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Published online 1 March 2007
Published in Crop Sci 47:811-818 (2007)
© 2007 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

A High-Density Map and PCR Markers for Russian Wheat Aphid Resistance Gene Dn7 on Chromosome 1RS/1BL

Nora L. V. Lapitan*, Junhua Peng and Vinod Sharma

Dep. of Soil and Crop Science, Colorado State Univ., Fort Collins, CO 80523-1170. All authors contributed equally to this study

* Corresponding author (nlapitan{at}lamar.colostate.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Russian wheat aphid (RWA) [Diuraphis noxia (Kurdjumov)] is an economically significant insect pest of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) in the USA. Since 1986, only one biotype existed in the USA. In 2003, a new biotype appeared which was virulent to all known resistance genes in wheat, with the exception of Dn7. Dn7 is a rye gene transferred into a wheat background via 1RS/1BL translocation. It was previously mapped; however, no PCR-based markers were developed for marker-assisted selection (MAS) and the map was sparse. This study presents a higher density map of Dn7 containing 19 markers. The markers spanning Dn7 are XHor2 and Xscb241 at distances of 1.4 and 1 cM, respectively, from Dn7. PCR markers were developed and tested on 19 wheat cultivars. Two markers which amplified rye-specific fragments proved to be useful for MAS. Xrems1303 amplified a 320-bp band only in cultivars with high-level resistance to biotype 2 and is effective for MAS of Dn7. Xib267 is linked to the susceptible locus and amplified a fragment specific for rye Petkus 1RS and would be a good marker for selecting against the susceptible locus. The value of Dn7 as a source of resistance has increased because of the broad spectrum resistance it provides against several biotypes. The markers developed in this study will be useful for facilitating the transfer of Dn7 into wheat cultivars with or without translocation chromosomes containing 1RS.

Abbreviations: AFLP, Amplified fragment length polymorphism • MAS, Marker-assisted selection • PCR, Polymerase chain reaction • RFLP, Restriction fragment length polymorphism • RWA, Russian wheat aphid • SSR, Simple sequence repeats • 1RS, rye chromosome 1R short arm.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE RUSSIAN WHEAT APHID is an important pest of small grains, particularly wheat and barley. RWA was introduced in the USA in 1986 and has caused direct and indirect losses estimated at more than $800 million in the western USA from 1987 to 1993 (Morrison and Peairs, 1998). The damage caused by the RWA has been mitigated with the widespread adoption of resistant wheat cultivars, particularly in areas with consistent infestations (Berzonzksy et al., 2002). Between 1986 and 2003, only one RWA isolate was known to exist in the USA. However, in 2003, a new biotype was reported in Colorado which was virulent to all known RWA resistance sources currently deployed in commercially available cultivars in the western Great Plains region of the USA (Haley et al., 2004). The germplasm tested represented most known resistance genes in wheat including Dn1, Dn2 (Du Toit, 1987), Dn3 (Nkongolo et al., 1991b), Dn4 (Nkongolo et al., 1991a), Dn5 (Du Toit et al., 1995), Dn6 (Harvey and Martin, 1990), Dn8, and Dn9 (Liu et al., 2001). The only germplasm that showed resistance to the new biotype (biotype 2) were wheat cultivars containing the rye gene Dn7 (Marais et al., 1994), previously shown to provide a high level of resistance to the original U.S. RWA biotype (biotype 1) (Anderson et al., 2003). Genetic mapping indicated that resistance to RWA biotype 2 was also conferred by Dn7 (Sharma, 2005). Furthermore, additional new biotypes of RWA were recently discovered, to which Dn7 was also resistant (Haley et al., 2004).

Dn7 originated from rye cultivar Turkey 77 and was transferred to a wheat background via recombination between the 1RS telosome of Turkey 77 and 1RS/1BL translocation chromosome from a susceptible spring wheat ‘Veery’ series cultivar, Gamtoos (Marais et al., 1994). The Veery wheat cultivars developed in CIMMYT (Rajaram et al., 1983) were derived from a cross between a spring wheat Mexican semidwarf and the winter wheat cultivar Kavkaz containing 1RS chromosome from ‘Petkus’ rye (Schlegel and Korzun, 1997; Zeller, 1973). The resulting Dn7-containing germplasm, 94M370, also contains resistance genes Sr31 and Lr26 from Petkus which confer resistance to stem rust (Puccinia graminis Pers.:Pers.= P. graminis Pers.:Pers. f. sp. tritici Eriks. & E. Henn.) and leaf rust [Puccinia triticina Eriks. = P. recondita Roberge ex Desmaz. f. sp. tritici (Eriks. & E. Henn.) D.M. Henderson], respectively (Marais et al., 1998).

Dn7 was genetically mapped using restriction fragment length polymorphic (RFLP) markers (Anderson et al., 2003). Xbcd1434 and Xksud14 flanked Dn7 with map distances of 1.4 and 7.4 cM, respectively. However, no PCR markers have yet been developed that would be useful for MAS of Dn7. Because of the Dn7 gene's high level and wide spectrum of resistance to RWA, this gene is also a target for map-based cloning. Hence, a saturated molecular map for the 1RS segment harboring Dn7 is necessary.

The development of different types of molecular markers and genomic tools in rye and in related Triticeae species provides reagents for saturating the 1RS chromosome. This includes rye simple sequence repeats (SSR) (Hackauf and Wehling, 2002; Khlestina et al., 2004; Saal and Wricke, 1999) and over 2500 expressed sequence tags (ESTs) for group 1 chromosomes in the Triticeae (Lazo et al., 2004; Peng et al., 2004; Qi et al., 2004). ESTs can be used directly as RFLP probes or as source of EST-derived SSR (eSSR) markers (Hackauf and Wehling, 2002; Peng and Lapitan, 2005) or single nucleotide polymorphisms (SNPs) (Picoult-Newberg et al., 1999; Useche et al., 2001). The conserved motifs in cloned plant resistance genes have provided primers to isolate resistance gene analogs (RGAs), which have been linked to plant resistance loci (Yan et al., 2003; Zhang et al., 2004). Rice genomic sequences can also provide markers for mapping in Triticeae species, provided synteny exists between rice and the Triticeae region of interest (Ahn et al., 1993; Sorrells et al., 2003; Van Deynze et al., 1995).

The objectives of this study were to construct a higher-density map of Dn7 and develop PCR-based markers for MAS. Synteny of the 1RS chromosome region containing Dn7 with rice was also investigated to explore the feasibility of using rice genomic sequences to saturate the region.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials
The mapping population consisted of 98 recombinant inbred lines (RILs) and was made by advancing F2–derived F3 families from a cross between wheat line 94M370 (resistant) and wheat cv. Gamtoos (susceptible) to the F6 generation by single seed descent. The pedigree of 94M370 was described by Anderson et al. (2003). A second mapping population consisting of 182 F2 individuals/F3 families, also made from a cross between 94M370 and Gamtoos, was used to validate the map from the RILs.

Nineteen wheat cultivars, including 94M370 and Gamtoos, were used to test the usefulness of PCR-based markers linked to Dn7 for MAS of the gene.

Phenotyping of Reaction to RWA Infestation
RWA screening and scoring for the RIL mapping population were performed at the Colorado State University (CSU) Insectary in the winters of years 2002 and 2003. The F2–F3 population was screened in Spring 2005 (F2 individuals) and Spring 2006 (F3 families). Ten to 15 seeds for each of the RILs and F3 families were planted in flats (52.0 x 25.5 cm) in a single row and were infested with RWA biotype 2 (Nkongolo et al., 1989). Parents, Gamtoos, and 94M370, were included in each flat while ‘Carson’ served as a susceptible control. Reaction to RWA infestation was rated at 7, 14, and 21 d after infestation and scored as previously described (Ma et al., 1998).

DNA Extraction, Bulk Segregant Analysis, and Southern Blotting
Total genomic DNA was extracted from each of the 98 RILs and 182 F2 plants, as well as from the parental lines, 94M370 and Gamtoos, according to Peng et al. (2000b). Resistant and susceptible bulks (Michelmore et al., 1991) were constructed by combining equal amounts of DNA from eight resistant and eight susceptible recombinant inbred lines, respectively. Four restriction enzymes (EcoRI, XbaI, EcoRV, HindIII) were used to digest the genomic DNA from parental lines, resistant and susceptible bulks, and the 98 RILs. Blotting and Southern hybridizations were performed as previously described (Peng et al., 2004). The parents and bulk segregants were screened for polymorphism in 59 probes and 56 PCR-based markers. Probes and PCR markers showing polymorphism between both the parents and bulks were used to genotype the mapping population. Although BE590674, BE442682, and mwg77 showed polymorphism only between the parents, and not the bulks, they were also mapped because of their known location in the short arm of chromosome group 1 (Peng et al., 2004).

RFLP Probes and PCR Primers
Probes and primers for the mapped markers in Fig. 1 were obtained from the following sources: mwg938, mwg36, and mwg77 from A. Graner (Graner et al., 1991); ksuD14 and ksuF43 from B. S. Gill (Gill et al., 1991); bcd1434 from M. Heun (Heun et al., 1991); Hor2 from Anders Brandt (Carlsberg Laboratory, Copenhagen, Denmark); Wheat ESTs BE403717, BE438866, BF475048, BE590674, BE442682, and BE405778 from O. Anderson (Peng et al., 2004); scb241 and SSR marker rems1303 from V. Korzun (Khlestina et al., 2004); iag95 from P. Wehling (Philipp et al., 1994); iB267 from R. Mago (Mago et al., 2002); and cwaP56170 and cwaP57173, from Lapitan laboratory (this study).


Figure 1
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  		Figure 1. (A) Genetic map of Dn7 in rye chromosome arm 1RS and (B) physical map location of markers on wheat group 1 chromosome based on previous studies (Sandhu et al., 2001; Peng et al., 2004).

 
Polymerase Chain Reaction (PCR) Analysis
PCR reactions were performed in a PTC-200 MJ Thermocycler (MJ Research, Inc., Waltham, MA). The PCR procedure was the same as in Peng et al. (2000b). The amplification products were separated on 3% (w/v) agarose or 7% (w/v) polyacrylamide gels or on a 5% (w/v) denaturing polyacrylamide gel. The gels were visualized with ethidium bromide (0.5 µg/mL) or silver staining.

Development of AFLP-derived Markers
AFLP reactions with PstI and MseI restriction enzymes were first performed on the parental lines as well as the two bulks. AFLP analysis was performed using the standard protocol (Peng et al., 2000a; Vos et al., 1995). For selective amplification, PstI and MseI primers with three additional nucleotides were used. Silver staining was used to visualize the gel. The primers and adaptor sequences used in this study were as described in Peng et al. (2000a).

AFLP fragments that were polymorphic between both the parents and the bulks were carefully excised from the 5% (w/v) denaturing polyacrylamide gel. Gel fragments containing the DNA were re-hydrated in 100 µL of deionized H2O for 3 min, crushed, and incubated in boiling water for 10 min. The supernatant was transferred to a new 1.5-mL Eppendorf tube after being centrifuged at 10700 g for 15 min. DNA was precipitated overnight at –20°C by adding 10 µL of 3 M sodium acetate (pH 5.2) and 2.5 volume of ethanol. The pellet was washed in 70% (v/v) ethanol and resuspended in 10 µL of deionized H2O. Two microliters of the supernatant was used as template to re-amplify the fragment using primers and reaction conditions similar to those for AFLP. The PCR products were separated on a 1.1% (w/v) agarose gel, excised, and purified with QIA quick gel extraction kit (Qiagen, Inc., Valencia, CA). The PCR product was cloned into pGEM-T easy vector (Promega, Madison, WI) according to the manufacturer's instructions. Because of the simultaneous migration of the different AFLP fragments of the same size, it was difficult to identify the correct fragment. Therefore, 15 clones were selected on the basis of their insert size after being digested with EcoRI and were sequenced at SeqWright (Houston, TX). These clones were divided into four groups on the basis of sequence similarities. Oligonucleotide primers were designed from each of the two largest groups using the primer3 software program http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi; verified 8 Jan. 2007) and were rescreened on the parents and the bulks.

Genetic Mapping
Mapmaker/EXP 3.0b (Lander et al., 1987; Lincoln et al., 1992) was used to construct the linkage map. A threshold LOD score of 3.0 was used in the mapping analysis. Centimorgan units were calculated using the Kosambi mapping function (Kosambi, 1944).

Evaluation of PCR-Based Marker-Assisted Selection for RWA Resistance
The accuracy of markers linked to Dn7 for MAS of resistance to RWA was calculated using the empirical formula described in Peng et al. (2000b). Accuracy refers to the percentage of homozygous resistant plants among the total number of plants containing the resistant parent-type marker in the F2 generation.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Genetic Map of Dn7
A genetic map of RWA resistance gene Dn7 located in chromosome 1RS/1BL was constructed (Fig. 1A). The map contains 19 markers consisting of RFLPs, wheat ESTs, RGAs, rye SSRs, and AFLP-derived STS markers. Fifteen of the markers are clustered around Dn7 in an 8-cM region (Fig. 1A, Table 1). The markers closest to Dn7 are XHor2 on one side and Xscb241 on the other side. XHor2 is 1.4 cM, and Xscb241 is 1 cM, from Dn7. Xiag95 was previously mapped distal to Xmwg36 (Mago et al., 2002), and it can be deduced that XHor2 is distal, and Xscb241 is proximal, to Dn7.


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  		Table 1. Markers linked to Dn7 and genetic distances from the gene.

 
Previous physical mapping in chromosome 1RS located Xiag95 and Xmwg36 in the distal end of the chromosome arm (Mago et al., 2005). Eight of the markers mapped in this study were physically mapped in wheat using deletion lines (Peng et al., 2004; Sandhu et al., 2001) and were also located in a terminal region (called IS-0.86–1.00) of the short arm of wheat consensus group 1 chromosome (Fig. 1B). Wheat and rye chromosomes are highly syntenic (Devos et al., 1993), and on the basis of this, it can be inferred that Dn7 is at the terminal region of chromosome arm 1RS. Genes for resistance to leaf rust, stripe rust (caused by Puccinia striiformis Westend.), stem rust, and powdery mildew (caused by Erysiphe graminis DC. f. sp. tritici Em. Marchal) have been mapped in the terminal region of group 1 chromosomes in wheat and chromosome 1 in rye (Devos et al., 1993; Naranjo et al., 1987). The rye rust resistance genes Sr31, Lr26, and Yr9 are proximal to Xmwg36 (Mago et al., 2005), and therefore to Dn7 as well.

Development of PCR-Based Markers for Marker-Assisted Selection of Dn7
Five PCR-based markers were linked to Dn7 (Table 2). Xib267 and Xiag95 were previously developed for MAS of stem rust resistance gene, SrR, from rye (Mago et al., 2002). Xrems1303 is a SSR marker developed from the rye EST database (Khlestina et al., 2004). Primers for the other two markers were designed in this study. BF475048 primers were developed from a wheat EST that contains a nucleotide-binding site leucine rich repeat domain (Dilbirligi et al., 2004) characteristic of many plant R genes (Huang et al., 2003; Mindrinos et al., 1994; van der Linden et al., 2004). The BF475048 primers (Table 2) amplified multiple bands, of which only the 1.7-kb band was polymorphic. This band mapped 5 cM from Dn7. XcwaP57173 was derived from an AFLP band generated from PstI/MseI enzyme–primer combinations and designated according to McIntosh et al. (2003). The DNA sequences of primers for these markers are included in Table 2.


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  		Table 2. PCR-based markers linked to Dn7 and primer sequences.

 
EST-derived SSR markers were also tested in this study. Eleven wheat eSSRs selected from the most distal end of wheat chromosome 1S (Peng and Lapitan, 2005) were tested for polymorphism between the mapping parents and the bulk segregants. Five eSSRs showed polymorphism between the parents but were monomorphic between the bulks, possibly indicating very small differences between resistant and susceptible progeny around the target region. It is also possible that the polymorphic fragments between the parents were far from Dn7 or even located in chromosomes other than 1RS.

We tested the usefulness of the markers for MAS of RWA resistance on 19 wheat cultivars, including the parents of the mapping populations (Fig. 2 , Table 3). The selection includes cultivars used at the CSU Wheat Breeding program and other cultivars containing 1RS/1BL or 1RS/1AL translocation chromosomes.


Figure 2
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  		Figure 2. Testing of PCR markers linked to Dn7 on 19 wheat cultivars. A) Xrems1303 and B) Xib26. Lanes 1 through 20 represent 50-bp DNA ladder (Invitrogen, Carlsbad, CA), 94M370, Gamtoos, Amigo, KS91WGRC14, MA1, MA2, Bobwhite ‘S’, Above, TAM107, ST-ARS02RWA2414-11, Prairie Red, CI2401-A2, Halt, Stanton, Ankor, Lakin, Avalanche, Betta, and Baviaans, respectively. R and S stand for resistance and susceptibility to Russian wheat aphid biotype 2, respectively.

 

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  		Table 3. Wheat cultivars and accessions screened with PCR-based markers linked to Dn7.

 
Xrems1303, which is 3.4 cM from Dn7 on the distal side (Fig. 1), amplified rye fragments from all the 12 cultivars containing 1RS (Fig. 2A, Table 3). A 290-bp band was amplified from all these 12 cultivars. A second band, 320 bp in size and possibly a duplication of the 290-bp band, was only present in 94M370 and in another highly resistant cultivar, 2414-11. The strong association of the 320-bp band with RWA resistance and the ease of distinguishing it from the 290-bp band makes it a useful marker for selection of RWA resistance. Xib267, which is proximal to Dn7, was linked to the susceptible locus in Gamtoos (or in repulsion to Dn7) at a distance of 2.6 cM (Fig. 1). A single band of 225 bp was amplified in five susceptible cultivars, all of which contain the Petkus 1RS chromosome (Fig. 2B, lanes 3, 5, 6, 7, and 8). Although the resistant lines 94M370 and 2414-11 also contain portions of 1RS from Petkus, the Xib267 band was missing from these lines (Fig. 2B, lanes 2 and 11). This indicates that the region containing Xib267 has undergone recombination with a 1RS chromosome containing Dn7 or another RWA resistance gene (in the case of 2414-11). The Xib267 band was not amplified either in the susceptible cultivars containing the 1RS/1AL chromosome. This band therefore appears to be very specific for the intact Petkus 1RS chromosome fragment.

Two other markers, Xiag95 and XcwaP57173, were also tested but proved to be less useful than Xrems1303 and Xib267 for MAS (not shown). Xiag95 primers amplified two closely associated rye fragments, approximately 1 and 0.95 kb, in the resistant parent 94M370 and only the 1-kb band in the susceptible parent. The 0.95-kb band was also present in 2414-11. However, the small size difference between the two bands made it difficult to distinguish the bands using horizontal gel electrophoresis. Furthermore, additional alleles were observed in other susceptible cultivars containing 1RS, making Xiag95 cumbersome and less informative for MAS. XcwaP57173 amplified a 173-bp fragment in Gamtoos that was linked to the susceptible locus. However, not all susceptible cultivars containing 1RS contained this band. In addition, this marker amplified four wheat bands in all the cultivars tested and hence is not informative for MAS.

Synteny between the 1RS Region Containing Dn7 and Rice
To explore the feasibility of using rice genomic sequences to find additional markers for the Dn7-containing region of 1RS, we first determined whether there is synteny between this region and the rice genome. The group 1 chromosome of the Triticeae shares conserved linkage groups with rice chromosomes 5 and 10 (Ahn et al., 1993; Gale and Devos, 1998; Peng et al., 2004). Sequences for five mapped ESTs (BF475048, BE403717, BE438866, BE590674, and BE442682) and Hor2 (Accession No. X03103) were used to conduct BLASTn homology searches against all rice BAC and PAC sequences in GenBank. On the basis of a criterion of E ≤ e –10, only two of the six markers had hits with rice genomic sequences. BF475048 and BE403717 showed significant homology with rice chromosomes 11 and 10, and rice chromosomes 6, 2, and 1, respectively. On the basis of this limited number of markers, it appears that there is no synteny between the Dn7-containing 1RS region and the rice genome.


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This paper presents a high-density genetic map for the Dn7 gene conferring resistance to RWA biotypes 1 and 2. The map contains 15 markers in addition to those included in the map of Anderson et al. (2003). Four markers (Xmwg2062, Xbcd1434, Xksud14, and Xmwg36) were also previously mapped in Anderson et al. (2003) and have the same order in the two maps. However, there is a discrepancy in the location of Dn7 relative to Xksud14 in the two studies. While Dn7 mapped distal to Xksud14 in the previous study (Anderson et al., 2003), it mapped proximal to Xksud14 in this study. Misscoring of phenotype or marker genotype in the previous or in the current study may explain this difference. Another discrepancy between the two maps was the difference in genetic distances between the markers and Dn7. The region between Xmwg2062 and Xmwg36 was over two times greater in distance in Anderson et al. (2003) compared with the current map (19 vs. 8 cM, respectively). These conflicting results may be explained in part by the difference in the mapping populations used in each study. F2–derived F3 families were used by Anderson et al. (2003), whereas we employed recombinant inbred lines. To resolve these contradictions, a new F2 population was developed from a cross between 94M370 and Gamtoos, and PCR-based markers in the current map (Table 2) were mapped using this population. The order of markers and genetic distances relative to Dn7 in this new F2 population are similar to those obtained for the recombinant inbred population used in the current study (not shown). On the basis of this additional data, we believe that the current map presented reflects the true order and distances of markers around the Dn7 gene.

The current map for the 1RS chromosome region containing Dn7 is consistent with previous maps for this chromosome (Khlestina et al., 2004; Korzun et al., 2001; Mago et al., 2002, 2005). The order of markers previously mapped (i.e., Xbcd1434, Xksud14, XHor2, and Xmwg36) in wheat (Gill et al., 1996; GrainGenes, 2006) is also consistent with their order in the current map. Four wheat ESTs were previously located on the most distal region of the short arm of wheat group 1 chromosome (Peng et al., 2004), and their orders relative to each other were determined in this study.

The resistant parent, 94M370, also contains genes for Lr26 and Sr31 (Marais et al., 1998). Marais et al. (1998) mapped Dn7 14 cM from Lr26, but the orientation of the genes in the chromosome was not known. Mago et al. (2005) mapped Lr26, Sr31, and Yr9 proximal to Xmwg36, indicating that Dn7 is distal to this rust resistance gene complex. The 1RS segment harboring Dn7 gene presented in the map appears to be physically located at the distal region of the chromosome, on the basis of comparison with physical maps of Triticeae group 1 chromosomes (Peng et al., 2004; Sandhu et al., 2001).

Recent comparative mapping studies between wheat and rice have revealed little or no synteny between the distal ends of Triticeae chromosomes and the corresponding homeologous rice chromosome (Ahn et al., 1993; Gale and Devos, 1998; Peng et al., 2004). This is what we also observed when we performed a BLASTn homology search of six markers linked to Dn7 against the rice genomic sequence database. Using a relatively high stringency of E ≤ e–10 to declare a hit, only one marker (BF475048) had a hit with the homeologous rice chromosome 10. This same marker also showed significant homology with another rice chromosome (rice 11). Interestingly, the amplified 170-bp wheat fragment from the BF475048 primers in Table 2 shared homology with a 170-bp fragment from rice chromosome 11 (88697–88525 bp in BAC AC119670), indicating that these wheat and rice fragments are likely orthologous. The 1.7-kb BF475048 fragment that mapped to the distal end of 1RS appears to be a duplicated fragment. The lack of homology between the ends of wheat chromosomes with rice agrees with previous studies and may indicate that gene evolution, particularly duplications, occurs preferentially at the ends of chromosomes (Akhunov et al., 2003a; See et al., 2006). This may explain why many genes for species-specific resistance to diseases and insects are located at chromosome ends (Leister et al., 1998; Schnable et al., 1998). The ends of wheat chromosomes have also been shown to have higher recombination frequencies than the regions closer to the centromere (Akhunov et al., 2003b). This would mean that 1 cM in this region would be much less than the predicted size of 4 Mb [total map length {approx}4200 cM (Messmer et al., 1999); genome size {approx}1.7 x 1010 bp], which is an advantage in terms of map-based cloning. The closest marker to the Dn7 gene (Xscb241) is still 1.0 cM away. Additional markers will need to be developed for this region. While it would be more practical to construct a saturated map of Dn7 in diploid rye, we and other researchers (C.M. Smith, personal communication) have not been able to find resistance to RWA in the reported donor, Turkey 77 (Marais et al., 1994). One explanation for the lack of success in identifying resistance in Turkey 77 is that it is heterogeneous and the resistant genotype occurs at a low frequency. It may also be possible that outcrossing occurred between Turkey 77 and other rye cultivars grown out in the field (for quarantine purposes) before screening for RWA resistance (Marais, personal communication). Until the rye donor is found, saturation of the Dn7 map will have to be done in hexaploid wheat.

Four PCR markers within 5 cM of Dn7 were tested for MAS of Dn7. Three of these markers (Xiag95, Xrems1303, and Xib267) amplified bands from rye fragments only. The AFLP-derived marker, XcwaP57173, amplified bands from both wheat and rye. The most effective of these markers for MAS of Dn7 is Xrems1303. While it amplifies a band (290 bp) from all wheat cultivars tested that contains a 1RS chromosome, the 320-bp band associated with resistance was only present in the highly resistant cultivars 94M370 and 2414-11. The presence of this band in 2414-11 was surprising. Even though this line contains the 1RS/1BL chromosome, its resistance has been assumed to come from wheat because the 1RS donor, ‘Custer’, is susceptible to RWA. Whether 2414-11 has undergone recombination with another wheat containing Dn7 is not known. At the phenotype level, resistance in 2414-11 to RWA biotype 2 is similar to that in 94M370. Studies are underway to map the resistance gene in 2414-11. On the basis of its genetic distance from Dn7, Xrems1303 will accurately predict a homozygote resistant F2 86% of the time. Xib267 is useful for selecting against susceptible genotypes containing the 1RS/1BL translocation chromosome, but not those containing 1RS/1AL. When Xib267 is used in conjunction with Xrems1303, the accuracy of identifying a homozygote resistant F2 is 98%.

With the development of new biotypes of RWA, the importance of Dn7 as a source of resistance has increased because of the broad-spectrum resistance it provides against several biotypes. The markers developed in this study will be useful for facilitating the transfer of Dn7 into wheat cultivars with or without translocation chromosomes containing 1RS. Although wheat cultivars containing the 1RS chromosome arm have not been used in many wheat breeding programs because of its undesirable effects on bread making quality, the 1RS/1BL and 1RS/1AL chromosomes occur in 11.4% of 210 American entries in the 1989 major wheat nurseries in the US (Lukaszewski, 1990). Of these, 4.3% contained the 1RS/1AL chromosome and 7.1% contained 1RS/1BL. Ten percent of wheat cultivars grown in Great Britain from 1980 to 1985 contained the 1RS/1BL chromosome, while 22.8% of cultivars grown in West Germany from 1983 to 1984 had the 1RS/1BL. The reason for the widespread occurrence of the 1RS/1BL chromosome may be attributed to the disease resistance genes located on it and the yield advantage associated with this translocation chromosome (Lukaszewski, 1990). Markers reported in this study may also be used for validation of the presence of the 1RS/1BL translocation.


   ACKNOWLEDGMENTS
 
This study was partially supported by the US Department of Agriculture under Cooperative Agreements USDA Contract No. 2001-52100-11293, USDA Contract No. 2003-34205-13636, USDA Contract No. 2006-55606-16629, the Colorado Wheat Research Foundation, and Hatch Funds. We are grateful to Dr. Frank Peairs and Jeff Rudolph for providing Russian wheat aphids and valuable advice. We thank Hong Wang and Dr. M. Tahir for technical assistance, and Drs. Dan Papa and Ahmad Arzani for their aid in developing the recombinant inbred lines.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Received for publication August 18, 2006.


   REFERENCES
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 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