**9. The role of diagnostic molecular markers in the detection of allelic variation among the major growth habit genes influencing the flowering time of bread wheat**

The fact that current wheat germplasm has not been characterized fully in terms of impor‐ tant agronomic traits limits the use of wheat germplasm to a certain extent. Identifying the alleles of these genes and estimating the effects of their combination on growth, heading date, and ultimately grain yield will enhance the selection of cultivars with wide adaptability to a set of environments [57]. This knowledge can help accelerate the introgression of adapt‐ ability and yield‐contributing genes by predicting the best combinations for enhanced yield potential and adaptation [28]. Moreover, the identification of alleles of growth habit genes subsequently leads to the development of a series of molecular markers (allele‐specific DNA markers) for improved identification of these alleles in future [46, 86, 94].

The development of allele‐specific DNA markers has allowed for efficient detection of exten‐ sive allelic variation existing among genes controlling flowering time in bread wheat [35, 46, 95]. Through these markers, it has been revealed that the allelic variation at the *Vrn‐A1* locus (*Vrn‐A1a, Vrn‐A1b*, and *Vrn‐A1c*) results from mutations within the promoter sequence [26] and/or deletions within the first intron of this gene [26, 95]. For the *Vrn‐B1* and *Vrn‐D1* loci, their allelic variation is determined only by deletions within the first intron sequence of the gene [95]. Diagnostic markers are available to differentiate among these forms and conse‐ quently, significant progress in understanding the molecular basis of vernalization has been made in wheat and barley species [15, 16, 94, 96].

For photoperiod response genes, photoperiod insensitivity is induced by indels in the 5' upstream region of pseudoresponse regulator (PPR) genes, which do not exist in photoperiod‐sensitive varieties [41, 46, 49]. For instance, a 2 kb deletion in the *Ppd‐D1* promoter region of chromosome 2D results in a semi‐dominant, photoperiod‐insensitive allele (*Ppd‐D1a*). The semidominant *Ppd‐ D1a* mutation has been identified as the major source of earliness in wheat varieties globally. This most potent allele upregulates the expression of the *Vrn‐3* gene, which is a homologue of the *flowering locus T* (*FT*) of *A. thaliana* [9], under both SD and LD conditions, therefore confers early flowering in wheat [13, 43].

Similar studies have been conducted successfully whereby high temperatures and drought stress during anthesis and grain filling were avoided through tailoring flowering time of

The potential advantage of tailoring flowering time can be used to escape environmen‐ tal conditions that lead to yield loss, such as high temperatures or conditions that lead to poor wheat quality, such as rain during harvest time. This could contribute to reducing the worldwide physiological phenomenon of preharvest sprouting (PHS). Preharvest sprouting, which is the germination of seed grains in the mother ear before harvest due to humid condi‐ tions, is prevalent in wheat‐growing regions experiencing high rainfall during the period of grain maturity and ripening [90]. This results in significant losses in the wheat production industry such as the downgrading of premium milling quality wheat to feed quality [91]. Resistance to PHS is a highly desirable trait sought by plant breeders globally [92, 93]. In addition to breeding for resistance of this trait, tailoring flowering time for the production of early flowering cultivars, which will escape conditions favorable to PHS, could help reduce

**9. The role of diagnostic molecular markers in the detection of allelic variation among the major growth habit genes influencing the flowering** 

The fact that current wheat germplasm has not been characterized fully in terms of impor‐ tant agronomic traits limits the use of wheat germplasm to a certain extent. Identifying the alleles of these genes and estimating the effects of their combination on growth, heading date, and ultimately grain yield will enhance the selection of cultivars with wide adaptability to a set of environments [57]. This knowledge can help accelerate the introgression of adapt‐ ability and yield‐contributing genes by predicting the best combinations for enhanced yield potential and adaptation [28]. Moreover, the identification of alleles of growth habit genes subsequently leads to the development of a series of molecular markers (allele‐specific DNA

The development of allele‐specific DNA markers has allowed for efficient detection of exten‐ sive allelic variation existing among genes controlling flowering time in bread wheat [35, 46, 95]. Through these markers, it has been revealed that the allelic variation at the *Vrn‐A1* locus (*Vrn‐A1a, Vrn‐A1b*, and *Vrn‐A1c*) results from mutations within the promoter sequence [26] and/or deletions within the first intron of this gene [26, 95]. For the *Vrn‐B1* and *Vrn‐D1* loci, their allelic variation is determined only by deletions within the first intron sequence of the gene [95]. Diagnostic markers are available to differentiate among these forms and conse‐ quently, significant progress in understanding the molecular basis of vernalization has been

For photoperiod response genes, photoperiod insensitivity is induced by indels in the 5' upstream region of pseudoresponse regulator (PPR) genes, which do not exist in photoperiod‐sensitive varieties [41, 46, 49]. For instance, a 2 kb deletion in the *Ppd‐D1* promoter region of chromosome

markers) for improved identification of these alleles in future [46, 86, 94].

made in wheat and barley species [15, 16, 94, 96].

wheat to local climatic conditions [42, 89].

84 Wheat Improvement, Management and Utilization

the problem.

**time of bread wheat**

The *Ppd‐D1* gene is said to exist in several forms. Six haplotypes (alleles) of this gene have been identified [40], four of which were common in bread wheat. The same study provided molecular markers to distinguish among these alleles and elucidated that they have differ‐ ent levels of expression. Haplotype I, which is equivalent to *Ppd*‐*D1a* in Beales et al [46] and Eagles et al [57], had the highest level of expression, and it was suggested that Haplotype II is a progenitor of the others and probably photoperiod‐sensitive. To further their work, Guo et al. [40] developed a method which identified allelic variation within a locus that Eagles et al. [57] and Fischer [97] labeled as *Ppd*‐*D1b*, eventually dividing that single classification into three alleles.

The alleles of *Ppd*‐*D1* were also identified by Cane et al. [98] using allele‐specific molecu‐ lar markers. Lines carrying *Ppd*‐*D1a* were identified with a large deletion in the promoter region by the method described in references [46] and [60]. Lines with a deletion in exon 7 were classified as *Ppd*‐*D1d* carriers. Lines harboring *Ppd*‐*D1c* alleles manifested a *mariner*‐ like transposable element in intron 1 and lines not characterized as either *Ppd*‐*D1a*, *Ppd*‐*D1c*, or *Ppd*‐*D1d* were designated as *Ppd*‐*D1b*. Frequent alleles of *Ppd*‐*D1*, *Vrn*‐*A1*, *Vrn*‐*B1*, and *Vrn*‐*D1* genes can be accurately identified using current molecular techniques. Inaccurate classification of alleles could reduce the accuracy of estimation of their effects on flowering time [57, 98].

Besides the *Ppd*‐*D1* gene, two more loci namely, *Ppd‐A1* and *Ppd‐B1*, have been identified and shown to have effects as strong as that of *Ppd‐D1* in accelerating flowering time in bread wheat [20, 41, 42, 99]. In a study to investigate the effect of *Ppd‐B1a* allele on flowering time in wheat, it was shown that *Ppd‐B1* displays copy number variation (CNV) [20]. Wheat geno‐ types with only one copy allele are photoperiod‐sensitive whereas an increased copy num‐ ber (2–4 copies) results in a day‐neutral, early flowering phenotype. A complementary study [100] confirmed that wheat genotypes with the three‐copy allele (termed *Ppd‐B1a*) and the four‐copy allele (termed *Ppd‐B1c*) exhibit reduced days to heading as compared to the one‐ copy allele (termed *Ppd‐B1b*) whereas the two‐copy allele (termed *Ppd‐B1d*) displays increased days to heading (late flowering). These results indicate that the CNV at the *Ppd‐B1* locus con‐ tributes in fine‐tuning the adaptation of wheat to local climatic conditions, in addition to the major effect of *Ppd‐D1*.
