Published online 1 February 2006
Published in Crop Sci 46:512-527 (2006)
© 2006 Crop Science Society of America
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REVIEW & INTERPRETATION
Spontaneous Hybridization between Bread Wheat (Triticum aestivum L.) and Its Wild Relatives in Europe
M. Zaharieva*,a and
P. Monneveuxb
a Instituto de Ecología, Universidad Nacional Autónoma de México (UNAM), A.P. 70-725, 04510 México D.F., México
b ENSA, UMR-DGPC, 2 place Viala, 34060 Montpellier, France
* Corresponding author (zaharieva{at}ecologia.unam.mx)
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ABSTRACT
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In Europe, wild wheat relatives of the Triticum–Aegilops complex grow in sympatry with cultivated bread wheat (Triticum aestivum L.) and spontaneous hybridization is known to occur. With the development of transgenic wheat, an understanding of the likelihood and occurrence of hybridization and introgression between wheat and its relatives is needed for use in risk assessment. To assess the probability of wheat to wild relative gene introgression, the distribution and biology of wheat wild relatives and their genome affinity and crossability with bread wheat were reviewed. Twelve of the 22 known Aegilops species, as well as one wild Triticum species, T. monococcum L. subsp. aegilopoides (Link) Thell., are known to occur in Europe near or within wheat cultivation. Five tetraploid species, Ae. cylindrica Host., Ae. triuncialis L., Ae. geniculata Roth., Ae. neglecta Req. ex Berthol., and Ae. biuncialis Vis., have wide distribution in most European countries. Bread wheat, wild Aegilops species, and Triticum species are predominantly autogamous (except Ae. speltoides Tausch, typically allogamous), but outcrossing among species is possible depending on species sympatry, concurrent flowering, and sexual compatibility. Spontaneous hybridization with wheat was reported for most of the tetraploid Aegilops species. The probability of gene transfer and gene retention in hybrid progenies is, however, higher when a gene is located on a shared genome, particularly on the D genome shared with Ae. cylindrica and Ae. ventricosa Tausch. Case-by-case and region-by-region assessments are needed to evaluate the risk associated with production and competitiveness of hybrids and their progeny.
Abbreviations: GISH, genomic in situ hybridization • GM, genetically modified • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat
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INTRODUCTION
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HYBRIDIZATION and introgression are natural biological processes that can occur among closely related species. For instance, it has long been recognized that cultivated plants naturally hybridize with their wild relatives (de Candolle, 1882). The major crops of wheat, rice (Oryza sativa L.), maize (Zea mays L.), soybean [Glycine max (L.) Merr.], cotton (Gossypium hirsutum L.), sorghum [Sorghum bicolor (L.) Moench], bean (Phaseolus vulgaris L.), sunflower (Helianthus annuus L.), and sugarcane (Saccharum officinarum L.) all have wild relatives to which they can cross (Ellstrand et al., 1999). Wild or weedy relatives may be able to acquire genes from commercial cultivars when they co-occur, have overlapping flowering periods, or share genomes and when no strong reproductive barrier exists that could prevent hybridization and introgression (Ellstrand et al., 1999). Until recently, there has been little interest in gene transfer from crops to their wild relatives. However, with the advent of genetically modified (GM) crops there has been renewed interest in understanding the implications of gene transfer from a cultivated crop to a wild relative.
Wheat is the most widely grown crop in the world and GM cultivars of wheat are in the process of development (Waines and Hegde, 2003). Wheat is cultivated on approximately 23 million ha in Europe (FAO, 2004). Natural hybridization between wheat and some of its wild relatives is known to occur in Europe, in some cases resulting in viable seeds (van Slageren, 1994). Gene transfer requires sympatry of the cultivated and wild species, synchronous pollen emission by the donor and stigma receptivity of the recipient species, and viability of the cross progenies.
The objective of this review is to summarize the scientific literature regarding the hybridization between bread wheat and related species in Europe as a first step to assess the likelihood of introgression of a gene from GM cultivated wheat into its wild relatives. This paper examines (i) the distribution, biology, and flowering phenology of wild wheat relatives (particularly those of genus Aegilops) in Europe, (ii) the genome affinity and crossability of bread wheat and its wild relatives, and (iii) the probability of spontaneous hybridization and gene transfer between wheat and its relatives.
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Taxonomy of Wheat Wild Relatives Present in Europe
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Wild relatives of cultivated wheat in Europe that may hybridize with wheat belong to the Triticum and Aegilops genera, family Poaceae Barnhart, subfamily Pooideae, tribe Triticeae Dumort, subtribe Triticinae Griseb (van Slageren, 1994). The Aegilops species have been described and classified by Linnaeus (1753), Jaubert and Spach (1851), Boissier (1884), Zhukovsky (1928), and Eig (1929). Taxonomic approaches to classification based on cytogenetics were developed by Kihara (1940, 1954) and Sears (1948). The latest revision of the classification of the Aegilops genus was made by van Slageren (1994) and represents a synthesis of the nomenclature and the development of clear keys to identify the species. The classification developed by van Slageren will be used in this paper. Currently, there is no consensus on how to classify Triticum sensu stricto, therefore the classification elaborated by van Slageren (1994) will be also used.
Twelve of the twenty-two Aegilops species are present in Europe (van Slageren, 1994). These include Ae. biuncialis, Ae. caudata L., Ae. columnaris Zhuk., Ae. comosa Sibth. and Sm., Ae. cylindrica, Ae. geniculata, Ae. neglecta, Ae. speltoides, Ae. triuncialis, Ae. umbellulata Zhuk., Ae. uniaristata Vis., and Ae. ventricosa (Table 1). Three Aegilops species, Ae. peregrina (Hack.) Maire and Weiller, Ae. bicornis (Forssk.) Jaub and Sp., and Ae. kotschyi Boiss., are present in Cyprus but not in continental Europe and will not be considered. One wild Triticum, T. monococcum subsp. aegilopoides is also considered in this review. The scientific names, synonyms, and common names of the species, according to van Slageren (1994), are presented in Table 1.
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Biology of Wheat-Related Species
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The Aegilops species are annual grasses that grow in tufts (Fig. 1 ). The culms are not ramified, have one to five nodes, and are generally semi-erect, geniculate and prostrate at the base, then ascending, particularly in Ae. umbellulata, Ae. geniculata, Ae. biuncialis, Ae. columnaris, and Ae. neglecta (Fig. 2
). Table 2 summarizes information on various morphological characteristics of the selected species based on van Slageren (1994) and our personal observation. Plant height is generally between 10 and 60 cm, but can reach 75 to 80 cm in Ae. speltoides and Ae. cylindrica. The plants can have up to several hundred tillers (Morishita, 1996; Zaharieva et al., 2001b). Spikes (Fig. 3
) are narrowly ovoid and short in Ae. umbellulata, Ae. geniculata, Ae. biuncialis, Ae. columnaris, and Ae. neglecta; narrowly cylindrical in Ae. caudata, Ae. comosa, Ae. speltoides, and Ae. cylindrica; subcylindrical in Ae. triuncialis; and moniliform in Ae. uniaristata and Ae. ventricosa. Three types of spike disarticulation are observed within the Aegilops and wild Triticum species present in Europe, that is, at the base of the rachilla (wedge-type), at the top of the rachilla (barrel-type), and at the base of the lowest fertile spikelet (whole-spike type). Aegilops speltoides var. ligustica has a wedge-type, Ae. cylindrica and Ae. ventricosa have the barrel-type, while Ae. speltoides var. speltoides and the remaining species reviewed possess the whole-spike type disarticulation. Aegilops glumes are coriaceous, rounded on the back (no distinct keel present, as in the case of Triticum), toothed and/or 1 to 5 awned. Lemmas are elliptic, thin, with 1 to 3 teeth and/or 1 to 3 awns. Triticum monococcum subsp. aegilopoides is an annual grass, with long stems reaching 135 cm. This species has slender and bearded spikes, with a wedge-type disarticulation (Fig. 4
). Each spikelet has only one seed (Peterson, 1965).
Aegilops plants produce a considerably greater number of spikes and seeds than wheat. The average seed number in Ae. geniculata plants grown under Mediterranean conditions is around 1400 (Zaharieva et al., 2001b), while under U.S. conditions jointed goatgrass plant (Ae. cylindrica) produces up to 100 spikes and up to 3000 seeds (Morishita, 1996). Wild diploid Triticum plants were also found to produce more seeds than wheat (Guzy et al., 1989). Seed dormancy allows Aegilops species to survive the hot summer. It has been shown in Ae. cylindrica (Morishita, 1996) and Ae. neglecta (Marañon, 1987) that subterminal seeds of an individual spikelet have greater dormancy than the basal seeds. An important reserve of seeds remains in the soil (Marañon, 1987) and can persist there for 3 to 5 yr (Morishita, 1996), allowing the wild population to escape unfavorable situations in the time and to rapidly colonize new areas.
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Distribution, Ecology, and Sympatry with Bread Wheat in Europe
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The Aegilops species occurring in Europe belong to the Mediterranean group of Triticeae (Sankary, 1990) and are mainly distributed in countries characterized by hot and dry summers and winter rainfall. These species grow in Europe at altitudes up to 1200 m (van Slageren, 1994). An understanding of the distribution of the different wheat relatives in a given country is useful in assessing the potential for hybridization and subsequent introgression. The ecogeographical distribution of the Aegilops species has been described in detail by Sankary (1990) and van Slageren (1994), and of T. monococcum subsp. aegilopoides by Perrino et al. (1996). Triticum monococcum subsp. aegilopoides is present and abundant in southeastern Europe. The presence and abundance of the Aegilops species in Europe are summarized in Table 3.
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Table 3. Distribution and abundance by country of wild Triticum and Aegilops species in Europe (van Slageren, 1994; Perrino et al., 1996). (Data only refer to countries where at least one Aegilops species is native. Austria, Belgium, Czech Republic, Germany, Netherlands, Slovakia, and Sweden where Ae. cylindica is adventive are not included).
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The diploid Aegilops species have a restricted distribution (van Slageren, 1994). Aegilops caudata, Ae. comosa, and Ae. uniaristata are typically Mediterranean species. Aegilops caudata occurs primarily in the southern Balkans (Greece, Bulgaria, Croatia) and is adventive (introduced, but not fully naturalized) in the South of France. Aegilops comosa is present in Bulgaria (Zaharieva, 1993), Greece, and Albania (van Slageren, 1994), while Ae. uniaristata is primarily found in Croatia, Greece, Albania, and southern Italy. Aegilops speltoides and Ae. umbellulata are classified by Sankary (1990) as western Asiatic and Mediterranean–western Asiatic, respectively. In Europe, Ae. speltoides is present only in Greece and southern Bulgaria (Delipavlov, 1983; Zaharieva, 1993). It is adventive in Belgium, France, Germany, Italy, and Spain (van Slageren, 1994). Aegilops umbellulata distribution is restricted to Greece (van Slageren, 1994) and southeastern Bulgaria (Zaharieva, 1993). Triticum monococcum subsp. aegilopoides is common in southern Balkans, including Bulgaria, Greece, Albania, and the former Yugoslavia (Perrino et al., 1996).
The tetraploid species are more widely distributed than the diploids. In Europe, Ae. cylindrica is present in Bulgaria, Hungary, Macedonia, Romania, Serbia-Montenegro, Croatia, Greece, and Slovenia. It has been introduced in Italy, France, Germany, and Switzerland and is adventive in many other European countries such as Austria, Belgium, Czech Republic, Netherlands, Spain, and Sweden (van Slageren, 1994). Aegilops ventricosa occurs in the Iberian peninsula, southern France, Croatia (van Slageren, 1994), and Italy (Larghetti et al., 1992). Aegilops geniculata, Ae. triuncialis, Ae. biuncialis, and Ae. neglecta occur throughout southern Europe. Aegilops neglecta is frequent in southern France, while Ae. biuncialis is particularly frequent in south and southeastern Europe. Aegilops ventricosa, Ae. triuncialis, and Ae. neglecta are adventive in Belgium, Germany, and Switzerland (van Slageren, 1994). Aegilops columnaris is present only in Greece (van Slageren, 1994). The countries where Aegilops species naturally occur have a Mediterranean climate. Among those countries, southeastern Europe (Balkan region) appears richer in Aegilops species compared to the western Mediterranean countries. Greece and Bulgaria have the greatest number of native Aegilops species in Europe (11 and 9 species, respectively) (Zaharieva, 1993; van Slageren, 1994). Some tetraploid species, such as Ae. geniculata, Ae. triuncialis, Ae. biuncialis, Ae. neglecta, and Ae. cylindrica, are native or adventive in a great number of European countries (Table 3).
The abundance of Aegilops species considerably varies among countries. Tetraploid species are more abundant than diploid species. Aegilops triuncialis, Ae. geniculata, Ae. neglecta, and Ae. biuncialis are the most abundant in the South of Europe, with Ae. geniculata being abundant mainly in islands and coastal regions (Sankary, 1990). Information on the abundance of Aegilops species in Slovenia, Croatia, Bosnia, Macedonia, Romania, and Serbia-Montenegro is insufficient. Van Slageren (1994) suggests a widespread distribution of Aegilops species in these countries. In Greece, the diploid species Ae. caudata, Ae. comosa, Ae. uniaristata, and Ae. umbellulata are abundant, while these are scarce or absent in most other European countries.
Most Aegilops species grow in dry and disturbed places such as field edges, roadsides, and sometimes in grassland vegetation, but Ae. caudata, Ae. umbellulata, Ae. ventricosa, and Ae. biuncialis are also found in field margins, and Ae. comosa, Ae. speltoides, Ae. cylindrica, Ae. geniculata, Ae. triuncialis, and Ae. neglecta grow near or within cultivated areas (van Slageren, 1994). Triticum monococcum subsp. aegilopoides occurs along roadsides and on the borders of cultivated fields.
All Aegilops species are colonizers, able to rapidly invade new territories (van Slageren, 1994). Aegilops biuncialis, Ae. cylindrica, Ae. geniculata, and Ae. triuncialis are the best colonizers and develop large stands (van Slageren, 1994). Although invasive, Aegilops species are not considered as weeds in Europe (Hanf, 1982). However, some Aegilops species (Ae. cylindrica, Ae. triuncialis, and Ae. geniculata) introduced in the USA at the end of the 19th century (Johnston and Parker, 1929) are now troublesome weeds (Barkley, 1986; Donald and Ogg, 1991; Hitchkock, 1951).
The reported distribution and ecology information indicates that wheat and some of its wild relative species grow in proximity in Europe. Wheat is cultivated in all European countries where Aegilops species are present and occupies an average of 11% of the total cultivated area (FAO, 2004). Where wheat and a wild relative co-occur there is the possibility for outcrossing if the other requirements are met (e.g., overlapping flowering periods and no genetic barriers).
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Synchronous Flowering with Bread Wheat
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Sympatry is not sufficient for two species to cross—the species must also have similar phenological characteristics and overlapping flowering periods. All Aegilops and Triticum species are annual, probably as a result of their adaptation to the seasonal rainfall of the Mediterranean region (Calder, 1966). As wheat, Aegilops species were divided by Tanaka (1954) into three groups, that is, winter, spring, and intermediate types, according to their needs in vernalization, defined by Chouard (1960) as the acquisition or acceleration of the ability to flower by chilling treatment. Most European Aegilops species, as well as T. monococcum subsp. aegilopoides, have a winter growth habit, thus requiring vernalization for flower induction. Aegilops columnaris and Ae. geniculata are characterized as having intermediate (or facultative) growth habit. In Ae. geniculata, normal flowering was observed in greenhouse planting or spring sowing, that is, without vernalization. However, in those conditions plants produced less productive tillers and seeds (Zaharieva, personal observation, 2002).
The flowering time of Aegilops and T. monococcum subsp. aegilopoides in Europe is from April–May until June–July, depending on the species and their ecogeographical location (van Slageren, 1994) (Table 2), while cultivated wheat flowers between the beginning of May and the end of June (Azzi, 1954). Aegilops and bread wheat spikes require from 3 to 5 d to complete flowering (Peterson, 1965; Boguslavski, 1978a), but the duration of the whole plant flowering is longer in Aegilops than in wheat, due to asynchroneous tillering (van Slageren, 1994). This trait enables wild species to escape environmental stress during flowering and ensure seed production. The continuous flowering period of Aegilops species extends the time that cross-pollination and hybrid production with bread wheat could occur.
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Pollination Mode, Glume Opening, and Pollen Movement
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The success of gene flow depends on pollination mode (Hamrick et al., 1979; Govindaraju, 1988), flower structure, and pollen dispersal (Waines and Hegde, 2003). Bread wheat as well as T. monococcum subsp. aegilopoides and most Aegilops species are considered to be autogamous (self fertilized) (Peterson, 1965; Hammer, 1980). Pollen grains mature before the floret opens. Most of the pollen falling within the flower becomes lodged on the stigma. However, D'Souza (1970) and Beri and Anand (1971) reported that 30 to 80% of the pollen can be shed outside the flower where it may be carried by wind. Occasionally, some pollen grains from one floret can reach the stigma of a different floret (or spike). Although not well documented, the rate of out-crossing of autogamous Aegilops species should be similar to wheat (Boguslavski, 1979). In wheat, outcrossing rates of up to 10% (depending of cultivar and year) were observed on some varieties (Enjalbert et al., 1998). However, outcrossing rates of 0 to 2% are commonly accepted. Among the Aegilops species present in Europe, Ae. speltoides is considered by Hammer (1980) as typically allogamous (cross fertilized) and Ae. caudata as facultative allogamous. In Ae. speltoides, stamen growth begins at blooming and the anthers disperse most pollen out of the floret. When the floret closes, from 200 to 500 pollen grains can be found on the stigma of selfing species, and very few or no pollen grains in cross-pollinating species (Boguslavski, 1979). Few or no seed were produced on Ae. caudata and Ae. speltoides when the spikes were isolated from receiving pollen from other plants.
Cross-fertilization and potential gene transfer depend, in part, on the degree and duration of glumes opening at anthesis, anther size, and pollen production and viability (Hucl, 1996; Waines and Hegde, 2003). In Aegilops species, glumes, lemmas, and paleas open at flowering (Boguslavski, 1978a) (Fig. 5
). Florets open in the morning, between 0600 and 1000–1100 h. In cultivated wheat, floret opening reaches its peak between 0900 and 1100–1200 h (Virmani and Edwards, 1983) and the duration that a floret remains open ranges from 8 to 60 min (de Vries, 1971).
According to Beri and Anand (1971), there is a positive association between the amount of pollen produced per anther and anther size. In cultivated wheat, anther length is approximately 2.5 to 4 mm (Picard, personal communication, 2003). In Aegilops species, anther length varies from 2.8 to 3.7 mm in the autogamous species to 5.1 to 7.3 mm in the allogamous species (Boguslavski, 1979). As a consequence, the amount of pollen produced in predominantly autogamous species is substantially lower compared with allogamous species (Waines and Hegde, 2003).
There are few published studies on pollen dispersal in Triticeae and most are on cultivated wheat. Pollen load in the air at a given time is a function of anther extrusion and the amount of pollen produced by an anther (Joppa et al., 1968). Pollen-mediated gene flow in wheat has been reviewed elsewhere (Waines and Hegde, 2003). Pollen viability was observed to range between 15 and 30 min depending on environmental conditions (de Vries, 1971), a duration long enough to allow cross-fertilization with a nearby wild relative (Hegde and Waines, 2004). Studies indicate that wheat pollen can move up to 60 m (Khan et al., 1973). However, Hucl and Matus-Cádiz (2001) reported that gene flow rate decreased with greater distance from the pollen source and highly depended on the wheat genotype and the wind direction. There is no published information on pollen transport in Aegilops species.
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Genome Constitution of Wild Wheat Relatives and Bread Wheat
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Aegilops and Triticum belong to a complex of wild and domesticated species whose allopolyploid members evolved via hybrid speciation (Kimber and Sears, 1987). Kihara (1919) and Sax (1922) showed that in the genus Triticum there are three different genomes, each composed of seven chromosomes. Kihara (1937) and Sears (1941) used karyological studies on different Aegilops species to demonstrate that their base chromosome number (n = 7) was the same as in wheat. They also demonstrated that the Aegilops genus can be divided, as the Triticum genus, into three ploidy levels (i.e., diploid 2n = 2x = 14, tetraploid 2n = 4x = 28 and hexaploid 2n = 6x = 42). The Aegilops species present in Europe are primarily diploid or tetraploid, with the exception of Ae. neglecta which also exists in a hexaploid form. The ploidy level and genome formulae of the selected species according to Waines and Barnhart (1992) are presented in Table 1.
Bread wheat, T. aestivum (2x = 6x = 42, BBAADD) is an allohexaploid with B, A, and D genomes. These genomes are chromosome sets (seven chromosome pairs for each genome) originated from different species and combined together during the evolution of wheat (Feldman et al., 1995). Two out of the three wheat genomes (B and D) came from diploid Aegilops species, while the A genome originated from a diploid Triticum ancestor, T. urartu Tum. ex Gandil. (MacFadden and Sears, 1946; Dvorak, 1988). The wheat D genome originated from Ae. tauschii Coss. (Pathak, 1940). This has been confirmed by the synthesis of hexaploid wheats from crosses between this species and tetraploid wheats (Kihara, 1944; MacFadden and Sears, 1946), by cytological analysis (Gill and Kimber, 1974), and by RFLP (restriction fragment length polymorphism) analysis (Rayburn and Gill, 1987). Conversely, the origin of the B genome is still very controversial as different diploid Aegilops species of the Sitopsis section (S genome species) have been proposed as potential donors, including Ae. bicornis (Sears, 1956), Ae. sharonensis (Kushnir and Halloran, 1981), Ae. longissima (Tsunewaki and Ogihara, 1983), Ae. searsii (Kerby and Kuspira, 1988), and Ae. speltoides (Chen and Gill, 1983; Bahrman et al., 1988; Dvorak and Zhang, 1990; Fernández-Calvín and Orellana, 1990).
Wheat possess 21 pairs of homologous chromosomes. The term homologous refers to a pair of chromosomes within a genome that contain identical linear order of gene sites (or loci) along their lengths and show synapsis during pachynema (Singh, 2003). These homologous chromosomes are classified into seven homeologous groups, each group containing one pair of chromosomes from the B, A, and D genomes (Sears, 1954). Homeologous group 1, for example, contains the pairs 1B, 1A, and 1D. The 21 wheat chromosomes can be readily identified by heterochromatic banding (Gill et al., 1991) or in situ hybridization patterns using repetitive DNA probes (Pederson and Langridge, 1997).
Since the different diploid species of the Triticinae subtribe have been derived from a common ancestor, their chromosomes still retain some degree of similarity, as shown by comparative mapping (Devos and Gale, 2000). A closer examination of synteny among hexaploid wheat homeologous chromosomes by southern hybridization of expressed sequence tags with deletion stocks, however, revealed numerous synteny perturbations with translocations, deletions, inversions, and duplications (Tikhonov et al., 1999; Keller and Feuillet, 2000). Most of these rearrangements originated during the evolution of the diploid ancestors of hexaploid wheat (Akhunov et al., 2003).
The five European Aegilops diploid species Ae. caudata, Ae. comosa, Ae. speltoides, Ae. umbellulata, and Ae. uniaristata have the C, M, S, U, and N genomes, respectively (Waines and Barnhart, 1992). The tetraploid species Ae. cylindrica, Ae. ventricosa, Ae. geniculata, and Ae. triuncialis have the CD, DN, MU, and UC genomes, respectively, while Ae. biuncialis, Ae. columnaris, and Ae. neglecta have the UM genome formula. The hexaploid form of Ae. neglecta has UMN genome composition. The polyploid species of Triticum and Aegilops constitute a classic example of evolution through amphiploidy. The diploid species are at the origin of the tetraploid species, which consequently have common genomes with their diploid ancestors (Table 1). For example, it has been postulated by Kimber et al. (1988) that Ae. geniculata (MU genome) is an amphiploid between Ae. comosa (M genome) and Ae. umbellulata (U genome). According to their genome Kimber (1988) distinguished three clusters among the polyploid Aegilops and Triticum species. In each cluster polyploid species have in common a genome called "pivotal" (A, U, or D) which has changed little throughout the allopolyploidisation and speciation process (Zohary and Feldman, 1962), while their second (or third) genomes (M, N, C, and S) have been modified. The polyploids in group A, for example, share the genome of the common diploid ancestor T. urartu, those in group D share the genome of Ae. tauschii, and those in group U share the genome of Ae. umbellulata. The presence of the pivotal genome may facilitate introgression among the species belonging to a given cluster. This genetic structure (one common genome and one or two different ones) may explain the comparatively high rate of successful spontaneous hybridization (and hence gene flow) between the polyploids (Kimber and Yen, 1988). Hybridization is facilitated by the shared genome, which acts as a buffer, ensuring some fertility in the resulting hybrids. The different genomes, which are brought together from different parents, can exchange genetic material and form a new mixed genome.
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Barriers to Hybridization Between Wheat and Its Wild Relatives
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Hybridization between wheat and its wild relatives, with the goal of introgression of useful traits into the cultivated species, has been the subject of considerable work (Sharma and Gill, 1983; Mujeeb-Kazi and Kimber, 1985; Mujeeb-Kazi, 1993, 1995). The three most important obstacles to wide hybridization are (i) incompatibility between parent species, (ii) nonviability of F1 hybrids, and (iii) sterility of F1 hybrids or their progeny (Sharma and Gill, 1983; Baum et al., 1992).
Cross incompatibility preventing hybridization arises when a pollen grain does not germinate, the pollen tube does not reach the ovary, or the male gamete does not fuse with the female gamete. When wheat is used as the female parent the success of the cross highly depends on the wheat cultivar (Ye et al., 1997).
The nonviability of F1 hybrids can be caused by the lack of pairing between genomes of the parental species. Homeologous chromosomes are normally prevented from pairing by two major pairing homeologous genes, Ph1 located on the long arm of chromosome 5B (Riley and Chapman, 1958) and Ph2 (Mello-Sampayo, 1971), and some minor genes (Sears, 1976). Consequently, only homologous partners pair in tetraploid and hexaploid wheats, what ensures diploid-like pairing and disomic inheritance. When Ph gene is deleted or its action neutralized, most Triticeae chromosomes can be induced to pair with their wheat homeologues (Wang, 1990, 1995; Jauhar et al., 1999). Riley et al. (1961) reported a genetic system in Ae. speltoides that can counteract the presence of Ph gene. Aegilops speltoides chromosomes can induce or promote meiotic chromosome pairing and the consequent genetic recombination between related chromosomes from different genomes (homeologous chromosomes) in hybrids with tetraploid and hexaploid wheats.
When chromosomes do not pair in the F1 hybrid, gametes receive different numbers of chromosomes leading, in general, to sterility. In this case, hybrid sterility can be overcome by chromosome doubling or backcrossing to one of the parents (Mujeeb Kazi, 1995). The addition of an entire genome from a wild species (i.e., the production of a synthetic amphiploid) has been an attractive approach for many wheat geneticists. Tschermak and Bleier (1926) were the first to obtain an amphiploid species as a result of the spontaneous doubling of the chromosome number of a wheat hybrid produced by crossing T. turgidum dicoccoides with the wild grass Ae. geniculata. The amphiploids obtained are usually fully fertile. The production of such amphiploids, in which the adaptive hybrid combinations are stabilized by polyploidy and the segregation of parental characters is prevented, opens up many possibilities for the production of new crops. A good example of a successful synthetic amphiploid level is the manmade crop triticale (xTriticosecale Wittm.). Bread wheat arising from hybridization of tetraploid wheat and Ae. tauschii is a good example of success of natural allopolyploidy. Chromosome doubling during meiosis is believed to contribute significantly to the widespread occurrence of polyploids in nature (Harlan and de Wet, 1975; Veilleux, 1985; Jauhar, 1993). In a recent study David et al. (2004) reported a high frequency (estimated at 10–6) of spontaneous amphiploidy between T. turgidum L. subsp. durum (Desf.) Husn. and Ae. geniculata in sympatric populations. By using genomic in situ hybridization, they showed that fertile amphiploids had arisen through unreduced gametes and that some of them carried wheat–Aegilops geniculata recombinant chromosomes. The authors concluded that amphiploids can provide a route for gene flow, including that of transgenes, to the wild.
During the last two decades, molecular data have provided new insights into polyploid evolution, leading to significant progress in understanding the mechanism and evolutionary aspects of polyploidy. Ozkan et al. (2001) studied the rate and time of elimination of several low-copy DNA sequences, existing at diploid level, in F1 hybrids and newly formed allopolyploids of Aegilops and Triticum. They found that rapid elimination of genome and chromosome specific sequences is a general phenomenon in newly synthesized allopolyploids. Dvorak et al. (2004) suggested a role of this phenomenon in augmenting the differentiation of homeologous chromosomes at the polyploid level, thereby providing the physical basis for the diploid-like meiotic behavior of newly formed allopolyploids. The resultant strict bivalent pairing prevents intergenomic recombination and brings about higher fertility and permanent heterosis between homoeoalleles, thus fostering the successful establishment of the newly formed allopolyploid species in nature. It was assumed that these genomic changes may have contributed to the successful establishment of newly formed allopolyploids as new species (Sasakuma et al., 1995; Feldman et al., 1997; Liu et al., 1998a, 1998b; Ozkan et al., 2001). In nature, amphiploids can also serve as an effective bridge over the interspecific and intergeneric cross breeding barriers.
Finally, another barrier to hybridization is the reduced development of embryo and/or endosperm. Hybrids with normal embryo and reduced endosperm were reported in crosses between wheat and Ae. umbellulata by Mujeeb-Kazi and Kimber (1985). The nonviability of F1 hybrids also can be caused by incompatibility between the genome(s) of one species and cytoplasm of the other. For example, reduced seed viability induced by cytoplasmic male sterility was observed by Maan (1979) in T. monococcum subsp. aegilopoides x T. aestivum hybrids.
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Crossability and Genome Affinity between Bread Wheat and Its Wild Relatives
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Hybrids between wheat and different Aegilops species have been produced and described (Kihara, 1937; Knobloch, 1968; Kimber and Abu Bakar, 1979; Sharma and Gill, 1983). An important database on pairing during meiosis in hybrids between wheat and its relatives was developed by Kimber and Abu Bakar (1979) and was used for assessing genome affinity and determining species relationships (Kimber et al., 1981; Mujeeb-Kazi and Kimber, 1985; Gill and Chen, 1987). Chromosome pairing is used to describe the occurrence of one or more chiasmata (points of attachment between homologous chromosomes resulting from crossing over) between two or more entire chromosomes or between two chromosome arms (Kimber et al., 1981). The lack of chromosome association during meiosis results in the formation of two univalents. Association of two pairing chromosomes results in the formation of a rod bivalents (pairing in only one arm) or ring bivalents (pairing in two arms). The evidence for the high chromosome homology is given by the formation of seven bivalents (complete chromosome pairing). Chromosome pairing can be evaluated by assessing the mean chromosomal associations per cell (Kimber et al., 1981). Based on chromosome pairing information, numerical methods of assessing genome affinity or similarity of the B, A, and D genomes of T. aestivum to related genomes of other species have been developed (Kimber and Hulse, 1978; Driscoll et al., 1979; Alonso and Kimber, 1981; Kimber and Alonso, 1981; Kimber et al., 1981).
Genomic affinity of individual chromosomes can also be determined by sequential banding and genomic in situ hybridization (Jiang and Gill, 1993, 1994b). Staining techniques were used to analyze the substructures of cereal chromosomes, and to develop a cytogenetic karyotype of wheat (Gill and Kimber, 1974; Gill et al., 1991). Nonisotopic methods of mapping DNA sequences in situ on chromosomes were used to construct a molecular karyotype of wheat (Rayburn and Gill, 1985; Jiang and Gill, 1994a). These methods have greatly facilitated cytogenetic analysis in wheat and related species, especially the analysis of alien transfers (Friebe et al., 1991, 1996). More recently, molecular markers such as RFLP (restriction fragment length polymorphism) or microsatellites (SSRs, simple sequence repeats) have been used for identification of alien chromosomes in wheat background and estimation of genome homeology (Zhang et al., 1998; Iqbal et al., 2000; Zaharieva et al., 2003b).
Data obtained from these different approaches allowed a description and better understanding of the relationships between the Aegilops and wheat genomes. The D genomes of Ae. cylindrica (CD) and Ae. ventricosa (DN) show complete homology with the D genome of T. aestivum and the D genome of the donor Ae. tauschii (Abu Bakar and Kimber, 1982; McGuire and Dvorak, 1982; Kimber and Zhao, 1983). These genomes have apparently changed little during their evolution in the polyploid species (Zhao and Kimber, 1984). A high-density genetic linkage map of the Ae. tauschii genome constructed by Boiko et al. (1999) demonstrated that most of the markers shared between the Ae. tauschii and T. aestivum physical maps were colinear. However, the discrepancy in the order of five markers on the 3D of Ae. tauschii genetic map versus the 3D T. aestivum physical map indicated a possible inversion. C-banding analysis of Ae. cylindrica genomic constitution made by Linc et al. (1999) showed that banding patterns of the D- and C-genome chromosomes of Ae. cylindrica are similar to those of D- and C-genome chromosomes of the diploid progenitor species Ae. tauschii and Ae. caudata, respectively. Genomic in situ hybridization analysis detected intergenomic translocation in three of the five Ae. cylindrica accessions. The A genome of T. monococcum subsp. aegilopoides has close homology to the A genome of T. aestivum (Kimber et al., 1981). However, the affinity between A genomes of bread wheat and other cultivated polyploid wheats (e.g., durum wheat, BA genome) is higher than between the A genome of wheat and T. monococcum subsp. aegilopoides, probably as a consequence of evolutionary divergence (Kimber et al., 1981). Hybrids between T. monococcum and its wild form subsp. aegilopoides are fully fertile, but hybrids between T. monococcum and T. urartu are fully sterile, although their chromosomes pair as ring bivalents indicating identical genomic constitution (Johnson and Dhaliwal, 1976). Those who wanted to improve T. monococcum by crosses with 4x and 6x wheats were disappointed as such transfers are impossible (Sharma and Waines, 1981). There has been some differentiation between the A genome of diploid and polyploid wheats as evidenced from the reduced level of pairing, or absence of pairing in the case of chromosome 4A (Gill and Chen, 1987) and the amount of C-heterochromatin (Friebe and Gill, 1996). The S genome of Ae. speltoides is closely related to the B genome of wheat (Friebe et al., 2000) and meiotic analysis data on hybrids between T. aestivum and Ae. speltoides have shown preferential pairing between A and D, and between B and S chromosomes (Maestra and Naranjo, 1998).
By analyzing the chromosome pairing of the diploid hybrids Ae. comosa x Ae. uniaristata (MN), Ae. umbellulata x Ae. tauschii (UD), and Ae. uniaristata x Ae. tauschii (ND), Cuñado and Santos (1999) found closer relationships between M and N, and U and D genomes than between N and D genomes (MN > UD > ND). Iqbal et al. (2000) using SSR marker analysis have demonstrated the close homeology of the N genome of Ae. uniaristata and the D genome of wheat. By comparing Ae. umbellulata and hexaploid wheat maps, Zhang et al. (1998) confirmed the homeology between Ae. umbellulata U and wheat D genome chromosomes. Based on the pairing affinities between Ae. geniculata (MU) and wheat genomes (B, A, and D), Fernández-Calvín and Orellana (1992) revealed that the wheat A and D genome chromosomes more frequently associated with M and U genome chromosomes of Ae. geniculata than did the B genome chromosomes. Similarity between U and M genome of Ae. geniculata and D genome of hexaploid wheat was also observed by Zaharieva et al. (2003b) using wheat D genome microsatellites. These experimental results suggest that the relationships between the bread wheat D genome and most of the Aegilops genomes are closer, compared to the A and B genomes.
Results of chromosome pairing (chromosomal associations and chiasmata formation per cell during meiosis) and crossability rate (seed set produced by pollinated florets, expressed as percentage) between bread wheat and different Aegilops species, are summarized in Table 4. In cross combinations, the first mentioned species (or cultivar) is the female parent and the second one the male parent. Normal cross combination refers to the case when bread wheat is used as the female parent and the reciprocal cross to the case when Aegilops or wild Triticum is used as the female parent. These data provide an estimation of the probability for the given wild species to cross with wheat. They have been obtained, however, by manual pollination techniques and sometimes using embryo rescue, leading to overestimation of crossability rate in comparison with hybridization in natural conditions. Crosses such as diploid x hexaploid or tetraploid x hexaploid reduced the fertility of the F1 generation substantially. Hybrid seed viability was low in crosses involving the diploid Aegilops species, either used as male or female parent, while the seed viability of hybrids involving tetraploid species was more than 50% (Bochev, 1993). After backcrossing with the wheat parent, crossability rate increases (Meissner, 1991; Zaharieva et al., 2003a). Higher seed set was obtained when Aegilops species was used as the female parent. The fact that both species share a genome facilitates the production of hybrids and successful backcrossing. Chromosome pairing in these hybrids is generally high, allowing recombination and introgression events. When the two parents do not share a genome, chromosome pairing is low and crossability is likely to depend on the similarity between the specific genomes. Although wheat x Aegilops hybridization depends to a large extent on how closely related the Aegilops species is to wheat, considerable genetic variation for crossability exists within wheat cultivars and wild relative species (Sharma and Gill, 1983; Farooq et al., 1989; Bochev, 1993).
Transfers from wheat to Aegilops species is also affected by the presence of gametocidal genes (Gc) in some Aegilops species. These genes induce breakage in wheat or Aegilops chromosomes (Endo, 1990). Repair and fusion of the broken ends result in the generation of deletions and translocations and finally in a variety of chromosome restructuring reducing pairing frequency (Nasuda et al., 1998). Gametocidal genes were identified on chromosome 3C of Ae. caudata L. (Endo and Katayama, 1978), 2C of Ae. cylindrica (Endo, 1979; Endo, 1996), 4M of Ae. geniculata (Friebe et al., 1999), 3C of Ae. triuncialis (Endo and Tsunewaki, 1975), and 2S and 6S of Ae. speltoides (Tsujimoto and Tsunewaki, 1983, 1988; Kota and Dvorak, 1988).
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Spontaneous Hybridization (Wild Relatives x Bread Wheat)
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The occurrence of hybridization under natural conditions in Europe has been documented. Ae. geniculata x bread wheat spontaneous hybrids were reported first by Requien in 1825 (van Slageren, 1994). Natural hybrids between Ae. neglecta and wheat and between Ae. triuncialis and wheat were described in South of France by Godron (1854) and in Spain by Lange (1860), respectively. Van Slageren (1994) listed natural hybrids between bread wheat and Ae. columnaris (Italy), Ae. cylindrica (Hungary), Ae. neglecta (France, Greece, Italy), Ae. triuncialis (France, Spain), and Ae. ventricosa (France). Recently, Guadagnuolo et al. (2001) reported that the mean hybridization rate between Ae. cylindrica and wheat under field conditions in Switzerland was 3%. The documented natural hybrids were limited to crosses between tetraploid Aegilops spp. and wheat and were observed between roads and adjacent wheat fields, or next to the fields where wheat had been cultivated the previous year. These Aegilops x Triticum hybrids were vigorous and usually attained a height of 50 to 90 cm. The observed tough rachis and keeled glumes were thought to come from the wheat parent. Because of the though rachis, the spike disarticulation was whole-spike type. Spike morphological traits were inherited from the Aegilops parent (Fig. 6
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Fig. 6. Aegilops geniculata x Triticum aestivum natural hybrid in the edge of wheat field (Montpellier, South of France).
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Most natural hybrids between Aegilops and Triticum species were found to be sterile, but seeds were occasionally found. Fertile progenies of an Ae. geniculata x wheat hybrid were described in 1838 by Fabre, a farmer from Agde, South of France (van Slageren, 1994). After a few years of cultivation, the farmer obtained seed-producing fertile plants that increasingly looked like wheat.
The first Aegilops cylindrica x wheat hybrid was described in 1917 by the Hungarian botanist von Degen on the St. Andrew Danub island as a new species he called Aegilops sancti-andreae Deg. (van Slageren, 1994). This late description is quite surprising, since Ae. cylindrica x wheat is the natural hybrid most frequently encountered by van Slageren (1994) during his collecting. Plants fitting the description of Ae. sancti-andreae were later found by Rajhathy (1960) at the same location, along wheat fields in places where Aegilops cylindrica was growing close by, and were identified as Ae. cylindrica x wheat hybrids. This has been confirmed by a chromosome count of 2n = 35. Of 300 plants, six seeds were found, providing partially fertile plants (originated from spontaneous backcrosses with wheat as revealed by cytological examinations) and fertile hybrids were found in the field borders confirming continuous spontaneous crossing. Natural hybrids (0.5%) between male sterile lines of the bread wheat ‘Bezostaya 1’ and Ae. cylindrica were reported in Bulgaria by Gotsov and Panayotov (1972). Manual pollination of the same lines resulted in similar rates of hybrid plants (0.7%) as the natural pollination. A higher rate (12%) of hybrid seed production was obtained by the same authors in a reciprocal cross (i.e., with Ae. cylindrica as the mother plant). According to van Slageren (1994), the sterility of most hybrids is caused primarily by the lack of viable pollen, so that willful pollination with the Triticum parent may restore fertility and lead to further backcross generations that increasingly resemble wheat.
Boguslavski (1978b) conducted a 6-yr study on spontaneous hybridization between species of the Aegilops and Triticum genus at the Vavilov Institute experimental station in Daghestan. Twenty-two Aegilops species accessions were cultivated together with different wheat cultivars. Aegilops x wheat hybrids were mainly observed with tetraploid Aegilops species (Ae. cylindrica, Ae. biuncialis, Ae. neglecta, and Ae. triuncialis). However, a few hybrids were found with the diploid species Ae. speltoides. The number of hybrids obtained was highly year and species dependant, and hybrid fertility ranged from 0.2 to 6%. The fertility of the progeny of these hybrids ranged from 6 to 48%.
More recently, viable seeds on Ae. cylindrica x bread wheat hybrid plants grown under field conditions in USA were reported (Mallory-Smith et al., 1996; Seefeldt et al., 1998; Snyder et al., 2000). Zemetra et al. (1998) and Wang et al. (2001) reported that experimental wheat x jointed goatgrass hybrids were male sterile but had about 1 to 2% female fertility. Snyder et al. (2000) observed that the mean hybrid fertility ranged from 2 to 4% and that viable seeds can be produced on hybrids with either jointed goatgrass or wheat as a pollen donor. What determines the preponderant pollen donor under field conditions is, however, still unclear and further assessments are needed to address this issue. A low rate of backcrosses to jointed goatgrass has been reported by Morrison et al. (2002). Zemetra et al. (1998) also found a low fertility in backcrossed plants (2%). However, the fertility rate increased with each successive backcross, both with Ae. cylindrica (15.1% for the BC1, 37.4% for the BC2) and bread wheat (4.8% for the BC1, 13.7% for the BC2, 55.8% for the BC3). Wang et al. (2001) showed that, on average, a BC1 plant had 2% male fertility and 4% female fertility. These findings suggest that BC1 and/or BC2 plants could serve as either the male or female parent in subsequent crosses. When jointed goatgrass was the recurrent parent, chromosome number decreased with each successive backcross, approaching the 28-chromosome number of jointed goatgrass. After two backcrosses and one or two selfings, individuals with chromosome numbers ranging from 29 to 34 were observed (Morrison et al., 2002).
Kroiss (2001) noted the presence of 14 wheat D genome–specific microsatellite markers in wheat x jointed goatgrass BC1, BC2, and BC2S1 derivatives. This indicated that if a gene is present on the D genome of wheat it has the potential to be retained in the jointed goatgrass backcrosses after self-fertility is restored (Zemetra et al., 2002). In a recent review on hybridization and introgression between bread wheat and jointed goatgrass in USA, Hegde and Waines (2004) estimated that the probability of recovering BC2 seed with jointed goatgrass as a recurrent parent would be approximately one plant out of 1.54 million plants if a transgene is present on D genome of wheat. It is expected that chromosomes of unshared genomes (in this case B and A) will be lost with each subsequent generation of selfing. However, cytological studies (Wang et al., 2001) showed that some B or A genome chromosomes can be retained in advanced derivatives. In addition, Wang et al. (2000) observed a translocation involving wheat B or A, and Ae. cylindrica C genome in BC2S2 plant. However, it was not retained in the following generation, indicating that gene retention by translocation may not occur at a sufficiently high frequency between unshared genomes. As a consequence, the potential of retention of a wheat gene is reduced when located on a unshared genome (B or A), compared to that of a gene of the shared D genome (Zemetra et al., 2002). The same authors considered that further assessments of the effect of selection pressure (herbicide treatment in this case) on gene(s) retention on shared and unshared genomes are warranted. Unfortunately, similar assessments are still lacking with other Aegilops species. On the basis of the findings to date, the risk of introgression of wheat genes into jointed goatgrass populations may be tied to (i) the potential for fertility restoration, (ii) the overall numbers of hybrid plants that potentially can produce seed, and (iii) the viability of the seed as well as its ability to successfully establish. Selection pressures in the form of the wheat harvest and wild-type dispersal biology will play a role in the movement of wheat genes into jointed goatgrass.
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Conclusion and Future Prospects
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The transfer of a transgene from genetically modified crops to related species could take place during hybridization. The formation of hybrids between crops and their wild relatives depends on a number of factors including the proximity of the parental plants, their mating system (outcrossing rate), and the synchroneous flowering and genetic compatibility (ploidy level, genome similarity, and chromosome pairing) of the crop and the wild species. In Europe these factors are present as shown by the occurrence of spontaneous hybridization, even at relatively low rates.
In Europe, Aegilops species and the wild emmer wheat T. monococcum subsp. aegilopoides occur in sympatry with cultivated wheat. Tetraploid species sharing the U genome (Ae. geniculata, Ae. triuncialis, Ae. biuncialis, and Ae. neglecta) are present in many European countries and are locally abundant. Among the Aegilops species carrying the D genome, Ae. cylindrica has wide distribution either as a native or introduced plant and shows weedy behavior more than most species of the genus, occupying large stands after recent disturbances. An overview on the distribution patterns of wild wheat relatives and bread wheat cultivation areas shows that some countries, such as those of southeastern Europe (e.g., Greece and Bulgaria) may have a higher likelihood of interspecies hybridization and gene transfer from wheat to its wild relatives, because of the presence and abundance of related species, and environmental conditions favoring outcrossing.
Aegilops species have synchroneous flowering with bread wheat. Aegilops speltoides is allogamous, while the remaining species are predominantly autogamous. However, in these species outcrossing may be able to reach levels similar to other autogamous species like
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ABSTRACT
INTRODUCTION
