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The application of biotechnology to wheat improvement
D. Hoisington, N. Bohorova, S. Fennell, M. Khairallah, A. Pellegrineschi, J.M. Ribaut


Today, the world’s population is increasing at the most rapid rate ever. Two hundred people are being added to the planet every minute. It is forecast that by the year 2050, the world’s population will double to nearly 12 billion people. To feed this population, these people will require a staggering increase in food production. In fact, it has been estimated that the world will need to produce more than twice as much food during the next 50 years as was produced since the beginning of agriculture 10 000 years ago.

How will researchers continue to develop improved wheat varieties to feed the world in the future? At least for the foreseeable future, plant breeding as it is known today will play a primary role. What will change are the tools that can be employed. This chapter focuses on current approaches for the use of modern molecular-based technologies to develop improved varieties and discusses areas for future applications.

Biotechnology can be defined in many different ways, but for the purpose of this chapter, all areas that use molecular approaches to understand and manipulate a plant genome will be considered. However, for the sake of discussion, the techniques are divided between those that make use of molecular markers for studying the genetic material already present within the wheat plant and genetic engineering aimed at the introduction of novel genetic material. It is the latter that often raises concern and that many believe represents ‘modern biotechnology’.

WHEAT MOLECULAR GENETICS

Molecular genetics, or the use of molecular techniques for detecting differences in the DNA of individual plants, has many applications of value to crop improvement. The differences are called molecular markers because they are often associated with specific genes and act as ‘signposts’ to those genes. Such markers, when very tightly linked to genes of interest, can be used to select indirectly for the desirable allele, and this represents the simplest form of marker-selection (MAS), whether used to accelerate the back-crossing of such an allele or in pyramiding several desirable alleles. Markers can also be used for dissecting polygenic traits into their Mendelian components or quantitative trait loci (QTL), thus increasing understanding of the inheritance and gene action for such traits and allowing the use of MAS as a complement to conventional selection procedures. Molecular markers are also used to probe the level of genetic diversity among different cultivars, within populations, among related species, etc. The applications of such evaluations are many, including varietal fingerprinting for identification and protection, understanding relationships among the units under study, efficiently managing genetic resources, facilitating introgression of chromosomal segments from alien species, and even tagging of specific genes. In addition, markers and comparative mapping of various species have been very valuable for improving the understanding of genome structure and function and have allowed the isolation of genes of interest via map-based cloning.

Several molecular marker types are available and they each have their advantages and disadvantages. In Table 10.1, the characteristics and usefulness of the most commonly used marker systems are shown. Restriction fragment length polymorphisms (RFLPs) were the first to be developed (some 15 years and have been widely and successfully used to construct linkage maps of various species, including wheat. With the development of the polymerase chain reaction (PCR) technology, several marker types emerged. The first of those were random amplified polymorphic DNA (RAPD), which quickly gained popularity over RFLPs due to the simplicity and decreased costs of the assay. However, most researchers now realize the weaknesses of RAPDs and use them with much less frequency. Microsatellite markers, or simple sequence repeats (SSRs), combine the power of RFLPs (codominant markers, reliable, specific genome location) with the ease of RAPDs and have the advantage of detecting higher levels of polymorphism. The amplified fragment length polymorphism (AFLP) approach takes advantage of the PCR technique to selectively amplify DNA fragments previously digested with one or two restriction enzymes. Playing with the number of selective bases of the primers and considering the number of amplification products per primer pair, this approach is certainly the most powerful in terms of polymorphisms identified per reaction. For more details of the marker types discussed, refer to Hoisington et al. (1998).

TABLE 10.1
Characteristics and usefulness of molecular marker types for wheat molecular geneticsa

Useb

RFLPs

RAPDs

SSRs

AFLPs

Varietal fingerprinting

++

-/+

+++

+++

Genetic diversity

++

-

++

+

Qualitative gene tagging

++

++

++

+++

QTL mapping

++

-/+

++

++

MAS

+

-

++

+/++

Comparative mapping

++

-

-

-

Principle

Endonuclease restriction; Southern blot; hybridization

DNA amplification with random primers

Amplification of simple sequence repeats using specific primers

Endonuclease restriction; use of adapters and specific primers

Types of probe/primers

gDNA, cDNA

Random 9- or 10-mer oligo-nucleotides

Specific 16- to 30-mer primers

Specific adapters and selective primers

Type of polymorphism

Single base changes; insertions/deletions

Single base changes; insertions/deletions

Changes in length of repeats

Single base changes; insertions/deletions

Genomic abundance

High

Very high

Medium

Very high

Level of polymorphism

Medium

Medium

High

High

Inheritance

Co-dominant

Dominant

Co-dominant

Dominant

Number of loci detected

3-9

1-10

1-3

70-140

Need for

No

No

Yes

No

sequence information





Technical difficulty

Medium

Low

Low

Medium/High

Reliability

High

Intermediate

High

Medium/High

Quantity of DNA required

10-15 mg

10-50 ng

50-100 ng

100-1 000 ng

Use of radioisotopes

Yes/No

No

Yes/No

Yes/No

Start-up costs

Medium

Low

Medium

High

Development costs

Medium

Low

High

Medium/High

aRFLP = restriction fragment length polymorphism; RAPD = random amplified polymorphic DNA; SSR = simple sequence repeat; AFLP = amplified fragment length polymorphism; - = not useful; +/- = somewhat useful; ++ = useful; +++ = very useful.

bQTL = quantitative trait locus; MAS = marker-assisted selection.

Source: Adapted from Rafalski and Tingey, 1993.

The developments in molecular genetics in wheat have been relatively slow, especially when compared to other crops, such as maize, rice or tomato, due to wheat’s ploidy level, the size and complexity of its genome, the very high percentage of repetitive sequences and the low level of polymorphism (Table 10.2). Much fewer maps exist in wheat and far fewer QTL studies have been reported when compared to other grass species. However, due to the large number of disease and pest resistances controlled by major genes, the mapping of such genes has dominated the research activities in wheat molecular genetics. On the other hand, the hexaploid nature of wheat and its amenity to cytogenetic manipulation have offered unique tools for molecular geneticists of wheat. These include the use of various aneuploid stocks, such as nullitetrasomic and ditelosomic lines, to assign molecular markers to specific chromosome arms (Anderson et al., 1992; Plaschke et al., 1996), of chromosomal deletion stocks (Endo and Gill, 1996) for the physical mapping of markers (Röder et al., 1998a) and of single chromosome substitution lines to map genes of known chromosomal location (e.g. Galiba et al., 1995; de la Peña et al., 1997).

TABLE 10.2
Characteristics of the bread wheat genome that explain the slow progress in mapping as compared to a diploid, highly polymorphic species such as maize

Characteristica

Wheat

Maize

Ploidy level

6x

2x

Number of chromosomes

21

10

Genome size (number of base pairs x 106)

16 000

4 500

Polymorphism level

Low

High


· RFLPs: probe x enzyme combinations (%)

20-30

80-85

· SSRs: primer pairs (%)

40-50

50-60

Repetitive sequences (%)

>80

60

To construct linkage maps of same density (15 markers/chromosome):



Number of loci needed

315

150

Number of RFLP probes needed

1 000-1 500

200-250

Number of SSR primer pairs needed

700-800

250-300

aRFLP = restriction fragment length polymorphism; SSR = simple sequence repeat.

Wheat molecular linkage maps

The establishment of genetic linkage maps provides the basis for mapping the gene(s) responsible for the expression of traits of interest. In wheat, such maps have also corroborated cytological evidence of major chromosome rearrangements (Devos et al., 1995; Nelson et al., 1995a) and have allowed the comparative mapping among related species (e.g. Ahn et al., 1993; Börner et al., 1998; Devos et al., 1994).

The first RFLP maps were reported by Chao et al. (1989) for the group 7 homoeologous chromosomes. Using mapping populations developed at the John Innes Centre, Norwich, England, Devos et al. (1992) published the group 3 maps. These were followed by group 2 (Devos et al., 1993), group 5 (Xie et al., 1993), groups 4, 5 and 7 (Devos et al., 1995) and group 6 (Jia et al., 1996) maps. The Norwich wheat RFLP linkage map has also been published altogether (Gale et al., 1995) and now contains over 500 loci.

Another important mapping population was developed at the International Maize and Wheat Improvement Center (CIMMYT) by crossing a synthetic (amphihexaploid) wheat (Aegilops tauschii [syn. Triticum tauschii] x Altar 84 durum) to a spring bread wheat cultivar Opata 85 and was genotyped at Cornell University in the United States. The use of such a non-intervarietal cross resulted in a very dense map (about 1 000 RFLP loci) due to the higher polymorphism level. Maps of group 1 (Van Deynze et al., 1995), group 2 (Nelson et al., 1995b), group 3 (Nelson et al., 1995c), groups 4, 5 and 7 (Nelson et al., 1995a) and group 6 (Marino et al., 1996) have been published. Recently, Röder et al. (1998b) placed 279 SSR loci on the map also referred to as the ITMI (International Triticeae Mapping Initiative) map.

In addition to two other linkage maps in wheat (Liu and Tsunewaki, 1991; Cadalen et al., 1997), a number of RFLP physical maps have been constructed using Chinese Spring deletion lines (e.g. Kota et al., 1993; Hohmann et al., 1994; Gill et al., 1996). These deletion lines were also used to construct group 2 SSR physical maps (Röder et al., 1998a). In general, genetic maps have revealed a lower level of polymorphism in the D genome (Liu and Tsunewaki, 1991; Cadalen et al., 1997).

Furthermore, a large number of RFLP loci (Anderson et al., 1992; Devey and Hart, 1993) and a fair number of microsatellite loci (Plas-chke et al., 1996; Bryan et al., 1997) have been assigned to chromosome arm locations using nullisomic-tetrasomic and ditelosomic lines.

Mapping of single or major genes

In the last five years, a large number of genes of various functions have been mapped to specific wheat chromosomal regions. Table 10.3 includes a vast majority of those genes controlling disease and pest resistance, stress tolerance, quality and other traits. Several mapping/tagging strategies using mostly RFLPs and RAPDs have lead to these results. As seen in Table 10.3, several RFLP and RAPD linked markers were then converted to PCR-based, more robust markers, such as sequence tagged sites (STSs), sequence characterized amplified regions (SCARs) or allele specific amplicons (ASAs).

The existence of numerous sets of wheat near-isogenic lines (NILs) differing in the presence or absence of a resistance allele has facilitated the mapping of genes for which such lines exist (e.g. Hartl et al., 1993, 1995 for Pm1, Pm2 and Pm3; Schachermayr et al., 1994, 1995 for Lr9 and Lr24; Demeke et al., 1996 for Bt-10; Sun et al., 1997 for Yr15). Dweikat et al. (1997) screened a series of NILs in Newton for Hessian fly resistance alleles using 1 600 random 10-mer primers. One to three RAPD markers were identified for each of the 11 genes being tagged, and linkage determined by screening F2 populations segre-gating for each individual gene. On the other hand, Feuillet et al. (1995) screened Thatcher NILs for Lr1 (on 5DL) with 37 RFLP probes mapping to group 5 chromosomes and found three to be linked to the gene after testing on F2 populations between Thatcher and Lr1/Thatcher. The same approach was used by Williams et al. (1994) who found two RFLP markers flanking the Cre (Cre1) gene on the long arm of 2B.

When the chromosomal location of a particular gene is known from previous genetic studies but no NILs are available, one can still utilize the markers mapped to that chromosome (Anderson et al., 1992) to score the parental lines for polymorphisms, construct a single chromosome map and determine which marker is closest to the gene of interest. This strategy was followed by Dubcovsky et al. (1996) to tag the Kna1 locus in wheat responsible for higher potassium/sodium (K+/Na+) accumulation in leaves, a trait correlated with higher salt tolerance. Single chromosome maps and markers for genes on these chromosomes have also been developed using single-chromosome recombinant lines (Galiba et al., 1995 for Vrn1 and Fr1; de la Peña et al., 1996, 1997 for Pch2; Korzun et al., 1998 for Rht8). These mapping populations were derived according to Law (1966) by crossing lines of the same background but differing for a single chromosome, back-crossing to a monosomic line for the chromosome under study, identifying the monosomic plants with a hemizygous recombinant chromosome, selfing those and detecting disomic recombinants. Despite the difficulties of producing such mapping populations, the main advantage they offer is that they allow the scoring of the phenotypic effect of the gene of interest without the confounding effects of other genes (on other chromosomes) involved in the expression of the same trait.

Bulk segregant analysis (BSA), developed by Michelmore et al. (1991) to tag disease resistance genes in lettuce, has been successfully applied in wheat. This approach has been mostly used with RAPDs (e.g. Hartl et al., 1995 for Pm1 and Pm2; Hu et al., 1997 for Pm1) although it is now being used with AFLPs (Goodwin et al., 1998; Hartl et al., 1998). Either marker technique is used to screen two bulks of DNA samples from individuals identified in the two opposite tails of a segregating population for a target trait. For a major gene, all loci in the genome should appear to be in linkage equilibrium, except in the region of the genome linked to the target gene. To overcome the problems of limited repeatability of RAPDs, and the fact that repetitive sequences are often amplified (Devos and Gale, 1992), Eastwood et al. (1994) and William et al. (1997) used BSA and RAPDs on DNA enriched for low-copy sequences. In both cases, there was a noted increase in repeatability and levels of polymorphism detected compared with non-enriched DNA. The AFLP technology offers the advantage of the high number of DNA fragments amplified with one primer combination, and the problem of highly repetitive DNA is overcome by using methylation sensitive endonucleases, such as PstI and SseI.

The fact that several of the resistance genes mapped in wheat have been introgressed from alien species explains the success of tagging them since a higher level of polymorphism is detected compared to segments where no alien DNA is transferred.

Quantitative trait mapping

The low number of quantitative traits dissected into their QTL in wheat is a reflection of the focus given to simply inherited traits and the difficulty of building comprehensive genetic linkage maps. In addition, more work is involved to generate good quality, reliable phenotypic data from replicated field (or greenhouse) evaluations of the trait under study.

TABLE 10.3
Published markers for important genes in wheat

Traita

Locusb

Sourcec

Markerd

Chromosome

Reference

Disease resistance







Leaf rust

Lr1

Triticum aestivum

RFLP/STS

5DL

Feuillet et al., 1995

Lr3

T. aestivum

RFLP

6BL

Parker et a/., 1998

Lr9

Aegilops

RAPD/STS

6BL

Schachermayr et a/., 1994


umbellulata

RFLP


Autrique et a/., 1995

Lr10

T. aestivum

RFLP/STS

1 AS

Schachermayr et a/., 1997

Lr13

T. aestivum

RFLP

2 BS

Seyfarth et a/., 1998

Lr18

T. timopheevii

N-band

5BL

Yamamori, 1994

Lr19

Thinopyrum

RFLP

7DL

Autrique et a/., 1995



Isozyme


Winzeler et al., 1995

Lr20

T. aestivum

RFLP

5 AL

Parker et a/., 1998

Lr23

T. turgidum

RFLP

2BS

Nelson et a/., 1997

Lr24

Agropyron

RFLP

3DL

Autrique et a/., 1995


elongatum

RAPD/STS


Schachermayr et a/., 1995



RAPD/SCAR


Dedryver et a/., 1996

Lr25

Secale cereale

RAPD

4BL

Procunier et a/., 1995

Lr27

T. aestivum

RFLP

3BS

Nelson et a/., 1997

Lr29

Ag. elongatum

RAPD

7DS

Procunier et a/., 1995

Lr31

-

RFLP

4BL

Nelson et a/., 1997

Lr32

Ae. tauschii

RFLP

3DS

Autrique et a/., 1995

Lr34

T. aestivum

RFLP

7DS

Nelson et a/., 1997

QTL

T. aestivum

RAPD/RFLP

7BL, 1BS, 1DS

William et a/., 1997

Suppressor

SuLr23

-

RFLP

2DS

Nelson et a/., 1997

Stem rust

Sr2

T. turgidum

RFLP/STS

3BS

Johnston et a/., 1998

Sr5

T. aestivum

RFLP

6DS

Parker et a/., 1998

Sr9e

T. aestivum

RFLP

2BL

Parker et a/., 1998

Sr22

T. monococcum

RFLP

7AL

Paull et a/., 1995

Sr36

T. timopheevii

RFLP

2BS

Parker et a/., 1998

Stripe rust

Yr15

T. dicoccoides

RFLP/RAPD

1BS

Sun et a/., 1997

Powdery mildew

Pm1

-

RFLP

7AS

Ma et a/., 1994



RFLP


Hartl et a/., 1995



RAPD-STS


Hu et al., 1997

Pm2

-

RFLP

5D

Ma et al., 1994; Hartl et a/., 1995



RFLP, STS


Mohler and Jahoor, 1996

Pm3

-

RFLP

1A

Ma et a/., 1994;



RFLP


Hartl et a/., 1993

Pm4a

-

RAPD

.

Li et a/., 1995

Pm4b

-

AFLP

-

Hartl et a/., 1998

Pm12

Ae. speltoides

RFLP

6B/6S

Jia et a/., 1994

Pm18

-

RFLP

7AL

Hartl et a/., 1995

Pm21

Haynaldia villosa

RAPD

6VS, 6AL

Qi et a/., 1996

Pm25

T. monococcum

RAPD

1A

Shi et a/., 1998

Suppressor

SuPm8

-

Storage protein

1AS

Ren et a/., 1996

Wheat streak mosaic virus

Wsm1

Ag. elongatum

STS

-

Talbert et a/., 1996

Common bunt

Bt-10

-

RAPD


Demeke et a/., 1996

Loose smut

Ut-X (T10)

-

RFLP/RAPD-

-

Procunier et a/., 1997



STS



T19

-

Antibody

6A

Knox and Howes, 1994

Eyespot

Pch1

-

RFLP/lsozyme

7DL

Chao et a/., 1989

Pch2

T. aestivum

RFLP

7AL

de la Pena et a/., 1997

Tan spot

QTL

-

RFLP

1AS, 4AL, 2DL

Faris et a/., 1997

Fusarium

QTL

T. aestivum

AFLP/RFLP

3BS, 2AL,

Anderson et a/., 1998

scab




6BS, 4BL


Karnal bunt

QTL

T. turgidum

RFLP

3BS, 5AL

Nelson et a/., 1998

Pest resistance

Hessian fly

H3,5,6,9,

-

RAPD

1A, 5A

Dweikat et al., 1997

10, 11, 12,





13, 14, 16, 17





H9

-

RAPD

-

Dweikat et a/., 1994

H21

S. cereale

RAPD

2RL

Seo et al., 1997

H23, H24

Ae. tauschii

RFLP

6D, 3DL

Ma et al., 1993

H27

Ae. ventricosa

Isozyme

4MV

Delibes et al., 1997

Cereal cyst nematode

Cre1

T. aestivum

RFLP-STS

2BL

Williams et al., 1994, 1996

Cre2

-

RFLP

6BL

Paull et al., 1998

Cre3

Ae. tauschii

RAPD

2DL

Eastwood et al., 1994

(Ccn-D1)





Stres tolerance

Cadmium uptake

-

-

RAPD

-

Penner et al., 1995

Aluminium

Alt2

-

RFLP

4D

Luo and Dvorak, 1996

tolerance



RFLP

4DL

Riede and Anderson, 1996

Drought

-

-

RFLP

5A

Quarrie et al., 1994

induced ABA






Na+/K+

Kna1

T. aestivum

RFLP

4D

Alien et al., 1995

discrimination



RFLP

4DL

Dubcovsky et al., 1996

Qualit traits

Kernel

Ha


RFLP

5D

Nelson et al., 1995a

hardness

Hn, QTL

.

RFLP

5DS, 2A, 2D,

Sourdille et al, 1996





5B, 6D


Grain protein

QTL

T. turgidum

RFLP

4BS, 5AL, 6AS,

Blanco et al., 1996





6BS, 7BS


High protein

-

T. dicoccoides

ASA

6B

Humphreys et a/., 1998

LMW glutenin

-

T. turgidum

-

1B

D'Ovidio and Porceddu, 1996

HMW glutenin

Glu-D1-1

T. aestivum

ASA

1DL

D'Ovidio and Anderson, 1994







Flour colour

-

-

RFLP/AFLP

7A

Parker et al., 1998

Other trait

Pre-harvest sprouting

QTL

T. aestivum

RFLP

-

Anderson et al., 1993

Vernalization

Vrn1

.

RFLP

5AS

Galiba et a/., 1995; Nelson et al., 1995a; Korzun et al., 1997; Kato et al., 1998


Vrn3

-

RFLP

5DS

Nelson et al., 1995a

Photoperiod

Ppd1

T. aestivum

RFLP

2DS

Worland et al., 1997


Ppd2

T. aestivum

RFLP

2BS

Worland et al., 1997

Dwarfing

Rht8

-

SSR

2DS

Korzun et al., 1998


Rht 12

-

SSR

5AL

Korzun et al., 1997

Fertility

Rf1, Rf3

-

RFLP

6BS, 1BS

Ma and Sorrells, 1995

restoration

Rf4





Meiotic

ph1b

-

RFLP/STS

5BL, 5BL

Gill and Gill, 1996

pairing

Deletion

-

AFLP/STS


Qu et al., 1998

aABA = abscisic acid; K+/Na+ = potassium/sodium; LMW = low molecular weight; HMW = high molecular weight.

bQTL = quality trait locus.

cAe. tauschii = T. tauschii.

dRFLP = restriction fragment length polymorphism; STS = sequence tagged site; RAPD = random amplified polymorphic DNA; SCAR = sequence characterized amplified region: AFLP = amplified fragment length polymorphism; ASA = allele specific amplicon; SSR = simple sequence repeat.

Source: Modified from Langridge and Chalmers, 1998.

The ITMI map is one of the densest available, and the population from which it was developed is segregating for a number of traits. It has therefore been used to map some important traits in addition to several major genes. Known genes include vernalization (Vrn1 and Vrn3), red-coleoptile (Rc1), kernel hardness (Ha) and powdery mildew (Pm1 and Pm2) genes (Nelson et al., 1995a), as well as genes conferring and suppressing leaf rust resistance (Nelson et al., 1997). QTL mapped for kernel hardness (Sourdille et al., 1996), resistance to tan spot (Faris et al., 1997) and Karnal bunt resistance (Nelson et al., 1998) are included in Table 10.3.

Aside from the ITMI population, Anderson et al. (1993) reported on QTL for pre-harvest sprouting (PHS) after using around 40 RFLP markers on two segregating populations of recombinant inbred lines (RILs), which were evaluated for PHS in up to seven environments. Using a combination of RFLP markers on most of a RIL population and selective genotyping with AFLPs on a sub-set of the population, Anderson et al. (1998) identified five putative QTL associated with Fusarium scab resistance (see Table 10.3).

At CIMMYT, efforts in breeding for disease resistance in general, and leaf rust resistance in particular, have focused on the use of durable resistance (van Ginkel and Rajaram, 1993). Such resistance is controlled by a number of minor genes also referred to as adult plant resistance (APR) genes. In order to determine the number and location of these genes, and find tightly linked markers that will enhance the breeding efforts for such resistance, CIM-MYT has been involved in mapping APR loci in the leaf rust resistant cultivars Parula and Frontana. William et al. (1997), using BSA on RILs from a cross between Parula and Siete Cerros, identified three RAPD markers associated with two leaf rust resistance loci. Nullisomic-tetrasomic analysis showed that these are located on 7BL and 1BS or 1DS. CIMMYT has also constructed a genetic linkage map using RFLP, SSR and AFLP markers in a segregating population of Frontana x INIA66 in order to map primarily durable leaf rust resistance but also other important traits that are segregating in the same population. Although the map now includes about 450 marker loci, some gaps still exist and efforts are focused on filling those with the SSR markers that are becoming available (e.g. Röder et al., 1998b). With the current map and using composite interval mapping, CIMMYT has identified five and seven QTL for leaf rust resistance and barley yellow dwarf virus (BYDV) tolerance, respectively (Khairallah et al., 1998).

Marker-assisted selection

Three factors are required for the effective implementation of molecular markers in breeding programmes: (i) the availability of ‘user-friendly’ markers (cheap, easy and reliable); (ii) the validation of markers across different genetic backgrounds; and (iii) the possibility of implementing them within a breeding programme (Langridge and Chalmers, 1998).

RFLPs, RAPDs and AFLPs do not fit the first requirement. However, techniques are available to turn them into user-friendly markers. RFLP clones can be sequenced, and primers designed to amplify the DNA fragments are shown by hybridization to be polymorphic. However, the resulting STS or SCAR does not always turn out to be polymorphic, and further manipulations are needed if this is the case. The amplified fragment is usually digested with one or two restriction endonucleases to detect small length differences, or the fragment from two or more cultivars is cloned and sequenced again to create ASAs. ASAs are usually based on single nucleotide differences. RAPD and AFLP fragments can be isolated from the gel, cloned and sequenced to generate STSs or SCARs, and if needed, ASAs. Attempts to generate such markers for wheat are neither always successful nor easily achieved. However, when they are, they represent very robust markers. SSRs, on the other hand, if tightly linked to genes of interest are probably the most attractive markers since no further manipulations are needed for implementation.

Despite the large number of markers for wheat genes listed in Table 10.3, few of those markers are close enough to the genes of interest to be useful in breeding applications. Some markers have been tested across a number of cultivars as a first step towards marker validation (e.g. Feuillet et al., 1995 for Lr1; Hartl et al., 1995 for Pm1 and Pm2; Demeke et al., 1996 for Bt-10; Dweikat et al., 1997 for Hessian fly resistance genes; Ogbonnaya et al., 1998 for Cre1 and Cre3). Although, in general, not a large enough number of varieties are tested, some markers seem to be promising, such as Lr9 (Schacher-mayr et al., 1994), Lr 10 (Schachermayr et al., 1997) and Lr19 (Winzeler et al., 1995). Another factor contributes, though to a lesser extent, to the scarcity of markers used in breeding programmes. Often the scientists developing the markers are not directly connected with breeding activities and/or their laboratories are not set up to handle the numbers that would come out of a breeding programme.

The authors are aware of very few me presently using markers as additional selection tools. Examples of those include the use of ASAs or SCARs for cadmium uptake, high protein (HP) content and the 1B/1R translocation at Agriculture and Agri-Food Canada’s Cereal Research Centre at Winnipeg in Canada (G.A. Penner, D.G. Humphreys and J.D. Procunier, personal communication, 1997). Ogbonnaya et al. (1998) report on very robust markers for both Cre1 and Cre3 and will be able to use those to pyramid the two resistance alleles in Australian material at the Victorian Institute for Dryland Agriculture, Horsham, Australia.

CIMMYT is presently in the process of validating a number of markers for genes of interest (e.g. Ph1, Sr2, Lr1, 9, 10, 24 and HP) across CIMMYT’s germplasm and of designing a marker service facility for the easy and reliable implementation of validated markers in the breeding programmes. The facility is being designed to implement only PCR-based markers and will be equipped with a DNA sequencer and an automated pipetting station in order to be able to handle the large number of samples coming from the breeding activities.

The future of wheat molecular genetics

There is little doubt that wheat has been a difficult species for the application of molecular genetics. The low level of polymorphism between elite varieties coupled with the hexaploid nature of the crop provide significant challenges for those attempting to develop molecular markers and to use then in genetic studies. With the development of AFLP and microsatellite marker systems, renewed studies are underway to analyse the genetic basis of many important traits in wheat.

What does the future hold? While always difficult to predict, there are some significant developments in marker technologies and functional genomics worth mentioning. While the PCR-based marker systems have allowed more effective and efficient genotyping, DNA-array technology offers to increase substantially the number of genes that can be analysed (Shalon 1995; Schena et al., 1995; Shalon et al., 1996). Currently, the cost of the arrayer (to develop the chips containing the desired genes), the array reader (to detect the presence of each gene) and a set of gene sequences (to develop primers to be arrayed) have limited the application of this new technology to wheat. Both the arrayer and reader are decreasing in price and this will make this technology available to many laboratories in the near future. Efforts are also underway to develop complete expressed sequence tag (EST) databases for wheat and related species. If this data can remain in the public sector (such databases for wheat are currently available in the private sector), chips containing a significant number of wheat genes will be produced and used in the not too distant future.

Perhaps the next challenge facing wheat researchers will be gene isolation. Examples of transposon-based cloning and even map-based cloning are available in many species. A few researchers have had success in wheat, although the approaches possible are by no means routine. What is promising is the availability of large DNA libraries (yeast artificial chromosomes and bacterial artificial chromosomes) for Ae. tauschii (D genome, E. Lagudah, personal communication, 1997) and T. monoccocum (A genome, R. Wing, J. Dubcowsky and B. Keller, personal communication, 1997). These represent valuable resources for the identification and isolation of genes from wheat. The use of degenerate primers and probes from other species can readily provide candidate sequences. The only components lacking, or at least limiting, are a reverse-genetic system for wheat and a reliable and efficient genetic engineering system. The later is becoming more effective, while the former will require significant work to develop. The insertion of Ac/Ds into rice indicates that it is feasible, but the hexaploid nature of bread wheat will make it more complex. It may be more practical to develop the system in a diploid species, such as Ae. tauschii or T. monoccocum.

Wheat has been and will continue to be a difficult species to investigate at the molecular level; however, recent innovations in technology have opened the door for renewed efforts to use wheat for molecular genetic investigations. While Arabidopsis and rice may provide interesting model systems, each plant species will require a certain level of study, hopefully utilizing what is known in other species. One can predict that researchers will have in their hands all the genes from most of the major crop species, including wheat, in the near future. The challenge then will be to determine the function of each and how to use this information to develop improved wheat varieties for feeding the world’s growing population.

WHEAT GENETIC ENGINEERING

Cereals, including wheat, have been prime targets for genetic manipulation since the first reports of successful production of transgenic plants. However, the progress towards efficient cereal transformation has been slow, mainly due to difficulties encountered in the establishment of embryogenic cell culture methods and a lack of efficient DNA delivery systems.

Several different methods have been attempted for transforming wheat. Direct transfer of DNA into protoplasts mediated by polyethylene glycol (Mass and Werr, 1989; Potrykus, 1990) and electroporation (Larkin et al., 1990; Zhou et al., 1993) have proven to be ineffective. A more recent and versatile method for cereal transformation is microprojectile bombardment or biolistics. In this method, the DNA transfer process is genotype and tissue independent, although the regeneration of transformed cells still requires competent cells, which do demonstrate genotype differences. The biolistics method involves shooting cells with micro-particles coated with the desired DNA. By a still somewhat unknown process, the DNA is removed from the particles and ultimately inserts itself into the cell’s (usually nuclear) genome. Usually, the insertion events are random and characterized by multiple copies and a certain degree of rearrangements (Jenes et al., 1993).

The most recent cereal transformation method involves the use of a naturally occurring bacterium, Agrobacterium tumefaciens. For several years, cereals were classified as non-hosts for Agrobacterium, as they were not infected in vivo or in vitro. Recent investigations have shown that Agrobacterium can attach to cereal cells, that these cells produce factors that induce Agrobacterium virulence genes and that the bacterium can transfer its T-DNA into the cell (Tinland, 1996).

The first reports of successful Agrobacterium-mediated transformation of wheat were those of Hess et al. (1990), which involved pipetting Agrobacterium onto the spikelets of wheat. Mooney et al. (1991) reported the first transformed cells from wheat embryos co-cultivated with Agrobacterium tumefaciens. These reports were considered promising, but Agrobacterium-mediated transformation of wheat was not considered practical until the recent reports by Cheng et al. (1997). Agrobacterium-mediated transformation has now been demonstrated for rice, maize and barley (Hiei et al., 1994; Rhodera and Hodges, 1996; Ishida et al., 1996; Tingay et al., 1997).

Whatever the method used, an effective selection regime is required for the isolation of transformed cells. Often, cereal tissue culture cells show a high level of natural resistance to the antibiotics or herbicides commonly used for selection (Hauptmann et al., 1988; Vasil et al., 1991). Currently, the most common selectable markers systems in use are PPT (phosphinothrycin) and bialaphos for the bar gene, G418 and paromomycin for the nptII gene, and hygromycin for the aphIII-IV (or hpt) gene. None of these are as selective as required, and usually large numbers of regenerants must be screened to identify the few that are transformed. Even so, fertile transgenic wheat plants presenting herbicide resistance have been produced in several laboratories (Vasil et al., 1992; Weeks et al., 1993; Nehra et al., 1994; Becker et al., 1994; Zhou et al., 1995; Takumi and Shimada, 1996). Hygro-mycin-resistant wheat plants have also been produced (Sautter et al., 1991; Ortiz et al., 1996).

The availability of strong promoters, such as rice actin or maize ubiquitin, active in most cell types, are providing useful alternatives to the less active cauliflower mosaic virus 35S promoters (McElroy et al., 1991; Christensen and Quail, 1996; Taylor et al., 1993). Chamberlain et al. (1994) reported a new promoter (Emu) which drives very high levels of gene expression in wheat. Attention is also given to promoters that can regulate the spatial and/or temporal expression of a gene (McElroy et al., 1994). The modification of various characters, such as grain quality and disease resistance, will depend also on the availability of promoters to regulate gene expression in specific tissues. Recent reports have demonstrated that the use of a native wheat glutenin promoter effectively controls the expression of an introduced high molecular weight wheat glutenin gene (Shewry et al., 1995; Blechl and Anderson, 1996; Alt-peter et al., 1996; Barro et al., 1997; Vasil and Anderson, 1997). Expression of the barnase gene under the control of a tapetum specific promoter resulted in male sterile wheat plants (De Block et al., 1997).

For practical application of genetic engineering technology, it is essential that transformed plants have continued expression and stable inheritance of the inserted transgene(s). There are a number of reports describing non-Mendelian inheritance and inactivation of the foreign genes (McElroy et al., 1994; Flavell, 1994). Such non-Mendelian inheritance and loss of expression appear to be independent of the cereal species transformed and the nature of the introduced genes. Current evidence indicates that the copy number and insertion position in the genome influence the level of stability and expression (Brettel and Murray, 1995). A better understanding of transgene inactivation is needed to improve the efficiency of the transformation process and the stability of the transgenes under field conditions. In addition, the development of site-specific recombination and transposon-mediated delivery systems may provide for improved transgene stability (Bretell and Murray, 1995).

Candidate genes for wheat genetic engineering

Genetic engineering has opened up new opportunities for plant breeders by enabling them to incorporate genes isolated from organisms outside the gene pools to which they usually have access. This broadens the possibilities they have for overcoming a number of biotic and abiotic stresses. This section describes some of the gene strategies that are being considered to provide useful products for breeders in a relatively short period of time.

TABLE 10.4
Candidate genes and targetted traits for wheat genetic engineering

Target trait

Candidate gene(s)a

Effects

Quality

Bread-making quality

HMW-GS 1Ax1
HMW-GS Dx5B
HMW-GS Dy10A

Increased levels of HWM-GS proteins in the endosperm of those varieties lacking these alleles; an increase in dough elasticity and strength

Nutritional quality

a, b and g zeins

Increased levels of proteins in the endosperm providing enhanced nutritional quality

Biotic stresses

Fungal diseases

b1,3-glucanase, chitinase

Degradation of fungal cell walls

Osmotin

Disruption of fungal membranes

Ribosome-inhibiting protein

Disruption of fungal protein synthesis

Insects

Lectins

Induction of plant defence responses; enhanced resistance to certain grain weevils and aphids

a-amylase inhibitors

Prevention of weevil growth and development

Nematodes

Cysteine proteinase inhibitors

Resistance to cereal cyst and root knot nematodes

Viruses

Coat protein genes

Prevention of disassembly and movement of viruses

Abiotic stresses

Drought, heat, cold Aluminium

Peroxidases
Citrate synthase

Protection against oxidative stress
Binding to aluminium leading to prevention of aluminium from entering the roots

aHMW-GS = high molecular weight glutenin subunits.

Table 10.4 highlights a few of the genes and respective traits that are available or that have already demonstrated usefulness in wheat or other crop plants. The list is by no means exhaustive, as there is an increasingly large number of genes being cloned that will provide new opportunities in the future.

Quality traits

Other than reporter genes, perhaps the most targetted trait for genetic engineering in wheat is quality. Seed storage proteins (SSP) are contained in the seed of higher plants. These proteins have been classified as albumins, globulins and glutenins on the basis of their solubility in solvents. The high molecular weight glutenin subunits (HMW-GS) genes in wheat are located on the long arm of the homeologous chromosomes 1A, 1B and 1D. Bread-making properties are particularly associated with variation at the Glu-D1 and Glu-A1 loci. The HMW-GS 1Ax1, 1Ax2, 1Dx5 and 1Dx10 have been shown to be associated with stronger dough, better elasticity and, hence, improved bread-making quality. Many elite wheat varieties lack the desired studies have demonstrated that the introduction of one or two HMW-GS genes results in a stepwise increase in dough elasticity. The transgenic lines produced so far have also demonstrated a very high level of expression and stability over several generations. This may imply that native genes are more tolerated by a plant genome.subunits and, thus, many research groups are attempting to introduce these via genetic engineering (Shewry et al., 1995; Blechl and Anderson, 1996; Altpeter et al., 1996; Barro et al., 1997; Vasil and Anderson, 1997). These -

In addition to increasing the bread-making quality, altered amino acid composition of the SSP is feasible and could result in improved nutritional properties. For example, the in-sertion of genes for proteins, such as zeins or albumins, could lead to an increase in the desired amino acid. Other approaches are also being considered, such as reducing the level of anti-nutritional factors and modifying starch and oil composition and content.

Induced fungal resistance

The fungal infection process is a complex mechanism that usually includes three stages: (i) prior-to-entry relationships; (ii) penetration; and (iii) establishment of the pathogen in the host. The plant resistance mechanisms involve the interaction of several factors, such as the environment, morphological peculiarities of the host and, in particular, biochemical defence genes. The later factors can be improved by the introduction of genes that modify the reaction of the plant metabolism to the infection. Examples are the introduction of the â1,3-glucanase gene that may stop the penetration of the fungus or the introduction of chitinase and osmotin genes that interact with the development of the fungal haustorium by changing the chitin structure and the osmolarity of the membrane.

Since single genes encode many of the active anti-microbial/anti-fungal factors, these defence systems are amenable to manipulation by gene transfer. The first report of success with such a strategy was the expression of a bean vacuolar chitinase gene in tobacco and Brassica napus that resulted in decreased symptom formation of Rhizoctonia solani (Broglie et al., 1991). Since this initial study, several other research groups have found similar results by transforming various anti-fungal genes into a range of crop plants including tobacco, tomato, canola and rice. These have resulted in enhanced resistance to a range of fungal pathogens, including R. solani, Fusarium oxysporum, Cercospora nicotianae and Cladosporium fulvum (Broglie et al., 1991; Logemann et al., 1992; Zhu et al., 1994; Jach et al., 1995; Jongedijk et al., 1995; Lin et al., 1995; Wubben et al., 1996).

An interesting observation is that the combination of different anti-fungal proteins can lead to synergistic protection against a broad range of phytopathogenic fungi (Zhu et al., 1994). Zhu’s study demonstrated that coexpression of a rice chitinase and a â1,3-glucanase derived from alfalfa gave substantially higher protection against the pathogen C. nicotianae than either transgene alone. Similar results have been reported with tomato (Jongedijk et al., 1995) and tobacco (Jach et al., 1995). It is likely that this battery of inducible defences represents a series of complementary mechanisms for protection against both the initial attack and possibly secondary, opportunistic infections.

Other possible targets

Many other targets exist for improving wheat via genetic engineering. Several strategies are available for engineering resistance to insects, for example, the use of lectins for resistance to aphids and lepidopteran pests (Down et al., 1996; Gatehouse et al., 1996, 1997) and á-amylases for resistance to various coleopteran pests (Ishimoto et al., 1996). In addition, proteinase inhibitors have been used to engineer both insect (Duan et al., 1996) and nematode (Vain et al., 1998) resistance. Viral coat protein genes for enhanced resistance to viruses (Grumet, 1994), citrate synthase for tolerance to aluminium (de la Fuente et al., 1997) and even various options for improving the tolerance of wheat to drought, heat salinity and waterlogging are also being investigated. Often the most advanced studies are in the private sector and, thus, current information regarding their success or failure remains confidential. Hopefully, the reports, or at least the products, will be made available for use in developing new approaches for the multitude of stresses that wheat is subjected to in its growing environment.

CIMMYT’s efforts in wheat genetic engineering

CIMMYT’s initial activity in wheat genetic engineering has been to identify a range of elite wheat cultivars that are not only regenerable, but also transformable. From these studies, the bread wheat genotypes Attila, Kauz, Baviacora and Bobwhite and the durum wheat genotypes Minimus, Ariza, Altar and Bajio were found to be highly regenerable and excellent candidates for transformation efforts (Bohorova et al., 1995). Efforts are now focused on the production of transgenic plants via biolistics and Agrobacterium-mediated methods. Current target traits are enhanced fungal resistance via various pathogen-related proteins, including chitinase, glucanase and ribosome-inhibiting proteins, and enhanced quality via HMW-GS genes. The first putative transgenic plants are being produced and investigated in a biosafety greenhouse. As these are confirmed, progeny are produced and analysed for the inheritance of each transgene. Ultimately, the best events will be taken to the field under appropriate biosafety regulations for proper evaluation of the expression of the inserted transgene and for effects on other agronomic characteristics.

CONCLUDING REMARKS

Although the potential of biotechnology has often been exaggerated, a high level of optimism is clearly justified for its use in the improvement of wheat. Undoubtedly, functional genomics, as it is now termed, will revolutionize the way in which plant breeding is undertaken in the future. Basic research is leading to an improved understanding of the genetic mechanisms operating within a plant in response to the diverse stresses that it is exposed to, as well as the overall production of biomass and grain. New methodology, such as automated marker systems and array technology, will allow the large number of samples to be processed that are encountered in a typical breeding programme. In fact, perhaps the biggest challenge facing scientists is how to store, process and access the vast amounts of information generated using these new marker systems. This knowledge base offers promise for making germplasm improvement faster, cheaper and more effective.

Emerging genetic engineering techniques are providing breeders with the never-be-fore-seen capability to create novel plants by combining genetic material from a wide array of sources. Although not without controversy, the options seem limitless and, with the proper oversight and understanding, should provide extremely powerful options to develop durable and highly productive plant varieties for almost any production environment.

The challenge for developing countries is to tap as much of this emerging technology as possible. This does not necessarily mean that countries must establish inhouse capabilities. What is required is that nations recognize the importance of the new approaches and ensure that appropriate legislation and regulations are enacted to allow the country to acquire, evaluate and most importantly deploy the new plant varieties produced via biotechnology. All available tools to ensure an adequate supply of food for the world must be employed.

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