Genome-specific introgression between wheat and its wild relative Aegilops triuncialis


  • C. Parisod,

    Corresponding author
    • Laboratory of Evolutionary Botany, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland
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  • C. Definod,

    1. Laboratory of Evolutionary Botany, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland
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  • A. Sarr,

    1. Laboratory of Evolutionary Botany, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland
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  • N. Arrigo,

    1. Laboratory of Evolutionary Botany, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland
    2. Department of Ecology and Evolution, University of Arizona, Tucson, AZ, USA
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  • F. Felber

    1. Laboratory of Evolutionary Botany, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland
    Current affiliation:
    1. Musée et Jardins botaniques cantonaux, Lausanne, Switzerland
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Correspondence: Christian Parisod, Laboratory of Evolutionary Botany, Institute of Biology, Rue Emile-Argand 11, University of Neuchâtel, 2000 Neuchâtel, Switzerland.

Tel.: +41 32 718 2344; fax: +41 32 718 3001; e-mail:


Introgression of sequences from crop species in wild relatives is of fundamental and practical concern. Here, we address gene flow between cultivated wheat and its widespread polyploid relative, Aegilops triuncialis, using 12 EST-SSR markers mapped on wheat chromosomes. The presence of wheat diagnostic alleles in natural populations of the barbed goatgrass growing in proximity to cultivated fields highlights that substantial gene flow occurred when both species coexisted. Furthermore, loci from the A subgenome of wheat were significantly less introgressed than sequences from other subgenomes, indicating differential introgression into Ae. triuncialis. Gene flow between such species sharing nonhomeologous chromosomes addresses the evolutionary outcomes of hybridization and may be important for efficient gene containment.


Reproduction between genetically distinct taxa, producing offspring of mixed ancestry (i.e. hybridization), plays a crucial role in evolution (Arnold, 2006). However, interspecific gene flow has been generally overlooked, and the factors determining the outcome of hybridization remain poorly understood (Abbott et al., in press). In particular, the impact of spontaneous hybridization followed by backcrosses leading to introgression of genetic material into related species deserves further consideration. Introgression of loci from domesticated plants to wild relatives is of fundamental, practical and economic interest (Ellstrand et al., 1999) and therefore represents a convenient model to explore the evolutionary significance of hybridization. The majority of cultivated plants indeed potentially hybridize at low level with wild species, and at least 27 cases of crop-to-wild gene transfer have been reported in European agro-ecosystems (Felber et al., 2007). With the advent of genetically modified crops, the consequences of introgression on local biota are receiving growing attention (Chapman & Burke, 2006; Kwit et al., 2011).

The Triticum/Aegilops species complex represents an outstanding model to evaluate crop-to-wild gene flow. The cultivated durum wheat (Triticum turgidum; 2n = 4x = 28; genome BA) is an allotetraploid species with subgenomes originating from T. uratu (A genome) and Ae. speltoides (B genomes), whereas the bread wheat (T. aestivum; 2n = x = 42; genome BAD) is an allohexaploid species with an additional subgenome (D genome) originating from Ae. tauschii (Feldman & Levy, 2009). In addition to those species that participated in wheat evolution, the genus Aegilops presents a great diversity of diploid and polyploid species that have been classified in three natural clusters (i.e. the U, D and A genome clusters) based on conserved karyotypes (Zohary & Feldman, 1962; Kilian et al., 2011). Cultivated wheat and several wild relatives are crossable, resulting in hybrids that occasionally produce viable seeds (reviewed in Kilian et al., 2011). Most of the tetraploid Aegilops grow in geographical proximity and present phenological overlap with wheat, sustaining occasional hybridization. Spontaneous hybridization was indeed reported for most of the species from the cluster U (Ae. columnaris, Ae. geniculata, Ae. neglecta and Ae. triuncialis) and the cluster D (Ae. cylindrica, Ae. ventricosa; reviewed in Zaharieva & Monneveux, 2006), and wheat is considered as a ‘moderate risk crop’ for transgene escape (Stewart et al., 2003). However, empirical studies conclusively documenting the magnitude of introgression in natural Aegilops populations are scarce, precluding formal risk assessment.

A recent molecular survey of European populations of three common relatives belonging to the cluster U using anonymous markers provided indirect evidence of gene flow from wheat (Arrigo et al., 2011). In particular, the tetraploid barbed goatgrass (Ae. triuncialis; genomes UC) showed signs of introgression from cultivated wheats. These species only share homeologous chromosomes, which expectedly hampers pairing and recombination in resulting hybrids (Sears, 1976), and would be predicted to show limited introgression. As targeting transgenes to specific subgenomes not shared by wild relatives was suggested as a strategy to limit their escape, the molecular mechanisms underlying the unpredicted introgression reported in Ae. triuncialis need to be further addressed. The present work thus uses suitable interspecific genetic markers mapped on wheat chromosomes to: (i) demonstrate and quantify introgression of cultivated wheat sequences into the barbed goatgrass genome in Europe, but also in the United States where the species is presently invasive and (ii) document genome-specific patterns of introgression. The presence of wheat diagnostic alleles in natural populations of Ae. triuncialis offers conclusive evidence of substantial gene flow in the course of species coexistence and reveals that wheat subgenomes introgress with differential effectiveness.

Materials and methods

Surveyed populations

Eleven and 19 populations of the barbed goatgrass (Aegilops triuncialis) were collected in Spain (Europe) and in California (United States of America; Fig. 1). The present study characterizes 351 individuals using wheat genome-specific markers (Table 1). The distance from the nearest cultivated field was recorded (irrespective of the crop currently grown), allowing to distinguish between populations collected ‘close to wheat’ and ‘distant from wheat’ (i.e. more than 50 m away from field). As pollen-mediated gene flow rarely exceeds 30 m in wheat (Zaharieva & Monneveux, 2006; Matus-Cadiz et al., 2007), this partition is assumed conservative.

Table 1. Sampled populations of Aegilops triuncialis (Pop) with geographical coordinates (NDD: latitude; EDD: longitude) and introgression of cultivated wheat subgenomes (B, A or D)
PopNDDEDDDistaNbNintrobGenome-specific introgressionc
  1. a

    *Distance from nearest cultivated field: close to wheat (C) and distant from wheat (D; i.e. more than 50 m away from field).

  2. b

    Number of individuals sampled (N) and individuals showing introgression (Nintro).

  3. c

    Number of wheat diagnostic alleles (mapped EST-SSR) amplified in addition to Ae. triuncialis alleles.

Spain, Europe
California, USA
 Total35162 40 5 22
Figure 1.

Gene flow from wheat in naturally occurring populations of its wild relatives Aegilops triuncialis in Spain (Europe) and in California (USA). The proportion of individuals showing introgression from wheat as detected with genome-specific EST-SSR markers is shown in black. The size of the pie chart is proportional to the number of individuals sampled, and populations collected ‘close to wheat’ are marked by a star.

Molecular survey

Naturally occurring individuals of Ae. triuncialis were genotyped with twelve EST-SSR loci mapped on wheat chromosomes using nulli-tetrasomic wheat lines (Zhang et al., 2007). Such genic molecular markers are appropriate for introgression assessment and allow evaluating genome-specific gene flow. The effectiveness of the EST-SSR markers in assessing gene flow between wheat and Ae. triuncialis was evaluated by genotyping 25 cultivated wheat references (tetraploid and hexaploid accessions collected during field sampling and from the ‘Agroscope Changins-Wädenswil’ germplasm) and three accessions of pure Ae. triuncialis, Ae. geniculata and Ae. cylindrica chosen to represent much of the genetic diversity present in the species. A total of 37 EST-SSR were initially examined and showed almost no intraspecific allelic variation. Four EST-SSRs markers showing clearly distinguishable alleles in wheat and Ae. triuncialis were selected for each of the wheat subgenomes. Those twelve genome-specific markers are presented with species-specific allele sizes in Table 2. Selected markers also presented alleles distinguishing Ae. triuncialis from other common Aegilops species to ensure that wheat diagnostic alleles amplified in the barbed goatgrass did introgress from wheat (data not shown). Allele additivity was confirmed in experimental F1 hybrids between the barbed goatgrass and wheat. Accordingly, naturally occurring individuals were considered introgressed when they presented both Ae. triuncialis and wheat alleles.

Table 2. Discriminating EST-SSR alleles considered for assessing introgression of genomic regions from cultivated wheat in Aegilops triuncialis
MarkerLocalizationaBand sizes in cultivated wheat (bp)Band sizes in Ae. triuncialis (bp)
  1. a

    Marker localization on chromosomes (1–7) of the different wheat subgenomes (B, A and D) following Zhang et al., 2007.

cfe1884B234 255
cfe2085A247 268
cfe1326A143 137
cfe662D222 229
cfe207D125 112

PCR amplifications were performed on 5 ng of DNA in 10 μL, with 5× GoTaq reaction buffer, 0.2 mm dNTPs, 0.5 mm of each primer (one primer was fluorescently marked with FAM, YYE or AT550) and 0.5 U of GoTaq DNA Polymerase (Promega, Dübendorf, Switzerland). Amplification was performed as follows: 120 s at 94 °C + 30x (30 s, 94 °C; 30 s, 60 °C; 30 s, 72 °C) + 8x (30 s, 94 °C; 30 s, 56 °C; 30 s, 72 °C) + 300 s at 72 °C. Resulting products were diluted five times, separated on a capillary 3500 Genetic Analyser (Applied Biosystems) and scored using GeneMapper 4.1.

Data analyses

The proportions of individuals presenting wheat diagnostic alleles in populations of Ae. triuncialis located in Spain (Europe) and in California (USA) were compared taking the distance to nearest cultivated field into account, using Generalized Linear Mixed Models (GLMM) fit by the Laplace approximation to the deviance and with binomial error distribution. Populations were treated as random effect.

Differential introgression of wheat subgenomes (B, A and D) was tested by comparing the proportions of individuals within Spanish populations showing signs of introgression with wheat B, A or D diagnostic alleles using GLMM (Laplace approximation; binomial error distribution) with populations as random effect. Statistical analyses were performed with R 2.11, using the lme4 package. This analysis of the patterns of genome-specific introgression into the barbed goatgrass focused on populations from Spain to avoid bias due to founder effects after the introduction of the species in the New World.


Among the 351 individuals of Ae. triuncialis from 30 natural populations surveyed here with 12 EST-SSR marking the different wheat subgenomes, 62 individuals (17.7%) from 20 populations (66.6%) presented wheat diagnostic alleles in addition to their own and thus showed patent signs of introgression from wheat (Fig. 1; Table 1). Eleven Californian populations of 19 (57.9%) present introgressed individuals as compared with 9 Spanish populations of 11 (81.8%). GLMM showed that populations of the barbed goatgrass collected in California (United States of America) presented significantly lower proportions of introgressed individuals than in Spain (Europe; z1,29 = −2.114, = 0.034). Across all populations, fewer introgressed individuals were detected in sites located more than 50 m away from any crop field than ‘close to wheat’ (z1,29 = −3.317, P < 0.001). A similar trend was also observed when only considering the Spanish populations where both situations (i.e. ‘close to wheat’ and ‘distant from wheat’) commonly occurred (z1,10 = −1.873, P = 0.061). In Spain, three of the seven populations growing close to cultivation fields (i.e. SP1, SP3 and SP9) indeed show high proportions of introgressed individuals. Noticeably, only six introgressed individuals, all collected in Spain, presented wheat diagnostic fragments from multiple loci (i.e. 2, 1, 1, 2 individuals with 5, 4, 3, 2 wheat alleles, respectively). These individuals were mostly collected in the population SP1 that was located within a wheat field.

Focusing on Spanish populations, where wheat and the barbed goatgrass coexist for a long time and where introgressed individuals were common, diagnostic alleles from the distinct wheat subgenomes were reported in different proportions in Ae. triuncialis. Alleles from the B subgenome occurred frequently, in four Spanish populations, representing 77.7% of the introgressed individuals. Individuals with alleles from the D subgenome represented 24.4% of the introgressed individuals and occurred in six populations. Two remote populations showed alleles from the A subgenome, accounting for 8.9% of the introgressed individuals. Taking nonindependence within populations into account, GLMM confirmed that the proportions of individuals showing signs of introgression were significantly lower for the A subgenome alleles than for the B or D subgenomes of wheat (Table 3).

Table 3. Generalized linear mixed models (with binomial error distribution and populations as random effect) of the effect of wheat subgenomes B, A and D on the proportion of introgressed individuals of Aegilops triuncialis
Explanatory variablesaEffect sizeSE z P
  1. a

    Cultivated wheat subgenomes.



Extensive gene flow from cultivated wheat to the barbed goatgrass

Spontaneous introgression of genetic material from crop species to wild relatives is of great concern for further understanding the consequences of hybridization on species evolution, but also for assessing the risk of spreading genetically modified organisms and developing efficient strategies of transgene containment. The present results firmly conclude that specific wheat genomic regions commonly introgress and segregate within natural populations of the barbed goatgrass.

The presence of wheat diagnostic alleles in 17.7% of individuals of Ae. triuncialis indicates that introgression is a frequent process having potential consequences for the species evolution. Noticeably, different wheat sequences were amplified in distinct populations of the barbed goatgrass in Spain, suggesting that multiple hybridization events have succeeded in producing introgressed individuals. Crosses between Ae. triuncialis and cultivated wheat have indeed been shown to regularly produce hybrids with relatively high fertility (Claesson et al., 1990). As 98.3% of the introgressed individuals showed only one wheat diagnostic allele, our results indicate successive backcrossing with Ae. triuncialis. Accordingly, introgression of wheat loci in Ae. triuncialis is an effective process with possible long-term impact on natural populations.

Although wheat sequences can be recovered from individuals currently growing in isolation from any cultivation due, for instance, to long distance dispersal of seeds, the proportion of introgressed individuals is significantly associated with the geographical distance to the nearest cultivated field (also see Arrigo et al., 2011). This suggests significant pollen-mediated gene flow when the species grow in sympatry, stimulating the recurrent hybridization and introgression of wheat sequences into the barbed goatgrass. Accordingly, introgressed individuals were more frequently reported in Spain than in California (27.3% vs. 9.14%, respectively), probably as a consequence of the repeatedly encountered sympatry between species in the Old World. American populations of Ae. triuncialis were indeed never observed along wheat fields in this work, and the proportion of introgressed individuals collected away from cultivation is not different in Spain and in California, suggesting that microgeographical isolation generally limits gene flow from wheat. Given that the barbed goatgrass has been invading the United States for the last century (Zaharieva & Monneveux, 2006), it could be that some of the originally introduced individuals in the New World were already introgressed. Accordingly, only a subsample of the wheat loci frequently observed in Spanish populations of Ae. triuncialis are segregating in Californian populations. Would the barbed goatgrass and wheat regularly occur in sympatry in the United States, massive introgression, as otherwise observed in Europe, should be further monitored.

Genome-specific introgression into the barbed goatgrass

Among introgressed individuals from Spain, wheat loci originating from the A subgenome occurred significantly less frequently in barbed goatgrass populations than loci from either the B or the D subgenomes. Pairing of Ae. triuncialis and wheat chromosomes is common in hybrids and results in remarkably high number of chiasmata (Claesson et al., 1990). Accordingly, intergenomic rearrangements involving the recombination of wheat subchromosomal regions in the Aegilops genome likely underlies the long-term maintenance of wheat alleles in Ae. triuncialis (Dvorak, 2009). In addition, multiple EST-SSR from the same wheat chromosome did not consistently amplify (data not shown), further suggesting introgression of chromosomal fragments rather than retention of wheat chromosomes in the barbed goatgrass.

The A subgenome may introgress less frequently because it originates from a Triticum species and may thus present lower recombination with Ae. triuncialis chromosomes than the B and D subgenomes coming from Aegilops species. While the A subgenome shows higher density of homeologous recombination events per physical units than the B subgenome (Peng et al., 2000), expectedly buffering hybrid fertility and supporting introgression of foreign sequences from taxa sharing homeologous chromosomes (e.g. wheat and Ae. cylindrica; Schoenenberger et al., 2005), it may hardly recombine and thus stably introgress sequences from structurally divergent genomes. Challenging such interpretation of uneven introgression based on specific failure of chromosome pairing, it should be noticed that the A subgenome shows considerable structural stability over evolutionary timescale (Zohary & Feldman, 1962) and high colinearity with other Triticeae genomes (Devos & Gale, 2000). In particular, the barbed goatgrass genomes show limited affinity with any of the wheat progenitors (Aegilops section Sitopsis, genome S, close to genome B; Triticum monococcum, genome A; Aegilops tauschii, genome D), suggesting that none of the wheat subgenomes presents a considerably higher probability of pairing and recombining with Ae. triuncialis (summarized in Kilian et al., 2011). Accordingly, the causes of the observed unequal, genome-specific introgression may deserve further explanation. As the A subgenome maintained a complete set of functional genes over long-term evolutionary scales and dominates the phenotype of all polyploid wheats of the A genome cluster (Peng et al., 2003), it may give reason for deficient maintenance of introgressed sequences in the A. triuncialis genetic background in the long term. To what extent loci of the A subgenome fail to stably introgress or show limited retention within Ae. triuncialis as compared with those from the wheat B or the D subgenomes remains an open question, and the mechanisms underlying differential introgression of distinct genomes deserve further attention.

Conclusions for genetically modified wheat

Presently, field tests with genetically modified wheat are under process but no transgenic variety is commercialized. Documenting extensive introgression, our work anticipates wheat as a worldwide risky crop for transgene escape. It further questions strategies for efficient transgene containment. In particular, we conclude that placing a transgene on the A subgenome might reduce, but will not be sufficient to exclude its escape in wild populations of widespread relatives. The evolutionary outcomes of hybridization between species remain hardly predictable yet and the environmental risk associated with transgene escape could only be minimized with the advent innovative containment strategies precluding introgression in nontarget organisms.


We thank R. Slobodeanu for statistical assistance. This work was funded by the National Research Program (NPR-59, grant 405940-115578) and the National Centre of Competence in Research ‘Plant Survival’ from the Swiss National Science Foundation.