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Crop Science 40:518-524 (2000)
© 2000 Crop Science Society of America

CELL BIOLOGY & MOLECULAR GENETICS

Development of PCR-Based Markers for a High Grain Protein Content Gene from Triticum turgidum ssp. dicoccoides Transferred to Bread Wheat

I.A. Khana, J.D. Procunierb, D.G. Humphreysb, G. Tranquillic, A.R. Schlatterc, S. Marcucci-Poltric, R. Frohbergd and J. Dubcovskya

a Dep. of Agronomy and Range Science, University of California, Davis, CA 95616-8515 USA
b Cereal Research Centre, Agriculture and Agri-food Canada, Winnipeg, Manitoba, Canada
c Instituto de Recursos Biológicos, INTA, Villa Udaondo, (1712) Castelar, Buenos Aires, Argentina
d Dep. of Plant Sciences, North Dakota State Univ., Fargo, ND, 58105 USA

jdubcovsky{at}ucdavis.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results
 Discussion
 REFERENCES
 
Grain Protein Content (GPC) of wheat (Triticum aestivum L. and T. turgidum L.) is important for improved nutritional value and is also one of the major factors affecting breadmaking and pasta quality. A quantitative trait locus (QTL) for high GPC was detected a few years ago in the short arm of chromosome 6B from accession FA15-3 of Triticum turgidum L. var. dicoccoides. New molecular markers are presented here to facilitate the transfer of this high GPC gene into tetraploid and hexaploid wheat cultivars. Two sets of PCR (polymerase chain reaction) primers were designed to amplify regions of the non-transcribed spacer of the XNor-B2 locus. This locus was selected because it mapped on the peak of the QTL for GPC. The first pair of allele-specific primers produced an amplification product only when the T. turgidum var. dicoccoides XNor-B2 allele was present. The second pair of primers amplified fragment(s) of similar length in the different genotypes that after digestion with the restriction enzyme BamHI allowed differentiation of the T. turgidum var. dicoccoides allele. Four microsatellites markers were mapped on the short arm of chromosome 6B at both sides of the QTL peak and two on the long arm. Five additional amplified fragment length polymorphism (AFLP) markers were mapped into the QTL region on 6BS. These PCR markers together with 10 restriction fragment length polymorphism (RFLP) markers showed that the hexaploid cultivar Glupro, selected for high GPC, carries a distal segment of chromosome 6BL and a proximal segment of 6BS from dicoccoides accession FA15-3 encompassing the segment with highest LOD score for the GPC QTL.

Abbreviations: AFLP, amplified fragment length polymorphism • ASA, allele specific amplification • BC, backcross • CAPS, cleavage amplified polymorphic sequence • cM, centimorgan • DIC, Triticum turgidum var. dicoccoides • GPC, grain protein content • LDN, Langdon • NOR, nucleolar organizing region • PCR, polymerase chain reaction • QTL, quantitative trait loci • RFLP, restriction fragment length polymorphism • RSL, recombinant substitution line


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results
 Discussion
 REFERENCES
 
GRAIN PROTEIN CONTENT of wheat is important for improved nutritional value and is also one of the major factors affecting breadmaking and pasta quality (Dick and Youngs, 1988; Finney et al., 1987). In spite of its importance, progress in breeding for high GPC has been slow and difficult. The first limitation is that genetic variation for protein content is small compared with variation due to differences in growing environments. The second limitation is that there is a strong negative correlation between GPC and grain yield; cultivars with high GPC tend to be low yielders. There are, however, exceptional genotypes that combine excellent yield potential and high GPC, probably by a more efficient relocation of nitrogen from senescing tissues to grain, or by a more efficient uptake of nitrate and ammonia from the soil (Blackman and Payne, 1987).

A promising source of high GPC was detected years ago in a survey of wild populations of tetraploid Triticum turgidum var. dicoccoides (accession FA15-3 from Israel; Avivi, 1978), referred to as dicoccoides hereafter. Substitution lines of the chromosomes of this dicoccoides accession in the cultivated durum cultivar Langdon (Triticum turgidum var. durum) showed that a gene for high protein content was present on chromosome 6B (Joppa and Cantrell, 1990). Recombinant substitution lines (RSLs) from a cross between the LDN(DIC 6B) substitution line and Langdon were developed by Joppa et al. (1997). These lines were used to map a QTL for GPC on the proximal region of the short arm of chromosome 6B that accounted for 66% of the variation in GPC present in that particular cross (Joppa et al., 1997).

Other researchers crossed the same dicoccoides 6B-substitution line with high yielding durum cultivars from North Dakota (Chee et al., 1998) and Canada (Kovacs et al., 1998) to introgress the high-GPC gene. Results from the North Dakota recombinant lines showed a single gene effect and no interaction between the increase in GPC content and the genetic background or growing environments. The effect of this gene was independent of protein quality, plant height, heading date, and yield (Chee et al., 1998). Results from the Canadian recombinant lines showed a positive effect on protein and quality (Kovacs et al., 1998). The incorporation of the high-GPC gene from dicoccoides into the Canadian lines resulted in increased protein content and a positive effect on pasta cooking quality. Kovacs et al. (1998) concluded that this gene would be a valuable resource to increase the protein level for durum wheat breeding programs.

The high-GPC gene from dicoccoides has also been transferred to hexaploid wheat. Hexaploid cultivar Glupro was developed by Dr. R. Frohberg from a three-way cross between two bread wheat cultivars and the same dicoccoides accession used to develop the substitution line LDN(DIC6B). Mesfin et al. (1999) showed that Glupro and dicoccoides accession FA15-3 shared five RFLP markers from a 15-cM proximal region of chromosome 6B. The positive effect of this segment on grain protein content in a bread wheat genetic background was recently demonstrated in double haploid lines (Humphreys et al., 1998) and recombinant inbred lines (Mesfin et al., 1999) developed from crosses between Glupro and bread wheat cultivars from Canada and USA.

The objectives of this work were to (i) develop PCR-based markers to facilitate the manipulation of the gene for high GPC in marker assisted selection programs, (ii) detect additional molecular markers in the GPC gene region, and (iii) extend the molecular comparison between Glupro and dicoccoides accession FA15-3 to determine the size of the dicoccoides chromosome 6B segment transferred to Glupro.


   Materials and methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results
 Discussion
 REFERENCES
 
Dr. L. Joppa provided the RSLs of chromosome 6B from dicoccoides and chromosome 6B from Langdon in a Langdon genetic background. In this study, only the RSLs showing at least one recombination event between RFLP markers Xcmwg652 and Xcdo534 were included (Joppa et al., 1997). The 25 selected RSLs (5, 8, 11, 14, 19, 28, 30, 32, 36, 41, 44, 47, 48, 50, 54, 56, 58, 59, 60, 63, 65, 67, 68, 77, and 78) have additional crossovers in other regions of chromosome 6B. Hexaploid wheat cultivar Glupro (`Columbus'/T. turgidum var. dicoccoides//`Len') and its progenitor cultivars Len and Columbus were tested with the different molecular markers developed in this study. Tetraploid cultivars and breeding lines (`Kronos', `Kofa', `Westbred Turbo', UC908, UC1112, UC1113, and UC1114) and hexaploid cultivars and breeding lines (`Express', `RSI5', `Anza', `Yecora Rojo', UC1036, UC1037, and UC1041) were tested only with probe pTA250.15.

Probes and RFLP Procedures
DNA was extracted from the previous cultivars and RSLs by the method described by Dvorak et al. (1988). A small scale DNA isolation protocol was used for the marker assisted backcrossing program (Weining and Langridge, 1991). Procedures for Southern blots and hybridization were as described by Dubcovsky et al. (1994). Clones used in this study were kindly provided by J. Dvorak (University of California, Davis), M. Gale (John Innes Centre, Norwich, UK), A. Graner (Institute for Plant Genetics & Crop Plant Research, Gatersleben, Germany), and M. Sorrells (Cornell University, Ithaca, NY). Probe pTA250.15 is a 750-bp HhaI fragment from the spacer region of the wheat rDNA and was used to detect the XNor-B2 locus (Appels and Dvorak, 1982).

PCR Procedures
The sequence of the non-transcribed spacer of theXNor-B2 locus (Barker et al., 1988) was used to design two sets of PCR primers with the computer program PRIMER (Version 0.5, Lincoln and Daly, 1991). One primer set (NTS#2F: 5'-ATG ATG GTC AAC AAA CGG TGC-3' and 18S#2R: 5'-TTT ATT GTC ACT ACC TCC CCG-3') amplified a region of the non-transcribed spacer having the highly polymorphic BamHI site (Kim et al., 1992). This primer set is designated as a cleavage amplified polymorphic sequence (CAPS, Jarvis et al., 1994). PCR cycling conditions were 94°C 3 min; (94°C 1 min, 58°C 1 min, 72°C 2 min) 35 cycles; 72°C 10 min in a volume of 25 µL. The final concentration of the different products used in the PCR reaction were: 1x Taq polymerase buffer, 2.5 U of Taq polymerase, 1.5 mM MgCl2, 1 ng/µL of each primer, 250 µM each dNTP, and 50 to 100 ng of genomic DNA. Fifteen microliters of the amplification product were then digested with 10 U of the restriction enzyme BamHI in a 1x final concentration of the commercial buffer provided with the enzyme. The product was then loaded in a 0.8% (w/v) agarose gel, run for 4 h to permit good separation of the bands and the gel was stained with ethidium bromide.

The second pair of primers amplified a sequence specific to theXNor-B2 non-transcribed spacer of dicoccoides. These are designated as allele specific amplification (ASA) primers and their sequences are ASA#2R: 5'-CTA CCA TCG AAA GTT GAT AGG GA-3' and ASA#1F: 5'-TTC ACA AAC TAA GGG GAG GGA-3'. Cycling conditions and final concentrations in the PCR reaction were similar to those for the first primer set except for the annealing temperature of 57°C. The PCR product was visualized as mentioned above.

Seven microsatellites (Xgwm193, Xgwm219, Xgwm361, Xgwm508, Xgwm518, Xgwm626, and Xgwm644) were used in this study. Primer sequences and PCR conditions for these microsatellites are published (Röder et al., 1998). PCR reactions were performed in a volume of 25 µL and amplification products were separated on denaturing 6% (w/v) polyacrylamide gels at 45 W for 3 h; the products were visualized by silver staining. Size of each band was estimated by a molecular weight standard (25-bp ladder, Gibco-BRL, Rockville, MD).

AFLP assays were performed by a modified version of the method described by Milbourne et al. (1997). Briefly, 500 ng of wheat genomic DNA were subject to restriction–ligation in a single step during 6 h in a 30-µL reaction mix (10 mM Tris-acetate pH 7.5, 10 mM Mg-acetate, 50 mM K-acetate, 5 mM dithiothreitol, 50 ng/mL BSA, 5U PstI, 5U Mse, 1U T4 DNA ligase (Gibco), 5 pmol PstI adaptors, 50 pmol MseI adaptors, and 12 pmol ATP). Five microliters of each adaptor-ligated template DNA were preamplified in a 25-µL PCR reaction containing 75 ng of both P01 and M01 AFLP primers (5'-GAC TGC GTA CAT GCA GA-3' and 5'-GAT GAG TCC TGA GTA AA-3' respectively) , 0.2 mM dNTPs, 1x PCR buffer (1.5 mM MgCl2) and 1U of Perkin Elmer Amplitaq LD (PE Corporation, Norwalk, CT). Selective amplifications were performed with 1 µL of non-diluted preamplification product and 30 ng of each selective non-labeled + 3 primer and the cycling conditions described by Vos et al. (1995). Ten microliters of formamide dye were added to the 20-µL PCR reactions and amplification products were separated in 6% (w/v) denaturing polyacrylamide gels and silver stained (Promega Sequencing Silver staining kit; Promega, Madison, WI). Selective primer combinations assayed were P36=ACC, P37=ACG, P40=AGC, P41=AGG against M31=AAA, M37=ACG, M38=ACT, M39=AGA, M40=AGC, M41=AGG, M42=AGT, M43=ATA, M44=ATC, M45=ATG .

Mapping Procedures
The 25 RSLs with at least one recombination event between RFLP markers Xcmwg652 and Xcdo534 were scored for the polymorphic markers and the data was incorporated in the data matrix used by Joppa et al. (1997) kindly provided by G. Hart (Texas A&M University). Linkage maps were constructed with the aid of the computer program Mapmaker/EXP 3.0 (Lander et al., 1987) using the Kosambi function.


   Results
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
Discussion
 REFERENCES
 
Development of a PCR Marker for the XNor-B2 Locus
The XNor-B2 locus was mapped in the same population of RSLs of chromosome 6B used by Joppa et al. (1997). In these RSLs, XNor-B2 was completely linked to Xmwg79, the locus with highest LOD score for the QTL for high GPC (Joppa et al., 1997) (Fig. 1) . When probe pTa250.15 was hybridized with wheat genomic DNA digested with TaqI, LDN(DIC 6B) substitution line showed a 2.9-kb restriction fragment that differed from the two restriction fragments of 2.7 and 3.1 kb present in the original Langdon DNA (Fig. 2) . The 2.9-kb band was a useful marker to differentiate the dicoccoides segment from most of the tetraploid and hexaploid cultivars tested. However, a few cultivars like tetraploid Kronos or hexaploid Columbus showed restriction fragments of similar mobility to the dicoccoides 2.9-kb band. In those cases, autoradiographs were overexposed and the LDN(DIC6B) allele was traced by the presence of two additional 7- and 14-kb restriction fragments that cosegregate with the 2.9-kb restriction fragment. These two additional restriction fragments were not detected in any of the tetraploid or hexaploid cultivars tested in our marker assisted selection program (data not shown).



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  		Fig. 1 Left: 6B map developed by Joppa et al. (1997) with QTL analysis for high GPC. Center: molecular markers added in this work. Right: inferred distribution of dicoccoides and bread wheat chromosome segments in hexaploid cultivar Glupro based on the same molecular markers. RFLP markers indicated as present in dicoccoides 6B substitution line and Glupro are also absent in Columbus and Len, the hexaploid progenitors of Glupro. Genetic distances are inferred from the map on the left. Distances between markers are proportional to genetic distances. Centromeres are indicated with a circle

 


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  		Fig. 2 Hybridization patterns of clone pTa250.15 (segment of the XNor-B2 locus) with TaqI digested DNAs from hexaploid and tetraploid wheat DNAs. Left side: First three lanes are T. aestivum cultivars and breeding lines (Yecora Rojo, UC1036, and UC1037) without the high GPC gene followed by cultivar Glupro carrying the high GPC gene. Right side: T. turgidum RSL #68 and substitution line of dicoccoides complete chromosome 6B in Langdon both carrying the high GPC gene followed by three lanes of durum cultivars and breeding lines (Langdon, UC908, Westbread Turbo) without the high GPC gene

 
To facilitate the use of this marker in breeding programs, PCR-based markers were developed for the XNor-B2 locus of dicoccoides. PCR with the CAPS primers yielded a single band for all genotypes that after cutting with BamHI gave a doublet when the dicoccoides allele was present and a singlet when this allele was absent (Fig. 3) .



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  		Fig. 3 PCR markers for theXNor-B2 locus. In both panels the first lane is bread wheat Canadian Prairie Spring line HY616 (T. aestivum allele at the XNor-B2 locus) and the second lane is Glupro (T. turgidum var. dicoccoides allele at theXNor-B2 locus) . Left panel: PCR reaction using cleavage amplification polymorphic sequence (CAPS) primers followed by digestion with BamHI. A doublet is observed when the dicoccoides allele is present. Right panel: PCR reaction using allele specific amplification (ASA) primers. A PCR product is observed only when the dicoccoides allele is present

 
A second pair of allele specific amplification primers (ASA) was designed to differentiate the dicoccoides allele without further digestion with a restriction enzyme. The ASA primers amplified a band only when the dicoccoides allele was present. No PCR product was observed when the dicoccoides allele was absent (Fig. 3). However, this PCR reaction was less robust than the reaction using the CAPS primers; occasional false positives (a light band present) were detected.

Microsatellite Markers
All microsatellite markers tested, with the exception of Xgwm518, showed polymorphism between LDN(DIC6B) and LDN (Fig. 4) . The molecular weights of the amplified fragments on LDN(DIC6B) and LDN were 136 and 138 bp for Xgwm508 (Fig. 4), 194 and 174 bp for Xgwm193 (Fig. 4), 138 and 142 bp for Xgwm361, 152 and 150 bp for Xgwm644, 135 and 105 bp for Xgwm626, and 150 and 154 bp for Xgwm219 (Fig.4).



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  		Fig. 4 Silver stained polyacrylamide gels containing PCR amplification products of microsatellite primers for loci Xgwm508 (left) and Xgwm193 (center) on the short arm of chromosome 6B, and Xmwg219 (right) on the long arm of the same chromosome

 
Polymorphic microsatellite markers were mapped in the population of RSLs developed by Joppa et al. (1997) (Fig. 1). Microsatellite markers Xgwm193, Xgwm361, and Xgwm644 were completely linked with each other and with RFLP marker Xpsr113 (Fig. 1). The LOD score for the QTL for GPC at this locus was approximately four units lower than the LOD score at the XNor-B2 locus (Fig. 1). Microsatellite Xgwm508 was 4.8 cM distal to the XNor-B2 locus and its LOD score was approximately 2 units lower than the LOD score at the XNor-B2 locus. These results indicated that there is a large probability that the gene for high GPC is located within the interval delimited by these microsatellite markers. Two microsatellite markers were mapped on the long arm of chromosome 6B. Microsatellite Xgwm626 was completely linked to RFLP Xcdo516 and microsatellite Xgwm219 was mapped within the Xcmwg669–Xglk762 interval (Joppa et al., 1997).

Microsatellite markers detected non-parental alleles in a few RSLs. RSL#30 and RSL#32 showed alleles different from Langdon and dicoccoides for microsatellite loci Xgwm193, Xgwm361, Xgwm644, and Xgwm219. The non-parental allele found with microsatellite Xgwm219 in RSLs #30 and #32 was also present in RSL#60.

AFLP Markers
Forty PstI–MseI primer combinations differing in the three 3' end nucleotides were tested for polymorphism between Langdon and RSL#68 that carries a 30-cM dicoccoides segment between Xcmwg652 and Xcdo534. Five of the 2400 AFLP fragments detected with the selected primers were mapped in this region.

AFLP locus XP36M43.137 was completely linked to microsatellite marker Xgwm508 (Fig. 1). The bands for the two alleles of this locus were one base pair apart and this marker was scored as codominant. Locus XP40M41.425 was mapped in repulsion with the dicoccoides allele within the Xgwm508–XNor-B2 interval (Fig. 1). Two AFLP loci were mapped in the XNor-B2–Xabg387 interval, XP36M39.97 in repulsion with the dicoccoides allele and XP37M42.220 in coupling. Finally, locus XP36M44.200 was mapped in coupling with the dicoccoides allele completely linked to RFLP marker Xpsr113 and microsatellite markers Xgwm193, Xgwm361, and Xgwm644.

One unexpected result was the detection of 28 segregating AFLP fragments that did not map in the targeted region of chromosome 6B. Seven of these markers were polymorphic between Langdon and RSL#68 and 21 were not polymorphic between these lines. These results suggest that LDN(DIC 6B) and LDN have genetic differences in chromosomes other than chromosome 6B.

Characterization of the dicoccoides segment transferred to Glupro using RFLP
All previous polymorphic microsatellite and AFLP markers and 14 RFLP markers previously mapped on homoeologous group 6 chromosomes of the Triticeae (Joppa et al., 1997; Graner et al., 1991; Marino et al., 1996; Dubcovsky et al., 1996) were tested on DNA samples from Glupro, Len, Columbus, LDN(DIC 6B), and LDN. The presence of common polymorphism in LDN(DIC 6B) and Glupro, and absence in Len and Columbus were used as criteria to establish the boundaries of the dicoccoides segment present in Glupro.

Five AFLP, five microsatellite and seven RFLP markers showed the same polymorphism in LDN(DIC 6B) and Glupro. The only marker tested for the short arm that did not show a dicoccoides allele was Xpsr946, indicating that a recombination event took place in the Xpsr964–Xcmwg652 interval during the development of Glupro (Fig. 1). Two additional recombination events were detected in intervals Xcdo534–Xcdo507 and Xgwm626–Xgwm219 (Fig. 1). These data indicate that two separate chromosome segments from dicoccoides were transferred to Glupro chromosome 6B. One in the distal region of the long arm and the other one in the proximal region of the short arm encompassing the 6BS segment with highest LOD score for the GPC QTL (Fig. 1). RFLP locus Xcdo507 and microsatellite locus Xgwm626 showed identical polymorphisms in Glupro and Len that were absent in Columbus and LDN(DIC 6B).


   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
 Discussion
REFERENCES
 
The XNor-B2 locus is a useful RFLP marker to trace the gene for high GPC not only because of its tight linkage with the peak of the QTL but also because thousands of copies of the rRNA gene (2C) are present in this locus (Flavell and O'Dell, 1979). The large number of copies reduces the amount of DNA that needs to be digested with restriction enzymes to 2 or 3 µg/lane, and the exposure time of the autoradiographs to less than 1 h. Though this RFLP is relatively easy to use, RFLP technology is more expensive and cumbersome than PCR technology. Consequently, the RFLP marker was converted into a PCR marker to facilitate its use in breeding programs.

The ASA primer pair was used to generate a dominant marker in coupling with the dicoccoides high GPC gene. However, since the specificity of this marker was based on differences of a few base pairs, occasional false positives were amplified when the PCR conditions were not perfectly optimized. The CAPS primers combined with digestion of the PCR product with restriction enzyme BamHI provided a more reliable marker. A possible strategy to minimize costs is to use the ASA marker for the initial testing and then use the CAPS marker to confirm the genotype of the critical plants.

These PCR-based markers have already provided a valuable tool for the detection of the high GPC gene from dicoccoides. Double haploid lines from BC2F1 from crosses between Glupro and two high yielding Canadian Prairie semi-dwarf advanced breeding lines were selected using these PCR markers. Double haploid lines carrying the PCR marker for the dicoccoides allele showed a significantly higher protein content than lines without the dicoccoides allele (P < 0.001, Humphreys et al., 1998).

Though the XNor-B2 locus is closely linked to the peak of the QTL, recombination between this marker and the high-GPC gene may result in the loss of the targeted gene during the marker assisted selection process. The distal microsatellite marker Xgwm508 can be used in combination with any of the three tightly linked proximal microsatellites Xgwm193, Xgwm361, and Xgwm644 to monitor the transfer of a segment of dicoccoides chromatin (Fig. 1) that has a high probability of including the high-GPC gene. To reduce the linkage drag of dicoccoides chromatin of the long arm during the backcrossing process, long arm microsatellites markers can be used to select BC plants carrying the high-GPC gene on the short arm and a reduced dicoccoides chromosome segment on the 6BL arm. By this procedure, short-stature, hard-red-spring cultivars carrying the high-GPC gene and reduced dicoccoides 6BL arm are being developed as an alternative source of the high-GPC gene for new marker assisted selection programs.

An alternative source of the same GPC gene in hexaploid wheat may be line ND683 [`Stoa sib'/ND645 (Columbus/T. dicoccoides var. dicoccoides//`Coteau')] developed by R. Frohberg (Mesfin et al., 1999). The small size of the dicoccoides chromosome segment present in this line was indicated by the fact that none of the RFLP or PCR markers analyzed in this study showed the characteristic dicoccoides alleles in ND683. Mesfin et al. (1999) indicated only one RFLP locus, Xcdo365, showing a dicoccoides allele in ND683. Though ND683 has the advantage of a reduced linkage drag that may eliminate potentially undesirable linked traits, it also has the disadvantage that none of the PCR markers developed here can be used to trace the GPC genes in marker assisted selection programs.

At the tetraploid level, RSL#68 can be used as a starting material to transfer the high-GPC gene to tetraploid cultivars. This line has a 6BS dicoccoides segment including RFLP markers Xcmwg652 and Xcdo534 (Joppa et al., 1997) that can be traced with the PCR markers presented in this study. However, RSL#68 is in a Langdon genetic background and has poor agronomic and quality characteristics. By means of the molecular markers developed in this work, short-stature durum cultivars carrying the high-GPC gene and reduced dicoccoides 6BL arm are being developed as alternative sources for the high-GPC gene.

A final objective of this work was to explore the possibility of targeting additional markers to the GPC gene region by AFLPs. Of the 2400 AFLP fragments detected by 40 primer combinations, five were mapped in the targeted region. This result suggests that selection of polymorphic AFLPs between Langdon and RSL#68 is a viable strategy to saturate this region with additional molecular markers, providing a preliminary step towards the positional cloning of the high-GPC gene.


   ACKNOWLEDGMENTS
 
The authors express their gratitude to Dr. Joppa for providing the seeds of the tetraploid lines and for the critical reading of the manuscript, and to M.D. Gale, A. Graner, G. Hart, A. Kleinhofs, M.E. Sorrells, J. Dvorak, and O.D. Anderson (Wheat Probe Repository, ARS-USDA, Albany) for supplying clones. J. Dubcovsky acknowledges financial support from USDA-Fund for Rural America competitive grant 97-36200-5272. G. Tranquilli expresses gratitude to the Argentinean Research Council (CONICET) for a fellowship during this work.

Received for publication June 8, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
 Discussion
 REFERENCES
 




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