Cytogenetics, phylogeny and evolution of cultivated wheats
B.S. Gill, B. Friebe

A wealth of data on the genomic structure of cultivated wheats has accumulated after almost a century of research, beginning with the pioneering genetic experiments of NilssonEhle (1909) and the cytological studies of Sakamura (1918) and Sax (1918). The tools for studying genetics have evolved over the years. Beginning in the 1920s, the method of nuclear genome analysis based on chromosome pairing behaviour in interspecific hybrids (Kihara, 1919; Sax, 1922) provided information on genome constitution, phylogeny and the evolution of Triticum and Aegilops species (summarized in Lilienfield, 1951). In the 1930s, Sears (summarized in Sears, 1954, 1966; Sears and Sears, 1978) began studies with wheat aneuploids that ushered in the era of formal cytogenetic analysis and gene mapping of individual chromosomes and arms in wheat (a catalogue of mapped genes is published each year, see McIntosh et al., 1998 for the latest edition). In the 1970s, modern staining techniques were used to analyse the substructures of cereal chromosomes, and a cytogenetic karyotype of wheat was developed (Gill and Kimber, 1974a; Gill, B.S. et al., 1991). Non-isotopic methods of mapping DNA sequences in situ (in situ hybridization) on chromosomes on a glass slide were used to construct a molecular karyotype of wheat (Rayburn and Gill, 1985; for review see Jiang and Gill, 1994a). These so-called molecular cytogenetic methods of genome analysis have greatly facilitated cytogenetic analysis in wheat and related species, especially the analysis of alien transfers (Friebe et al., 1991, 1996a). The technique of DNA restriction fragment length polymorphism (RFLP) analysis and linkage mapping was applied to wheat in the 1980s (Chao et al., 1989; Kam-Morgan et al., 1989). The genetic linkage maps of common wheat (Liu and Tsunewaki, 1991; Cadalen et al., 1997; Devos and Gale, 1993; Van Deynze et al., 1995; Nelson et al., 1995a, 1995b, 1995c; Marino et al., 1996), durum wheat (Blanco et al., 1998) and two of the progenitor species have been developed (Gill, K.S. et al., 1991; Dubcovsky et al., 1996).

The century has ended with the production of powerful deletion stocks (Endo and Gill, 1996) that were used to develop cytologically based physical maps of molecular markers for the 21 chromosomes of wheat (Gill et al., 1993, 1996a, 1996b; Delaney et al., 1995a, 1995b; Mickelson-Young et al., 1995; Werner et al., 1992; Hohmann et al., 1994). These cytogenetic maps pave the way for targetted mapping and eventual cloning of agronomically significant genes in polyploid wheat. The recently constructed microsatellite map of wheat (Röder et al., 1998) is a major milestone that will promote wide application of molecular markers in wheat breeding research. Building upon an earlier review (Morris and Sears, 1967), the last comprehensive review of wheat cyto-genetics appeared in 1987 (Morris, 1987). This chapter will highlight main advances since then, woven in a historical context that is most relevant to wheat crop improvement efforts worldwide.

CULTIVATED WHEATS BELONG TO THREE PLOIDY GROUPS

By the 1920s, it was known that cultivated wheat species of the genus Triticum have chromosome numbers of 2n=14, 28 and 42 (Sakamura, 1918). This suggested a basic 1x chromosome number of 7 and the occurrence of diploid (2n=2x=14), tetraploid (2n=4x=28) and hexaploid (2n=6x=42) wheat species. Numerous studies since then have confirmed that 1x=7 is the basic chromosome number of the tribe Triticeae (or the basic Triticeae genome is organized into seven chromosomes). Furthermore, a specific chromosome or part of a chromosome in a basic genome is genetically related to a specific chromosome or a part of it in all other genomes of the Triticeae species. This is because gene synteny has been conserved throughout genome evolution and speciation of the genera in the Triticeae tribe and Poaceae family (Ahn et al., 1993). These genetically related chromosomes in different genomes are called homoeologous as compared to essentially genetically identical homologous pairs of chromosomes in diploid species. Poly-ploid wheat contains homologous as well as homoeologous chromosomes. The whole cytogenetic enterprise rests on the recognition of homoeology and gene content of individual chromosomes in different genomes of the Triticeae species. This information is used to formulate strategies for the manipulation of unique genes in different genomes of diverse species for wheat crop improvement. In addition, this information is useful in studies of the natural phenomenon of genome evolution and speciation that has generated a wondrous species diversity.

TAXONOMIC STATUS OF TRITICUM AND AEGILOPS SPECIES

The most recent taxonomic treatment of Triticum and Aegilops (van Slageren, 1994) is followed here with some modifications (Table 4.1, Table 4.2). One major disagreement is the treatment of Amblyopyrum (formerly Ae. mutica) as a separate genus; in Table 4.2 it is retained in genus Aegilops as Ae. mutica.

Two valid biological species of Triticum exist at each ploidy level (Table 4.1). Among diploid wheats, T. monococcum (einkorn wheat) is still cultivated to a limited extent, and its wild form ssp. aegilopoides (also known as T. aegilopoides) is widely distributed in the Middle East. Hybrids between the two are fully fertile. The second diploid wheat species T. urartu, previously thought to be endemic to Armenia, is sympatric with T. aegilopoides and wild tetraploid Triticum species throughout their range of distribution in the Middle East (Johnson, 1975). Johnson and Dhaliwal (1976) showed that hybrids between T. monococcum and T. urartu were fully sterile (although their chromosomes paired as ring bivalents indicating identical genomic constitution) and unequivocally established T. urartu as a valid biological species.

Both tetraploid species T. turgidum and T. timopheevii have cultivated as well as wild forms. The hybrids between T. turgidum and T. timopheevii are sterile, establishing them as valid biological species. The hulled wheat T. turgidum ssp. dicoccum (emmer wheat) was one of the ancient cultivated wheats. However, it is the free-threshing macaroni or durum wheats that are more widely cultivated now. Triticum timopheevii is of little economic importance. The wild forms of both tetraploid wheats T. turgidum ssp. dicoccoides (also known as T. dicoccoides) and T. timopheevii ssp. armeniacum (also known as T. armeniacum or araraticum) are widely distributed in the Fertile Crescent. Triticum dicoccoides is found exclusively in Israel, Syria and Lebanon, T. armeniacum in Azerbaijan and Armenia, and both overlap in Turkey, northern Iraq and possibly Iran.

Both hexaploid species T. aestivum and T. zhukovskyi are only found in cultivation. Triticum zhukovskyi is a botanical curiosity. However, T. aestivum, also known as common, bread or dinkel wheat, is of paramount economic importance and, along with rice, is a staple food of humankind.

TABLE 4.1
Species of genus Triticum and their genomic constitution

Aegilops is the most closely related genus to Triticum and both share annual growth habits (Table 4.2). Two of the three genomes present in bread wheat were donated by two different Aegilops species (see below). The genus Aegilops is comprised of 11 diploid species and 12 polyploid species, including tetraploids and hexaploids. Seven distinct genomes are present in diploid species genomes, and all, except the T genome of Ae. mutica, are also present in polyploid Aegilops species.

PHYLOGENY OF POLYPLOID WHEATS

One wild diploid Triticum species and two species of the closely related taxon Aegilops are involved in the phylogeny of polyploid wheats (Figure 4.1). Kihara (1919) and Sax (1922) presented cytological data on the pairing behaviour of interspecific hybrids between 2x/4x and 4x/6x wheats indicating that T. monococcum and T. turgidum share one genome in common and T. turgidum and T. aestivum share two genomes in common. The cytological data did not discriminate between T. monococcum and T. urartu genomes (Johnson and Dhaliwal, 1976), but the molecular evidence showed that T. urartu actually is the Agenome donor of both polyploid wheats (Dvorak et al., 1988, 1993; and references cited therein), erroneously proposed as the Bgenome donor by Johnson (1975).

There has been much controversy regarding the origin of the B and G genomes of poly-ploid wheats since the early proposal of Sarkar and Stebbins (1956) supported by Riley et al. (1958) that Ae. speltoides was the donor of the second genome of tetraploid wheats. Recent molecular evidence is convincing that the B and G genomes of tetraploid wheats were donated by Ae. speltoides (Dvorak and Zhang, 1990). Cytoplasmic genome hetero-geneity within Ae. speltoides indicates that it may be the maternal (=cytoplasmic) donor of all polyploid wheats (Wang et al., 1997). Jiang and Gill (1994b) reported cytological evidence supporting a diphyletic origin of the two tetraploid wheat species.

TABLE 4.2
Species of genus Aegilops and their genomic constitution

^aUnderlined genomes are modified at the polyploid level and those in brackets were deduced from DNA analysis.

Source: Modified from Dvorak, 1998, based on chromosome pairing and DNA analysis.

Whereas the tetraploid wheats are of ancient origin, as evident from their wide distribution in nature, both hexaploid wheats are of recent origin under cultivation. McFadden and Sears (1944, 1946) and Kihara (1944) demonstrated that Ae. tauschii was the D-genome donor of bread wheat, which arose from a hybridization of T. turgidum and Ae. tauschii ssp. strangulata, about 7 000 years ago (see Dvorak et al., 1998 for latest review). Triticum zhukovskyi arose from the hybridization of T. timopheevii with T. monococcum (Upadhya and Swaminathan, 1963).

DOMESTICATION OF CULTIVATED WHEATS

The Fertile Crescent is considered the birth-place of cultivated wheats about 8 000 to 10 000 years ago. Pure stands of wild diploid einkorn and wild tetraploid emmer are found there and may have been harvested and cultivated as such. Recent genetic evidence indicates that einkorn wheat (T. monococcum) may have been domesticated from wild einkorn (T. monococcum ssp. aegilopoides) in the region of the Karacadag mountains in southeast Turkey (Heun et al., 1997). Both wild and cultivated einkorn seed remains have been excavated in the nearby archaeological sites dating from 7500 to 6200 BC.

FIGURE 4.1
Phylogeny of polyploid wheats

The remains of cultivated emmer (T. turgidum ssp. dicoccum) have been discovered at several archaeological sites in Syria dating to 7500 BC (Zohary and Hopf, 1993). The free-threshing forms arose by mutation from primitive emmer wheats.

Bread wheat arose farther northwest, away from the Fertile Crescent, in the corridor extending from Armenia in Transcaucasia to the southwest coastal areas of the Caspian Sea in Iran (Dvorak et al., 1998). In this region, Ae. tauschii var. strangulata is predominant, which evidently hybridized with cultivated emmer to produce T. aestivum. Perhaps several independent hybridization events occurred that constitute the gene pool of bread wheat (Talbert et al., 1998). The first bread wheats may have looked similar to T. aestivum ssp. spelta found growing in Iran from which free-threshing types were derived by mutation (McFadden and Sears, 1946). The European spelt wheats may have been derived secondarily from a hybridization involving T. compactum and emmer wheat (Ohtsuka, 1998).

CYTOGENETIC STRUCTURE OF CULTIVATED WHEATS

Triticum aestivum

Bread wheat has a genome size of 16 billion base pairs (bp) of DNA organized into 21 pairs of chromosomes, seven pairs belonging to each of the genomes A, B and D (Sears, 1954; Okamoto, 1962). Sears (1954) identified individual chromosomes of wheat by monosomy and made some observations on their gross morphology, although most chromosomes were cytologically indistinguishable. Sears and Sears (1978) isolated marker telocentric chromosomes to expedite their identification. Chinese Spring aneuploid stocks have been converted to locally adapted varieties by numerous cytogeneticists (Law, 1993) for the mapping of phenotypic traits. Wheat chromosomes are most efficiently identified based on their unique heterochromatic banding (C-banding) patterns (Gill, B.S. et al., 1991) (Plate 2) and molecular karyo-typing (Pederson and Langridge, 1997). In Plate 2, the C-banding patterns of each of the 21 chromosomes of wheat is unique as shown for each chromosome at somatic metaphase (left) or meiotic metaphase (univalent) (right) stages. The C-banding technique reveals the location of two classes of chromatin, namely heterochromatin (dark-staining regions) and euchromatin (light-staining regions), along the chromosome axis. The rapid identification of somatic chromosomes from readily available root meristems has revolutionized cytogenetic research in wheat. Genomic affinity of individual chromosomes is determined by meiotic pairing analysis (Naranjo et al., 1987; Gill and Chen, 1987) or by sequential banding and genomic in situ hybridization (Jiang and Gill, 1993, 1994b).

Although gross chromosome homologies are conserved, structural changes involving chromosomes 4A, 5A and 7B are apparently present in all hexaploid and tetraploid wheats (Naranjo et al., 1987; Naranjo, 1990). Furthermore, chromosome 4A has undergone complex structural rearrangements (Mickelson-Young et al., 1995) and fails to pair with its ancestral 4A of diploid wheat or 4A of T. timopheevii (Gill and Chen, 1987). Such a chromosomal restructuring event coinciding with the origin of polyploid species is called a species-specific translocation, and it is postulated to restore fertility in the raw amphiploid (Gill, 1991). Chromosome 4B has undergone an inversion in Chinese Spring and other wheats but not in winter wheats (Endo and Gill, 1984; Mickelson-Young et al., 1995). In addition, some translocation polymorphisms have been documented in different wheat cultivars (Friebe and Gill, 1994).

Although polyploid, wheat shows disomic inheritance due to diploid-like pairing regulated by two major pairing homoeologous genes Ph1 (Riley and Chapman, 1958) and Ph2 (Mello-Sampayo, 1971) and some minor genes (see Sears, 1976 for review). Mutants of Ph genes have been produced (Sears, 1977) and genes epistatic to Ph genes (PhI) have been identified in related diploid species and transferred to common wheat (Chen et al., 1994). PhI, as well as Ph gene mutants, can be used in alien gene transfers by induced homoeologous pairing (Koebner and Shepherd, 1986; Sears, 1993).

As mentioned earlier, cytogenetically based physical maps have been constructed for all 21 chromosomes of wheat. In Figure 4.2, the physical map (left) shows an ideogram of C-banded chromosome 5D. The position of deletion breakpoints is indicated on the left. Thus, 5DS-2 (top left) is a deletion stock with breakpoint at 78 percent of fraction length from the centromere in the short arm. The molecular markers mapped in deletion intervals are indicated on the right. The 5D genetic map shows centimorgan (cM) distances on the left and the position of molecular markers on the right. The group 5 consensus physical map is an integrated map based on deletion mapping data of chromosomes 5A, 5B and 5D of wheat (see Gill et al., 1996a for details). Common markers joining the genetic and physical maps are indicated. The integrated map clearly shows that recombination is suppressed in the proximal centromeric regions of wheat chromosomes. The consensus physical map also shows the paucity of markers in the centromeric region. The approximate position of some major genes including Ha (hardness gene), Vrn (vernalization response), Lr1 (leaf rust resistance), Fr1 (frost resistance) and others are indicated on the map (see Boyko et al., 1999).

The cytogenetic maps show that molecular markers, most of which are cDNA (copy DNA of actively transcribing genes), are non-randomly distributed in clusters in the distal regions of the chromosomes. Recombination is also mostly restricted to gene cluster regions. The gene density and bp/cM ratio in cluster regions may vary from 55 to 120 kilo-bases (Kb) of DNA. Moreover, agronomically important genes are also mostly located in gene cluster regions and may be amenable to map-based cloning using targetted mapping strategies (Gill et al., 1996a, 1996b).

Triticum turgidum

Durum wheat has a genome size of 10 billion bp of DNA organized into 14 pairs of chromosomes. The structure of A- and B-genome chromosomes of durum wheat is essentially identical to the corresponding homologues in bread wheat. Durum wheat is far less tolerant of aneuploidy than bread wheat, and monosomic analysis was difficult due to poor fertility of the monosomics. However, the development of D-genome chromosome substitution lines and double ditelocentric stocks have greatly expedited chromosome and arm mapping of genes (Joppa, 1987). A Ph1 gene deletion mutant (Giorgi and Cuozzo, 1980) and a DS5B(5D) stock (Joppa, 1987) are useful stocks for alien gene transfers in durum wheat. Wild T. dicoccoides shows extensive chromosome polymorphism, in addition to the primitive chromosome arrangement similar to Chinese Spring in some accessions (Kawahara, 1988).

Triticum timopheevii

The individual chromosomes of T. timopheevii were cytogenetically identified by banding analysis of meiotic pairing configurations of T. timopheevii/T. turgidum hybrids (Gill and Chen, 1987) and substitution lines of T. timopheevii chromosomes in common wheat (Brown-Guedira et al., 1996). There has been some differentiation of the A genome of T. timopheevii as compared to the A genome of diploid and polyploid wheats. Badaeva et al. (1994) have presented extensive data on chromosome polymorphisms in wild populations of T. timopheevii. A species-specific founder translocation involving chromosomes 6At, 1G and 4G (Jiang and Gill, 1994b) distinguishes T. timopheevii from T. turgidum, which contains a founder translocation involving chromosomes 4A, 5A and 7B (Naranjo, 1990). Triticum timopheevii and T. turgidum have a common origin from the same diploid progenitor species, share the Ph1 gene (Dhaliwal, 1977) and their B and G genomes share more homology with each other than with any other genome of the Triticeae (Gill and Chen, 1987). The cytogenetic data indicate that genes can be transferred from T. timopheevii into wheat by direct crosses and recombination.

FIGURE 4.2
Integrated physical and genetic map for the group 5 chromosomes of wheat

Triticum monococcum

The A genome of diploid wheat consists of 5 billion bp of DNA organized into seven pairs of chromosomes. Kuspira and his colleagues (see Friebe et al., 1990) carried out cytogenetical studies and developed a primary trisomic series that proved to be of limited value due to its high sterility. Friebe et al. (1990) constructed the standard karyotype, and all chromosomes were individually identified. In addition to cytological polymorphism associated with different geographical populations, both T. monococcum and T. urartu contain a translocation involving chromosomes 4A and 5A (Dubcovsky et al., 1996). 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), in the amount of C-heterochromatin (Friebe and Gill, 1996) and other structural features (Jiang and Gill, 1994c). Genetic transfers are feasible from diploid to polyploid wheats by bridging crosses or direct crosses (Cox et al., 1991). However, because of genome differentiation, linkage drag is likely, and methods to enhance recombination may need to be deployed (Dubcovsky et al., 1995). 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).

STANDARD KARYOTYPES OF AEGILOPS AND OTHER TRITICEAE SPECIES

Because wild species are excellent sources of useful genes, interspecific hybridization and cytogenetic identification of individual chromosomes of wild species is a necessary first step in the exploitation of wild species in wheat improvement. Based on cytogenetic analysis, each analysed chromosome must be individually identified (by banding or molecular karyotyping), assigned to one of the seven basic homoeologous groups of the Triticeae and assigned as to its genomic affinity. The cytogenetic identification of chromosomes of progenitor species is straightforward and is based on comparative banding analysis using the standard karyotype of bread wheat (see Friebe and Gill, 1996 for review).

The cytogenetic analysis of non-progenitor species that carry a different genome than those of bread wheat is more time-consuming. The classical cytogenetic approach is illustrated in Figure 4.3. A synthetic amphiploid is used to isolate all alien chromosomes as single chromosome additions in wheat. The genetic identity (homoeology grouping) of individual chromosomes is determined by molecular markers, especially RFLPs. Gametophytic and sporophytic compensation experiments may follow, leading to the production of a set of individual alien chromosome substitution lines.

In the second approach, the cytogenetic identity and homology of an unknown alien chromosome is determined from its pairing behaviour with a wheat chromosome of known homology. The alien chromosome is induced to pair with a wheat chromosome by manipulating the Ph-gene system. The paired chromosomes in meiotic configurations at metaphase I of meiosis (MI) are identified by banding analysis (Naranjo et al., 1987; Gill and Chen, 1987). This approach was used to detect some of the species-specific translocations in wheat and has also been used to construct standard cytogenetic karyotypes and detect structural changes in the genomes of several alien species. Using this approach, one can also determine the relative feasibility of genetic transfer between specific wheat and alien chromosomes.

The third approach, which is not fully developed but individual pieces are in place, allows rapid molecular cytogenetic identification of individual chromosomes in situ (on a glass slide). Technical advances in DNA probe labelling, in situ hybridization and microscopy allow repeated hybridization, mapping and processing of chromosome images for multiprobe mapping on a single metaphase figure. A sequential banding/in situ hybridization procedure also can be used to determine the genetic and/or genomic affinity of cytologically identifiable individual chromosomes of a species using appropriate probes (Jiang and Gill, 1993, 1994b). A procedure for rapid cytogenetic mapping and assigning of genomic affinity of individual chromosomes of polyploid Ae. cylindrica using the tools and techniques of wheat cytogenetic analysis are demonstrated in Plate 3. The metaphase cell (Plate 3a) was processed for in situ hybridization, and ribosomal genes were mapped as indicated (Plate 3b-c). The probe was stripped and hybridized with repetitive DNA probes as indicated (Plate 3d-e). Repetitive DNA probe pAs1 is D-genome specific (Rayburn and Gill, 1986), and it distinguished the D-genome chromosomes from the C-genome chromosomes. Plate 3f is an overlay that depicts the physical location of all the mapped DNA sequences. All D-genome chromosomes and some C-genome chromosomes and their genomic affinities (and phylogenetic origin) were cytogenetically identified in a experiment in a few days.

FIGURE 4.3
Scheme for systematic cytogenetic analysis of alien species and introgression of alien genetic variation into wheat

TABLE 4.3
Some examples of species for which standard cytogenetic karyotypes have, been constructed and a large number of alien-wheat chromosome additions, substitutions and translocation lines have been isolated

Some of the species of Aegilops and other genera for which standard karyotypes have been constructed and a large number of wheat-alien addition, substitution and translocation lines have been isolated are listed in Table 4.3. As an example, all seven chromosomes of Ae. longissima have been isolated as addition and substitution lines (Friebe et al., 1993). The next urgent task is the isolation of individual arms as compensating translocation lines (Figure 4.3) (Friebe et al., 1994a, 1994b). All wheat and alien chromosomes where gene synteny is conserved are candidates for the production of translocations. Structurally modified wheat chromosomes, such as 4A, 4B and arms 5AL, 7BS and others where fertility and other pivotal genes are located, should not be used in such manipulations. In general, all D-genome chromosomes are good candidates for the production of compensating translocations as they are the least modified among the three genomes of wheat.

The compensating translocation lines then need to be evaluated for biotic and abiotic stress factors and physiological, agronomic and other desirable traits in greenhouse and field experiments. The donor species accessions can be screened, and useful genetic factors transferred to wheat using compensation translocations in bridging crosses (Friebe et al., 1994a, 1994b). Thus, once a complete set of compensating translocation stocks incorporating the entire donor species genome have been isolated, any genetic variation in that species can be accessed for wheat improvement via bridging crosses.

TABLE 4.4
Alien transfer in production agriculture

CHROMOSOME ENGINEERING

Because of polyploidy, the wheat genome is highly buffered, and the transfer of chromosome segments or large linkage blocks is more important than single genes. In general, compensating translocations involving exchanges of short arms may be agronomically desirable as is the case of transfers of the short arm of rye chromosome 1R substituting for short arms of wheat chromosomes 1A or 1B (Table 4.4). For translocations involving large alien chromosome arms, the unwanted chromatin in the translocation lines must be removed by chromosome engineering.

Already, there are spectacular examples of alien chromosome segments transferred to wheat, either by physical means (irradiation) or by genetic manipulation, that have immensely improved the productivity of the wheat crop (Table 4.4). Sears (1993) has suggested some refinements in both induced homoeologous pairing and irradiation treatments to enhance the recovery of desirable transfers. Jiang et al. (1994b) have discussed strategies for the production and identification of desirable alien transfer segments.

C-banding is used to identify the alien chromosome with the target gene, the recipient wheat chromosome and the translocated chromosome. Genomic in situ hybridization using labelled alien DNA in excess of unlabelled wheat DNA is used to determine the size of the transferred alien segment and the size of the wheat segment that it replaced. A typical example of cytogenetic analysis for an alien transfer is shown in Plate 4. The C-banding pattern of a donor chromosome 4Ai#2 from Thinopyrum intermedium is shown on top. Gene Wsm1, which conditions resistance to wheat streak mosaic virus (WSMV), a devastating disease of wheat for which wheat lacks resistance, is located on the short arm of 4Ai#2. A subterminal C-band is diagnostic for the identification of the short arm. Three different transfer lines involving wheat chromosomes 6A, 4A and 4D were analysed. Translocations involving 6A (left) and 4A (a highly rearranged chromosome) (middle) were non-compensating and agronomically undesirable. However, T4DL.4Ai#2S is a whole arm compensating translocation as donor DNA painting assay shows (right bottom). This germplasm is being used in many breeding programmes to develop WSMV-resistant wheat cultivars.

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