**4.1. Grain hardness**

located during recent years [29]. The first QTL for type II resistance was identified in the spring wheat 'Sumai 3' on chromosome 3BS. This QTL was named *Fhb1* and characterised by molecular markers [35–37]. Recently, *Fhb1* was cloned from Sumai 3 and a pore-forming toxin-like (PFT) gene was found to confer FHB resistance [38]. *Fhb1* has been found to reduce FHB disease severity tremendously and MAS is employed to incorporate the resistance in breeding programs [29]. A QTL, named *Fhb2,* on chromosome 6BS was found to confer type II FHB resistance [39, 40]. Additionally, *Fhb4* was identified and located on chromosome 4B [41]. **Table 2** lists all FHB-resistant genes identified by molecular markers. Currently, breeders are pyramiding *Fhb1, Fhb2* and *Fhb4* in single breeding lines to obtain optimal FHB resistance [34].

Several additional QTL have been identified and located in numerous studies [29].

**Table 2.** Overview of the FHB-resistant genes identified in wheat using molecular markers.

**Resistance gene Marker type Marker name Location Reference** *Fhb1* SSR Xgwm493, Xgwm533 3BS [42] *Fhb2* SSR Xgwm133, Xgwm644 6BS [40] *Fhb4* SSR Xhbg226, Xgwm149 4B [41] *Fhb5* SSR Xgwm304, Xgwm415 5A [43] *Fhb6* KASP Wg1s\_snp1 1AS [44] *Fhb7* SSR XsdauK66, Xcfa2240 7DS [45]

Wheat stripe rust, mostly designated as 'yellow rust' (YR), causes major yield losses every year. The disease is caused by *Puccinia striiformis,* which belongs to the family *Pucciniaceae* of rust fungi. The most devastating epidemics occur in temperate areas with cool and humid summers or in warmer areas with cool nights. The fungus is heteroecious, i.e., it requires at least two hosts in order to proliferate. *P. striiformis* uses cereals as a primary host and *Berberis* spp. as a secondary host for sexual recombination. Typical, yellow stripes develop on the leaf in lesions. Spores continue to be produced as stripes spread longitudinally on the leaf. After the onset of senescence, *P. striiformis* will produce teliospores. Teliospores can infect the secondary host, *Berberis* spp., and initiate onset of pycnia infection of the *Berberis* leaf [46].

Breeding for YR resistance was initiated in 1905 by Biffen [47]. To date, more than 70 genes (*Yr* genes) conferring YR resistance have been identified [48]. Most of the catalogued genes confer seedling resistance, while relatively few confer adult plant resistance. In general, studies have shown that seedling resistance is conferred by single genes and the resistance is therefore easily overcome by the pathogen by mutations in virulence genes. Adult plant resistance is generally thought to be more durable [49]. High-temperature adult plant (HTAP) genes are expressed as the plants grow older and the weather becomes warmer [50]. HTAP genes confer a non-specific, quantitative resistance. Studies have proven that varieties with HTAP genes display resistance

**3.3. Wheat stripe rust (yellow rust)**

8 Next Generation Plant Breeding

Grain hardness influences milling, flour and end-use properties of wheat. Flour from grain with hard endosperm texture has higher water absorption than flour from soft grain and is therefore preferred for bread-making. A soft endosperm texture leads to less starch granule damage during the milling and consequently to lower water absorption, which is preferred in the production of biscuits and cakes. Grain hardness is primarily controlled by the *Hardness* locus on chromosome 5DS. This locus consists of three small genes: *Pina-D1*, *Pinb-D1* (*Puroindoline a/b*) and *grain softness protein-1 (Gsp-1*). Wheat varieties with the wild-type alleles *Pina-D1a* and *Pinb-D1a* normally have soft grain, while deletions or other loss-of-function mutations in one or both *Pin* genes cause harder grain (**Table 4**) [67, 68]. *Pinb-D1* mutations are positively associated with many quality traits, but the alleles are not equally useful in breeding for improved quality. *Pinb-D1d* has been reported to have a lower effect on gluten quality and loaf volume than the *b-* or *c-*allele [69]. Alleles of *Pinb-D1* can be detected using PCR primers that target a specific mutation (*Pinb-D1b*), using a restriction enzyme on the amplified *Pinb-D1* gene (*Pinb-D1c*), or by sequencing the amplified gene (*Pinb-D1d-g*) [67, 70, 71].

**4.2. Gluten**

**4.3. Wheat-rye translocation and falling number**

The characteristic viscoelastic properties of wheat dough are due to a network of gluten proteins that is formed when flour is mixed with water. Thus, gluten is a major factor contributing to wheat quality. High grain protein content is typically associated with high quality, since roughly 80% of the grain protein is gluten [76]. However, both the amount and the composition of gluten affect wheat quality. Gluten consists of two types of proteins: polymeric glutenins and monomeric gliadins. Glutenins can be classified as low or high molecular weight (LMW or HMW) subunits, while gliadins can be classified as α, β, γ or ω types [77, 78]. The most important HMW glutenins, LMW glutenins, and gliadins are encoded by the *Glu-1*, *Glu-3* and *Gli-1* loci, respectively (**Table 5**). HMW glutenins generally have the largest impact on wheat quality. Each of the three *Glu-1* loci comprises two genes that can encode an x- or a y-type HMW subunit. In hexaploid wheat, only three to five of the HMW subunits are expressed (zero to two from *Glu-A1*, one to two from *Glu-B1*, and two from *Glu-D1*) [79]. The *Glu-1* alleles with the largest positive effect on baking quality are *Glu-D1d*, *Glu-A1a* or *Glu-A1b* and *Glu-B1al* [80, 81]. SDS-PAGE electrophoresis can be used to screen varieties for their HMW glutenin proteins. DNA markers have also been developed to discriminate between different alleles of *Glu-1*, *Glu-3* and *Gli-1* loci [82, 83]. For *Glu-A1* and *Glu-D1*, KASP markers are available that can be used to select varieties with the optimal alleles [84]. Each of the *Glu-3* loci (*Glu-A3*, *Glu-B3* and *Glu-D3*) contains several linked genes, and many alleles have been found for all three loci [85–89]. Markers are available for individual alleles of *Glu-A3* and *Glu-B3*, and multiplex PCR can be used to screen for certain combinations of alleles simultaneously [87]. However, the alleles of *Glu-3* loci with the largest effects are not consistent across studies [90–92]. The exact effects of the individual alleles on wheat quality traits are challenging to determine, since they can be influenced by genetic background, environment and G×E interactions [91, 93]. Furthermore, the alleles can have both additive effects and epistatic interactions [94, 95]. Ref. [93] showed that the *d*-allele of *Glu-B3* might increase the positive effects of the HMW loci *Glu-B1i* and *Glu-D1d*. The *Glu-A3b* or *d*-allele and *Glu-B3b*, *d-* or *g*-allele can possibly be used for improving dough strength and extensibility [90–92]. *Glu-B3i* has been reported to be positively associated with wheat quality in some lines and negatively associated in other lines. This discrepancy is possibly due to linkage with different *Gli-B1* alleles [90]. The *Gli-1* loci encode γ and ω gliadins and are linked to the *Glu-3* loci [96], while *Gli-2* loci encode α and β gliadins and are located on chromosome 6AS, 6BS and 6DS [78]. Overview of markers (including primer sequences) for more alleles of *Glu* loci and other quality genes can be found in [82].

Marker-Assisted Breeding in Wheat http://dx.doi.org/10.5772/intechopen.74724 11

The wheat-rye translocation 1BL.1RS has been employed in many breeding programs as it carries resistance genes against powdery mildew and rusts. Markers for the resistance genes can be used to test for the absence or presence of the translocation in wheat varieties [100]. Alternatively, markers for *Glu-B3* or *Gli-B1* might be used (**Table 6**), since many wheat varieties with the 1BL.1RS translocation do not have these two loci, but instead can have the rye secalin locus *Sec-1* [96]. Therefore, wheat quality can be negatively affected by the translocation [101]. Additionally, the 1BL.1RS translocation can have a negative effect on falling number. Falling number is an indirect measure of α-amylase enzyme activity. The α-amylases are encoded by


Wild type alleles confer soft endosperm; mutations confer hard endosperm. For additional alleles, see reviews [68, 72].**\*** Digest with restriction enzyme *Pvu*II to cut other alleles into 264 bp and 184 bp. *Pinb-D1c* is not cut.

**Table 4.** Alleles of *Pina-D1* and *Pinb-D1* and the change in amino acid sequence of the encoded protein.

## **4.2. Gluten**

during the milling and consequently to lower water absorption, which is preferred in the production of biscuits and cakes. Grain hardness is primarily controlled by the *Hardness* locus on chromosome 5DS. This locus consists of three small genes: *Pina-D1*, *Pinb-D1* (*Puroindoline a/b*) and *grain softness protein-1 (Gsp-1*). Wheat varieties with the wild-type alleles *Pina-D1a* and *Pinb-D1a* normally have soft grain, while deletions or other loss-of-function mutations in one or both *Pin* genes cause harder grain (**Table 4**) [67, 68]. *Pinb-D1* mutations are positively associated with many quality traits, but the alleles are not equally useful in breeding for improved quality. *Pinb-D1d* has been reported to have a lower effect on gluten quality and loaf volume than the *b-* or *c-*allele [69]. Alleles of *Pinb-D1* can be detected using PCR primers that target a specific mutation (*Pinb-D1b*), using a restriction enzyme on the amplified *Pinb-D1* gene (*Pinb-D1c*), or by sequenc-

**Primer sequences, 5′–3′ PCR product References**

448 bp [73, 74]

Null (0 bp) [73, 74]

240 bp [70, 73]

240 bp [70, 73]

448 bp\* [67, 73]

[67, 71]

[71, 75]

[71, 75]

[71, 75]

300 bp for pyrosequencing

300 bp for pyrosequencing

300 bp for pyrosequencing

300 bp for pyrosequencing

ing the amplified gene (*Pinb-D1d-g*) [67, 70, 71].

Wild-type F: ATGAAGGCCCTCTTCCTCA

Wild-type F: ATGAAGACCTTATTCCTCCTA

R: TCACCAGTAATAGCCAATAGTG

F: ATGAAGGCCCTCTTCCTCA R: TCACCAGTAATAGCCAATAGTG

R: CTCATGCTCACAGCCGC**C**

F: ATGAAGACCTTATTCCTCCTA R: CTCATGCTCACAGCCGC**T**

F: ATGAAGACCTTATTCCTCCTA R: TCACCAGTAATAGCCACTAGGGAA

F: TGCAAGGATTACGTGATGGA R: TCACCAGTAATAGCCACTAGGGAA

F: TGCAAGGATTACGTGATGGA R: TCACCAGTAATAGCCACTAGGGAA

F: TGCAAGGATTACGTGATGGA R: TCACCAGTAATAGCCACTAGGGAA

F: TGCAAGGATTACGTGATGGA R: TCACCAGTAATAGCCACTAGGGAA

Wild type alleles confer soft endosperm; mutations confer hard endosperm. For additional alleles, see reviews [68,

Digest with restriction enzyme *Pvu*II to cut other alleles into 264 bp and 184 bp. *Pinb-D1c* is not cut.

**Table 4.** Alleles of *Pina-D1* and *Pinb-D1* and the change in amino acid sequence of the encoded protein.

**Allele Change in protein**

10 Next Generation Plant Breeding

Large deletion

Gly to Ser pos. 46

Leu to Pro pos. 60

Trp to Arg pos. 44

Trp to stop codon pos. 39

Trp to stop codon pos. 44

Cys to stop codon pos. 56

*Pina-D1a*

*Pina-D1b*

*Pinb-D1a*

*Pinb-D1b*

*Pinb-D1c*

*Pinb-D1d*

*Pinb-D1e*

*Pinb-D1f*

*Pinb-D1g*

72].**\***

The characteristic viscoelastic properties of wheat dough are due to a network of gluten proteins that is formed when flour is mixed with water. Thus, gluten is a major factor contributing to wheat quality. High grain protein content is typically associated with high quality, since roughly 80% of the grain protein is gluten [76]. However, both the amount and the composition of gluten affect wheat quality. Gluten consists of two types of proteins: polymeric glutenins and monomeric gliadins. Glutenins can be classified as low or high molecular weight (LMW or HMW) subunits, while gliadins can be classified as α, β, γ or ω types [77, 78]. The most important HMW glutenins, LMW glutenins, and gliadins are encoded by the *Glu-1*, *Glu-3* and *Gli-1* loci, respectively (**Table 5**). HMW glutenins generally have the largest impact on wheat quality. Each of the three *Glu-1* loci comprises two genes that can encode an x- or a y-type HMW subunit. In hexaploid wheat, only three to five of the HMW subunits are expressed (zero to two from *Glu-A1*, one to two from *Glu-B1*, and two from *Glu-D1*) [79]. The *Glu-1* alleles with the largest positive effect on baking quality are *Glu-D1d*, *Glu-A1a* or *Glu-A1b* and *Glu-B1al* [80, 81]. SDS-PAGE electrophoresis can be used to screen varieties for their HMW glutenin proteins. DNA markers have also been developed to discriminate between different alleles of *Glu-1*, *Glu-3* and *Gli-1* loci [82, 83]. For *Glu-A1* and *Glu-D1*, KASP markers are available that can be used to select varieties with the optimal alleles [84]. Each of the *Glu-3* loci (*Glu-A3*, *Glu-B3* and *Glu-D3*) contains several linked genes, and many alleles have been found for all three loci [85–89]. Markers are available for individual alleles of *Glu-A3* and *Glu-B3*, and multiplex PCR can be used to screen for certain combinations of alleles simultaneously [87]. However, the alleles of *Glu-3* loci with the largest effects are not consistent across studies [90–92]. The exact effects of the individual alleles on wheat quality traits are challenging to determine, since they can be influenced by genetic background, environment and G×E interactions [91, 93]. Furthermore, the alleles can have both additive effects and epistatic interactions [94, 95]. Ref. [93] showed that the *d*-allele of *Glu-B3* might increase the positive effects of the HMW loci *Glu-B1i* and *Glu-D1d*. The *Glu-A3b* or *d*-allele and *Glu-B3b*, *d-* or *g*-allele can possibly be used for improving dough strength and extensibility [90–92]. *Glu-B3i* has been reported to be positively associated with wheat quality in some lines and negatively associated in other lines. This discrepancy is possibly due to linkage with different *Gli-B1* alleles [90]. The *Gli-1* loci encode γ and ω gliadins and are linked to the *Glu-3* loci [96], while *Gli-2* loci encode α and β gliadins and are located on chromosome 6AS, 6BS and 6DS [78]. Overview of markers (including primer sequences) for more alleles of *Glu* loci and other quality genes can be found in [82].

## **4.3. Wheat-rye translocation and falling number**

The wheat-rye translocation 1BL.1RS has been employed in many breeding programs as it carries resistance genes against powdery mildew and rusts. Markers for the resistance genes can be used to test for the absence or presence of the translocation in wheat varieties [100]. Alternatively, markers for *Glu-B3* or *Gli-B1* might be used (**Table 6**), since many wheat varieties with the 1BL.1RS translocation do not have these two loci, but instead can have the rye secalin locus *Sec-1* [96]. Therefore, wheat quality can be negatively affected by the translocation [101]. Additionally, the 1BL.1RS translocation can have a negative effect on falling number. Falling number is an indirect measure of α-amylase enzyme activity. The α-amylases are encoded by


the loci *α-Amy-1*, *α-Amy-2* and *α-Amy-3* located on the homoeologous chromosome groups 6, 7 and 5, respectively. High falling number reduces the risk of pre-harvest sprouting, which has a considerable negative impact on quality. Environmental conditions around the time of harvest influence falling number, but it is also influenced genetically. The *b-*allele of the *Rht-D1* (*reduced height*) gene on chromosome 4D is correlated with increased falling number [102].

Marker-Assisted Breeding in Wheat http://dx.doi.org/10.5772/intechopen.74724 13

Ref. [103] identified a gene, *wheat bread making (wbm*), that was highly expressed in developing seeds of wheat varieties with good bread-making quality. Polymorphisms in the promoter region sequence were identified between good- and poor-quality varieties. The allele identified in the good quality varieties was positively associated with gluten and bread-making quality in

Genes from wild wheat relatives might also be used for improving quality in modern cultivars. Backcrossing can be used to transfer the genes into breeding material. In this case, MAS is useful since offspring containing the desired genes easily can be detected, and linkage drag can be reduced. One example of such a gene is *Gpc-B1* (*grain protein content*), which was found in wild emmer (*Triticum turgidum* L. ssp. *dicoccoides*). This gene has been used for increasing grain protein content in both durum and common wheat [105]. Markers tightly linked to *Gpc-B1* were identified*,* but require digestion with restriction enzymes. Therefore, [105] recommends the use of the marker shown in **Table 6** for MAS, although it is not completely linked to *Gpc-B1*.

Trait-linked DNA markers have been identified for numerous traits in wheat, including disease resistance and grain quality. Employing such markers in MAS offers several advantages to wheat breeding compared to conventional phenotypic selection and laborious analysis of grain quality. These advantages include the fixation of desirable traits at an early stage of the breeding program and marker-assisted backcrossing in order to transfer agronomically

In addition, DNA markers are neutral to both environment and tissue type. Thus, they can be employed at any plant developmental stage and independent on environmental conditions during selection. This is particularly relevant for selection for disease resistance. DNA markers further offer the possibility for targeted pyramiding of several resistance genes, a task impossible by phenotypic selection due to complex host-pathogen interactions. To secure durable resistance, it is important to combine qualitative and quantitative resistance in a given

As DNA markers have been correlated to numerous traits, they can be employed to combine, e.g., resistance and grain quality in the early generations. Consequently, DNA markers are being employed in early generations to select for several traits, in turn reducing the number of lines entering replicated, multi-location trials. Similarly, the number of samples for laboratory analysis of grain quality can be reduced. In effect, the application of MAS can lead to an

CIMMYT (The International Maize and Wheat Improvement Center) germplasm [104].

**4.4. Other genes for improving quality**

**5. Conclusion and perspectives**

important genes from wild relatives to cultivated wheat.

line. Here, molecular markers can be used to combine both resistances.

**Table 5.** Important HWM and LMW glutenin loci, their chromosomal location and primer sequences for detection of alleles with positive effects on wheat quality.


**Table 6.** Additional loci influencing wheat quality traits.

the loci *α-Amy-1*, *α-Amy-2* and *α-Amy-3* located on the homoeologous chromosome groups 6, 7 and 5, respectively. High falling number reduces the risk of pre-harvest sprouting, which has a considerable negative impact on quality. Environmental conditions around the time of harvest influence falling number, but it is also influenced genetically. The *b-*allele of the *Rht-D1* (*reduced height*) gene on chromosome 4D is correlated with increased falling number [102].
