Published in Crop Sci. 44:1429-1433 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
Marker-Assessed Retention of Wheat Chromatin in Wheat (Triticum aestivum) by Jointed Goatgrass (Aegilops cylindrica) Backcross Derivatives
L. J. Kroissa,
P. Tempallia,
J. L. Hansenb,
M. I. Valesa,
O. Riera-Lizarazua,
R. S. Zemetrab and
C. A. Mallory-Smitha,*
a Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331
b Department of Plant, Soil, and Entomological Sciences, University of Idaho, Moscow, ID 83844-2339
* Corresponding author (Carol.Mallory-Smith{at}oregonstate.edu).
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ABSTRACT
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With the advent of herbicide-resistant wheat (Triticum aestivum L.), there is a concern with the potential for gene migration between wheat and jointed goatgrass (Aegilops cylindrica Host.). This is especially true for genes on the D genome, since this genome is shared by wheat and jointed goatgrass. To study the potential for gene migration, BC1 and BC2 plants were produced with jointed goatgrass as the male recurrent parent. To determine if wheat chromatin was retained at expected Mendelian frequencies in these backcrosses, 14 simple sequence repeats (SSR) markers associated with the long and short arms of the D genome chromosomes were used. Chi-square analysis showed that 12 of the 14 markers fit the expected ratio for retention of wheat alleles in the BC1 generation. In the BC2 generation, 11 of the 14 markers fit the expected frequencies for the retention of wheat alleles. The markers not fitting the expected frequencies in both generations deviated in the direction of more heterozygotes than expected, indicating a higher than expected retention of wheat alleles. Furthermore, recombination between the D genome chromosomes of the two species was observed. On the basis of these results, it appears that it is possible for a gene on the D genome of wheat to move into jointed goatgrass if the BC1 and BC2 generations were produced in the field.
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INTRODUCTION
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IMAZAMOX-RESISTANT WHEAT (2n = 6x = 42; AABBDD) cultivars are being commercially produced in the USA (Haley et al., 2003; Lazar et al., 2003) and glyphosate-resistant cultivars are being developed (Zhou et al., 2003). Since there is no selective control for jointed goatgrass (Aegilops cylindrica Host; 2n = 4x = 28; CCDD) in wheat, these herbicide-resistant cultivars allow the use of a herbicide to control jointed goatgrass. However, it is possible that these herbicide-resistant cultivars will have limited utility, if hybridization occurs between jointed goatgrass and the herbicide-resistant wheat. These hybrids may serve as a bridge for the introgression of herbicide-resistance genes from wheat to jointed goatgrass populations.
Partially female fertile wheat x jointed goatgrass F1 hybrids are possible in part because wheat and jointed goatgrass have the D genome in common (Kimber and Sears, 1987). The presence of homologous D genomes in the hybrid allows for normal bivalent formation and chromosome segregation of the D genome chromosomes at meiosis. Partial female fertility in wheat x jointed goatgrass hybrids has been observed in both the field and greenhouse (Mallory-Smith et al., 1996). The presence of seven bivalents, on the average, in the hybrid indicates that recombination and gene exchange could occur between the wheat and jointed goatgrass D genome chromosomes (Kimber and Zhou, 1983; Riley and Law, 1965; Zemetra et al., 1998).
A number of investigations have already examined fertility of the hybrids and backcross generations (Snyder et al., 2000; Zemetra et al., 1998; Wang et al., 2000b), retention of A and B genome chromosomes (Wang et al., 2000a), and retention of glutenin genes (Morrison et al., 2002). Conclusions from these studies suggested that it would take only two backcrosses to jointed goatgrass for partial restoration of self-fertility and that A and B genome chromosomes appear to be slowly eliminated as self-fertility and the normal chromosome number of jointed goatgrass is restored. These results also indicate that there is a greater potential for a gene on the D genome to be retained and transferred because of the homologous pairing of D genome chromosomes from the two species. In field trials involving imazamox [(RS)-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methoxymethylnicotinic acid]-resistant wheat, viable BC1 seed were found on wheat x jointed goatgrass hybrids from non-sprayed control plots (Seefeldt et al., 1998). Among seven BC1 plants, six were resistant to imazamox, demonstrating both the partial female fertility of the wheat x jointed goatgrass hybrid and the potential for a gene from wheat to migrate to jointed goatgrass via backcrossing in the field.
Theoretically, herbicide-resistant jointed goatgrass populations may appear through either natural selection of a plant that carries a mutation that imparts resistance or via a resistant-wheat x jointed goatgrass hybrid-bridge. In order for herbicide resistance to be transferred from wheat to a jointed goatgrass population via a hybrid bridge, wheat alleles in the F1 hybrids must be transmitted and retained in the backcross generations that are produced by backcrossing with jointed goatgrass until partial self-fertility is restored. To assess the risk of gene migration from herbicide-resistant wheat to jointed goatgrass, more information about the genetics of the wheat x jointed goatgrass hybrids and successive backcross generations is needed.
It has been speculated in sunflower (Helianthus spp.) that there is a genic preference for the weedy or wild chromosomes–alleles in subsequent generations after the initial formation of the hybrid (Rieseberg et al., 1995). On the other hand, Fritz et al. (1995) reported that D genome chromosome segments from either T. aestivum or Aegilops tauschii (2n = 2x = 14; DD) could be preferentially transmitted in experimental T. aestivum x Ae. tauschii populations. Thus, it also would be of interest to determine the pattern of retention of D genome chromosomes or chromosome segments over backcross generations in wheat x jointed goatgrass derivatives.
The utility of wheat SSRs to assay D genome products in Ae. tauschii and to monitor D genome chromosomes of jointed goatgrass in wheat x jointed goatgrass F1 hybrids and BC1 derivatives has been demonstrated (Guadagnuolo et al., 2001; Lelley et al., 2000). Thus, D genome wheat SSRs also can be used to track wheat and jointed goatgrass D genome alleles in recurrent backcrosses to jointed goatgrass. SSR markers also can be used to determine if recombination has occurred between the D genome chromosomes of the two species.
The objective of this study was to use SSR markers to determine the pattern of inheritance of wheat D genome alleles in the progeny of wheat x jointed goatgrass hybrids backcrossed to jointed goatgrass for two generations.
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MATERIALS AND METHODS
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Plant Material
The F1, BC1, and BC2 seed were produced using the approach crossing method (Zemetra et al., 1998). The female parent (designated FM) for the production of the F1 was a backcross between ‘Fidel’ and ‘Madsen’ (Allan et al., 1989) wheat where Madsen was the recurrent parent and the Fidel carried the imidazolinone-resistance gene. The pollen donor for the F1 was jointed goatgrass, designated JGG, from an Idaho field collection maintained at the University of Idaho. The BC1 and BC2 seed were produced using jointed goatgrass as the pollen donor. Twenty-three BC1 plants were analyzed. Five randomly selected BC2 progeny of each of 10 of the BC1 plants comprised the BC2 sample, for a total of 50 BC2 plants. One BC2 family with 18 individuals, designated 890, was selected for further analysis.
Genetic Analysis
Each of the 23 BC1 and 50 BC2 plants was considered an independent sample. The SSR markers used to analyze the DNA samples were those described by Röder et al. (1998) and Pestsova et al. (2000). Fourteen distinct SSR markers, two per chromosome of the D genome, one per arm, were utilized (Table 1). Each marker had previously been placed into an existing linkage map of wheat (Röder et al., 1998; Pestsova et al., 2000). The 14 markers used in this study were selected by screening single-copy D-genome SSR markers with five DNA standards: two jointed goatgrass accessions (JGG and FC13, which is an Oregon field collection), the F1 hybrid, FM, and Madsen, one of the parents of FM. The amplification product(s) of each primer pair were assessed for polymorphism between jointed goatgrass and FM. The criterion for choosing a marker for the analysis of the BC1 and BC2 generations was visual distinctness of jointed goatgrass versus F1 banding patterns. Observed molecular weight estimates of FM and Madsen were used as controls for the primer pairs and compared to published values (Röder et al., 1998; Pestsova et al., 2000).
Polymerase chain reactions (PCR) were performed in a volume of 10 µL in a Primus96 plus (MWG Biotech, Inc., High Point, NC) thermocycler. The reaction mixture contained 1x PCR buffer, 250 nM of each primer, 0.2 mM of each deoxynucleotide, 1.5 mM MgCl2, 1 unit of Taq DNA polymerase, and 50 ng of template DNA. The PCR reactions were set up in a 384-well format and the thermocycler was programmed to an initial 3 min at 94°C, 45 cycles with 1 min at 94°C, 1 min at 50, 55, or 60°C (depending on the marker), 1 min at 72°C, and a final extension step of 5 min at 72°C (Röder et al., 1998; Pestsova et al., 2000). For visualization, amplification products were loaded on 4% (w/v) agarose gels containing 0.3 µg mL–1 ethidium bromide, and electrophoresed at 100 V for 2 to 3 h. For each marker, a gel with the five controls (two jointed goatgrass accessions, F1, FM, and Madsen), and all samples for that backcross generation were scored for their wheat or jointed goatgrass allele constitution. The BC1 or BC2 plants were either homozygous or heterozygous for the jointed goatgrass allele at each locus.
Data Analysis
The number of plants with wheat alleles at the marker locus were evaluated. In addition, plants were evaluated for the number of wheat alleles they were estimated to contain as an indication of the percentage of their D genome derived from wheat. Chi-square tests for goodness of fit were used to compare observed marker retention rates to expected retention rates. Yates correction for continuity was used to adjust the Chi-square values due to sample size and having only one degree of freedom (Little and Hills, 1978). The number of plants scored as having a particular wheat allele was compared to the number of plants expected to have a particular wheat allele in that backcross generation. In this analysis, 50% of BC1 plants were expected to have a wheat allele at any particular locus. For the BC2 generation, 25% of plants were expected to have a wheat allele at any particular locus. A significance level of 
= 0.05 was used for all analyses.
A Chi-square test of homogeneity was used to assess variation in the percentage of D genome alleles that migrated from wheat. If there was no evidence to suggest that the plants were different with respect to percentage of D genome alleles that originated from wheat, a Chi-square goodness of fit test was performed for the population as a whole. In this study, the expected percentage of the D genome derived from wheat was 25% for the BC1 generation and 12.5% for the BC2 generation. Linkage analysis and two-point recombination frequencies were evaluated with GMendel v.3.0 (Holloway and Knapp, 1994).
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RESULTS
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Retention of Wheat Alleles in the BC1 Generation
Overall, the 14 wheat SSRs tested, as unselected markers for wheat alleles were present as if they were inherited in a Mendelian fashion in the BC1 generation. An example of the segregation observed can be seen in Fig. 1
. Chi-square analysis showed that 12 of the 14 markers fit the expected 1 heterozygote to 1 homozygote ratio in the BC1 generation. Two markers, gwm190 and gwm44, located on 5DS and 7DS, respectively, deviated from the expected 1:1 ratio in the direction of more heterozygotes than expected (Table 1) indicating a higher than expected retention of wheat alleles.
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Fig. 1. PCR-based assays of wheat (FM), jointed goatgrass (JGG and FC13), an F1 hybrid between jointed goatgrass and wheat, and nine BC1 and BC2 derivatives with the wheat simple sequence repeat (SSR) marker gdm98. Lanes labeled with an M contain a 100-bp marker ladder.
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The average number of wheat alleles retained in the BC1 generation was 8.8 (out of a possible 14) with a range of 4 to 12. This is equivalent to 31.3% (range of 13.3–42.9%) of the D genome alleles in the BC1 generation being retained from wheat. The expectation was that 25% of the total alleles in the BC1 generation would have descended from wheat. Because the plants were found to be homogeneous for the percentage of D genome alleles originating from wheat, a Chi-square goodness of fit was performed for the population. The analysis indicated that the population does not fit the expectation of 25% alleles of wheat origin. The deviation was due to two plants, which deviated significantly from the expected 25%. When markers used to assay BC1 derivatives were subjected to linkage analyses, no statistically significant (LOD > 2.0) linkage between markers was detected.
Retention of Wheat Alleles in the BC2 Generation
In the BC2 generation, the expectation is of a 1 in 4 chance that a wheat allele will be present at any given D genome locus. The inheritance of 11 of the 14 markers did not deviate from the model of 1 heterozygote to 3 homozygotes. The remaining three markers, gwm106, gwm349, and gwm44, located on 1DS, 2DL, and 7DS, respectively, deviated (P < 0.05) from this model (Table 2). In all three cases, the deviation was due to a higher percentage of wheat alleles than expected.
The mean number of wheat D genome alleles in the BC2 generation was 4.4 with a range of 0 to 9. It was not surprising to observe some BC2 plants with no wheat alleles since the potential to lose an allele from the nonrecurrent species should increase with each subsequent backcross. The retention of wheat D genome alleles in this backcross generation was 15.7% with a range of 0 to 32.1%. For the BC2 generation, the expectation would be that the genome of a BC2 plant would be comprised of 12.5% wheat alleles. Because the plants were again homogeneous for the percentage of D genome alleles originating from wheat, a Chi-square for goodness of fit was performed for the population and showed that the population did not fit the expectation of 12.5% alleles of wheat origin. This deviation was due to BC2 plants that originated from BC1 plants that had a higher percentage of wheat alleles than expected. As was the case with the BC1 derivatives, we failed to detect a statistically significant (LOD > 2.0) genetic linkage between markers used to assay the BC2 derivatives, which indicates that the markers had segregated independently.
Analysis of a BC2 Family
A BC2 family, designated 890, was chosen because it had 18 individuals derived from one BC1 plant and thus comprised a good sample size to analyze on a per family basis. A Chi-square goodness of fit was performed to compare the expected 1 heterozygote to 1 homozygote ratio at any given D genome locus. Six of 14 markers were segregating in this BC2 family. All segregations fit the expected ratio of 1 heterozygote to 1 homozygote (p < 0.05). Alleles from the D genome of wheat also were assessed as a percentage of total D genome alleles in the BC2 generation. In this generation, the expectation would be that the genome of a BC2 plant would be comprised of 12.5% wheat alleles. The average percentage of loci with wheat alleles was 10%. A Chi-square for goodness of fit for this population showed that the population fit the expectation of 12.5% alleles of wheat origin.
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DISCUSSION
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Wheat SSRs were used as a tool to track the retention of specific regions of the wheat D genome in wheat x jointed goatgrass hybrids over successive generations of backcrossing to jointed goatgrass. The wheat SSR markers appeared to be selectively neutral in the BC1 generation, although two markers located on 5DS and 7DS, deviated from the expected inheritance (P < 0.05). Because of the small sample size used in this study, it is difficult to determine if the segregation distortions observed have a biological basis. On the other hand, multiple markers showing the expected 1 heterozygote to 1 homozygote pattern of segregation suggests that the inheritance of wheat SSR loci were not preferentially retained or lost. In the BC2 generation, 11 of the 14 markers fit the expected frequencies for the retention of wheat alleles in the BC2 generation. Three markers, located at 1DS, 2DL, and 7DS deviated from the expected 1 heterozygote to 3 homozygote ratio of wheat to jointed goatgrass alleles. Wheat alleles for these three markers were present in more plants than would have been expected, which could mean that selection is acting to maintain wheat alleles in these regions of the chromosome. Further study with a larger population of BC1 and BC2 plants is needed to determine if the higher than expected retention of wheat chromatin on these chromosomes is real or an artifact of the sample size of this experiment.
The retention rates of unselected wheat D genome alleles in the BC1 and BC2 generations did not differ significantly from the expected inheritance for the majority of unselected makers. Thus, wheat D genome alleles are neither preferentially inherited nor lost when wheat x jointed goatgrass F1 hybrids are backcrossed to jointed goatgrass. This implies that when wheat D genome alleles are selectively neutral to jointed goatgrass alleles in backcross generations where jointed goatgrass is the recurrent parent, they may be maintained in a population.
With each arm of the D genome chromosomes represented by one marker, it is possible to determine whether recombination is occurring between the D genome chromosomes of wheat and jointed goatgrass. Because of the placement of each of the markers used and the known genetic distance between them, ranging from 65 to 141 cM (Röder et al., 1998; Pestsova et al., 2000), recombination was expected to result in the independent segregation of markers and the lack of linkage. Thus, our inability to detect linkage between the markers in the same chromosomes that were used in this study suggested that recombination between the D genomes of wheat and jointed goatgrass in our BC1 and BC2 derivatives had occurred. This indicates that a gene in wheat on the D genome, such as one for herbicide resistance, could be retained and integrated into the D genome of jointed goatgrass. Although expected from cytological observation (Zemetra et al., 1998), results of this study demonstrate the integration of wheat D genome chromatin into jointed goatgrass through normal genetic recombination. The integration of wheat D genome chromatin into jointed goatgrass provides evidence to support the concept of targeting the unshared genomes (A and B) between the two species to reduce the migration of novel genes that may enhance jointed goatgrass competitiveness.
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ACKNOWLEDGMENTS
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Partial funding for this project was provided by United States Department of Agriculture National Research Initiative Grant 9801039 and the United States Department of Agriculture National Jointed Goatgrass Research Initiative.
Received for publication July 15, 2003.
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ABSTRACT
INTRODUCTION
