Keywords

1 Learning Objectives

  • What is an introgression?

  • Chemical and radiation versus recombination.

  • How to generate introgressions via homologous recombination and homoeologous recombination.

  • How to generate introgressions from addition and substitution lines.

  • How to use molecular tools for the detection of wheat/wild relative introgressions.

  • Why is the phenotyping of introgression lines important?

  • Understanding the case study.

2 Introduction

The rapidly increasing global population, set to pass the nine billion mark by 2050, presents one of the greatest challenges that humanity has faced – how to feed all the extra people? Major crops such as bread wheat, which provides 20% of the world’s total calories and protein [1], will have to play a major role in feeding the population of the future. However, instead of increasing, wheat yields have recently been starting to plateau.

The plateauing of yields presently observed is most likely due to two compounding factors. Intensive breeding in the past, although very successful, has led to the exploitation and erosion of a proportion of the genetic variation available. This gradual erosion of genetic variation means that in time it will become increasingly more difficult for breeders to generate and identify new gene combinations to develop higher yielding varieties. In addition, the slowing of production increases is being further exacerbated by adverse environmental conditions resulting from climate change, e.g. heat, drought etc.

A major game changer for the production of wheat varieties adapted to climate change that would meet the needs of the increasing global population, is to dramatically increase the available gene pool. In order to achieve this, a new source of donor genetic variation that can be transferred into wheat, needs to be identified.

Wheat is related to a large number of wild species that grow in a wide range of very varied environments, e.g. in fields of cereals, deserts, salt inundated sand dunes, at high and low altitudes etc. These wild relatives, many of which evolved millions of years ago [2], provide a vast reservoir of genetic variation for potentially most, if not all, traits of agronomic and scientific importance, e.g., they carry completely novel forms and levels of genetic variation above and beyond that observed in cultivated wheat (See Chap. 17).

The transfer of genetic variation from the wild relatives in the past, while limited, has had a major impact on wheat production. While conventional breeding produces slow, but gradual, increases in yield production, the successful introgression of genetic material/genes from the wild relatives frequently results in substantial jumps in production and improvement. As a result, many commercial breeders believe that the transfer of genetic variation from the wild relatives may be the only way by which the increases in yield production required by 2050 can be achieved.

There are a number of examples of previously successful introgressions from wild relatives into wheat [3]. These include (1) An introgression from Aegilops umbellulata that carried a resistance gene to the disease leaf rust [4]. In 1960, this introgression saved US wheat production from catastrophic failure. (2) A spontaneous introgression from rye to wheat resulted in a substantial increase in grain yield and disease resistance [5]. The advantages that this introgression conferred over normal wheat were such that it was present in most wheat varieties in the 1990s (it is still present in many modern-day varieties). (3) A high proportion of present-day varieties carry an introgression from Aegilops ventricosa [6] that confers a yield advantage of circa 4% and also carries the only effective sources of resistance to the diseases eye spot and wheat blast [7]. As a result, the Ae. ventricosa introgression is present in nearly 90% of all new CIMMYT varieties. (4) Recent work has revealed that many past and present-day varieties carry Triticum dicoccoides introgressions that have been unconsciously selected for over time due to the advantage they confer over lines that lack them, e.g. the variety Robigus. (5) 30% of all wheat lines bred at CIMMYT are derived from crosses between normal wheat and “synthetic” wheat [8]. The latter is derived from crosses between Aegilops tauschii (DD genome) and tetraploid durum wheat (AABB genome) followed by chromosome doubling using colchicine. Since synthetic and bread wheat have the same genomic constitution, they can be readily hybridized to transfer novel alleles and genes from different accessions of Ae. tauschii, the D-genome progenitor.

From a physiological perspective, there have also been some clear benefits associated with introgressions. The erectophile leaf trait originated from Triticum sphaerococcum can be seen in many modern wheats, especially high yielding spring durums [9]. Evidence for genetic variation in source:sink balance and its importance in boosting yield and radiation use efficiency (RUE) has come from various sources, including studies with cytogenetic stocks [10]. Substitution of the long arm of chromosome 7D in hexaploid wheat with the homologous chromosome from Agropyron elongatum resulted in a significant increase in yield and biomass in six elite lines associated with increased spike fertility and post-anthesis RUE [10]. Synthetic wheats are also present in the pedigrees of lines with high yield potential and have contributed to outstanding expression of stress adaptive traits under heat and drought stress including more vigorous root systems and accumulation of stem carbohydrate reserves [11, 12].

Even though genetic variation from the wild relatives has delivered dramatic increases in wheat improvement, to date only a tiny fraction of the genetic variation available has been exploited. The reasons for this are the direct result of the difficulty in transferring genetic variation from the wild relatives to wheat (specific crossing schemes are required) and the difficulty in identifying plants which carry introgressions. In addition, introgressions carrying genes of interest also frequently carry undesirable genes. The removal of these genes using past technology proved extremely difficult. As a result of these difficulties, the use of wild relatives for wheat improvement went into decline. Thus, where there were many 100s of researchers in the field in the 1970s and 1980s, today only a handful of scientists globally, with the requisite expertise required to transfer genetic variation from the wild relatives to wheat, now remain. However, the advent of new molecular genetic technologies is reinvigorating the exploitation of wild relatives, i.e. it is now possible to transfer and characterise large numbers of introgressions, from a wide range of species, into wheat. These technologies coupled with specific crossing strategies are facilitating, for the first time, the large scale and systematic transfer of genetic variation of genetic variation from the gene pools of the wild relatives into wheat [13,14,15,16,17,18,19].

2.1 Different Classes of Wheat/Wild Relative

Before discussing introgression further it is first essential to establish the relationship between the different types of wild relative and wheat. Hexaploid wheat is an allohexaploid with 42 chromosomes (2n = 6x = AA BB DD). It has seven pairs of A genome chromosomes derived from Triticum urartu [20], seven pairs of B chromosomes from a species thought to be related to Aegilops speltoides [21,22,23] and seven pairs of D chromosomes derived from Ae. tauschii [24].

The wild relatives of wheat effectively fall into three classes or gene pools. The primary gene pool contains species which in several cases could more correctly be called ancestral species, have the same or very similar genomes to wheat. These species include T. urartu and Triticum monococcum (AA genome), Triticum turgidum (AABB genomes) and Ae. tauschii (DD genome). Species in the secondary gene pool also carry at least one genome very closely related to wheat although they show modifications, e.g. they might carry translocations or inversions relative to wheat. Species in the secondary gene pool include Ae. speltoides (SS genome) and Triticum timopheevii (AAGG genomes).

The genomes of the species in the primary and secondary gene pools thus have the equivalent gene content to that of a wheat genome although there may be some allelic differences as well as the structural changes. Thus, the genomes in the primary and secondary gene pools are said to be homologous to the genomes of wheat.

The genomes/chromosomes of the tertiary gene pool of wild relatives, although related, have diverged significantly from those of wheat, often with regard to both DNA content and chromosome structure and morphology. Thus, the chromosomes of these species are said to be homoeologous to those of wheat, i.e. related but not identical. There are a large number of these species from several different genera, e.g. Aegilops caudata (CC), Ae. umbellulata (UU), Aegilops uniaristata (NN), Amblyopyrum muticum (TT), Secale cereale (RR), Thinopyrum bessarabicum (EbEb), Thinopyrum elongatum (EeEe), Thinopyrum intermedium (StStJrJrJvsJvs) and Thinopyrum ponticum (EeEeEbEbExExStStStSt).

2.2 Transferring Genetic Variation from Wild Relatives into Wheat

How is genetic variation from wild relatives transferred to wheat? The first step in the process requires that wheat is hybridised with a wild relative to produce an F1 interspecific hybrid, e.g. pollen from a wild relative is used to pollinate wheat. These F1 hybrids carry the haploid genomes of wheat and the haploid genome(s) of the wild relative and provide the starting point in the transfer of genetic variation from wild relatives.

Transfer of genetic variation from wild relatives has to date been achieved using two different methods. The first involves the use of chemicals or radiation to induce random breakage of chromosomes in the F1 interspecific hybrids or their derivatives (e.g. addition or substitution lines – see later) (e.g. [25, 26]). These broken chromosome segments are said to have sticky ends and they can re-join with other broken chromosome segments. Wheat/wild relative translocations occur when a wheat chromosome segment fuses with a chromosome segment from a wild relative and thus results in the production of interspecific translocations. This process was used very successfully in the past by Ernie Sears to transfer leaf rust from Ae. umbellulata into wheat [4]. However, chromosome breakage induced by chemicals or radiation occurs at random, i.e. translocations frequently occur between completely unrelated chromosomes. A direct result of this is that the progeny derived from translocations are frequently genetically unbalanced, e.g. they carry gene deletions (from the lost wheat chromosome(s)) and duplications (from the added wild relative chromosome(s)) which consequently have deleterious effects on plant vigour.

The second method is via recombination, i.e. the chromosomes of wheat and those of a wild relative recombine in the gametes of the F1 interspecific hybrids or their derivatives at meiosis to produce interspecific wheat/wild relative chromosomes commonly known as introgressions*. These introgressions are then transmitted to the next generation through the gametes. Unlike translocations, because recombination occurs between related chromosomes, they are less likely to give rise to gene deletions and duplications (although deletions and duplications have been observed if the genomes of the wild relatives are translocated relative to wheat or unequal crossing over occurs).

Much of the work discussed in this chapter is applicable to the generation of introgressions either via translocation or recombination. However, because of the problems associated with the production of unbalanced gametes derived from translocations, the remainder of this paper is directed at the induction of introgressions via recombination.

*It should be noted that sometimes introgressions that have been generated via recombination are referred to as translocations which is not strictly correct. The term translocation refers to the phenomenon of chromosome breakage and reunion that is not associated with recombination at meiosis. Thus, introgressions generated via recombination should be referred to as “interspecific recombinant chromosomes” or “recombinant chromosomes.”

3 Generation of Introgressions

The transfer of genetic variation to wheat from wild relatives whose genomes are homologous to one or more of those of wheat is relatively straight forward. In F1 hybrids and their derivatives, the chromosomes of wheat and those of the wild relative are able to pair and recombine during meiosis, leading to the generation of interspecific recombinant chromosomes/introgressions which are recovered in the progeny.

For example: if hexaploid wheat (AABBDD) is crossed with diploid T. urartu (AuAu) the resulting interspecific hybrid’s genomic constitution will be AuABD (Fig. 18.1). At meiosis, recombination between the A and Au chromosomes would be expected to be nearly normal. Thus, a large proportion of the gametes would be expected to carry a balanced number of 7 A/Au recombinant chromosomes (although the A and Au genomes are homologous a level of chromosome failure would still be expected in such crosses). In contrast to the A genome, the B and D genome chromosomes of wheat will not have homologous partners to pair with to form bivalents at meiosis, i.e. they will form univalents and not segregate normally to the spindle poles at anaphase I. Thus, their inclusion in the nuclei at telophase and the resulting gametes will occur at random. As a result, there will be large variations in the number of B and D genome chromosomes carried by the individual gametes, so that many will be genetically unbalanced and inviable. However, a small percentage of sufficiently balanced gametes will be produced. In order to address the high level of infertility, very large numbers of crosses are made to the F1 interspecific hybrid, using the F1 as the female parent, with normal wheat. This increases the likelihood that any viable gametes are fertilised, and the progeny produced will normally carry large numbers of introgressions. These plants are then recurrently crossed to wheat until lines carrying only a single A/Au introgression are isolated.

Fig. 18.1
figure 1

Wheat/wild relative crossing strategy where the genome of the wild relative is homologous to one of the wheat genomes. The example shown is T. urartu, genome Au, which is homologous to the A genome of wheat

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Single introgressions, generated via backcrossing, will be in a heterozygous state and hence if they are self-fertilised, they will segregate for plants that carry the introgression and those that do not. Thus, plants homozygous for the introgressions need to be generated in order to ensure that they are stably inherited to the next generation. This can be achieved by taking heterozygous lines and either using the doubled haploid (DH) procedure [27] or simply by self-fertilizing and screening subsequent progenies with genetic markers for the presence of introgression homozygotes (see Sect. 18.3).

In contrast to species that have genomes homologous to wheat, the generation of introgressions from species with genomes that are homoeologous to those of wheat is more complicated. This is because recombination between homoeologous chromosomes is inhibited at meiosis by the Ph1 locus located on the long arm of chromosome 5B [28] (See Chap. 16). One strategy to overcome this problem is to use lines that lack chromosome 5B or more commonly to use a mutant line in which the wild type Ph1 locus has been deleted, i.e. the ph1 mutant. In F1 interspecific hybrids derived from crosses between a wild relative and wheat homozygous for the ph1 mutation, recombination can occur between the chromosomes of wheat and chromosomes from the wild relative resulting in the generation of introgressions. However, the level of recombination observed is generally very much lower than that seen in interspecific hybrids between wild relatives with homologous genomes, with the result that the frequency of genetically unbalanced and hence inviable gametes is very much higher. The fertility of F1 interspecific hybrids between wheat and wild species with homoeologous genomes is can be extremely low depending on the species, e.g. 16% of crosses to F1 hybrids produced between wheat and Am. muticum generated seed while 29% of crosses to F1 hybrids produced between wheat and Ae. speltoides generated seed [17, 18]. In order to generate sufficient progeny from the F1 interspecific hybrids involving these species, very large numbers of crosses need to be made with wheat, using the hybrid as the female parent. Although the use of these F1 interspecific hybrids is very labour intensive, their recent exploitation has led to the generation of very large numbers of new wheat/wild relative introgressions [17, 29].

An alternative strategy to generate introgressions from wild relatives with homoeologous genomes to wheat, is to use addition and/or substitution lines. Addition lines carry the full complement of wheat chromosomes + a pair of chromosomes from a wild relative, i.e. they carry 42 wheat chromosomes + 2 chromosomes from a wild relative = 44. In contrast, substitution lines have a single homologous pair of wheat chromosomes replaced by a homoeologous pair of chromosomes from a wild relative, i.e. 40 wheat chromosomes + 2 chromosomes from a wild relative = 42. Both of these types of lines are initially generated by chromosome doubling (using colchicine) a F1 interspecific hybrid to generate an amphidiploid, e.g. an amphidiploid between hexaploid wheat and a diploid wild relative such as rye has 56 chromosomes AABBDDRR. These amphidiploids normally show a significantly higher level of fertility compared to the F1 interspecific hybrids they were derived from.

Addition lines are generated (Fig. 18.2) by repeatedly backcrossing an amphidiploid to wheat until lines carrying a single chromosome from the wild relative have been isolated. These monosomic additions (42 wheat + 1 wild relative chromosome) are then allowed to self-fertilise and the progeny screened to identify plants carrying a pair of wild relative chromosomes. Although these disomic additions carry a pair of homoeologous wild relative chromosomes they are relatively unstable and thus require checking at each generation for their presence in order to maintain them.

Fig. 18.2
figure 2

Strategy for the production of a disomic addition line

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The generation of substitution lines (Fig. 18.3) first requires a line of wheat that has lost a single copy of one pair of chromosomes (a monosomic line), e.g. a wheat line monosomic for chromosome 1A would have 40 chromosomes + 1 x 1A = 41. This line is then pollinated with a wheat/wild relative disomic addition line where the pair of chromosomes from the wild relative are homoeologous to the chromosome of wheat present only as a single copy, e.g. (40 wheat + 1 x 1A) x (42 wheat + 2 x 1R). The progeny of this cross will all carry a copy of the chromosome from the wild relative (1R) but will segregate for the presence or absence of the wheat chromosome present as only a single copy in the monosomic line (e.g. +1A or -1A). The progeny are then screened to select plants that have lost the single wheat chromosome, e.g. 40 wheat + 1R = 41 chromosomes while plants still carrying the single wheat chromosome (40 wheat + 1A + 1R = 42 chromosomes) are discarded. The selected plants are called monosomic substitution lines and once identified are self-fertilised to produce disomic substitution lines, e.g. 40 wheat + 2x1R = 42 chromosomes. Even though disomic substitution lines have lost a complete pair of homologous wheat chromosomes, the homoeologous wild relative chromosomes are frequently able to compensate for their absence (providing that the homoeologous chromosomes carry a related gene compliment etc.). Substitutions have a big advantage over addition lines in that they are normally stably inherited from one generation to the next.

Fig. 18.3
figure 3

Strategy for the production of a disomic substitution line

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The generation of introgression lines from substitution and addition lines can be achieved by crossing them twice to the wheat ph1 mutant line followed by selection for lines that have lost the Ph1 wild type locus but still retain the wild relative chromosome (Fig. 18.4). In the absence of the Ph1 locus, homoeologous recombination can occur between the chromosomes of wheat and the wild relative leading to the generation of introgressions which can then be recovered in the progeny of crosses to normal wheat.

Fig. 18.4
figure 4

Generating introgressions from a disomic substitution line

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The decision to generate introgressions directly from interspecific F1 hybrids or from addition/substitution lines is dependent on what is trying to be achieved. Both strategies have advantages and disadvantages. One advantage of using interspecific F1 hybrids is that large numbers of introgressions can be quickly generated, potentially from the entire genome of a wild relative, without the need to generate a complete set of addition and/or substitution lines. Addition and substitution lines, however, are very useful if you are attempting to introduce genetic variation from a known area of the wild relative genome as they are considerably more fertile than F1 interspecific hybrids and efforts can be focussed on the required chromosome.

While the removal of the Ph1 locus is required for the induction of homoeologous recombination between wheat and the chromosomes of the majority of wild relatives there are exceptions, e.g. Am. muticum, Ae. speltoides and Aegilops geniculata [17, 18, 30]. These species carry a gene or genes that induce homoeologous recombination even in the presence of the wild Ph1 locus. The efficacy of the genes responsible in Am. muticum and Ae. speltoides has been demonstrated through the generation of very large numbers of wheat/Am. muticum and wheat/Ae. speltoides introgressions from interspecific F1 hybrids [17, 18] while chromosome 5Mg of Ae. geniculata recombined with both chromosome 5D of wheat and also with the group 5 chromosomes of other wheat wild relatives [30].

4 Tools for Detection of Wheat/Wild Relative Introgressions

The recent advent of next generation sequencing and the concomitant development of new genetic marker technologies has resulted in a revolution in the field of wheat/wild relative introgression. Previously, the lack of genetic markers was a major limiting factor in the detection and characterisation of introgressions. As a result, many introgressions could only be detected via phenotypic analysis and without characterisation, many were frequently very large and carried deleterious genes affecting plant vigour as well as the genes of agronomic importance.

Today the development of sequencing technologies is resulting in the generation of 1000s of molecular markers that can be exploited in wheat/wild relative introgression programmes. Single nucleotide polymorphism (SNPs) markers, in particular, have been valuable, i.e. SNP markers are based on a single base pair difference between wheat and a wild relative at a specific DNA sequence. The presence of an introgression is determined by screening individual plants to ascertain which bases are present. There are a number of platforms that can be used to screen plants with SNPs. The Axiom Wheat-Relative Genotyping array (Affymetrix), for example, has allowed large numbers of plants to be screened for the presence of circa 35,000 SNPs [14]. Alternatively, plants can be screened for introgressions via SNPs with Kompetitive Allele Specific PCR (KASP) markers, a genotyping technology based on allele-specific oligo extension and fluorescence resonance energy transfer for signal generation and found to be more cost-effective for large-scale projects (Fig. 18.5).

Fig. 18.5
figure 5

(a) KASP marker designed to be polymorphic for a SNP found on all three genomes of wheat and a wild relative. The signals for both a (i) heterozygous introgression and a (ii) homozygous introgression cluster between the signals for the wheat controls and the wild relative controls. (b) KASP marker designed to be polymorphic between a wheat chromosome specific SNP (in this instance the SNP occurs on 3B) and a wild relative. The signal for heterozygous introgressions (i) will cluster between the signals for the wheat controls and the wild relative controls. The signal for homozygous introgressions (ii) will cluster with the wild relative controls

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Recently, KASP markers polymorphic between wheat and ten of its wild relative species were developed [31]. These markers were designed not just to be polymorphic between a wild relative and wheat but to be polymorphic between a wild relative and a specific chromosome of wheat (Fig. 18.5). Thus, in addition to detecting the wild relative segment in the introgression line, these markers have the additional functionality in that they can indicate whether the segment is heterozygous or homozygous through the loss of wheat alleles for the KASP markers. These markers therefore firstly reduce the need for more labour-intensive laboratory techniques such as GISH (see end of this section) but secondly, and more importantly in a breeding programme, remove the need for logistically demanding progeny testing necessary to distinguish between plants with heterozygous or homozygous introgressions (In progeny testing, 10 to 20 progeny seed are germinated and tested with markers for the presence of the segment. Where all the progenies are found to contain the introgressed segment, it is assumed that the original plant was homozygous. Progeny showing segregation for the presence/absent will have been derived from a heterozygous parent. Testing of a second generation will validate the result). Moreover, in homozygous introgression lines these chromosome-specific KASP markers can indicate which genome of wheat (A, B or D) the recombination with the wild relative species has occurred. These markers have recently been used to characterise Ae. caudata introgressions in bread wheat [32] and D-genome introgressions from bread into durum wheat [33].

The continued developments in high-throughput sequencing and the reduction in costs are now allowing the generation of sequence data from wild relative species, e.g. Ae. tauschii [34], T. urartu [35], S. cereale [36]. Thus, chromosome-specific KASP markers are now being developed based on SNPs between wheat and the wild relative in single-copy regions of the wheat genome (unpublished results) taking away the cumbersome need to anchor the KASP primers to chromosome-specific alleles during their design. The sequencing of individual introgression lines is also enabling the detection of the site of recombination between wheat and the wild relative (unpublished data).

In addition to sequencing and marker technology, molecular cytological techniques and microscopy systems have also developed significantly. In the past, the detection of introgressions depended on cytological techniques which were very labour intensive and frequently provided limited information. However, techniques such as genomic in situ hybridisation, can now be used routinely to detect introgressed chromosome segments in wheat, while at the same time distinguishing the chromosomes of the three genomes of wheat from each other (Fig. 18.6). Furthermore, many systems have high levels of automation, e.g. large numbers of slides can be screened remotely for chromosome spreads and multiple fluorescent images taken.

Fig. 18.6
figure 6

Multi-colour GISH analysis of a homozygous introgression line. The wheat A genome chromosomes are shown in green, wheat B-genome chromosomes in blue and wheat D-genome chromosomes in red. The homozygous introgression from Am. muticum (white arrows) is shown in yellow. This introgression has recombined at both ends with the D-genome

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In combination, cytological analysis, markers and sequence data are increasingly providing new information within this field of research. For example, until recently it was thought that the majority of wild relative introgressions were very large and recombinant events restricted to the ends of chromosomes. However, recent research has clearly shown that recombination appears to occur throughout the length of the chromosomes and that very small introgressions are not uncommon [29, 30].

5 Reducing the Size of Introgressions

When introgressions are very large, they carry deleterious genes affecting plant vigour as well as the genes of agronomic importance. Thus, further work needs to be carried out to reduce the size of the introgression to as small as possible carrying the wild relative gene of interest. Ernie Sears, the father of wheat cytogenetics and wheat wild relative introgression, developed a strategy to reduce the size of large introgressions [37]. He used this strategy to remove deleterious genes from two wheat/Ae. umbellulata introgressions while retaining a gene for resistance to leaf rust. Essentially this strategy involved the inter-crossing of plants containing overlapping introgressions where the target gene was located in the overlap (Fig. 18.7). In the presence of Ph1, recombination freely occurred between the overlapping introgressions, resulting in some individuals among the progeny that had a significantly smaller introgression but still retained the gene for resistance to leaf rust. This strategy, although ground-breaking, was ahead of its time because the marker technology required to identify large numbers of overlapping introgressions and to identify smaller modified introgressions was not available at the time.

Fig. 18.7
figure 7

Strategy to reduce the size of a large introgressed segment

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Alternative strategies to reduce the size of introgressions such as the induction of further homoeologous recombination between wheat and a wild relative chromosome segment in the absence of Ph1 [38] have also previously proved difficult to undertake, again largely due to the lack of markers available to detect rare small recombinants.

6 Phenotyping

While this paper concentrates on the process of wheat/wild relative introgression, it is important to discuss the role of phenotyping in the exploitation of introgression lines in breeding programmes. In the past, wild relatives and in particular addition and substitution lines, have been screened to identify genetic variation for a specific trait. Introgressions have then been made to transfer genetic variation for the target trait. However, the resulting introgressions have been found to carry important genetic variation for additional characters. For example, an introgression from Ae. ventricosa was introduced into wheat that carried resistance to the disease eyespot [39]. However, at the time of writing, it has been shown that it also possesses the only source of resistance to the disease wheat blast and also confers a significant yield advantage [7].

Others have used a strategy where they have transferred the entire genome of a wild relative into wheat by generating large numbers of introgressions. Although the wild relatives used were selected as they were known to carry genetic variation for several target traits, the emphasis of the work was on screening any resulting introgressions for as many traits as possible. Irrespective of how they have been generated, in order for the potential of the 1000s of new wheat/wild relative introgressions being generated to be realised, it is essential that each is screened for a broad spectrum of traits in a wide range of environments. This will make it possible to determine what agronomic characters are affected by the genes present in each introgression. This large-scale screening is critical because without it each introgression will just be seeds in a packet of unknown agronomic value.

7 Case Study

In order to provide an insight into the workflow, disciplines and logistics required to undertake a present day introgression programme we here describe a case study of a wheat/wild relative programme carried out at the Nottingham BBSRC Wheat Wild Relative Centre involving Am. muticum (Fig. 18.8).

Fig. 18.8
figure 8

Case study: generating introgressions from Am. muticum

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7.1 Step 1 – Generation of Introgression Lines

Am. muticum was used to pollinate wheat (variety Paragon), which carried the ph1 mutation, to produce F1 interspecific hybrids. It is important to note two key facts with regard to these hybrids. Firstly, because each F1 lacked the wild type Ph1 locus, homoeologous recombination could occur during meiosis and in addition, Am. muticum carries a gene(s) which promotes homoeologous recombination. Secondly, since the interspecific hybrids only possessed the haploid genomes of wheat and Am. muticum, i.e. ABDT, and thus homologous pairs of chromosomes were not present, only homoeologous recombination could occur. As a result of this strategy large numbers of introgressions were generated. However, the drawback to this strategy was that the F1 hybrids showed very low fertility. In order to obtain progeny and hence isolate introgressions, each of the interspecific hybrids was extensively crossed to wheat.

7.2 Step 2 – Molecular Identification of Introgressions and Their Characterisation

The resulting progeny were screened using the Axiom® Wheat-Relative Genotyping Array (the array carries circa 35,000 SNP markers that show polymorphism between 10 wild relatives and wheat varieties such as Paragon) to detect potential introgressions. Plants carrying introgressions were recurrently crossed to wheat and the presence or absence of introgressions was confirmed using genomic in situ hybridisation.

The ultimate aim of the programme is to generate lines that are homozygous for a single introgression so the lines can be multiplied and distributed to collaborators for phenotypic analysis. As described earlier this can be achieved either by the DH procedure or simply by self-fertilisation. In order to test the potential of the DH procedure, several plants heterozygous for an introgression were selected from the BC3 generation and crossed with maize. In the resulting hybrids, the maize chromosomes were eliminated, and the resulting haploid plants were chromosome doubled to give rise to DH lines that were potentially homozygous for each introgression. The presence of homozygous introgressions was confirmed via GISH and by genome specific KASP markers, i.e. this also allowed the determination of which wheat chromosomes were involved in each introgression. This work led to the isolation of 66 wheat/Am. muticum introgressions which were multiplied and have been distributed to both the public and private sectors free of IP [27]. This programme is presently generating many additional new wheat/Am. muticum homozygous introgressions, via self-fertilisation, prior to their distribution.

7.3 Step 3 – Making Use of the Introgression Lines

An initial series of phenotypic analyses have been undertaken on 20 wheat/Am. muticum introgression lines for resistance to leaf, stem and yellow rusts by collaborators from the USDA at Kansas State University [40]. In each case introgressions were identified that conferred resistance. Furthermore, resistance to Wheat streak mosaic virus and powdery mildew was also observed. These introgressions are presently being introduced into US adapted germplasm for further testing. At the time of writing large numbers of further introgressions have been sent to Kansas, and distributed within the UK for large scale phenotypic analyses on a wide range of traits (all of the available homozygous lines developed at Nottingham are listed at the following web sites: Nottingham https://www.nottingham.ac.uk/wrc/home.aspx and Norwich https://www.jic.ac.uk/research-impact/germplasm-resource-unit/).

8 Key Concepts

This chapter has discussed the different strategies for the generation of introgressions from the wild relatives of wheat and their detection and characterisation.

  1. 1.

    Definition of an introgression and understanding the difference between an introgression and a translocation.

  2. 2.

    The benefits of generating an introgression via recombination rather than via random breakage and joining of chromosomes.

  3. 3.

    Advantages and disadvantages of generating introgressions via homologous recombination compared to homoeologous recombination.

  4. 4.

    The generation and use of addition and substitution lines.

  5. 5.

    How the developments in molecular biology over the last decade have enabled the detection and characterisation of introgressions.

  6. 6.

    The importance of phenotyping

9 Conclusions

  • Wheat evolved only once or twice about 8 to 10,000 years ago while many of its wild relatives, of which there are hundreds of different accessions, evolved several millions of years ago.

  • Wild relatives therefore provide a vast reservoir of genetic variation above and beyond anything seen in wheat for potentially all traits of agronomic importance.

  • Due to a lack of adequate technologies, it has been very difficult to exploit genetic variation in the wild relatives for wheat improvement. Recently, the development of new technologies, has enabled the large-scale transfer of genetic variation from the wild relatives into wheat. These technologies, combined with large-scale phenotypic analyses, will enable the genetic variation from wild relatives to have a major global impact on wheat production.

  • Future technological advances, such as a greater understanding of the genomes of the wild relatives, will further enhance our ability to transfer genetic variation into wheat.

  • A major concern for the exploitation of the wild relatives is the lack of scientists with the prerequisite expertise in wheat chromosome manipulation.