 host strain.

AFLP analysis:
All 256 pairwise combinations of ³³P-labeled (New England Nuclear Life Science Products, Boston, MA) EcoRI and MseI primers (listed in Table 1) were used to screen for polymorphisms between both the pools and the parents. AFLP analysis was performed as per the AFLP instruction manual (GIBCO BRL). For each pool, 3 µl of the preamplified products from each of the 16 individual lines was combined, diluted 50x, and selective amplification was carried out in the presence of [³³P-]ATP-labeled EcoRI primer and MseI primer (as shown in Table 1). The amplified fragments were size-fractionated through a 7% acrylamide gel (Long Ranger; FMC Bioproducts, Rockland, ME) and exposed directly (without drying the gel) to Biomax XR film (Eastman Kodak, Rochester, NY) at -80° for 16–24 hr.

Sequence-specific AFLP:
The AFLP preamplification products were obtained through the use of the E-A/M-C, E-A/M-T, E-G/M-C, and E-G/M-T primer pairs. For selective amplification, the [³³P-]ATP-labeled, long terminal repeat (LTR) sequence (5'-TGTTGGAATTATGCCCTAG-3') of the barley Bare-1-retrotransposon (WAUGH et al. 1997 ) was utilized in combination with one of the random AFLP primers (listed in Table 1).

Cloning and sequencing of AFLP DNA fragments:
AFLP fragments were identified by matching the target signal on the autoradiogram with its corresponding area in the acrylamide gel. The cut gel slices were dissolved in ddH₂O overnight at 4° and the fragments were enriched using only the EcoRI primers via 10 cycles of PCR, followed by amplification with both EcoRI and MseI primers for another 30 cycles. The resulting fragment was cloned into pGEM-T cloning vector and transformed into the E. coli TB1 host strain for selection of putative clones. The cloned inserts were prescreened by direct PCR with the T7-1 (5'-AATACGACTCACTATAG-3') and SP6 (5'-GATTTAGGTGACACTATAG-3') primer pairs. Comigration (via polyacrylamide gel electrophoresis) of the cloned AFLP insert as compared to the original genomic AFLP fragment was used for final verification. Two confirmed colonies from each cloning experiment were purified with Microcon-100 (Amicon, Bedford, MA) and sequenced using T7-2 (5'-CGACTCACTATAGGGCGAAT-3') and SP6-2 (5'-GCGTTGGGAGCTCTCCCATATGGT-3') vector primers. DNA sequencing and oligonucleotide synthesis were performed by the Iowa State University DNA sequencing and synthesis facility. PCR primers were designed according to the DNA sequences of the clones with the assistance of Oligo 5.0 software (PE Biosystems, Foster City, CA).

Preparation of chromosomal yeast/YAC DNA:
YAC clones were grown and maintained using Kiwi media (AUSUBEL et al. 1988 ). The AB1380 yeast host strain was grown and maintained using YEPD media [1% (w/v) bacto-yeast extract, 2% (w/v) bacto-peptone, 2% (w/v) glucose, 100 mg/l adenine]. Single-colony purified YAC clones were used to inoculate 25 ml of selective media and grown for 24–36 hr with shaking at 30°. Cells were harvested (5000 x g, 10 min at 4°) and resuspended in 5 ml 50 mM Na₂EDTA (pH 8.0). Yeast cell concentration was determined with a hemacytometer. Subsequently, cells were harvested and resuspended at a concentration of 1 x 10⁹ cells/ml in resuspension buffer (10 mM Tris, pH 7.2, 20 mM NaCl, 50 mM Na₂EDTA). The cell suspension was prewarmed to 50° briefly before adding lyticase (Sigma, St. Louis) to 1 mg/ml and gently mixing with prewarmed InCert agarose [FMC Bioproducts; 2% (w/v) in resuspension buffer], and removed to plug molds. Plugs were allowed to set for 10 min at 4° before being removed from the molds into 5 ml of lyticase buffer (10 mM Tris, pH 7.2, 50 mM Na₂EDTA, 1 mg/ml lyticase) per ml of plug and incubated for 1 hr at 37°. Plugs were washed once (10 min at RT) in 1x wash buffer (20 mM Tris, pH 8.0, 50 mM Na₂EDTA) before being transferred to 5 ml of proteinase K reaction buffer [100 mM Na₂EDTA, pH 8.8, 0.2% (w/v) sodium deoxycholate, 1% (w/v) sodium lauryl sarcosine, 1 mg/ml proteinase K] per ml plug and incubated for 48 hr at 50°. Plugs were washed three times (10 ml per ml of plug; 30 min at RT) in 1x wash buffer (1 mM PMSF was included in the second wash to eliminate residual proteinase K) before a final wash in 0.1x wash buffer and storage at 4°.

Pulsed-field gel electrophoresis analysis of YAC and BAC clones:
Pulsed-field gel electrophoresis (PFGE) conditions were as calculated by the auto-algorithm function of a Bio-Rad (Hercules, CA) Chef Mapper XA. The YAC and BAC clones were resolved by PFGE at 14° using 0.5x TBE buffer and 1% SeaKem LE agarose (FMC Bioproducts). PFGs were stained with ethidium bromide (1 µg/ml in ddH₂O), UV nicked (60 mJ/cm²), and DNA transferred to Hybond N⁺ membrane (Amersham Pharmacia Biotech, Piscataway, NJ) using 1.5 M NaCl, 0.4 M NaOH as the transfer buffer. YAC clones were further restriction mapped utilizing a partial digest strategy described by BURKE et al. 1987 , employing medium-rare and rare-cutting restriction endonucleases.

Generation of hybridization probes for the physical mapping of YACs:
Sequences specific for the right and left arms of the YAC vector were amplified via PCR using pBR322 as a template. The primers used were as follows: YAC-LA-as (5'-AAGCGAGCAGGACTGGGCGG-3') in conjunction with YAC-LA-s (5'-GTCAGCGGGTGTTGGCGGGT-3') amplified a 2.5-kb product specific for the left arm of the YAC vector. YAC-RA-as (5'-CGGTTTTTTCCTGTTTGGCT-3') in conjunction with YAC-RA-s (5'-TTGTTTCGGCGTGGGTATGG-3') amplified a 1.4-kb product specific for the right arm of the YAC vector. Amplification conditions for the PTC-100 programmable thermal cycler (MJ Research) were as follows: one cycle for 3 min at 94°; 34 cycles for 30 sec at 94°, 30 sec at 56°, 3 min at 72°; and one final extension cycle for 10 min at 72°.

Genetic mapping of YAC ends or other sequences derived from the Mla region:
Barley DNA was isolated from frozen tissue using a modified CTAB extraction. These DNA extractions, as well as DNA gel blot analyses, were conducted as previously described (WISE and SCHNABLE 1994 ). YAC ends or other sequences derived from the Mla region were first established as being low-copy by hybridization with strip blots of HindIII digested and resolved parental DNA. Once low-copy status had been determined, sequences were screened for RFLPs by Southern hybridization with parental DNA digested with a number of restriction enzymes to reveal which restriction endonuclease revealed a polymorphism. RFLPs were exhibited as differences between the two parental lines and were mapped by Southern hybridization with recombinants from our high-resolution mapping population (MANLEY 1993).

BAC AFLP fingerprinting:
BAC DNA was isolated by using the Clemson University Genomic Institute (CUGI; 1998) protocol. The standard AFLP analysis protocol was followed except that the preamplification and amplification primer pairs are identical. These primers do not contain a selective base. The sequence of the EcoRI primer (designated E-0) is 5'-AGACTGCGTACCAATTC-3' and the sequence of the MseI primer (designated M-0) is 5'-GATGAGTCCTGAGTAA-3'.

BAC-end cloning and sequencing:
BAC ends were cloned via double-end rescue. Briefly, the BAC DNA (0.5 µg) was digested for 4 hr at 37° with 20 units of NsiI (New England Biolabs, Beverly, MA), which does not cut within the vector, but cuts fairly frequently in the genomic-DNA insert. The reaction was inactivated for 20 min at 70°. The BAC DNAs were recircularized by self-ligation in 200 µl overnight at 16°. The ligated products were transformed into the E. coli TB1 host strain and plated on LB-chloramphenicol plates. For sequencing, "mini-BAC" DNA was prepared according to our standard BAC purification protocol and further concentrated through Microcon-100 columns (Amicon, Beverly, MA). Sequence data were obtained with ABI Big Dye terminators (PE Biosystems) using the T7-1 or M13 reverse (designated R1; 5'-GGAAACAGCTATGACCATG-3'), using 5 µg of total template DNA. The resulting sequence data were utilized for designing PCR primers for mapping and further library screening.

Low-pass BAC sequencing:
Cesium chloride-density gradient purified BAC DNA was used for sequencing library construction. Two milliliters of BAC DNA solution [20 µg DNA, 500 µl glycerol, 200 µl 10x TM (0.5 M Tris-Cl, 150 mM MgCl₂)] was nebulized with N₂ at 6 psi for 2 min. DNA fragments of 1.7–3.0 kb were agarose-gel purified and ligated to dephosphorylated SmaI-restricted pUC18 (Boehringer Mannheim, Indianapolis) overnight at 16°. One microliter of the ligation solution was transformed into 25 µl of E. coli electroMAX DH10B TM competent cells (GIBCO BRL) by electroporation with the Cell-Porator system (GIBCO BRL; 400 V, capacitance 330 µF, impedance low ohms, charge rate fast, voltage booster resistance 4 kohms). White colonies were picked into 96-well microtiter plates containing LB freezing medium and ampicillin, incubated overnight at 37°, and stored at -80°.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Four Mla specificities are inseparable by recombination in the C.I. 16151 x C.I. 16155 cross:
Over 30 specificities of Mla have been described (reviewed by JORGENSEN 1992 , JORGENSEN 1994 ; KINTZIOS et al. 1995 ). Two of these specificities are present in coupling in each of our mapping parents. The Franger-derived line, C.I. 16151, contains the Mla6 and Mla14 specificities as described previously (GIESE et al. 1981 ; JORGENSEN 1992 , JORGENSEN 1994 ). Likewise, the Rupee-derived line, C.I. 16155, contains the Mla13 and Ml-Ru3 specificities (JORGENSEN 1992 , JORGENSEN 1994 ; CAFFIER et al. 1996 ). Previously, we determined the position of the Mla6, Mla13, and Mla14 specificities (MAHADEVAPPA et al. 1994 ). The first objective of this study was to confirm the position of Mla14 and to determine the position of the fourth specificity in our mapping population, Ml-Ru3.

Unique ITs in response to characterized isolates of E. graminis are utilized to detect the different specificities in segregating populations. In our earlier studies, we had mapped the Mla14 specificity via inoculation of the recombinant population with isolate A27 (MAHADEVAPPA et al. 1994 ). As shown in Table 2, the C.I. 16151 line that contains Mla14 confers an IT of 2–3n in response to isolate A27, whereas the C.I. 16155 line containing Mla13 imparts an IT of 0 with the same isolate. However, the 0 IT in response to Mla13 would be predicted to be epistatic over the 2–3n IT displayed by Mla14. Thus, it was conceivable that some of the recombinant progeny in our segregating population would display a 0 IT in response to Mla13, but still contain Mla14. This epistasis could have complicated mapping of the Mla14 specificity.

To map the position of Mla14 and Ml-Ru3, 88 homozygous families that are recombinant between Xbcd249.1 and Xmwg036 were utilized. Four to six progeny from each homozygous recombinant family were inoculated separately with the R63, R189, A27, and 5874 isolates of E. graminis (shown in Table 2) and subsequently scored for IT using a 0–4 scale. Isolate R63 imparts a unique IT only in response to Mla14; thus, screening with this isolate would provide an unambiguous result. First, the 49 recombinant individuals that displayed an IT of 0 in response to A27 (confirming the presence of Mla13) also displayed an IT of 4 in response to R63, indicating that these individuals, in fact, do not contain Mla14. Conversely, the remaining 39 recombinant individuals that displayed an IT of 2–3n in response to A27 also displayed the identical IT in response to R63. This confirmed the presence of Mla14 in these 39 progeny. Therefore, these additional results from the inoculations with R63 confirmed that Mla14 specificity cosegregates (in repulsion) with Mla13.

To position Ml-Ru3, the recombinant population was inoculated with isolate R189. The C.I. 16155 parent that contains Ml-Ru3 displays an IT of 1–2n in response to this isolate. Forty-nine recombinant individuals displayed an IT of 1–2n in response to R189, indicating the presence of Ml-Ru3 (Table 2). These same 49 individuals also displayed an IT of 0 in response to A27, indicating the presence of the Mla13 specificity (in coupling). Importantly, the remaining 39 recombinant individuals that displayed an IT of 0 in response to 5874 (confirming the presence of Mla6), also displayed an IT of 2–3n in response to A27, R63, and R189, indicating the presence of Mla14. On the basis of these experiments, we established that the Ml-Ru3 specificity cosegregates (in repulsion) with Mla6 and Mla14. Thus, current observations indicate that all four specificities in this mapping population (of 3600 gametes) are at the same genetic position on chromosome 5 (1H). This observation could be viewed as advantageous, because it suggested that all four of these specificities could be physically close, which would facilitate their ultimate isolation.

We also further tested two putative recombinants that were reported previously in MAHADEVAPPA et al. 1994 . In that report, we had postulated recombination between the Mla6 and Mla13 specificities in two F₃ families of this same mapping cross. To review, both of these putative recombinant lines (H92S 6526 and H92S 6562 in Table 5 of MAHADEVAPPA et al. 1994 ) contained one or more recombination events between the flanking markers, Hor1 and Hor2, that we were using to screen the population. In addition, these progeny families displayed an IT ratio in response to infection with isolates A27 and 5874 that was consistent with a recombination event (or gene conversion) within Mla. However, lack of DNA markers tightly linked to Mla prevented the precise fingerprinting of recombination events in our previous work. Therefore, to follow up on our assumption, the putative recombinant lines H92S 6526 and H92S 6562 were subjected to several progeny tests with isolates 5874 and A27. However, when these H92S 6526 and H92S 6562 progeny were genotyped with our current tightly linked markers, the intra-Hor1-Hor2 recombination events appeared to be positioned on either side of the Mla locus. Hence, even though the original lines repeatedly displayed non-Mendelian IT ratios, at present, we are unable to confirm our original hypothesis of recombination between Mla6 and Mla13 at the molecular level. It is possible that a distorted segregation of parental chromosomes caused the altered IT ratios in the F₃ in these two families.

Bulk-segregant, RAPD analysis increases the genetic resolution flanking the Mla cluster:
Saturation of the target interval with DNA markers is a prerequisite for physical delimitation via large-insert clones in the complex barley genome. A 3-cM window, defined by the Xbcd249.1–Xmwg036 interval (DESCENZO et al. 1994 ), was established from our high-resolution population for bulk segregant (GIOVANNONI et al. 1991 ; MICHELMORE et al. 1991 ; CHURCHILL et al. 1993 ) RAPD, and AFLP (VOS et al. 1995 ) analyses.

A total of 739 RAPD primers were used to amplify DNAs from the defined bulks. Of these, 91 primers produced DNA fragments that were polymorphic between C.I. 16151 and C.I. 16155, or the bulks. Eighteen recombinant lines, each possessing a unique recombination breakpoint between the Hor1 and Hor2 loci, were used to quickly determine if markers were positioned near the Mla locus. Only 3 of the 91 primers that produced amplified polymorphisms mapped to the region between Hor1 and Hor2. Primer OPA-10 (5'-GTGATCGCAG-3') amplified a 1500-bp fragment in C.I. 16155, designated OPA-10₁₅₀₀, that mapped between Hor1 and XChs3. Primers UBC465 (5'-GGTCAGGGCT-3') and UBC165 (5'-GAAGGCACTG-3') amplified 950-bp and 1626-bp fragments, respectively, in pools containing the Mla6 or Mla14 specificities but not in pools containing the Mla13 or Ml-Ru3 specificities. The 950-bp fragment was designated UBC465₉₅₀ and mapped between XChs3 and Xmwg068. The 1626-bp fragment was designated UBC165₁₆₂₆ and cosegregated with Mla in the low-resolution, interval-mapping population described above.

Eighty-eight lines, each containing a unique recombination breakpoint in the Xbcd249.1–Xmwg036 interval, were used to fine-map UBC165₁₆₂₆ to a position 0.3 cM proximal to Mla6. The 1626-bp UBC165₁₆₂₆-derived fragment was subsequently cloned, sequenced, and a series of PCR primers were designed. The different pairwise combinations yielded a number of genomic-PCR products, which is likely due to the repetitive sequence represented by the 1626-bp UBC165₁₆₂₆-derived fragment. The combination of primers P0 (5'-GAAGGCACTGAATCGTTGATGG-3') and P954RC (5'-CAGTTTAGGGAAGTATTGCATC-3') produced a C.I. 16151-specific product that mapped 0.28 cM distal to Mla. Apparently, the primer pair P0 and P954RC uncovered a sequence-related, tightly linked copy of UBC165₁₆₂₆. This amplification product consistently yielded the most stable map position and was designated Fr1062. The Fr1062 PCR primers amplified the same fragment from Franka, the cultivar used in the construction of the Maltagen YAC library (KLEINE et al. 1993 , KLEINE et al. 1997 ).

AFLP analysis is used to further saturate the genetic map:
To further enrich the Mla region with markers for large-insert clone isolation, 256 AFLP primer pairs were used to screen for polymorphisms between the C.I. 16151 and C.I. 16155 mapping parents and the pools described above. Out of 22,500 AFLP fragments generated, 132 polymorphisms amplified from 104 primer pairs were observed. Seven of these polymorphic fragments mapped to the Xbcd249.1–Xmwg036 interval on our low-resolution, interval-mapping population. In the high-resolution analysis (Table 3), it was established that the FW108 AFLP marker was 0.14 cM distal from the Mla locus and allele-specific primers were developed for library screening as described below.

Development of allele-specific, AFLP-derived STS markers:
First-round PCR primers were designed according to the corresponding DNA sequence of the cloned AFLP fragment. Six of the primer pairs derived from the internal sequences of the seven markers did not display a polymorphism in amplification experiments of the parental DNAs (Table 3). However, the FW108-derived marker displayed a potential polymorphism, amplifying a strong band in parental DNA from C.I. 16151 but a weak band in parental DNA from C.I. 16155; this same pattern was observed among the recombinants in the high-resolution mapping population.

The FW108 PCR products from both the C.I. 16151 and C.I. 16155 parental DNAs (Figure 1A) were cloned and sequenced. Three single-nucleotide polymorphisms (SNPs) were detected in the 108-bp FW108 fragment (Figure 2). These SNPs facilitated the design of C.I. 16151- and C.I. 16155-specific forward primers (designated FW108.2 and FW108.3, respectively). As illustrated in Figure 1, it was established that when paired with FW108 reverse primer, the FW108.2 and FW108.3 primers amplified allele-specific polymorphisms that mapped to the site of the original AFLP marker, FW108. This approach was not useful for the development of STS primers for the other six AFLP markers. The original EcoRI/MseI polymorphism was lost when the amplified fragments were cloned and the internal sequence of the C.I. 16151 and C.I. 16155 parental fragments were 100% identical.

View larger version (66K): In this window In a new window Download PPT slide   Figure 1. PCR products amplified from C.I. 16155 and C.I. 16151 parental DNAs with FW108 allele-specific primers. (A) Parental-specific PCR products generated from first-round PCR primers designed according to the DNA sequence of the AFLP clone. (B) C.I. 16151-specific PCR products generated from FW108.2 primers. (C) C.I. 16155-specific PCR products generated from FW108.3 primers. The FW108.2 primers were used to screen the Maltagen (Franka) YAC library.

View larger version (19K): In this window In a new window Download PPT slide   Figure 2. Nucleotide sequence of the FW108 fragment from C.I. 16151 and C.I. 16155. The allele-specific primers FW108.2 and FW108.3 were designed on the basis of the three-nucleotide sequence polymorphism as shown.

Bare-1 retrotransposon, sequence-specific AFLP is used to identify additional markers:
Sequence-specific AFLP (S-SAP) was utilized to further screen for markers close to the Mla cluster. There are at least 3 x 10⁴ copies of the Bare-1 retrotransposon in barley, which is equivalent to 6.7% of the genome (SUONIEMI et al. 1996 ). Therefore, the Bare-1 inverted repeat primer was used in conjunction with 24 EcoRI- and 24 MseI-primers to amplify DNAs from the bulks and the parents. Two separate preamplifications were used for a total of 96 pairwise combinations. From the resulting 5700 amplified bands, 114 polymorphisms were detected. One DNA fragment, designated AGCBare, cosegregated with the map position of Fr1062, 0.28 cM distal to the Mla locus. The number of polymorphisms detected in the bulks suggests that there are multiple, near-identical copies of the Bare-1 retrotransposon in the Mla-flanking region.

The 236R end clone from YAC236 cosegregates with the Mla cluster:
Figure 3 illustrates the integration of all new RAPD, AFLP, and derived STS markers into the Hor1-Mla-Hor2 region of chromosome 5 (1H). The Fr1062- and FW108.2-derived primers amplified DNA from Franka, and thus, markers fulfilled the criteria for large-insert clone isolation. Therefore, these two primer sets were used to screen the Maltagen (Franka) YAC library (Table 4; KLEINE et al. 1993 , KLEINE et al. 1997 ). As shown in Figure 4, YAC clones were sized by PFGE followed by Southern hybridization with YAC vector-specific sequences. YAC terminal-end sequences were isolated by inverse PCR (LEISTER et al. 1997B ). It was established that two of these ends (234L and 236R) hybridized to low-copy fragments that were polymorphic between the C.I. 16151 and C.I. 16155 mapping parents. Subsequently, a combination of genetic and physical mapping established that one of the two copies of 236R cosegregates with the Mla locus. During the course of this investigation, we also mapped the RFLP markers, mwg2083 and mwg2197, previously shown to map between the Hor1 and Hor2 loci (http://wheat.pw.usda.gov/ggpages/maps.html; kindly provided by Dr. Andreas Graner, IPK, Gatersleben, Germany). One of the three copies of mwg2083 cosegregated with the Mla locus. The single-copy marker mwg2197 was positioned two crossovers distal (0.056 cM) to the Mla locus. We also positioned the RGH Hv-b6.1 (LEISTER et al. 1997A ). We hypothesized that by taking this candidate-gene approach, we might identify large-insert clones that contained the Mla gene family. This proved not to be the case as Hv-b6.1 cosegregated with XciwS10, which is 0.62 cM distal to the four specificities in our mapping population (see Figure 5).

View larger version (23K): In this window In a new window Download PPT slide   Figure 3. High-resolution genetic map of the Hor1–Hor2 region of barley chromosome 5 (1H). Fr and FW prefixes designate Wise laboratory RAPD- and AFLP-derived markers, respectively. Markers in brackets were mapped on the low-resolution interval population. An X prefix designates an RFLP marker; mwg markers are from Munich-Weihenstephan-Grunbach; bcd markers are from Cornell University; and ciw markers are from the Carnegie Institute of Washington.

View larger version (87K): In this window In a new window Download PPT slide   Figure 4. Analysis of Mla-linked YAC clones. High-resolution genetic analyses established that the Fr1062-RAPD- and FW108-AFLP-derived markers were 0.28 and 0.14 cM distal to the Mla locus, respectively, and allele-specific primers were developed to screen the Maltagen (Franka) YAC library. (A) YAC clones were resolved by PFGE using a CHEF Mapper XA system (Bio-Rad) in conjunction with a 14-cm gel. (B) Southern analysis of YAC clones. Filters were then hybridized with YAC vector-specific sequences to identify all YACs.

View larger version (29K): In this window In a new window Download PPT slide   Figure 5. Genetic and physical map of the Mla region. This comparison of physical to genetic distance in the Mla region was obtained by the use of common probes/primers on our high-resolution mapping population in addition to the overlapping Franka YACs and Morex BACs. Franka YACs are designated by a "Fr" prefix and are shown in dark red, whereas Morex BACs are designated only by their library addresses and are shown in black. A vertical red rectangle designates a cloned end-sequence from which the primers in Table 6 were developed and subsequently used for genetic and/or physical mapping. An orange filled-in circle designates that YAC/BAC was amplified by the respective end-clone primer set or it hybridized to the amplified product. An X under the top horizontal line represent crossovers in the recombinant mapping population. When BAC ends were sequenced, horizontal arrowheads designate the T7 side of the vector. Distances are in centimorgans across the top horizontal line and YACs/BACs in the 1080-kb contig are drawn to scale in kilobases below.

Additional overlapping YACs are identified with primers developed from 236R:
For the first step in our chromosome walk to span the Mla cluster, primers were developed from the sequence of 236R and mwg2197 and used to identify the three overlapping Franka YACs, 98IIF5, 99IIE7, and 120ID1 (Table 4). YACs 98IIF5, 99IIE7, and 120ID1 were mapped physically via a partial digest strategy. This physical analysis indicated that YACs 98IIF5 and 99IIE7 contained identical DNA inserts but were cloned in opposite orientations. YAC 120ID1 is identical to YACs 98IIF5 and 99IIE7 except for an additional ~10 kb that begins ~14 kb from the mwg2197 end of this YAC. It is likely that these three YACs correspond to YAC2197 A, B, and C reported by SCHWARZ et al. 1999 , as they were isolated from the same library (KLEINE et al. 1997 ) with primers developed from mwg2197.

DNA gel blots of total YAC digests were hybridized with probes 236R, mwg2197, and mwg2083 to physically position them on the contig. It was established that 236R and mwg2083 both lie within the same ~7-kb subregion located ~12 kb from the left end of YAC 120ID1. Similarly, it was determined that mwg2197 lies on a ~7-kb subregion positioned ~14 kb from the right end of YAC 120ID1 (Figure 5). These restriction analyses indicated that the physical distance between 236R/mwg2083 and mwg2197 is ~120 kb. In preparation for sequencing, YAC 120ID1 was further fractionated to create a subgenomic pBeloBAC11 library. The identified Franka BACs IV16.11, I6.24, I3.2, and VI12.7 all hybridize to 236R and Franka BAC III12.9 hybridizes to mwg2197.

Overlapping Morex BACs are identified to extend the physical contig:
Unfortunately, no additional YACs could be identified that allowed us to extend the Franka contig proximal to 236R. Therefore, for the next step in our chromosome walk, amplified products from 236R, 234L, mwg2083, and mwg2197, were used as hybridization probes on high-density filters of a new 6.3-genome-equivalent BAC library from the barley cultivar Morex [Clemson University Genomic Institute (CUGI)]. The Mla-cosegregating markers 236R and mwg2083 each hybridized to three classes of Morex BACs. These classes most likely originated from different regions of the genome and were designated class I (typified by 80H14, shown in Table 5), class II (typified by 192H7, not shown), and class III (not typified by 80H14 or 192H7, not shown). To determine which class of Morex BACs overlapped with the Franka YACs, a number of approaches were employed. First, representative members of the three classes of Morex BACs and the five YAC 120ID1-derived Franka BACs were digested with HindIII and EcoRI and the resulting DNAs were size fractionated via agarose-gel electrophoresis. Due to the sequence diversity between Morex and Franka, we were unable to visually determine the overlap via comigrating EcoRI and HindIII restriction fragments. When a class I or class II Morex BAC was used as a hybridization probe, it appeared that class I Morex BACs were more related to the Franka BACs. However, the frequency of repetitive sequences in the barley genome complicated the interpretation. Hence, we employed a BAC AFLP fingerprinting strategy to identify small, comigrating, amplified DNA fragments. We reasoned that comigrating amplified fragments would be sequence-related and would facilitate the identification of the overlapping region between the Franka and Morex BACs. Indeed, four comigrating AFLPs of 275, 281, 595, and 693 bp were observed among class I (80H14-like) BACs and the YAC 120ID1-derived Franka BACs. Sequence analysis of these comigrating DNA fragments revealed that the class I Morex BACs were 97–98% identical to the respective sequences from the Franka BACs. Furthermore, when the low-copy 693-bp AFLP-derived fragment was used to hybridize the initial DNA gel blots described above, only class I Morex BACs and the YAC 120ID1-derived Franka BACs showed any detectable signal. In contrast, comigrating AFLPs were not detected between class II and class III from Morex and any of the YAC 120ID1-derived BACs. These results indicated that Morex class I BACs shown in Table 5, in fact, overlapped with the YAC 120ID1-derived Franka BACs.

Additional overlapping Morex BACs are identified that physically encompass the Mla cluster:
For the third step in our chromosome walk, a low-copy probe developed from the 80H14-R1 end was used to identify 12 additional BACs from the Morex library. These 12 BACs all overlapped physically due to the existence of a second copy (80H14-R1.2; see encircled markers in Figure 5) ~50 kb proximal to the actual R1 end of 80H14. The MluI fingerprint of these BACs shown in Figure 6 illustrates the overlapping pattern and extension of the Mla-spanning contig.

View larger version (64K): In this window In a new window Download PPT slide   Figure 6. MluI fingerprint analysis of BAC clones. The low-copy 236R YAC end was used to hybridize filters of the Morex BAC library. BAC 80H14 was identified as cosegregating with the Mla locus and a low-copy end probe developed from 80H14 was used to rescreen the library to isolate additional BAC clones. Products of digestion were resolved by PFGE using the CHEF Mapper XA system (Bio-Rad) in conjunction with a 14-cm gel.

BAC-end sequences were used to develop primers (shown in Table 6) for genetic mapping on our high-resolution population. If an amplification polymorphism was detected with the first-round primers between our C.I. 16151 and C.I. 16155 mapping parents, then these same primers were used for mapping on every recombinant between Xmwg036 and Xbcd249.1 (Figure 3). This approach was utilized for the Mla-cosegregating STS marker, 721K19-R1.1. However, if no polymorphisms were observed but the first-round primers could be used to amplify a product from both mapping parents, then the fragments were cloned and sequenced to develop allele-specific STS primers. Three additional polymorphic markers were developed by this method. As shown in Figure 5, STS marker 80H14-R1.1 cosegregated with Mla, STS marker 175D16-T7 was 0.25 cM proximal to Mla, and STS marker 206I20-T7 was 0.40 cM proximal to Mla.

Additionally, individual BACs were used as template for direct PCR amplification and the products were utilized in DNA gel blot hybridization to verify the overlapping pattern. All of the primers positioned between 80H14-R1.1 and 80H14-R1.2 (Figure 5) amplified an additional fragment from BAC 80H14. This result suggests that there is a large tandem duplication of 80H14 sequences on the overlapping BACs proximal to 80H14-R1.1.

Low-pass and BAC-end sequencing reveals eight RGHs within a 240-kb interval:
Four 96-well plates were used to sequence 384 random subclones derived from Morex BAC 80H14. These data were combined with 24 BAC-end sequences to expedite gene discovery in the Mla-spanning region. A total of 144,000 nucleotides from random 80H14 subclones and 13,200 nucleotides from BAC ends were utilized for computational analyses via BLASTx searches of the NCBI nonredundant (nr) database. This approach revealed eight near full-length sequences that possessed highly significant amino acid similarity to the NBS-LRR class of cloned plant-resistance genes (RONALD 1998 ). Seven of these sequences originated from the random sequencing of 80H14 and one was revealed by the T7-end sequencing of BAC 714K1.

Pairwise comparisons of these NBS-LRR RGHs were performed using BLASTn, BLASTp (ALTSCHUL et al. 1997 ), and the GAP comparison of GCG (Wisconsin Package for sequence analysis; Oxford Molecular, Madison, WI). Initially, comparisons were delimited to sequences between and including the P-loop and the "GLPLA" motif (BAKER et al. 1997 ). Pairwise BLASTp comparisons of the deduced amino acid sequences indicate that these RGHs fall into three families. The RGH1 family consists of five members, the RGH2 family consists of two members, and the RGH3 family has one member. Intrafamily GCG-GAP comparisons revealed 80–98% deduced amino acid similarity between members of RGH families 1 and 2, whereas interfamily comparisons showed only 46–51% amino acid similarity.

We also compared the near full-length nucleotide sequences of the NBS-LRR-like RGHs. Interfamily BLASTn comparisons between members of RGH families 1, 2, and 3 revealed no significant similarity. However, the five members within the RGH1 family are at least 60–98% similar and the two members within the RGH2 family are 97% similar. Pairwise BLASTp comparisons revealed that members within a family contain 60–98% amino acid similarity, whereas pairwise interfamily comparisons among members of RGH families 1, 2, and 3 revealed 33% amino acid similarity or less. Pairwise intrafamily GCG-GAP comparisons revealed that members within a family were up to 87% similar at both the nucleic acid and amino acid level. Pairwise interfamily GCG-GAP similarity among members of RGH families 1, 2, and 3 was 47% or less at the nucleic acid level and 44% or less at the amino acid level. To the same extent, we did not observe interfamily cross-hybridization among the members of RGH1, RGH2, and RGH3 under high-stringency wash conditions (0.1% SDS, 0.1x SSPE at 65°).

Genetic mapping and physical organization of the RGH families:
Sequences corresponding to the RGHs from 80H14 were mapped back onto our high-resolution population. Allele-specific PCR primers and/or polymorphic-hybridization probes were developed for RGH1a, 1b, 1d, 1e, and 3a (Table 7). We were unable to obtain allele-specific primers for RGH1c and 2a due to the monomorphic feature of the respective products amplified from the C.I. 16151 and C.I. 16155 mapping parents. Intragenic DNA sequence similarities between the C.I. 16151- and C.I. 16155-derived amplified RGH fragments are >95%, whereas the intragenic DNA sequence similarities between C.I. 16151- or C.I. 16155-derived RGH amplicons and Morex are ~80%. However, each of the allele-specific RGHs was genetically positioned in the physical interval that cosegregated with Mla6, Mla14, Mla13, and Ml-Ru3. This demonstrates that most, if not all, Morex-derived RGHs are also present in lines that contain characterized Mla specificities and that these RGH copies map to syntenic positions.

We also used sequences representing these RGHs as hybridization probes on BAC DNA fingerprinting filters (Figure 7) to identify additional RGH members on the BAC contig and derive a model for the physical organization of the RGHs associated with the Mla cluster. Indeed, another three additional RGHs, each belonging to one of the three families, were discovered on the adjacent BACs proximal to 80H14. By combining these hybridization results with data from the low-pass and BAC-end sequencing, at least 11 RGHs were found in this region. These new RGHs fall into the three previously described families, which brings the total to 6 members in the RGH1 family, 3 members in the RGH2 family, and 2 members in the RGH3 family. Presently, 7 of these NBS-LRR-like RGHs cannot be separated from the Mla locus by recombination events. Due to the large duplication proximal to 80H14, we were unable to develop distinct polymorphic markers between 721K19-R1.1 and 175D16-T7 (Figure 5). Therefore, at this time, we cannot determine whether the proximal 4 RGHs are genetically within the Mla cosegregating interval. Figure 8 illustrates a model for the physical organization of the RGHs associated with the Mla cluster. The segment between 236R and 175D16-T7 contains all of the known RGHs, and therefore defines the physical limit of Mla-associated NBS-LRR gene families to 240 kb.

View larger version (51K): In this window In a new window Download PPT slide   Figure 7. DNA gel blot hybridization of RGH probes onto EcoRI-digested Morex BACs. All membranes were washed at high stringency (0.1x SSPE, 0.1% SDS for 30 min at 65°). Probe DNAs were amplified from the primer pairs listed in Table 7. (A–H) Probes representing RGH domains are shown at the bottom; at the top are the BAC clone designations. At the left of A and E are -HindIII DNA size standards. RGH member designations are indicated on the right side of A–H. (A) Probe 1a-LRR was amplified from primer pair 39F13 and 39B95 and corresponds to the LRR region of RGH1a. Four highly similar RGH1 members hybridized to this probe. (B) Probe 1a-P-loop was amplified from primer pair 39F236 and 39B318 and corresponds to the P-loop region of RGH1a. Four highly similar RGH1 members also hybridized to this probe. (C) Probe 1b-P-loop was amplified from primer pair 15B02F1 and 15B02B9 and corresponds to the P-loop region of RGH1b. The hybridization pattern revealed two highly similar members of the RGH1 family. (D) Probe 1d-middle was amplified from primer pair 15G06F34 and 15G06B21 and corresponds to the LRR region of RGH1d. The hybridization pattern indicates that there are five highly similar members of the RGH1 family. (E) Probe 1e-LRR was generated from primer pair 38F19 and 38B27 and corresponds to the LRR region of RGH1e. Four copies of the RGH1 family hybridized to this probe. (F) Probe 2a-middle was amplified from primer pair 38IF2 and 38IB4 and corresponds to the region between the P-loop and LRR of RGH2a. The hybridization result showed the existence of three highly similar members of the RGH2 family. (G) Probe 2a-5' was amplified from primer pair 15A08F2 and 15A08B3 and corresponds to the 5' end of the P-loop region of RGH2a. Three highly similar copies hybridized to this probe. (H) Probe 3a-LRR was generated from primer pair 80H14R1F30 and 80H14R135 and corresponds to the LRR region of RGH2b. The hybridization pattern indicated that there are two copies of this region.

View larger version (15K): In this window In a new window Download PPT slide   Figure 8. Physical model illustrating the minimum-tiling path of NBS-LRR resistance-gene homologues on BACs in the cosegregating Mla interval. This model was derived by hybridizing probes derived from the BAC-end primers listed in Table 6, the RGH primers listed in Table 7, and RFLP markers shown in Figure 5, to DNA-gel blots containing the EcoRI-digested BACs shown in Figure 7. There are six distinct copies of the RGH1 family dispersed over ~150 kb, three copies of the RGH2 family covering ~100 kb, and two copies of the RGH3 family also covering ~100 kb. The two markers, 236R and 721K19-R1.1, define the current Mla cosegregating interval (Figure 5). However, the segment between 236R and 175D16-T7 contains all of the known RGHs and therefore defines the physical limit of Mla-associated NBS-LRR gene families to 240 kb.

The use of the fingerprinting restriction endonuclease EcoRI simplified the interpretation of the physical organization of the RGH family members. During initial library construction, BAC inserts were ligated into the HindIII cloning site of pBeloBAC11. Restriction digestion of these BACs with the enzyme EcoRI releases asymmetric BAC-end fragments. Hence, migration of an EcoRI BAC-end fragment will be distinguishable from a BAC-internal fragment. This was advantageous because, due to the duplicated segments in the Mla region, additional members of a gene family could be exposed upon hybridization with various RGH domain probes. For example, in Figure 7A and Figure D, a RGH1e-comigrating fragment corresponding to RGH1f in BACs 711N16, 721K19, and 257G8 is revealed by the altered migration of the BAC end from 175D16. In F and G, the second member of the RGH2 family is revealed by BAC 714K1-end sequencing and is also shown by the altered migration of the end via the EcoRI digest of this BAC. Furthermore, a third RGH2 member is shown by hybridization of the non-714K1 overlapping BAC, 175D16. These additional copies were indistinguishable when DNA gel blots of HindIII-restricted BACs were probed with the RGH probes in Figure 7.

Suppressed recombination within the Mla cluster:
Generally, the ratio of physical to genetic distance is low in regions near the centromere and high in regions toward the telomere (SCHNABLE et al. 1998 ). The average relationship between genetic and physical distance in barley, based on a genome size of 5300 Mb and a genetic map of 1250–1453 cM, is 4.2–3.7 Mb/cM (GRANER et al. 1991 ; KLEINHOFS et al. 1993 ). On the basis of cytogenetic analysis, PEDERSEN and LINDE-LAURSEN 1995 and SOROKIN et al. 1994 reported a ratio of 1.0 and 2.0 Mb/cM, respectively, in the short arm region of barley chromosome 5 (1H).

As shown in Table 8, we compared physical to genetic distance ratios in eight intervals cosegregating with and adjacent to the Mla cluster. It appears that intervals closer to the Mla cluster undergo less recombination, at least in progeny of the C.I. 161561 x C.I. 16155 mapping cross. We observed no recombinants in the 236R to 721K19-R1.1 interval, which contains nearly all the NBS-LRR resistance-gene homologues. This lack of recombination delimits the physical to genetic distance ratio to 5 Mb/cM. However, regions immediately flanking the Mla cluster appeared to recombine at a higher rate than the average for the barley genome and the short arm of chromosome 5 (1H).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To determine the molecular processes that mediate host resistance, our aim is to isolate a number of resistance specificities of the Mla locus. In this article, we describe the identification of several tightly linked DNA markers and the establishment of a Mla-spanning YAC and BAC contig. This Mla-spanning contig has facilitated the discovery of 11 NBS-LRR resistance-gene homologues, at least 7 of which cosegregate with the Mla locus.

Highly dissimilar NBS-LRR resistance-gene-like families are physically associated with the Mla cluster:
In the past several years, major long-term efforts have reached fruition in the cloning of resistance genes in a variety of plant species (reviewed by MICHELMORE and MEYERS 1998 ; RONALD 1998 ). Although the isolated genes confer resistance to a diverse range of pathogens, those involved in gene-for-gene interactions between host and pathogen share various conserved motifs. These include a serine-threonine protein-kinase domain, a leucine zipper (LZ), a Toll and interleukin-like receptor domain (TIR), NBS, and LRRs. The most prevalent class of cloned plant-resistance genes contains the nucleotide-binding site combined with various lengths of a leucine-rich repeat. This NBS-LRR class is predicted to encode intracellular proteins (MICHELMORE and MEYERS 1998 ; RONALD 1998 ). The 11 RGHs that are physically present on the Morex BAC contig belong to three distinct families of the NBS-LRR class of resistance genes. Of these RGHs, 7 have been genetically delineated to the region that contains the Mla6, Mla14, Mla13, and Ml-Ru3 specificities. Previous reports have shown that NBS-LRR genes can be physically juxtaposed to genes defining additional components of the resistance response. Prf, a NBS-LRR gene, is located adjacent to Pto, encoding a serine-threonine kinase, both of which define essential components of race-specific resistance to bacterial speck disease in tomato (SALMERON et al. 1996 ). Among >150 kb of DNA sequence surveyed, no sequences exhibiting similarities to kinases were identified in the contig spanning Mla. Thus, the present data suggest that the Mla locus contains only NBS-LRR-type RGHs.

The majority of plant resistance genes appear to be organized as complex clusters. For example, the Xa21 resistance gene family of rice and the Cf-2 family of tomato are assembled as single, locally restricted clusters of homologous genes (SONG et al. 1995 , SONG et al. 1997 ; DIXON et al. 1996 , DIXON et al. 1998 ). The Dm3 locus of lettuce and the Cf4/Cf9 locus of tomato define two examples in which numerous related copies of resistance gene homologues are spread over several megabases within one chromosome (ANDERSON et al. 1996 ; PARNISKE et al. 1997 ; MEYERS et al. 1998A , MEYERS et al. 1998B ; SHEN et al. 1998 ). Finally, the related L and M genes of flax are located on different chromosomes (LAWRENCE et al. 1995 ; ANDERSON et al. 1997 ). In contrast, we have observed at Mla an interspersed arrangement of three unrelated NBS-LRR-like gene families (Figure 8). Additionally, these three Mla-cosegregating RGH families do not have significant similarity to the barley Hv-b6 RGH family, positioned 0.48–0.62 cM distal to the Mla6, Mla13, Mla14, and Ml-Ru3 specificities (Figure 5). This multifamily organization of resistance genes and resistance-gene homologues is comparable to the recent report of mixed clusters of NBS-LRR RGHs of rice, each harboring at least two highly dissimilar NBS-LRR genes (LEISTER et al. 1999 ).

RGH families and Mla resistance specificities:
The physical organization of the NBS-LRR-like sequences associated with the Mla locus was obtained from cultivar Morex, a Manchuria-type barley (KLEINHOFS et al. 1993 ). The cultivar Manchuria does not have any known Mla specificity and Morex also does not confer resistance to our isolates used for mapping the Mla6, Mla13, Mla14, and Ml-Ru3 specificities. However, the cosegregating feature of the three RGH families within the genetically delimited (Mla6, Mla14, Mla13, and Ml-Ru3) interval indicates that they may be homologues of individual Mla resistance specificities. Indeed, it has been shown that susceptible cultivars or subspecies do harbor homologues of resistance genes at syntenic positions. The Cf0 locus in susceptible Lycopersicon esculentum contains a homologue of the Cf9 resistance gene that was introgressed from L. pimpinellifolium (PARNISKE et al. 1997 ). Likewise, a homologue of the Xa1 resistance gene in the resistant cultivar IR-BB1 is present at the same locus in the susceptible near-isogenic line IR24 (YOSHIMURA et al. 1998 ). Because Mla6 and Mla14 were introgressed from the wild barley H. spontaneum nigr. (reviewed by JORGENSEN 1994 ), it is conceivable that the RGH families at Mla, derived from the susceptible cultivar Morex, represent homologues of individual Mla resistance specificities.

There are, however, also cases in which susceptible lines lack homologues of resistance genes. For example, the Xa21 bacterial-blight resistance locus that was introgressed from wild rice, Oryza longistaminata, does not exist in cultivated rice, O. sativa (SONG et al. 1995 , SONG et al. 1997 ). Similarly, RPM1 in Arabidopsis is present in ecotype Columbia but absent in at least six other naturally occurring accessions (GRANT et al. 1995 ). This does not appear to be the case for the Mla cluster. As described above, we have been able to amplify homologous sequences corresponding to several of the RGHs from C.I. 16151 (containing Mla6 + Mla14) and C.I. 16155 (containing Mla13 + Ml-Ru3) with the Morex-derived RGH primers described in Table 6. Additionally, these homologs genetically cosegregate with the Mla6, Mla13, Mla14, and Ml-Ru3 specificities in our high-resolution mapping population. Taken together, these data provide the possibility that the Morex-derived RGH families represent homologues of single Mla resistance specificities.

Mutational studies uncovered two genes, Rar1 and Rar2, required for Mla-specified resistance responses (TORP and JØRGENSEN 1986; JORGENSEN 1988 , JORGENSEN 1996 ; FREIALDENHOVEN et al. 1994 ). Rar1, located on barley chromosome 2, has been recently isolated and encodes a novel protein that is likely to function in disease resistance signaling (LAHAYE et al. 1998 ; SHIRASU et al. 1999). Rar1 and Rar2 are required for the function of some but not all tested Mla specificities (JORGENSEN 1988 , JORGENSEN 1996 ). Our finding of three unrelated RGH families at Mla could provide a simple explanation for the differential Rar-gene requirements if some of the Mla specificities are encoded by one RGH family and another set are encoded by a different RGH family. In this scenario, distinct NBS-LRR families would have the capacity to activate downstream signaling components. The availability of altered-specificity mutants for Mla1 (S. SOMERVILLE, unpublished results), Mla6 (R. P. WISE, unpublished results), and Mla12 (TORP and JØRGENSEN 1986), each exhibiting differential requirements for Rar1 and Rar2, is expected to facilitate the identification of individual Mla resistance specificities and to provide a molecular basis to test our hypotheses.

Recombination is suppressed in highly polymorphic regions of the genome:
The relationship between physical and genetic distance varies throughout the eukaryotic genome. This variation depends on many factors, including the composition of surrounding DNA sequences (GUSTAFSON et al. 1990 ; LEITCH et al. 1991 ; SCHWARZACHER and HESLOP-HARRISON 1991 ; WERNER et al. 1992 ; GILL et al. 1993 ; JIANG and GILL 1993 ; KOTA et al. 1993 ; LEITCH and HESLOP-HARRISON 1993 ; SCHMIDT et al. 1994 ; PEDERSEN and LINDE-LAURSEN 1995 ). As shown in Table 8, the ratio of physical to genetic distance varies >10-fold in intervals adjacent to and cosegregating with the Mla cluster. Indeed, recombination was not observed in the cosegregating physical interval that encompasses the Mla6, Mla14, Mla13, and Ml-Ru3 specificities and the three associated RGH families. This observation could be due to lack of pairing and subsequent strand exchange between homologous regions in the C.I. 16151 and C.I. 16155 parents of our mapping cross. These two accessions were originally chosen because of their high rate of hordein-polypeptide polymorphism and easily detectable differences in infection type (MAHADEVAPPA et al. 1994 ). However, it may be that suppression of recombination occurs within the Mla cluster because of this high rate of polymorphism. This recombination suppression contrasts with observations at the Rp1 rust-resistance cluster in maize (COLLINS et al. 1999 ), where high rates of recombination and unequal crossover have been shown to be a source of new resistance specificities (RICHTER et al. 1995 ).

The Mla6 allele was originally introgressed into cultivated barley from H. spontaneum, a wild relative of H. vulgare (JORGENSEN 1994 ). Suppressed recombination has been observed in other introgressed regions associated with disease resistance, such as the Mi (VAN DAELEN et al. 1993 ) and Tm2-a (GANAL et al. 1989 ) loci in tomato. There is a distinct difference in these two cases, however, as the Mi and Tm2-a loci are physically close to the centromere where regions of heterochromatin were postulated to suppress recombination in this area of the chromosome.

In summary, we have established a detailed physical map of the Mla-spanning region and presented the physical organization of different members of R-gene homologues within the contig. New Mla mutants should allow us to determine the location of different members of this resistance-gene family and ultimately define specific regions of the gene (and therefore, protein) that are important in host-pathogen recognition. Determination of the sequence differences among mutant alleles will provide important clues in our long-range goal to understand the evolution and molecular mechanisms of host-pathogen interaction among members of the Gramineae and obligate biotrophs.

	ACKNOWLEDGMENTS

The authors thank Dr. Thomas Baum for critical review of the manuscript. This research was supported in part by United States Department of Agriculture (USDA)-National Research Initiative Competitive Grants Program grant 98-35300-6170 and facilitated by the North American Barley Genome Mapping Project. D.L. was supported by a long-term European Molecular Biology Organization postdoctoral fellowship and J.K. was supported by a European Community biotechnology research grant. Research in the P.S.-L. lab is supported by a Gatsby foundation grant. Joint contribution was from the Corn Insects & Crop Genetics Research Unit, USDA-Agricultural Research Service, and the Iowa Agriculture and Home Economics Experiment Station. This is journal paper no. J-18538 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa, project no. 3368, and was supported by Hatch Act and State of Iowa funds.

Manuscript received July 19, 1999; Accepted for publication September 10, 1999.

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				R. Brueggeman, N. Rostoks, D. Kudrna, A. Kilian, F. Han, J. Chen, A. Druka, B. Steffenson, and A. Kleinhofs The barley stem rust-resistance gene Rpg1 is a novel disease-resistance gene with homology to receptor kinases PNAS, July 9, 2002; 99(14): 9328 - 9333. [Abstract] [Full Text] [PDF]

				D. Sandhu and K. S. Gill Gene-Containing Regions of Wheat and the Other Grass Genomes Plant Physiology, March 1, 2002; 128(3): 803 - 811. [Abstract] [Full Text] [PDF]

				A. Demirbas, B. G. Rector, D. G. Lohnes, R. J. Fioritto, G. L. Graef, P. B. Cregan, R. C. Shoemaker, and J. E. Specht Simple Sequence Repeat Markers Linked to the Soybean Rps Genes for Phytophthora Resistance Crop Sci., July 1, 2001; 41(4): 1220 - 1227. [Abstract] [Full Text] [PDF]

				D. Sandhu, J. A. Champoux, S. N. Bondareva, and K. S. Gill Identification and Physical Localization of Useful Genes and Markers to a Major Gene-Rich Region on Wheat Group 1S Chromosomes Genetics, April 1, 2001; 157(4): 1735 - 1747. [Abstract] [Full Text]

				J. Dubcovsky, W. Ramakrishna, P. J. SanMiguel, C. S. Busso, L. Yan, B. A. Shiloff, and J. L. Bennetzen Comparative Sequence Analysis of Colinear Barley and Rice Bacterial Artificial Chromosomes Plant Physiology, March 1, 2001; 125(3): 1342 - 1353. [Abstract] [Full Text]

				D. B. Chin, R. Arroyo-Garcia, O. E. Ochoa, R. V. Kesseli, D. O. Lavelle, and R. W. Michelmore Recombination and Spontaneous Mutation at the Major Cluster of Resistance Genes in Lettuce (Lactuca sativa) Genetics, February 1, 2001; 157(2): 831 - 849. [Abstract] [Full Text]

				F. Zhou, J. Kurth, F. Wei, C. Elliott, G. Valè, N. Yahiaoui, B. Keller, S. Somerville, R. Wise, and P. Schulze-Lefert Cell-Autonomous Expression of Barley Mla1 Confers Race-Specific Resistance to the Powdery Mildew Fungus via a Rar1-Independent Signaling Pathway PLANT CELL, February 1, 2001; 13(2): 337 - 350. [Abstract] [Full Text]

				N. Stein, C. Feuillet, T. Wicker, E. Schlagenhauf, and B. Keller Subgenome chromosome walking in wheat: A 450-kb physical contig in Triticum monococcum L. spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.) PNAS, November 8, 2000; (2000) 230361597. [Abstract] [Full Text]

				K. Shirasu, A. H. Schulman, T. Lahaye, and P. Schulze-Lefert A Contiguous 66-kb Barley DNA Sequence Provides Evidence for Reversible Genome Expansion Genome Res., July 1, 2000; 10(7): 908 - 915. [Abstract] [Full Text]

				K. M. Devos and M. D. Gale Genome Relationships: The Grass Model in Current Research PLANT CELL, May 1, 2000; 12(5): 637 - 646. [Abstract] [Full Text]

				N. Stein, C. Feuillet, T. Wicker, E. Schlagenhauf, and B. Keller Subgenome chromosome walking in wheat: A 450-kb physical contig in Triticum monococcum L. spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.) PNAS, November 21, 2000; 97(24): 13436 - 13441. [Abstract] [Full Text] [PDF]