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Published online 7 November 2007
Published in Crop Sci 47:2418-2429 (2007)
© 2007 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Molecular Mapping of the Russian Wheat Aphid Resistance Gene Dn2414 in Wheat

Junhua Penga,b,*, Hong Wanga, Scott D. Haleya, Frank B. Peairsc and Nora L.V. Lapitana,*

a Lapitan, Soil and Crop Sciences Dep., Colorado State Univ., Fort Collins, CO 80523-1170
b current address: Wuhan Botanical Garden/Institute, The Chinese Academy of Sciences, Wuhan, Hubei 430074, China
c Dep. Bioagricultural Sciences and Pest Mgmt., Colorado State Univ., Fort Collins, CO 80523-1177

* Corresponding authors (nlapitan{at}lamar.colostate.edu, jpeng{at}lamar.colostate.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Russian wheat aphid (RWA) [Diuraphis noxia (Kurdjumov)] is an important pest of wheat (Triticum aestivum L.) in several production areas of the world. The most effective and economical approach for controlling RWA is to use resistant cultivars. A wheat line, ST-ARS 02RWA2414-11 (2414-11), showed a high level of resistance to RWA biotype 2. Our objectives were to map the resistance gene and develop polymerase chain reaction (PCR)–based markers for marker-assisted selection (MAS). A mapping population of 212 F2 individuals was developed from a cross of 2414-11 and the susceptible cultivar Yuma. The F2 individuals and F2:3 families were infested using biotype 2 RWA. The RWA resistance of 2414-11 is controlled by a single major gene, provisionally designated as Dn2414. Using standard PCR, 30 marker loci were found to be linked to Dn2414 with recombination frequencies ({theta}) of 0.00 to 0.27 and logarithm of the odds to the base 10 (LOD) scores of 7.6 to 66.1. Of the 30 markers, 26 were tightly linked to Dn2414 with {theta} ≤ 0.05. A genetic map was constructed consisting of 31 loci spanning a region of 34.7 cM. The close linkage of Dn2414 with several rye chromosome 1R short arm (1RS)-specific simple sequence repeat markers and low {theta} values around the Dn2414 gene indicate that Dn2414 is located on chromosome 1RS.1BL (translocation chromosome with IRS and wheat chromosome 1B long arm). Phenotypic and marker profiles of 2414-11 and its relatives are the same as other lines known to carry Dn7. The Dn2414 gene is thus located on 1RS arm, and the large number of PCR markers will be valuable for MAS of this gene.

Abbreviations: MAS, marker-assisted selection • PCR, polymerase chain reaction • RWA, Russian wheat aphid • SSR, simple sequence repeat; STS, sequence tagged site; 1BL, long arm of the wheat chromosome 1B; 1BS, wheat chromosome 1B short arm; 1RS, rye chromosome 1R short arm

Molecular Mapping of the Russian Wheat Aphid Resistance Gene Dn2414 in Wheat

Junhua Penga,b,*, Hong Wanga, Scott D. Haleya, Frank B. Peairsc and Nora L.V. Lapitana,*

a Lapitan, Soil and Crop Sciences Dep., Colorado State Univ., Fort Collins, CO 80523-1170
b current address: Wuhan Botanical Garden/Institute, The Chinese Academy of Sciences, Wuhan, Hubei 430074, China
c Dep. Bioagricultural Sciences and Pest Mgmt., Colorado State Univ., Fort Collins, CO 80523-1177

* Corresponding authors (nlapitan{at}lamar.colostate.edu, jpeng{at}lamar.colostate.edu).

Russian wheat aphid (RWA) [Diuraphis noxia (Kurdjumov)] is an important pest of wheat (Triticum aestivum L.) in several production areas of the world. The most effective and economical approach for controlling RWA is to use resistant cultivars. A wheat line, ST-ARS 02RWA2414-11 (2414-11), showed a high level of resistance to RWA biotype 2. Our objectives were to map the resistance gene and develop polymerase chain reaction (PCR)–based markers for marker-assisted selection (MAS). A mapping population of 212 F2 individuals was developed from a cross of 2414-11 and the susceptible cultivar Yuma. The F2 individuals and F2:3 families were infested using biotype 2 RWA. The RWA resistance of 2414-11 is controlled by a single major gene, provisionally designated as Dn2414. Using standard PCR, 30 marker loci were found to be linked to Dn2414 with recombination frequencies ({theta}) of 0.00 to 0.27 and logarithm of the odds to the base 10 (LOD) scores of 7.6 to 66.1. Of the 30 markers, 26 were tightly linked to Dn2414 with {theta} ≤ 0.05. A genetic map was constructed consisting of 31 loci spanning a region of 34.7 cM. The close linkage of Dn2414 with several rye chromosome 1R short arm (1RS)-specific simple sequence repeat markers and low {theta} values around the Dn2414 gene indicate that Dn2414 is located on chromosome 1RS.1BL (translocation chromosome with IRS and wheat chromosome 1B long arm). Phenotypic and marker profiles of 2414-11 and its relatives are the same as other lines known to carry Dn7. The Dn2414 gene is thus located on 1RS arm, and the large number of PCR markers will be valuable for MAS of this gene.

Abbreviations: MAS, marker-assisted selection • PCR, polymerase chain reaction • RWA, Russian wheat aphid • SSR, simple sequence repeat; STS, sequence tagged site; 1BL, long arm of the wheat chromosome 1B; 1BS, wheat chromosome 1B short arm; 1RS, rye chromosome 1R short arm


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
RUSSIAN WHEAT APHID (RWA) is an economically important insect pest of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) in several production areas of the world. The RWA was first discovered in the United States in 1986 (Webster et al., 1987) and has caused extensive damage to wheat production. Yield losses and increased production costs associated with RWA infestations were estimated to be >$800 million in the first 10 yr after its introduction (Morrison and Peairs, 1998) and additional losses have occurred since then (F.B. Peairs, unpublished data, 2007). Control of this pest in the United States has largely relied on host-plant resistance genes effective against biotype 1 RWA (Smith et al., 2004). Most RWA-resistant wheat cultivars planted commercially in the United States have relied on the Dn4 gene from PI 372129 (Collins et al., 2005a). In Colorado, more than 25% of the wheat acreage has been planted to cultivars having the Dn4 gene over the past 5 yr (Colorado Agricultural Statistics Service, 2004). However, the emergence of a new biotype (biotype 2) of RWA in 2003 poses a threat to existing wheat cultivars containing resistance genes against biotype 1, particularly Dn4 and Dny (Haley et al., 2004a; Collins et al., 2005a; Jyoti and Michaud, 2005; Qureshi et al., 2006).

Although the identification of biotype 2 represents the first detectable change in RWA populations in the United States, it is not necessarily the last (Qureshi et al., 2006). The RWA populations from other parts of the world already show considerable biotypic variation (Shufran et al., 1997; Basky, 2003) and Puterka et al. (1992) recognized at least eight RWA biotypes worldwide. In the United States, at least eight RWA biotypes were also recently identified, and biotype 2 is currently the predominant biotype (F.B. Peairs, unpublished data, 2007). Therefore, biotypic variation in RWA, whether introduced from exotic sources or evolved in situ, will likely continue to pose a serious threat to wheat production in the United States, mostly the west central Great Plains (Qureshi et al., 2006). Host-plant resistance is the most cost-effective and environmentally safe means for controlling RWA. Continuous efforts are necessary to identify and introduce additional resistance genes into commercially acceptable cultivars.

Among the 11 RWA resistance genes identified in wheat, rye (Secale cereale L.), and goatgrass (Aegilops tauschii Coss.) (Smith et al., 2004), only the rye-derived Dn7 is resistant to all the U.S. biotypes including biotype 2 (Anderson et al., 2003; Haley et al., 2004a; Lapitan et al., 2007). The Dn7 gene has also been shown to provide effective protection from yield losses in field experiments (Collins et al., 2005b). However, Dn7 is located on a rye chromosome 1R short arm (1RS).1BL (long arm of wheat chromosome 1B) wheat–rye translocation (Marais et al., 1994, 1998) which is associated with poor bread-making quality (Graybosch et al., 1990). In an effort to identify additional wheat germplasm accessions that confer resistance to biotype 2 RWA present in the western and southern Great Plains regions, Collins et al. (2005a) reported that a USDA-ARS breeding line ST-ARS 02RWA2414-11 (2414-11 hereafter) is a promising resistance source. Among a group of biotype 1-resistant germplasm accessions tested for resistance to biotype 2 RWA, 2414-11 was among a small set of accessions that showed a similar level of resistance as the Dn7 gene (Collins et al., 2005a).

The availability of polymerase chain reaction (PCR)-based molecular markers and genomic tools in Triticeae species allows for mapping and marker development of RWA resistance in 2414-11. This resource includes a large number of wheat SSRs (Bryan et al., 1997; Guyomarc'h et al., 2002; Röder et al., 1998; Somers et al., 2004; Song et al., 2002), some rye simple sequence repeats (SSRs) (Hackauf and Wehling, 2002; Khlestina et al., 2004), and deletion bin system of wheat SSRs (Sourdille et al., 2004). Our objectives in the present study were (i) to map the gene conferring RWA resistance in 2414-11 and (ii) to develop PCR-based molecular markers for marker-assisted selection of the resistance gene.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Materials
A cross was made between the RWA resistant wheat line 2414-11 and the RWA susceptible wheat cultivar Yuma. The 2414-11 was developed by the USDA-ARS from the cross ‘Custer’*3/3/‘Karl 92’//‘Chisholm’/PI 366515 (C. Baker, personal commununication, 2006). An F2 mapping population consisting of 212 individuals was developed by bagging the heads of a single F1 plant of 2414-11/Yuma to ensure selfing. The F2–derived F3 families were obtained by bagging spikes of the F2 individuals. In addition, 46 wheat breeding lines or cultivars, including the mapping parents and sister lines or progeny of 2414-11, were used to test the usefulness of PCR-based markers for marker-assisted selection (MAS) of RWA resistance derived from 2414-11.

Phenotyping of Reaction to Russian Wheat Aphid Infestation
The F2 individuals, F2–derived F3 families of the mapping population, and the cultivars and breeding lines were subjected to RWA infestation in standard greenhouse seedling screening tests as described by Nkongolo et al. (1989). The RWA screening and scoring of the vernalized (2°C for 8 wk) F2 individuals were conducted in root-trainers (Hummert International, www.hummert.com; verified 7 Sept. 2007) in the autumn of 2005. After scoring, the F2 plants were immediately transplanted to the greenhouse to obtain the F3 families. For the F2–derived F3 families and the breeding lines and cultivars, 12 seeds were planted in flats (52.0 by 25.5 cm) in a single row and were infested in the spring of 2006 with RWA biotype 2 as described by Nkongolo et al. (1989). The 2414-11 and Yuma lines were included in each flat of the F3 families while ‘Gamtoos’ (susceptible to all RWA biotypes) and ‘Halt’ (containing Dn4 and susceptible to biotype 2) served as the susceptible controls. The wheat line 94M370 containing the Dn7 gene (Anderson et al., 2003; Marais et al., 1994) served as the resistant control. Reaction to RWA infestation was rated at 7, 14, and 21 d after infestation and plants were scored as resistant (R) or susceptible (S) based on leaf shape (rolling or flat).

Tissue Collection and DNA Extraction
Before the elongation stage of wheat plants, approximately 0.1 g of young leaf tissue was collected from each of the individual F2 plants, the mapping parents, and the wheat cultivars and breeding lines. The tissue was placed in a 2 mL Eppendorf tube, immediately frozen in liquid N, and stored in a –80°C freezer. Total genomic DNA was extracted using extraction buffer (pH 7.8–8.0) consisting of 500 mM NaCl, 100 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 0.84% (w/v) SDS, and 0.38% (w/v) sodium bisulfite, for each of the F2 individuals as well as the parental lines following the procedure modified from Edwards et al. (1991).

Bulked Segregant Analysis and Polymerase Chain Reaction Analysis
To find potential markers linked to the RWA resistance in 2414-11, bulked segregant analysis (Michelmore et al., 1991) was used. The resistant and susceptible bulks were prepared by pooling equal amounts of DNA from 10 homozygous resistant and 10 homozygous susceptible F2 individuals, respectively. The mapping parents and bulks were screened for polymorphisms using PCR-based molecular markers. The PCR reactions were performed in PTC-200 MJ Thermocyclers (MJ Research, Inc., Watertown, MA). The PCR procedure was the same as in Peng et al. (1999). The amplification products were separated on a 3% agarose or on a 5% denaturing polyacrylamide gel. The gels were visualized with ethidium bromide (0.5 µg/mL) or silver staining.

To date, most of the RWA-resistant genes have been mapped to group 1 or 7 Triticeae chromosomes (Anderson et al., 2003; Arzani et al., 2004; Lapitan et al., 2007; Liu et al., 2001, 2002, 2005). Thus, we initially tested 23 SSR markers located on group 1 and 7 chromosomes. We then tested an additional 80 wheat 1B or rye 1R SSRs. The markers detecting polymorphisms between both the parents and bulks were used to genotype the mapping population. Based on the proportions of homozygotes in the F2, the frequencies of both the Yuma and 2414-11 alleles were estimated for each locus. The primer sequences and sources of markers mapped in this study are shown in Table 1


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  		Table 1. Primer sequences of Dn2414-linked polymerase chain reaction (PCR) markers.

 
Genetic Mapping
All the markers and the Dn2414 gene were subjected to Chi-square analysis to test for segregation pattern, 3:1 or 1:2:1. Mapmaker 3.0/EXP 3.0 (Lander et al., 1987; Lincoln et al., 1992) was used to construct the linkage map. The ORDER and RIPPLE commands in Mapmaker were used to ensure a robust marker order. Two-point tests were also used to estimate the pair-wise linkage between markers and the RWA resistance gene in 2414-11. A threshold logarithm of the odds to the base 10 (LOD) score of 3.0 was used in the mapping analysis. CentiMorgan units were calculated using the Kosambi mapping function (Kosambi, 1944).

Marker Analysis of Wheat Cultivars and Breeding Lines
To detect the possible source of the RWA resistance gene in 2414-11 and determine the effectiveness of the PCR-based markers in MAS, four marker pairs, Xiag95 vs. Xgwm11, Xrems1303 vs. Xgwm11, Xiag95 vs. Xgwm18, and Xrems1303 vs. Xgwm18, were used to test 46 wheat cultivars or breeding lines containing different RWA resistance genes. Xiag95 and Xrems1303 are 1RS specific and linked to the resistance gene in coupling phase. Xgwm11 and Xgwm18 are 1BS specific and linked to the resistance gene in repulsion phase. The multiplex PCR procedure (Henegariu et al., 1997) was used in this experiment.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Genetic Analysis of Russian Wheat Aphid Resistance in 2414-11
The individual F2 plants and the corresponding F2–derived F3 families, as well as the parental lines of the mapping population were infested using biotype 2 RWA to determine the reaction of individual F2 plants to RWA infestation and also identify the genotype of individual F2 plants [e.g., homozygote resistant (RR = A), homozygote susceptible (rr = B), heterozygote resistant (Rr = H) (Table 2 )]. Among the 212 F2 individuals, 98 had the H, 43 had the A, and 71 had the B genotype. This segregation pattern showed a higher than expected frequency of the susceptible allele and significantly deviated from the expected 1:2:1 ratio (P < 0.05). However, the ratio of (H+B) to A fits the expected ratio 3:1 of a single dominant gene model ({chi}2 = 2.516, P > 0.05) (Table 2), indicating that the RWA resistance in 2414-11 is conferred by a single dominant gene. The gene for RWA resistance in 2414-11 is provisionally designated as Dn2414 according to the nomenclature of McIntosh et al. (2003).


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  		Table 2. Segregation and allele frequency of Dn2414 gene and its linked markers.

 
Segregation Patterns of Dn2414-linked Polymerase Chain Reaction Markers
Out of 30 marker loci linked to Dn2414, nine showed significant segregation distortion (P < 0.05) and the other 21 demonstrated the expected segregation ratio of 3:1 (Table 2). All nine segregation-distorted marker loci were either codominant or dominant with linkage to Dn2414 in coupling phase. All the dominant marker loci linked to Dn2414 in repulsion phase showed the expected 3:1 segregation (P > 0.05).

The frequencies for Yuma alleles were higher than those for 2414-11 for most of the markers. According to these estimated allele frequencies, an additional seven markers linked to Dn2414 in repulsion phase showed significant deviation from the expected ratio 1:2:1 (Table 2). Therefore, distorted segregation may be underestimated for dominant markers if we only take into consideration the phenotypic ratios rather than the allele frequencies.

Polymerase Chain Reaction-based Markers and Genetic Map for Dn2414
Among a set of 23 markers located on group 1 and 7 chromosomes, we found four group 1 markers including two Dn7-linked markers, Xrems1303 and Xiag95, which showed polymorphism between both the parents and the two bulks (Fig. 1a ). We then screened an additional 80 1B and 1R markers. Based on the results of bulked segregant analyses and known chromosome locations (Sourdille et al., 2004) of specific markers, we chose 32 SSR and sequence tagged site (STS) primer pairs to genotype the mapping population. In total, 50 PCR-based marker loci were generated. Figures 1b, 1c, and 1d show the banding patterns of PCR markers, Xgwm18, Xiag95, and Xrems1303, respectively, and the corresponding genotypes of Dn2414 gene in the two mapping parents and 23 F2 individuals.


Figure 1
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  		Figure 1. Amplification profiles of polymerase chain reaction (PCR)-based markers in the Dn2414 mapping population. (a) Bulked segregant analyses of four markers, Xgwm11, Xgwm18, Xrems1303, and Xiag95. Lanes 1 to 4 represent 2414-11, ‘Yuma’, resistant bulk, and susceptible bulk, respectively. Panels (b), (c), and (d) show the PCR patterns of Xgwm18, Xrems1303, and Xiag95, respectively. Lanes P1 and P2 represent 2414-11 and Yuma, respectively. The black or white arrows indicate the diagnostic band of specific markers. Lanes 1 to 23 represent 23 F2 individual plants. The genotypes of RWA resistance gene Dn2414 are shown for specific lanes/individuals: RR = homozygous resistant, Rr = heterozygous resistant, rr = homozygous susceptible.

 
Using two-point analysis and the NEAR command of Mapmaker/EXP 3.0 (Lander et al., 1987; Lincoln et al., 1992), 30 out of the 50 marker loci were found to be linked with the RWA resistance gene Dn2414 (Table 3 ). The recombination frequencies between Dn2414 and marker loci ranged from 0.00 to 0.27, and the LOD values for the linkage ranged from 7.6 to 66.1. The majority (26) of these 30 marker loci have a recombination frequency ≤0.05 and a map distance ≤4.7 cM from the Dn2414 gene (Table 3). The PCR-based markers closely linked with Dn2414 showed very good association with the RWA resistance gene in the mapping population as demonstrated in Fig. 1. A combination of repulsion-phase marker (e.g., Xgwm18) and coupling-phase marker (e.g., Xiag95 and Xrems1303) can be used together as a codominant marker to distinguish the heterozygous resistant genotypes (Rr) from the homozygous resistant genotypes (RR).


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  		Table 3. Linkage between molecular markers and the Russian wheat aphid resistance gene Dn2414.

 
Among the 30 markers linked with Dn2414, 27 are SSRs and 3 are STSs. Out of these Dn2414-linked markers, only Xbarc181 and Xbarc61 are codominant and the rest are dominant. Over half (16) of these markers can be analyzed using agarose gels (Table 3), which would facilitate their use in marker-assisted transfer of the Dn2414 gene into the susceptible wheat germplasm.

To develop markers that are of practical use for MAS of Dn2414 in wheat RWA resistance breeding, we focused solely on the PCR-based SSR and STS markers identified. With the aid of Mapmaker/EXP 3.0 (Lander et al., 1987; Lincoln et al., 1992), a genetic linkage map of the Dn2414 region was constructed (Fig. 2 ). The present genetic map consists of 30 PCR-based marker loci that showed linkage with the Dn2414 gene and covers a region of 34.7 cM. In this genetic map, 20 marker loci cosegregated with the Dn2414 gene. The Dn2414 gene and the 20 marker loci were flanked by a triplet marker cluster (Xpsp3000, Xwmc230, and Xbarc137b) at 1.1 cM and an SSR marker (Xbarc148a) at 0.4 cM of genetic distances. Thus, a majority (24) of the 30 markers and the gene are clustered in a region of 1.5 cM in length (Fig. 2). The number of markers with close genetic linkage to the Dn2414 gene may be the highest in comparison to all the published genetic maps of the RWA resistance genes (Anderson et al., 2003; Arzani et al., 2004; Heyns et al., 2006; Lapitan et al., 2007; Liu et al., 2001, 2002; Miller et al., 2001; Myburg et al., 1998).


Figure 2
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  		Figure 2. High-density polymerase chain reaction (PCR)-based genetic map of rye chromosome 1R short arm (1RS).1BL (long arm of wheat chromosome 1B) chromosome carrying Russian wheat aphid (RWA) resistance gene Dn2414. The interval length (cM) is indicated on the left side, and marker order and names are shown on the right side of the genetic map.

 
The 26 markers closely linked to the Dn2414 gene (Table 3) are user-friendly PCR-based markers that will be useful for the efficient transfer of the RWA resistance gene into a broad range of wheat cultivars or germplasm. As shown in Fig. 1 and 3 , using a single combination of a coupling-linked and a repulsion-linked marker, we could precisely select for the homozygous or heterozygous resistant individuals in the segregating progeny/population concerning the Dn2414 gene. The MAS for RWA resistance will allow screening of plants even during periods of the year when the weather is too hot for growth and reproduction of aphids. The biggest advantage of using MAS for the RWA resistance breeding, however, is that it facilitates transfer of two or more resistance genes, allowing for breeding for resistance to several biotypes, or simultaneous selection for other traits in addition to the pest resistance.


Figure 3
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  		Figure 3. Multiplex polymerase chain reaction (PCR) amplification patterns of Xrems1303 + Xgwm18 in 46 wheat cultivars, accessions, and breeding lines. The black or white arrows indicate the diagnostic band of specific markers. The lane codes are corresponding to that of cultivars or lines shown in Table 4. Resistant genotypes are shown based on the infestation results of biotype 2 Russian wheat aphid (RWA): RR = homozygous resistant, Rr = heterozygous resistant, rr = homozygous susceptible. The size difference among Xgwm18 bands reflects polymorphism among the wheat cultivars or recurrent parents of backcross-derived breeding lines.

 

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  		Table 4. Wheat cultivars/accessions and breeding lines screened with biotype 2 Russian wheat aphid and polymerase chain reaction (PCR)-based markers.

 
Dn2414 Is Located in Chromosome 1RS.1BL
Many of the SSR markers included in the Dn2414 genetic map were physically mapped to specific intervals/bins or arms of wheat chromosome 1B (Sourdille et al., 2004). This enables us to place the Dn2414 gene to a specific chromosome bin or arm. Linkage of Dn2414 with the 1RS-specific markers in coupling phase, and with the 1BS-specfic dominant markers in repulsion phase indicate that Dn2414 is located in chromosome arm 1RS. Some of the wheat SSR markers are transferable among grass species though the proportion is low (Röder et al., 1995). Thus a couple of wheat SSRs, for example, Xgwm582, could amplify rye DNA fragments and show cosegregation with the Dn2414 in coupling phase. The 20 markers cosegregating with the Dn2414 gene span nearly the entire 1R or 1B short arm and the proximal part of the long arm of wheat chromosome 1B. The cosegregation of 1BL (C-1BL6-0.32) SSRs, Xbarc187, Xgwm264, and Xgwm582b with Dn2414 and 1BS SSR markers may be explained by suppression of recombination around the centromere. It is also possible that the proximal part of 1RL is also involved in the 1RS.1BL translocation.

In populations segregating for 1RS.1BL translocation, the observed frequencies of the different genotypes (homozygous for 1RS.1BL translocation, heterozygous for 1RS.1BL translocation, and homozygous for normal chromosome 1B) significantly deviated from the expected ratio 1:2:1 (de Froidmont, 1998; Rayburn and Mornhinweg, 1988; Schlegel and Meinel, 1994). In the present study, the Dn2414 gene and many of its linked markers also showed significant segregation distortion (Table 2). This result further corroborates that the Dn2414 gene is located on chromosome 1RS.1BL. The obtained data indicate that the gametes carrying the Yuma alleles have stronger vigor and higher competition ability than those with the 2414-11 alleles. Therefore, the cause for distorted segregation on the Dn2414 region appears to be gametic competition (Table 2), or a selection against gametes carrying the translocated chromosome 1RS.1BL (Schlegel and Meinel, 1994).

In a wheat composite map (http://wheat.pw.usda.gov/cgi-bin/graingenes/report.cgi?class=mapdata;name=Wheat%2C%20Composite%; verified 10 Sept. 2007), the genetic distances between Xpsp3000 and Xbarc187, and between Xwmc85 and Xbarc187 were approximately 57 and 36 cM, respectively. In the present Dn2414 map, the distance between the same sets of markers are 1.1 and 0 cM, respectively (Fig. 2). In a wheat consensus SSR map (Somers et al., 2004), the genetic distances from Xgwm264 to Xbarc187, Xgwm582, Xcfd48 and Xgwm153 are 11, 16, 19, and 40 cM, respectively. The genetic distances between these same marker intervals in the present study are 0, 0, 16.4, and 33.6 cM, respectively (Fig. 2). In a tetraploid wheat genetic map (Peng et al., 2000), the genetic distances from Xgwm273b to Xgwm18 and Xgwm153a were 5.3 and 35.6 cM, respectively. These distances are 0 and 33.6 cM, respectively in the present study (Fig. 2). The rye 1RS specific Xiag95 and Xrems1303 cosegregated with Dn2414 in the present study (Fig. 2), but were 1.9 cM apart in a rye background (Khlestina et al., 2004) and 1.5 cM in 1RS.1BL translocation (Lapitan et al., 2007). These comparisons indicate that recombination in the Dn2414 region was greatly suppressed and consequently resulted in the significant clustering of PCR markers around the RWA resistance gene (Fig. 2). This extremely low recombination frequency would be expected if the F1 hybrid contained one copy of 1RS.1BL and one copy of normal 1B chromosome. This further supports the conclusion that the Dn2414 gene is located in chromosome arm 1RS.

There is usually no recombination between chromosomes 1R from rye and 1A, 1B, or 1D from wheat even though they can substitute for each other (Sears, 1966). However, the homeologous recombination between rye and wheat chromosomes could be induced by ph1b or absence of the Ph1 gene (Lukaszewski, 1995, 2000; Lukaszewski et al., 2004). In the present study, we observed recombination between chromosome arms 1RS and 1BS (Fig. 2). All these homeologous recombination occurred in the distal region of 1RS and 1BS (Fig. 2) similar as demonstrated by Lukaszewski (1995) and Lukaszewski et al. (2004) for group 1, 2, and 7 chromosomes when the Ph1 gene was absent. This low frequency of recombination detected by molecular markers might not be detected via conventional approaches. The fact that several wheat cultivars/lines derived from crosses involving 1RS translocation showed both the 1RS-specific bands and 1BS-specific bands (Lapitan et al., 2007; Fig. 3; Table 4 ) corroborate the recombination between 1RS and the homeologous wheat chromosome 1 arms.

Marker Test of Wheat Cultivars and Breeding Lines
Table 3 shows that the 1RS-specific markers such as Xrems1303 and Xiag95 are closely linked with Dn2414 in coupling phase, while the 1BS-specific SSRs such as Xgwm11 and Xgwm18 are closely linked with Dn2414 in repulsion phase. These results suggest that the Dn2414 gene may be of rye origin. To further investigate this hypothesis, two 1RS-specific markers (Xiag95 and Xrems1303) and two 1BS-specific markers (Xgwm11 and Xgwm18) were used to test 46 wheat cultivars and breeding lines including four parental lines of 2414-11, 18 sister lines or progeny of 2414-11, eight 2414-11 derived BC1F2–derived breeding lines, and several Dn7-containing lines. Biotype 2 RWA evaluations were also conducted in the greenhouse for these 46 entries. The multiplex PCR pattern for Xrems1303+Xgwm18 is shown as an example in Fig. 3, and the marker data are summarized in Table 4.

The wheat line Altus 034 contains the RWA resistance gene Dn7 from rye (Porter et al., 2005). Five BC1F2–derived breeding lines from Altus 034 (code/lane # 20–24) showed the same genotype of RWA resistance and similar marker profile of Xiag95, Xrems1303, Xgwm11, and Xgwm18 as in the 2414-11 derived breeding lines (code/lane # 6, 7, 8, 10, 11, 13, 14, and 16) (Table 4, Fig. 3).

The wheat breeding line 2414-11 was derived from a composite cross involving four genotypes, Custer*3/3/Karl 92//Chisholm/PI 366515 (C. Baker, personal communication, 2006; Collins et al., 2005a). Biotype 2 RWA infestation showed that none of the four genotypes is resistant. Marker tests indicated that Custer and Karl 92 contained Xrems1303 fragment (specific to 1RS) similar to the fragment contained by the biotype 2-susceptible lines Gamtoos, CO00554, and Halt. All the four parental genotypes (Custer, Karl 92, Chisholm, and PI 366515) contain the 1BS segment. None of the four genotypes has the resistance genotypes and marker profiles as shown in 2414-11 and its related 18 lines (Table 4, Fig. 3). The 2414-11 and its sister lines originated from the breeding line 2001RWA1121 (lane 29 in Fig. 3) showing segregation in response to biotype 2 RWA infestation. However, as revealed by Xiag95, Xrems1303, Xgwm11, and Xgwm18, this original breeding line of 2414-11 and all its 18 relatives showed exactly the same marker patterns as the Dn7-containing 94M370 and Altus 034 (Table 4, Fig. 3). Therefore, Dn2414 might not have originated from any of the four genotypes, but from the same source as the Dn7 gene.

Wheat is a typical self-pollinated crop species with outcrossing rates assumed to be <1%, although outcrossing rates more than 1% have been reported for wheat plants grown in close proximity (Waines and Hegde, 2003). Lawrie et al. (2006) observed 0 to 3.5% outcrossing rates and unusually high record of >10% for specific cultivars in greenhouse conditions. If the pedigree Custer*3/3/Karl 92//Chisholm/PI 366515 of 2414-11 is correct, it is possible that a plant carrying the Dn2414 gene may have outcrossed to one of these parents during development of 2414-11.

As demonstrated in Lapitan et al. (2007), both Xiag95 and Xrems1303 have two bands, but only the upper one is associated with RWA resistance conferred by the rye-derived Dn7 (Fig. 3). All the 2414-11–derived lines (CO05710, CO05711, CO05712, CO05714, CO05718, CO05722, CO05723, and CO05725) showed segregation for resistance conferred by the Dn2414 gene (Fig. 3, Table 4). The marker bands generated by Xgwm11 and Xgwm18 and the diagnostic bands generated by Xiag95 and Xrems1303 were present in all of these eight breeding lines. The pure recurrent parental lines CO00739, ‘Bond CL’ (Haley et al., 2006) and Halt only have Xgwm11 and Xgwm18 bands and do not have resistance-associated Xiag95 and Xrems1303 bands. Thus, genotypes carrying Dn2414 can be determined using these four markers: homozygous resistant plants only have 1RS-specific resistance-associated Xiag95 and Xrems1303 bands, homozygous susceptible plants have 1BS-specific Xgwm11 and Xgwm18 bands and occasionally the 1RS-specific resistance-associated Xiag95 band, and heterozygous resistant plants have both the 1RS- and 1BS-specific bands (Table 4). Therefore, the developed PCR-based markers are effective for MAS of RWA resistance conferred by the Dn2414 gene.

Relationships among the Russian Wheat Aphid Resistance Genes
To date, 11 RWA resistance genes have been reported in wheat, rye, and goatgrass (Smith et al., 2004). The chromosome locations for nine of these genes are known. The Dn1, Dn2, Dn5, Dn6, Dn8, and Dnx genes were located in chromosome arm 7DS (Liu et al., 2001, 2002, 2005; Miller et al., 2001). The Dn1, Dn2, Dn5, Dn6, and Dnx genes are either alleles of a single locus or are closely related members of a Dn gene family (Liu et al., 2005). The Dn4 gene is located in chromosome arm 1DS (Liu et al., 2002; Arzani et al., 2004). The Dn9 gene is located in chromosome arm 1DL (Liu et al., 2001). The Dn7 gene is rye-derived, located in chromosome arm 1RS, and is the only one resistant to all the U.S. biotypes including biotype 2 (Anderson et al., 2003; Haley et al., 2004a; Lapitan et al., 2007). In the present study, we have mapped the Dn2414 gene also to chromosome arm 1RS where the Dn7 gene resides. The two rye-specific markers, Xiag95 and Xrems1303, are common in both the Dn7 and Dn2414 genetic maps. In the Dn7 map, Xiag95 and Xrems1303 are 4.9 and 3.4 cM away from Dn7, respectively (Lapitan et al., 2007). In the Dn2414 genetic map, Xiag95, Xrems1303, and Dn2414 are cosegregating (Fig. 2). This linkage difference between the Dn7 and Dn2414 maps is because of the suppressed recombination between chromosome arms 1RS and 1BS in the Dn2414 population in the present study. Based on the phenotypic reactions to RWA infestation and marker profiles of Dn7-containing and Dn2414-containing lines (Fig. 3, Table 4; Lapitan et al., 2007), Dn2414 and Dn7 may be identical, or different alleles located in the same locus. The further experiments are underway to reveal the genetic/allelic relationship of Dn2414 with Dn7 through crosses between 2414-11 (carries Dn2414) and 94M370 (carries Dn7) and between 2414-11 and Gamtoos (susceptible to RWA but contains 1RS.1BL translocation).


   ACKNOWLEDGMENTS
 
This study was partially supported by the U.S. Department of Agriculture under Cooperative Agreements USDA Contract No. 2001-52100-11293 and USDA Contract No. 2003-34205-13636; the National Research Initiative of USDA's Cooperative State Research, Education and Extension Service, CAP Grant No. 2006-55606-16629; the Colorado Wheat Research Foundation, and Hatch Funds. We sincerely thank C. Baker of USDA-ARS, Stillwater, OK, for providing seeds of 2414-11 and its relative lines. We also thank J. Stromberger for providing the F1 hybrids, J. Rudolph for providing biotype 2 RWA aphids and assistance in our RWA infestation experiments, and Drs. P. Byrne and J.S. Quick for critical review of the manuscript.


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Received for publication March 10, 2007.


   REFERENCES
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 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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