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Crop Science 43:1484-1492 (2003)
© 2003 Crop Science Society of America

CELL BIOLOGY & MOLECULAR GENETICS

Development and Mapping of Markers Linked to the Rice Bacterial Blight Resistance Gene Xa7

B. W. Portera, J. M. Chittoorb, M. Yanoc, T. Sasakic and F. F. White*,a

a Dep. of Plant Pathology, Kansas State Univ., Manhattan, Kansas 66506
b Monsanto Company, Life Science Informatics, Mail Code: CV-BRG, 700 Chesterfield Parkway North, St. Louis, Missouri 63198
c National Institute of Agrobiological Resources 1-2, Kannondai 2-chome, Tsukuba, Ibaraki 305-8602, Japan

* Corresponding author (fwhite{at}ksu.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Markers were generated that are linked to the rice bacterial blight resistance gene Xa7. Amplified restriction fragment length polymorphism (AFLP) analysis of a segregating, near-isogenic F3 population of IR24 x IRBB7 revealed one polymorphic fragment, M1, which was mapped to position 107.3 centimorgans (cM) on the Rice Genome Research Program (RGP) map. Sequence comparisons of resistant and susceptible lines near M1 were used to develop additional markers. A sequence tagged site (STS) named M2 was mapped proximal to M1 and farther from Xa7, indicating that Xa7 lies distal to M1. On the distal side of M1, two simple sequence repeats (SSRs), M3 and M4, were mapped 0.5 and 1.8 cM, respectively, from Xa7. The pattern of recombinants was consistent with the order of M1–Xa7–M3–M4, and the map distances indicated that Xa7 is located in the region corresponding to the ends of the physically mapped Clemson University Genomics Institute (CUGI) bacterial artificial chromosome (BAC) contigs 96 and 143. A complex repeat was identified in the DNA sequence from rice (Oryza sativa L.) cultivars 93-11 and Nipponbare that matched the end of contig 96 and a previously mapped expressed sequence tag (EST) marker (C52865S). Amplification of the repeat and flanking sequences revealed the presence and absence of the repeat in IR24 and IRBB7, respectively. No recombinants were identified between Xa7 and the polymorphic repeat, which was named M5, in 277 F3 susceptible progeny of the IR24 x IRBB7 cross. Comparison of the physical and genetic maps of rice in this region indicates that Xa7 could lie within 40 kilobases (kb) of M5, a distance suitable for gene pyramiding efforts and Xa7 cloning strategies.

Abbreviations: AFLP, amplified restriction fragment length polymorphism • BAC, bacterial artificial chromosome • bp, base pairs • CUGI, Clemson University Genomics Institute • EST, expressed sequence tag • kb, kilobases • LOD, logarithm of odds • NBS, nucleotide binding sites • NLS, nuclear localization signal • LRR, leucine rich repeats • PCR, polymerase chain reaction • RFLP, restriction fragment length polymorphism • RGP, Rice Genome Research Program • SSR, simple sequence repeat • STS, sequence tagged site • Xoo, Xanthomonas oryzae pv. oryzae


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OVER THIRTY PLANT RESISTANCE GENES have been cloned from 11 different host plants (Hulbert et al., 2001), and a variety of resistance genes (R-genes) have been cloned from rice. Xa21, the first R-gene cloned from rice, confers resistance to a broad range of strains of Xanthomonas oryzae pv. oryzae (Ishiyama) Swings et al. [= X. campestris pv. oryzae (Ishiyama) Dye] (Xoo) (Song et al., 1995). Xa21 was isolated by map-based cloning and found to be a member of a complex locus located on chromosome 11 (Song et al., 1995). The predicted structure of Xa21 indicates that the protein has a cytoplasmic domain containing a serine–threonine kinase, a transmembrane domain, and an extracellular domain with leucine rich repeats (LRRs) (Song et al., 1995). Xa1, the second Xanthomonas R-gene to be positionally cloned from rice, is located on chromosome 4 and confers resistance to bacteria containing avrXa1 (Yoshimura et al., 1998). Unlike Xa21, Xa1 is a single copy gene and pathogen and wound inducible. Xa1 is predicted to code for a cytoplasmic protein with nucleotide binding sites (NBS) and a LRR domain (Yoshimura et al., 1998) and, therefore, a member of the large NBS–LRR class of resistance genes (Hulbert et al., 2001). The two most recent R-genes cloned from rice, Pib and Pi-ta, confer resistance to the fungal pathogen Magnaporthe grisea (Hebert) Barr and also contain NBS–LRR motifs (Wang et al., 1999; Bryan et al., 2000).

Xa7 is a dominant resistance gene directed against Xoo and was originally identified in rice cultivar DV85, International Rice Research Institute accession number 8839 (Sidhu et al., 1978). The gene was transferred to cultivar IR24, and the near-isogenic line IRBB7, with Xa7, was created by recurrent backcrossing (Ogawa et al., 1991). Xa7 is an example of a R-gene that is directed against an avirulence gene family (Hopkins et al., 1992) and is a potential source of broad resistance to bacterial blight of rice (Vera Cruz et al., 2000). The corresponding avirulence gene to Xa7, avrXa7, has been cloned and is a member of the avrBs3 gene family (Bonas et al., 1989; Hopkins et al., 1992; Leach and White, 1996, White et al., 2000). The products of this family are distinguished by the presence of a repetitive domain in the middle one-third of the protein (Bonas et al., 1989). The various members have distinct repetitive domains with regard to the number and specific sequence variation, and the repetitive domain is responsible for the specificity for the corresponding R-gene (Herbers et al., 1992; Yang et al., 1994; Yang and Gabriel 1995; Zhu et al., 1998). The repetitive coding domain of avrXa7 has 25.5 direct repeats (Yang et al., 2000).

AvrXa7, as well as the products of several other related avirulence genes, has several other peculiarities. The protein requires a functional nuclear localization signal (NLS) and an acidic transcriptional activation domain motif for avirulence activity, indicating that the interaction with Xa7 might occur within the host nuclei (Yang et al., 2000). The isolation and characterization of Xa7, therefore, represents an important step toward the evaluation of the models for the interaction of an avrBs3 family member and the corresponding R-gene or gene product.

AvrXa7 mediates two different responses in the host. In plants harboring Xa7, the protein elicits a resistance response. In the absence of Xa7, AvrXa7 is a virulence factor contributing to the ability of Xoo to multiply and spread in the host (Bai et al., 2000). Deploying an R-gene that is directed against a virulence factor of the pathogen may provide more durable resistance against the pathogen, if circumventing the resistance response requires the loss of the virulence factor activity. Field sampling experiments indicate that populations of bacterial strains containing avrXa7, which are presently the predominant strains in the Philippines, are unable to adapt readily to the deployment of Xa7 without concomitant loss of virulence (Vera Cruz et al., 2000). Therefore, mapping and characterization of Xa7 should provide insight into strategies to extend the life of R-genes in the field as well as identify markers for use in breeding programs.

Previous studies placed Xa7 on chromosome 6. Kaji and Ogawa (1995) determined a recombination value of 8.8% between Xa7 and G1091, which is located at 107.5 cM on the current RGP rice map (Kaji and Ogawa, 1995). Subsequently, Yoshimura et al. (1996) reported three additional probes, linked to Xa7, that provide restriction fragment length polymorphisms (RFLPs) at positions 107.3, 103, and 90.5 cM. In this study, we used AFLP, SSR, and STS analysis to identify additional markers linked to Xa7. These markers provide a significant contribution toward cloning and molecular characterization of Xa7.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Media
Xoo strain PXO99A(pZWavrXa7-F2H) was grown overnight at 28°C in plates containing TSA (tryptone sucrose agar) with 100 µg/mL spectinomycin. The bacterial lawn was suspended in sterile, distilled water at an optical density of 1.0 (OD600), determined with a DU 640B spectrophotometer (Beckman Coulter, Inc., Fullerton, CA) and immediately used for plant inoculations.

Mapping Populations and Plant Inoculations
Initial mapping of marker M1 to chromosome 6 was based on recombination values for 120 F2 plants from a Nippon-bare x Kasalath population as previously described by Harushima et al. (1998). Further mapping of marker M1 and markers M2, M3, M4, and M5 was conducted with a IR24 x IRBB7 population. IRBB7 (Ogawa et al., 1991), a near-isogenic line carrying Xa7 (Xa7/Xa7) in an IR24 genetic background (BC4F7), was used to pollinate IR24 (xa7/xa7). F3 seeds were harvested from 118 F2 progeny, and 20 F3 seedlings from each of the 118 F2 individuals were grown in growth chambers (12-h photoperiod, 28°C and 80% relative humidity). The second leaf of each seedling was inoculated at 12 d with a needleless syringe (Reimers and Leach, 1991). After 24 hours, plants that developed the brown, necrotic lesions associated with the hypersensitive response were scored as resistant. Plants that developed yellow, water-soaked lesions in 3 d were scored as susceptible. The scores for the F3 seedlings were used to identify the original F2 individuals as homozygous resistant, heterozygous resistant or homozygous susceptible. Chi-square analysis was used to test the significance of a single gene, 3:1, segregation ratio. Because plants that are heterozygous for Xa7 (Xa7/xa7) exhibit the same phenotype as homozygous resistant plants, only susceptible F3 plants from heterozygous families were used for recombination evaluation.

Plant DNA Extraction, Southern Blotting, and Recombinant DNA Techniques
Plant DNA was isolated according to Fulton et al. (1995) for PCR. Large scale DNA isolations were performed by the CTAB (cetyltrimethylammonium bromide) method (Murray and Thompson 1980). Southern blotting was performed as described previously (Kurata et al., 1994; Nagamura et al., 1995). PCR products were cloned with a TOPO TA Cloning Kit with pCR 2.1-TOPO vector (Invitrogen Life Technologies Corporation, Carlsbad, CA).

AFLP, STS, and SSR Analysis
AFLP analysis was conducted as described previously (Vos et al., 1995) with AFLP Analysis System II and AFLP Small Genome Primer Kit (Invitrogen Life Technologies Corporation, Carlsbad, CA). Two pooled template DNAs, a resistant pool and a susceptible pool, were used to conduct the screen of 64 primer combinations. Twelve DNA isolations of six homozygous resistant F3 families (20, 14-d-old plants per family) and six homozygous susceptible F3 families (twenty, 14-d-old plants per family) were used for pooling. The pools were created by combining 100 µL (0.5µg/µL) from each of the six large scale genomic DNA preparations. Adjustments were made to the AFLP Analysis System II protocol to accommodate the need to evaluate two template DNAs with multiple primer combinations. A 1:10 dilution of the stock EcoRI primers (27.8 ng/µL) was used for each 33P labeling reaction consisting of 1.8 µL of a 1:10 dilution of the stock EcoRI primer, 1 µL distilled water, 1 µL 5x kinase buffer, 0.5 µL [{gamma}-33P]ATP (PerkinElmer/New England Nuclear Life Sciences, Boston, MA) and 0.5 µL T4 kinase. After labeling, the following were added to each reaction: 4.5 µL of MseI primer (6.7 ng/µL, dNTPs), 5 µL template DNA (1:50 dilution from preamplification reaction), and 10 µL of a mix containing 158 µL distilled water, 40 µL 10x PCR buffer plus Mg, and 3 µL Taq polymerase (5u/µL). All reactions were fractionated on 6% (w/v) acrylamide gels with Long Ranger gel solution (FMC BioProducts, Rockland, ME). Microtube Tough-Tags (Diversified Biotech, Boston, MA) painted with Glo-It paint (DecoArt, Stanford, KY) were placed around the perimeter of the dried gel to orient the autoradiographs for fragment excision. Excised fragments were placed in 30 µL of distilled water and ground with a pipette tip. Five microliters of this solution was used as template DNA for reamplification. Five clones were sequenced for each potential polymorphic DNA fragment. PCR primers were designed on the basis of the candidate sequences or by extending the original EcoRI and MseI primers with A, T, G, and C nucleotides. STS markers were designed from sequence provided by the RGP High-Density Rice Genetic Map or other public rice genome databases. Primers were ordered from Invitrogen Life Technologies Corporation. The sequence of primers used for recombination analysis are provided in Table 1.


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  		Table 1. Primers used for markers M1 through M5.

 
Linkage Analysis
Linkage analysis was conducted with MAPMAKER/EXP (version 3.0b) with a threshold LOD score of 3.0 (Lander et al., 1987). Map distances (cM) are reported in Kosambi units (Kosambi 1944).


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Xa7 Segregation Analysis
Our approach to mapping Xa7 involved calculating genetic distance using recombination analysis of polymorphic sites linked to Xa7. This analysis was conducted with susceptible F3 individuals from the resistant and susceptible parents IRBB7 and IR24, respectively. Because plants that are heterozygous for Xa7 (Xa7/xa7) exhibit the same phenotype as homozygous resistant plants, only susceptible plants from segregating families were used for recombination evaluation. To obtain relatively large numbers of susceptible individuals, the F3 progeny from heterozygous F2 individuals were analyzed. The F2 progeny were derived from a cross of IR24 (susceptible) and IRBB7 (Xa7), and their genotypes, with respect to Xa7, were established by segregation analysis for Xa7 in F3 progeny. Twenty F3 progeny from each of the 118 F2 families were scored for resistance to bacterial blight with a strain of Xoo carrying avrXa7. The ratio of homozygous resistant:heterozygous resistant:homozygous susceptible families was 30:62:26, which is consistent with a 1:2:1, resistant:susceptible, Mendelian ratio for a single dominant gene (Chi square test, P > 0.95).

AFLP analysis was used to locate markers closely linked to Xa7 by identifying polymorphic amplification products between the near-isogenic resistant (IRBB7) and susceptible (IR24) parents. To reduce interference due to unlinked and loosely linked introgressed DNA, preliminary AFLP analysis was performed with pooled DNA from six homozygous resistant and six homozygous susceptible F3 families (20, 14-d-old plants per family). Evaluation of 64 primer combinations with AFLP analysis revealed candidate marker fragments. The fragment from the resistant pool was 129 base pairs (bp) in length (including adaptor sequence), and the smaller fragment, from the susceptible pool, was 127 bp (Fig. 1A). The primer combination producing this polymorphism was named AFLP31-10 with selective base pairs AC from the EcoRI primer (5'-GACTGCGTACCAATTCAC-3') and selective base pairs CTA from the MseI primer (5'-ATGAGTCCTGAGTAACTA-3'). Because the primers generate additional, nonpolymorphic fragments, reiterative PCR, with the addition of base pair extensions, was used to design primers that are specific for the polymorphic fragments. Single base pair extensions, at the 3' end, resulted in the identification of primers EcoRI 5'-GACTGCGTACCAATTCACG-3' and MseI 5'-ATGAGTCCTGAGTAACTAT-3' which reduced the complexity of the AFLP products to single polymorphic fragments (Fig. 1B and 1C, respectively).



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  		Fig. 1. AFLP analyses of pooled resistant (R) and susceptible (S) F3 families from a IR24 x IRBB7 cross. (A) Candidate marker AFLP31-10 using original adaptor–primers (see text). (B) The products of four AFLP31-10 EcoRI primers, each with unique one base extensions, and the original MseI primer are shown in lanes C, G, T, and A, respectively. Polymorphic fragments in lane G indicate that the next base on the EcoRI adaptor side, in the polymorphic fragments, is G. The products of the original AFLP31-10 primers are at left (control). (C) The products of four AFLP31-10 MseI primers with unique one base extensions and the original EcoRI. Polymorphic fragments in lane T indicate the next nucleotide on the MseI adaptor side is T. Products of original AFLP31-10 primers are at left (control). Amplified products were fractionated on 6% (w/v) polyacrylamide gels.

 
The DNA sequence from the single polymorphic fragments from susceptible and resistant genotypes was determined and used to design a set of primers named M1 (Table 1) for the amplification of polymorphic fragments directly from plant DNA. The M1 primers amplified fragments with sizes of 107 bp for resistant plants and 105 bp for susceptible plants, and, in addition, larger polymorphic fragments of 313 bp for resistant plants and 306 bp for susceptible plants (Fig. 2A). The 313 and 306 bp fragments of the resistant and susceptible individuals, respectively, contained a duplicate sequence with a second EcoRI site and forward primer site (Fig. 2B). The absence of the 313/306-bp fragments in initial AFLP analysis resulted from the AFLP technique. Restriction digestion of the genomic DNA, in preparation for AFLP analysis, cleaved both EcoRI sites in the duplicated sequence, and adapters were ligated to both ends. However, only EcoRI/MseI fragments are amplified in the AFLP analysis. Hence, fragments of 129 and 127 bp that correspond to the 107- and 105-bp EcoRI/MseI fragments of the resistant and susceptible genotypes, respectively, plus the 22-bp AFLP adaptor sequences are amplified. The first 207 and 202 bp of the resistant and susceptible genotypes are removed by digestion with EcoRI, and failed to amplify with the EcoRI primers alone because the second EcoRI site lacks the correct selective base pairs.



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  		Fig. 2. AFLP analysis and sequence comparison of IRBB7 (R) and IR24 (S) at marker M1. (A) Products of six susceptible (S) and six resistant (R) F3 individuals amplified with primer combination M1 were loaded in alternate lanes and fractionated on a 6% (w/v) acrylamide gel. (B) Sequence of M1 from IRBB7 (R) and IR24 (S). The forward and reverse primer sequences are underlined.

 
An F2 Nipponbare x Kasalath population of 120 individuals was used to map M1 by RFLP analysis. Polymorphic BamHI fragments of approximately 4 kb in Nipponbare and 6 kb in Kasalath are produced when the 313-bp fragment from IRBB7 is used as an RFLP probe (Fig. 3). The fragments cosegregated with fragments corresponding to RGP cDNA clone G1091, which is linked to Xa7 and maps at position 107.3 cM (data not shown).



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  		Fig. 3. Southern blot analysis of rice DNA probed with M1 (IRBB7). Genomic DNA from rice cultivars Nipponbare (N) and Kasalath (K) were digested with BamHI, fractionated on agarose gels, and blotted. Bacteriophage {lambda} DNA (S) was digested with HindIII and fractionated on the same gel, was used as a size standard.

 
Two hundred seventy-seven susceptible F3 progeny from heterozygous F2 individuals of the IR24 x IRBB7 cross were evaluated for recombination by AFLP analysis and scored on the basis of the upper fragments of 313/306 bp generated by M1 primers. Twelve recombinant plants, which contained the 313-bp fragment from the resistant parent, were identified. M1 was calculated to be approximately 2.2 cM from Xa7.

Identification of Markers Flanking Xa7
Primers that amplified polymorphic fragments from IR24 and IRBB7 were designed from the available rice genomic sequences of Nipponbare and used to determine the proximal or distal relationship of M1 to Xa7 in relation to the centromere (Table 1). Sequence approximately 295 kb from the proximal side of M1 and near EST E383S (105.3 cM) in sequence accession AP004744 was used to design primers for M2 (Table 1). M2 primers amplify fragments of 240 and 234 bp in IR24 and IRBB7, respectively (Fig. 4A), and an initial recombination analysis at M2 was conducted on 26 susceptible F3 progeny, including six of the recombinant F3 plants previously identified as recombinants at M1. All six individuals that were recombinant at M1 were also recombinant at M2, and two new recombinants for M2 were identified from the remaining 20 individuals, indicating that M2 is farther from Xa7 on the proximal side of M1. Markers in this direction were not pursued further.



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  		Fig. 4. Sequence comparison among lines at markers M2–M4. (A) Comparison of M2 from IRBB7 (R), IR24 (S), and Nipponbare (N). (B) Comparison of M3 from IRBB7 (R), IR24 (S), and Nipponbare (N). (C) Comparison of M4 from IRBB7 (R), IR24 (S), and Nipponbare (N). Only sequences corresponding to 480 to 814 bp of IRBB7 are shown for M4.

 
RGP EST marker C11635S (109.5 cM) lies distal to M1 and is located in RGP sequence accession AP003728. C11635S contained a polymorphic SSR sequence for IRBB7 and IR24 and was designated as M3 (Table 1). The 297-bp PCR product from resistant plants (IRBB7) contains 35 additional bases compared with the product from IR24 (Fig. 4B). The primer set for M4 was derived from another SSR on the distal side of M1, which is located in RGP accession AP003723 and approximately 210 kb distal from M3 (Table 1). M4 amplifies a fragment of 1311 bp from IRBB7 and 1197 bp from IR24 (Fig. 4C). Recombination analysis of the 277 F3 susceptible plants at M3 and M4 revealed three recombinant plants at M3, all of which were non-recombinant for M1. Ten recombinants were identified by means of M4. Three of the M4 recombinants were the same as those found by means of M3, and none were recombinant at M1. Recombination distances of 0.5 and 1.8 cM distal from Xa7 were calculated for M3 and M4, respectively.

Identification of a Marker Closely Linked to Xa7
The sequence analyses of the regions associated with M1 to M4 indicate that the genomes of japonica and indica subspecies of rice are, to a high degree, colinear in the region of Xa7. The mapping data indicated that Xa7 is located in a region spanning contigs 96 and 143 of the physically mapped BAC clones from Nipponbare (Chen et al., 2002). We attempted to develop additional markers from the region by identifying sequences in the databases of rice cultivars 93-11 (O. sativa subsp. indica) (Yu et al., 2002) and Nipponbare (Goff et al., 2002) from the extreme ends of either contig. The BAC clone OSJNBa0017K10 from Nipponbare occurs at the end of contig 96, and both ends of this BAC have been sequenced (Chen et al., 2002). The left (reverse) end is located within the proximal side of RGP sequence contig AP005192. The right (forward) end sequence matches sequence 11 within contig CL003847.142 of the Nipponbare database, which overlaps the indica database contig 2308 (Genbank accession AAAA01001203). Contig 2308, in turn, contains sequence that overlaps the distal end of AP005192. In addition, sequence in the contigs also matches RGP EST marker (C52865S), which was previously mapped to the region near M3. A complex repeat was identified within contig CL003847.142 consisting of seven and two-thirds, near perfect, 76- to 77-bp direct repeats. Primer combination M5 (Table 1) amplifies the region of the complex repeat producing fragments of approximately 1170 and 294 bp from IR24 and IRBB7, respectively (Fig. 5). Comparison of the IRBB7 and Nipponbare sequences indicates that the polymorphism is due to the absence of the repeat region in IRBB7 (Fig. 6). No recombinant banding patterns were seen by means of the M5 primer combination on the 277 susceptible F3 plants evaluated, indicating that Xa7 is highly linked (= 0.16 cM, 1 recombinant error factor) to M5 (Fig. 7).



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  		Fig. 5. Polymorphism produced with M5 primers using IR24(S) and IRBB7(R) template DNA. The DNA was amplified as described in Materials and Methods and fractionated on a 2.0% (w/v) agarose gel in TAE buffer. The left lane contains a 1-kb standard with base pair sizes indicated on the left side of the figure.

 


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  		Fig. 6. Comparison of IRBB7 (R) 294-bp and Nipponbare (N) 1170-bp M5 sequence. Similar sequence in the 5' region is single underlined and, in the 3' region, double underlined.

 


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  		Fig. 7. Map of chromosome 6 in the region of Xa7. (A) Genetic map distances of markers M1, M3, M4, and M5 from Xa7. (B) Physical map is based on Nipponbare and indica sequence databases showing approximate distances (kb) between marker M1, M2, M3, M4, M5, and G1091. Gray box between M3 and M5 indicate a gap region. (C) RGP contigs in the region of Xa7. Nipponbare sequence database contig CL003847.142 and indica sequence database contig 2308 extend distally toward the gap (hashed region) beyond RGP AP00519.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The identification of markers linked to Xa7 has been facilitated by the various efforts in sequencing the rice genome. For this study, the extensive degree of colinearity between the indica and japonica genomes permitted anchoring of sequence contigs to the physical map and tiling of orphaned contigs by means of alternate sequence databases. The available sequence also permitted estimation of the physical distance between M5 and Xa7, provided the indica and japonica chromosomes are colinear throughout the region. The physical distance between M1 and M5 and M3 and M4, respectively, generally reflect the genetic distances estimated by recombination. M1 and M5 are 2.2 cM apart in genetic distance and approximately 368 kb in physical distance, while the genetic and physical distances between M3 and M4 are 1.3 cM and approximately 210 kb, respectively. Thus, physical distance per centimorgan is calculated at 167 and 161 kb/cM, respectively. On the basis of the average physical distance between these makers, Xa7 is predicted to be 82 kb from M3 and ≤27 kb from M5, assuming a maximum of one recombinant in 300 susceptible progeny. The immediate region around Xa7, however, may not be collinear and could represent more, or less, DNA than the above prediction. Marker M5, which represents a discontinuity between Nipponbare and IRBB7 (Fig. 6), provides evidence that the region corresponding to Xa7 has the potential to be moderately diverged between the two lines. Cosmid library screening and sequencing is underway to expand the sequence surrounding M5.

The mapping of Xa7 also highlights one limitation of the present rice map. The region of Xa7 has a gap in the physical map extending from the ends of the CUGI BAC clones OSJNBa0017K10 on the left side to OSJNBa029C21 on the right. These BACs extend from the ends of the corresponding RGP draft sequence in contigs AP005192 on the right to AP004989 on the left. No BAC or yeast artificial chromosome clones span this region (Saji et al., 2001; Chen et al., 2002). In addition, sequence contigs from the region contain highly repetitive elements and prevent identification of contigs from shotgun library sequences with a high degree of confidence. Whether this region is unclonable in standard vectors or simply a gap due to chance is unknown. Further characterization of the region will aid the completion of chromosome 6 physical and sequence maps.

The cloning of Xa7 will be essential to understanding the unique aspects of the interaction. AvrXa7, an AvrBs3-related protein (Bonas et al., 1989), is an important virulence factor for Xoo in colonizing the host plant in the absence of the Xa7 gene (Bai et al., 2000). On the basis of the requirement for at least one of the three NLS sequences, the product of avrXa7 appears to be targeted to the host cell nuclei (Yang et al., 2000). Therefore, one possible model for resistance might involve the direct or indirect interaction of the Xa7 gene product with AvrXa7 in the nuclei. Whether AvrXa7 and AvrBs3 are recognized by the same mechanism remains to be shown, and efforts to clone Bs3 from pepper are also in progress. Markers have been identified which flank a 2.1-cM region containing Bs3 (Pierre et al., 2000), and the two systems should be available for comparison in the near future.

While the markers described in this paper have identified an interval for cloning Xa7, they also provide a tool for immediate application in breeding efforts such as gene pyramiding. A broad range of utility is suggested by the polymorphisms seen within and between two ecogeographical races. Because IR24, IRBB7, and DV85, the donor of Xa7, are all lines of the indica ecogeographical race (McKenzie et al., 1987), it would be expected that the introgressed region, containing Xa7, would be more similar among these lines than when compared to lines from another ecogeographical race (japonica). However, marker M3 contradicts this assumption. The SSR associated with this marker is present in IRBB7 and Nipponbare, but it is absent in IR24 (Fig. 4B). In addition, the major deletion found in IRBB7, by means of marker M2, is found in Nipponbare, but is missing in IR24 (Fig. 4A). These two polymorphisms, interestingly, would not have been found in the wider cross of Nipponbare x IRBB7. Regardless, M5 provides a readily scored STS for crosses with indica and japonica lines and is tightly linked to Xa7. M5 should prove useful in any breeding program involving Xa7.


   ACKNOWLEDGMENTS
 
We thank Drs. Randall Warren, Bikram Gill, and Scot Hulbert for critical review of the manuscript. This work was supported by funds provided by the Kansas Agriculture Experiment Station (KAES) and award 02-02349 from the NRI Competitive Grants Program/USDA.


   NOTES
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 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Contribution No. 02-479-J from the Kansas Agric. Exp. Stn.

Received for publication September 6, 2002.


   REFERENCES
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 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome