**2. First-generation sequencing resources for yield improvement in wheat**

#### **2.1. Restriction fragment length polymorphism (RFLP) markers**

bioethanol is made primarily from wheat in Europe. Wheat starch is a major component for the production of bread, porridge, cakes, biscuits, and cereals which is a highly versatile crop for the human diet. In 2013, wheat was the third most produced cereal crop (713 million tons), after maize (1016 million tons) and rice (545 million tons). There are two distinct types of wheat, spring wheat and winter wheat, cultivated in many countries based on growing seasons, of which spring wheat is planted in most countries except in the United States and Northern Europe where the predominant crop is winter wheat. The global consumption of wheat has increased at a much faster rate than all other crops, because of the scale-up cultivation in developing countries, particularly in China [2]. Currently, out of the total cultivation area of more than 217 million hectares, the European Union countries has the largest area, followed by China, India, Russia, United States, and Canada [3]. Therefore, there has been an extensive effort over the past decades to increase wheat production through the application of molecular techniques which are powerful tools for enhancing effectiveness

The most common or bread wheat species, *Triticum aestivum*, is an annual grass cultivated in temperate zones worldwide that belongs to the genus *Triticum* of the tribe *Triticeae*, and the family Poaceae. The most economically important cereals in the Poaceae family are maize, wheat, rice, barley, and millet. In the genus *Triticum*, there are approximately 25 species including wild and domesticated consisting of a series of diploid, tetraploid, and hexaploid forms, including the diploid einkorn wheat, *Triticum monococcum* (AA genome), the diploid wild wheat, *Triticum urartu* (AA genome), the allotetraploid emmer wheat, *Triticum turgidum* var. *durum* (AABB genome), the allohexaploid common wheat, *T. aestivum* L. (AABBDD genome), and the autoallohexploid *Triticum zhukovskyi* (AAAAGG genome). Of these, *T. aestivum* has 42 chromosome pairs that are derived from two rounds of polyploidization events. Its genome size is approximately 17 Gb, composed of A, B, and D genomes created from the hybridization of three different species. The first hybridization between *T. urartu* (2n = 2x = 14) and a B genome species that has not yet been identified occurred 0.20–1.3 million years ago (MYA) to form the tetraploid *Triticum dicoccoides* (2n = 4x = 48) [4, 5]. The B genome is still unclear and may be extinct, but cytological evidence suggests that the S genome of *Aegilops speltoides* is a closely related species or an ancestral progenitor to the B genome of wheat [6, 7]. The second hybridization event resulted in the complete form of the hexaploid genome *T. aestivum* (2n = 6x = 42), which occurred between the domesticated *Triticum dicoccum* or *T. durum*, a wild goatgrass, and *Aegilops Tauschii* about 8000–10,000 years ago [8–10]. Like most allopolyploid plants, wheat also has the diploid-like chromosome pairing behavior during meiosis, preventing multivalent formation created by multiple homologous or homoeologous chro-

In wheat breeding, a strong emphasis has been put toward the improvement of grain yield as it the most important goal in wheat breeding. There have been concerns about the stagnation or decline of the staple crops in some parts of the world. It has been detected that 37% of wheat areas have experienced the yield stagnation [12]. If the breeder develops an improved wheat variety, having a superior of trait, but produces low yields, producers unlikely will grow it because the yield is necessary for economic feasibility. The grain yield is a complex character with low heritability which is influenced not only by genes but also by the effects of the environment. In wheat, it has been documented that the higher yield is inversely related to

in breeding.

18 Wheat Improvement, Management and Utilization

mosomes [11].

Earlier molecular studies in wheat have shown that the RFLP markers are the common tools used as the oldest method of molecular markers for the construction of genetic maps. RFLP are typically inherited as simple Mendelian codominant markers, and are not influence by the environment, which could serve as highly heritable genetic markers for the study of inheritance of a trait. Chao et al. created the first linkage maps of the homoeologous group 7 chromosomes on the wheat genome using 18 cDNA clones across six mapping populations from ten varieties [14]. They mapped 31 RFLP loci on chromosomes 7A, 7B, and 7D. In 1991, detailed linkage maps were constructed with a total size of 1800 cm consisting of 197 RFLP loci [23]. Ma et al. [24] and Anderson et al. [25] identified RFLP markers associated with two Hessian fly resistance genes from *Triticum taushii*, and preharvest sprouting genes from two recombinant inbred populations of white wheat (*T. aestivum* L. em. Thell.). Based on comparative mapping among cereal species such as wheat, rye, and barley, the development of detailed RFLP maps of the homoeologous group-2 chromosomes has established and showed that they had collinear relationships, indicating the high degree of conservation in gene orders [26]. The RFLP markers flanking the resistance to cereal cyst nematode were identified to be *Xglk605* and *Xcdo588*, which were mapped at 7.3 and 8.4 cm from the *Cre* locus [27]. Later, RFLP-based genetic maps belonging to different homoeologous groups were generated including homoeologous group 1 [28], group 2 [29], group 3 [30], and group 5 [31], and group 6 [32]. During this period, the powdery mildew genes were detected by RFLP probes. Ma et al. [33] reported that the powdery mildew resistant gene *Pm2* was located at 3.5 cm away from the RFLP marker *BCD1871*, and both *Pm1* and *Pm4a* showed cosegregation of markers. In addition, the *Pm1a* and *Pm2* were positioned on the RFLP maps at 3 cm away from *Xwhs178-7A*, at 2.7 cm away from *Whs296*, respectively [34]. To increase the frequency of polymorphism detection in wheat, a non-intervarietal cross (W7984 X Opata85) developed at the International Maize and Wheat Improvement Center (CIMMYT) was used creating a dense map which consisted of about 1000 RFLP loci on group 1 [28], group 2 [29], group 3 [30], and group 6 [35]. Other disease-resistant genes of *Lr9* responsible for leaf rust and *Sr22* responsible for stem rust were characterized and mapped using RFLP markers [36, 37]. Furthermore, the feasibility of the 37 RFLP probes on group 5 chromosomes from Thatcher near isogenic lines (NILs) for leaf rust resistance gene *Lr1* was conducted by Feuillet et al., of which three were investigated to be linked the gene [38]. Galiba et al. analyzed the cross between a frost-sensitive, vernalization-insensitive substitution line, *Triticum spelta* 5A and a frost-tolerant, vernalization-sensitive line, Cheynne 5A and showed cosegregation with *Vrn1* and *Xpsr426* RFLP marker [39]. There was also a linkage detected between *Vrn1* and *Xwg644* in accordance to Korzun et al. [40]. The mapping of a single locus controlling the aluminum tolerance gene *Alt2* was completed on the Chinese Spring chromosome arm 4DL derived from homoeologous recombination between *T. aestivum* cv. Chinese Spring chromosome 4D and *Triticum turgidum* cv. Cappelli chromosome 4B.

Starting from 1998, QTLs for yield and yield-related traits were investigated. The dwarfing genes, *Rht-B1* and *Rht-D1*, associated with plant height in wheat were firstly mapped on the short arms of chromosomes 4BS (*Xfba1-4B*) and 4DS (*Xfba211-4D*) [41]. A year later, thirteen RFLP probes and one morphological marker locus, *Eps*, were used to develop a genetic linkage map and identified that an RFLP marker *Xcdo549* on the short arm of chromosome 3A was associated with plant height, kernel number spike-1, and 1000-kernel weight [42]. Araki et al [43] reported one QTL for yield, *Qyld.ocs-4A.1*, and other yield-related traits of spikelet number ear, *QSpn.ocs-4A.1*, and grain weight/ear *QGwe.ocs.-4A.1*, on chromosome 4A which were detected by the *Xbcd1738* marker. As wheat lodging can result in yield losses, nine QTLs for lodging resistance were detected with the genetic distance between the flanking RFLP markers, of which seven coincided with QTLs for morphological traits [44]. Since the year 2000, there have been many studies published for loci associated with grain yield, heading date, disease, and spike morphology. Kato et al. conducted mapping of QTLs for grain yield and its components on chromosome 5A and confirmed that the grain-yield QTLs were closely linked to QTLs for yield components [45]. They found RFLP markers associated with grain yield, tiller number/plant, ear grain weight, 50-grain weight, and spikelet number/ear based on a homozygous population of single-chromosome recombinant lines. Genetic mapping of QTLs conferring resistance to stripe (yellow) rust and powdery mildew was performed by Singh et al. [46] and Tao et al. [47]. The resistance gene, *Yr28*, derived from *Ae. tauschii* and the adult-plant resistance (APR) gene *Yr18* were mapped on chromosome arm 4DS and 7DS, respectively. They found that *Yr18* was closely correlated with leaf-rust gene Lr34. In addition, the RFLP *xbcd135* and *xbcd266* loci mapped at a genetic distance of 1.6 and 4.8 cm were identified to be closely linked to *Pm6*, a gene conferring resistance to the powdery mildew. Later, two of the powdery resistant genes, *Pm26* and *Pm29*, were mapped on the RFLP linkage map [48, 49]. Several RFLP markers within six major QTL regions have been identified to be tightly linked traits related to compactness such as spike length and number of spikelets [50]. Based on a set of 114 recombinant lines (RILs) of the 'International Triticeae Mapping Initiative' mapping population, morphological, agronomical, and disease resistance traits have been studied. Börner et al. [51] mapped 211 QTLs distributed over 20 chromosomes, of which they detected 64 major QTLs with a LOD score of >3.0 conferring glume color, leaf erectness, peduncle length, ear emergence time, flowering time, plant height, ear length, winter hardiness, grain-filling, grain number, thousand-grain-weight, fusarium resistance, powdery mildew resistance, and leaf rust resistance. The mapping of an earliness *per se* gene in wheat was carried out by Bullrich et al. [52]. This gene was located between the RFLP *Xcdo393* and *Xwg241* on the log arm of chromosome 1A, which showed a large effect on heading date with a phenotypic variance of 0.47. Despite RFLP markers are highly reproducible and used as the primary way for most genetic work in wheat, it has been difficult to use RFLP markers due to the low levels of polymorphism detected in wheat.

#### **2.2. Random amplified polymorphic DNA (RAPD) markers**

7 chromosomes on the wheat genome using 18 cDNA clones across six mapping populations from ten varieties [14]. They mapped 31 RFLP loci on chromosomes 7A, 7B, and 7D. In 1991, detailed linkage maps were constructed with a total size of 1800 cm consisting of 197 RFLP loci [23]. Ma et al. [24] and Anderson et al. [25] identified RFLP markers associated with two Hessian fly resistance genes from *Triticum taushii*, and preharvest sprouting genes from two recombinant inbred populations of white wheat (*T. aestivum* L. em. Thell.). Based on comparative mapping among cereal species such as wheat, rye, and barley, the development of detailed RFLP maps of the homoeologous group-2 chromosomes has established and showed that they had collinear relationships, indicating the high degree of conservation in gene orders [26]. The RFLP markers flanking the resistance to cereal cyst nematode were identified to be *Xglk605* and *Xcdo588*, which were mapped at 7.3 and 8.4 cm from the *Cre* locus [27]. Later, RFLP-based genetic maps belonging to different homoeologous groups were generated including homoeologous group 1 [28], group 2 [29], group 3 [30], and group 5 [31], and group 6 [32]. During this period, the powdery mildew genes were detected by RFLP probes. Ma et al. [33] reported that the powdery mildew resistant gene *Pm2* was located at 3.5 cm away from the RFLP marker *BCD1871*, and both *Pm1* and *Pm4a* showed cosegregation of markers. In addition, the *Pm1a* and *Pm2* were positioned on the RFLP maps at 3 cm away from *Xwhs178-7A*, at 2.7 cm away from *Whs296*, respectively [34]. To increase the frequency of polymorphism detection in wheat, a non-intervarietal cross (W7984 X Opata85) developed at the International Maize and Wheat Improvement Center (CIMMYT) was used creating a dense map which consisted of about 1000 RFLP loci on group 1 [28], group 2 [29], group 3 [30], and group 6 [35]. Other disease-resistant genes of *Lr9* responsible for leaf rust and *Sr22* responsible for stem rust were characterized and mapped using RFLP markers [36, 37]. Furthermore, the feasibility of the 37 RFLP probes on group 5 chromosomes from Thatcher near isogenic lines (NILs) for leaf rust resistance gene *Lr1* was conducted by Feuillet et al., of which three were investigated to be linked the gene [38]. Galiba et al. analyzed the cross between a frost-sensitive, vernalization-insensitive substitution line, *Triticum spelta* 5A and a frost-tolerant, vernalization-sensitive line, Cheynne 5A and showed cosegregation with *Vrn1* and *Xpsr426* RFLP marker [39]. There was also a linkage detected between *Vrn1* and *Xwg644* in accordance to Korzun et al. [40]. The mapping of a single locus controlling the aluminum tolerance gene *Alt2* was completed on the Chinese Spring chromosome arm 4DL derived from homoeologous recombination between *T. aestivum* cv. Chinese Spring chromosome 4D and

Starting from 1998, QTLs for yield and yield-related traits were investigated. The dwarfing genes, *Rht-B1* and *Rht-D1*, associated with plant height in wheat were firstly mapped on the short arms of chromosomes 4BS (*Xfba1-4B*) and 4DS (*Xfba211-4D*) [41]. A year later, thirteen RFLP probes and one morphological marker locus, *Eps*, were used to develop a genetic linkage map and identified that an RFLP marker *Xcdo549* on the short arm of chromosome 3A was associated with plant height, kernel number spike-1, and 1000-kernel weight [42]. Araki et al [43] reported one QTL for yield, *Qyld.ocs-4A.1*, and other yield-related traits of spikelet number ear, *QSpn.ocs-4A.1*, and grain weight/ear *QGwe.ocs.-4A.1*, on chromosome 4A which were detected by the *Xbcd1738* marker. As wheat lodging can result in yield losses, nine

*Triticum turgidum* cv. Cappelli chromosome 4B.

20 Wheat Improvement, Management and Utilization

The applications of RAPD markers have been beneficial to improve breeding programs in wheat because they are simple and fast PCR based, require no prior knowledge of target DNA sequence, and are analyzed either by the presence or absence of an amplicon via agarose gel electrophoresis. In wheat, RAPD has been used since 1990 [53]. Devos and Gale confirmed that a degree of polymorphism detected by six RAPD primers was comparable with RFLP markers [54]. They identified four RAPD markers with bread wheat cultivar 'Chinese Spring.' The application of both bulk segregant analysis (BSA) and RAPD has started in 1994 by Eastwood et al. [55]. Using BSA on DNA enriched for low-copy sequences by RAPD markers, the *Cre3* gene resistance to cereal cyst nematode (CCN) in *Triticum tauschii*, was mapped on the long arm of chromosome 2D (Ccn-D2) [55]. More efforts have been made to distinguish the wheat varieties resistant to cadmium stress [56], powdery mildew [57], and common bunt [58]. The RAPD marker *OPC20* was closely correlated with a gene controlling cadmium uptake in western Canadian durum wheat (*T. turgidum* L. var. *durum*) [56]. In addition, a RAPD marker *OPH17-1900* located on the chromosome arm 6VS, was detected that could be used for the detection of a powdery mildew resistance gene, *Pm21*, in breeding [57]. For common bunt, also known as stinking smut and covered smut, the polymorphic RAPD marker, *BW553*, was identified between resistant and susceptible NILs [58]. Furthermore, identification of RAPD markers linked to the *Yr15* gene controlling stripe rust resistance was conducted using 340 RAPD primers, six of which were detected to be polymorphic [59]. The *OPB13* RAPD marker was the only one that produced polymorphism in 123 F<sup>2</sup> individuals and showed that it was linked to *Yr15* through screening a series of NILs each consisted of a different gene for Hessian fly resistance using 1600 random 10-mer primers. Another RAPD marker, *OPE-13*, showed the complete cosegregation with the H21 Hessian fly resistance gene in wheat [60]. Dweikat et al. developed RAPD markers linked to 11 different Hessian fly resistance loci that could be used for determining the presence or absence of specific Hessian fly resistance genes [61]. More RAPD markers including *OPX061050, OPAG04950*, and *OPAI14600*, was observed to be linked to the new powdery mildew resistance *Pm25* gene, where the linkage distance between them was 12.8, 17.2, and 21.6 cm, respectively [62]. The application of the BSA approach on DNA enriched for low-copy sequences was used by Eastwood et al. [55] and William et al. [63], generating an increased level of polymorphism and in repeatability. Hu et al. [64] identified two RAPD markers, *UBC320420* and *UBC638550*, that cosegregated with *Pm1a* and one RAPD marker, *OPF12650*, tightly linked to the resistance gene. In these studies, it has been observed that RAPD markers also have been difficult to use in wheat like RFLP markers due to the very low level of polymorphism. Furthermore, the reproducibility of this RAPD bands have been found to be questionable. Despite this, continuous efforts have been made to develop RAPD genetic markers for the discrimination of species/cultivars from each other [65, 66].

#### **2.3. Amplified fragment length polymorphism (AFLP) markers**

For assessment of large numbers of polymorphic loci, the AFLP technology has been implemented as a powerful tool because of its advantage of having good levels of reproducibility, insensitivity, fast, and no need of sequence information required for primer design [67]. In 1995, a novel PCR-based assay for plant DNA fingerprinting using AFLP markers has resulted in high levels of DNA polymorphism [68]. In fact, the AFLP technique has observed to be more efficient and less expensive and less labor intensive compared to the RFLP technique in wheat [69]. Earlier AFLP-based marker studies have been found to be informative in assessment of genetic diversity in wheat varieties started in 1998 [70, 71]. Regarding the investigation of traits associated with yield, Goodwin et al. [72] and Hartl et al. [73] initiated the AFLP technique to develop an AFLP marker associated with resistance to Septoria tritici blotch and powdery mildew, respectively. Hartl et al. identified several AFLP markers closely linked to the *Pm1c* and *Pm4a* [73]. Out of 92 AFLP primer combinations, 31 polymorphic fragments detected between the resistance and susceptible lines, of which eight were found to be the most reliable polymorphic markers. One of the AFLP markers, *18M2*, was detected as being highly specific for the *Pm1c* gene, while the 4aM1 AFLP marker was identified at 3.5 cm from *Pm4a*. A couple of years later, an AFLP analysis was conducted using NILs of the strip rust resistance gene *Yr10* and designed AFLP primers that can be useful in detecting the *Yr10* gene [74]. Cao et al. analyzed the 119 individuals of H9020-17-5 x Mingxian169 F<sup>2</sup> population to detect AFLP markers linked to the strip rust resistance gene *YrHua* [75]. They found the two markers, *PM14(301)* and *PM42(249)*, of which *PM14(301)* was converted to PCR marker that could be a useful tool for MAS. In 2006, nine AFLP markers that showed polymorphism between the Argentinian wheat cultivar Sinvalocho MA and the rust leaf susceptible cultivar Gamma 6 were used to construct a linkage map of the *Lr3* gene for leaf rust resistance on chromosome 6BL of wheat [76]. The development of AFLP markers was carried out by Li et al. [77]. They detected seven markers linked to *Lr19* resistance to wheat leaf rust using *Mse I* and *Pst I* based on Thatcher, 23 NILs and F2 generation of TcLr19 x Thatcher. Dhillon et al. detected putative AFLP markers linked to leaf rust resistance genes *Lr9, Lr19*, and *KLM4-3B* using NILs of wheat [78]. More likely, the AFLP technique has been approached for developing polymorphic markers underlying a trait.

common bunt [58]. The RAPD marker *OPC20* was closely correlated with a gene controlling cadmium uptake in western Canadian durum wheat (*T. turgidum* L. var. *durum*) [56]. In addition, a RAPD marker *OPH17-1900* located on the chromosome arm 6VS, was detected that could be used for the detection of a powdery mildew resistance gene, *Pm21*, in breeding [57]. For common bunt, also known as stinking smut and covered smut, the polymorphic RAPD marker, *BW553*, was identified between resistant and susceptible NILs [58]. Furthermore, identification of RAPD markers linked to the *Yr15* gene controlling stripe rust resistance was conducted using 340 RAPD primers, six of which were detected to be polymorphic [59]. The

and showed that it was linked to *Yr15* through screening a series of NILs each consisted of a different gene for Hessian fly resistance using 1600 random 10-mer primers. Another RAPD marker, *OPE-13*, showed the complete cosegregation with the H21 Hessian fly resistance gene in wheat [60]. Dweikat et al. developed RAPD markers linked to 11 different Hessian fly resistance loci that could be used for determining the presence or absence of specific Hessian fly resistance genes [61]. More RAPD markers including *OPX061050, OPAG04950*, and *OPAI14600*, was observed to be linked to the new powdery mildew resistance *Pm25* gene, where the linkage distance between them was 12.8, 17.2, and 21.6 cm, respectively [62]. The application of the BSA approach on DNA enriched for low-copy sequences was used by Eastwood et al. [55] and William et al. [63], generating an increased level of polymorphism and in repeatability. Hu et al. [64] identified two RAPD markers, *UBC320420* and *UBC638550*, that cosegregated with *Pm1a* and one RAPD marker, *OPF12650*, tightly linked to the resistance gene. In these studies, it has been observed that RAPD markers also have been difficult to use in wheat like RFLP markers due to the very low level of polymorphism. Furthermore, the reproducibility of this RAPD bands have been found to be questionable. Despite this, continuous efforts have been made to develop RAPD genetic markers for the discrimination

For assessment of large numbers of polymorphic loci, the AFLP technology has been implemented as a powerful tool because of its advantage of having good levels of reproducibility, insensitivity, fast, and no need of sequence information required for primer design [67]. In 1995, a novel PCR-based assay for plant DNA fingerprinting using AFLP markers has resulted in high levels of DNA polymorphism [68]. In fact, the AFLP technique has observed to be more efficient and less expensive and less labor intensive compared to the RFLP technique in wheat [69]. Earlier AFLP-based marker studies have been found to be informative in assessment of genetic diversity in wheat varieties started in 1998 [70, 71]. Regarding the investigation of traits associated with yield, Goodwin et al. [72] and Hartl et al. [73] initiated the AFLP technique to develop an AFLP marker associated with resistance to Septoria tritici blotch and powdery mildew, respectively. Hartl et al. identified several AFLP markers closely linked to the *Pm1c* and *Pm4a* [73]. Out of 92 AFLP primer combinations, 31 polymorphic fragments detected between the resistance and susceptible lines, of which eight were found to be the most reliable polymorphic markers. One of the AFLP markers, *18M2*, was detected as being highly specific for the *Pm1c* gene, while the 4aM1 AFLP marker was identified at 3.5 cm

individuals

*OPB13* RAPD marker was the only one that produced polymorphism in 123 F<sup>2</sup>

of species/cultivars from each other [65, 66].

22 Wheat Improvement, Management and Utilization

**2.3. Amplified fragment length polymorphism (AFLP) markers**

#### **2.4. Simple sequence repeat (SSR) and intersimple sequence repeat (ISSR) markers**

Among all available markers, SSR (or microsatellite) markers have become the best suited tool in plant breeding programs, because they are practical, convenient, easy to use, and inexpensive. Moreover, SSRs with tandem repeats of a motif of <6 bp are the most polymorphic, codominant, easy for scoring banding patterns, and have wide genomic distribution, high reproducibility, and a multiallelic nature [79]. SSR analysis has been conducted in most of the QTL studies for mapping various traits. Up to 2015, there are more than 4000 SSR markers that have been developed and used for the construction of wheat genetic maps [80]. The high level of variability and Mendelian inheritance of SSR DNA markers have been first reported by Devos et al. [81] and Röder et al. [82]. Moreover, Röder et al. [83] and Stephenson et al. [84] placed SSR loci onto the genetic map, providing a starting point for developing a saturated map of the wheat genome. For example, SSR markers have been implemented for tagging and mapping important yield-related genes such as the dwarfing genes *Rh8* [85], *Rht12*, and the vernalization gene *Vrn1* [40], and a gene for preharvest sprouting tolerance [86]. SSR primers also have been used to detect some of the wheat resistance genes. Peng et al. found nine microsatellite loci found to be linked to *YrH52* with recombination frequencies of 0.2–0.35, and LOD scores of 3.56–54.22 [87]. The identification chromosomes with QTLs underlying 1000 grain weight (GW) has conducted by Varshney et al. [88]. They found the SSR marker Xwmc333 as being linked to GW and the major QTL for GW (*QGw1. ccsu-1A*) with a R<sup>2</sup> value of 0.1509. Moreover, the SSR markers of *Xgwm210, Xgwm296*, and *Xgwm455*, were detected to be polymorphic and linked to the leaf rust resistance gene *Lr39*, of which *Xgwm210* was the closest marker mapped 10.7 cm from *Lr39* [89]. Ammiraju et al. [90] found four intersimple sequence repeat (ISSR) markers, *UBC8181000, UBC842600, UBC8431200*, and *UBC814750*, controlling seed size in wheat, which can be measured indirectly by 1000-kernel weight (TKW). These markers showed a signification association with gene effects of 84.662.92, and 2.61%, contributing a total of 31% of the phenotypic variance in seed size. The following year, Zhou et al. [91] identified six SSR markers linked to the major QTL for scab resistance, which were *Xgwm389, Xgwm533, Xgwm493, Xbarc75, Xbarc88*, and *Xbarc147* spanning a region of approximately 20 cm on chromosome 3BS. Additional SSR markers have been reported for strip rust resistance genes, including the *Xgwm501* marker linked to *Yr5* [92], and the *Xpsp3000* marker linked to *Yr10* [93]. A microsatellite linkage map of the powdery mildew resistance gene *Pm5e* on chromosome 7B has constructed with 20 microsatellite loci, consisting of two codominant markers *Xgwm783* and *Wgwm1267* located close to *Pm5e* with a linkage distance of 11.0 cm and 6.6 cm, respectively [94]. The detection of QTL linked to FHB resistance has found that two microsatellite loci, *Xgwm533* and *Xgwm*, were significantly associated with QTL for FHB [95]. In 2004, a SSR-based consensus map has been completed by Somers et al. [96] that has been widely used a reference. Furthermore, five QTLs, *QYld.crc-1A, QYld.crc-2D, QYld.crc-3B, QYld.crc-5A.1*, and *QYld.crc-5A.2*, controlling grain yield have been identified on chromosomes 1A, 2D, 3B, and 5A [97]. Other QTLs associated with 1000 grain weight, spikes meter−2, seed number spike−1, average seed weight spike−1, harvest index, days to heading, days to maturity, and grain filling time have also been detected in the Superb/BW278 mapping population. Liu et al. [98] performed association mapping by genotyping wheat germplasm accession from China using SSR markers and EST-SSR markers, detecting 10 SSR markers on chromosome 4A associated with plant height, spike length, spikelets per spike, spikelet density, grains per spike and thousand-kernel weight. Even though studies involving RFLP, RAPD, and AFLP markers have only been used for identification and mapping of QTLs and genes, they are less likely to be applied into breeding programs because the application of these markers is likely to be less efficient for MAS [99].
