Distribution of Vernalization and Photoperiod Genes (Vrn-A1, Vrn-B1, Vrn-D1, Vrn-B3, Ppd-D1) in Turkish Bread Wheat Cultivars and Landraces

Vernalization and photoperiod response genes play a significant role in the geographical adaptation, agronomic performance and yield potential of crops. Therefore, understanding the distribution pattern and allelic diversity for vernalization and photoperiod genes are important in any wheat breeding program. In this study, we screened 63 bread wheat cultivars and 7 bread wheat landraces from Turkey for photoperiod (Ppd-D1) and vernalization genes ( Vrn-A1, Vrn-B1, Vrn-D1 and Vrn-B3 ) using diagnostic molecular markers. The photoperiod insensitive dominant allele, Ppd-D1a, was present in 60% of wheat cultivars and 42% of land-races, whereas, all other genotypes carried the photoperiod sensitive allele Ppd-D1b as recessive allele. Twenty-four cultivars and two landraces contained recessive alleles for all four VRN loci, whereas 39 wheat cultivars and 6 landraces contained one or more dominant VRN alleles. The highest percentage of Turkish wheat cultivars contained the dominant Vrn-B1 allele followed by Vrn-D1 and Vrn-A1 . Information for vernalization and photoperiod alleles in Turkish germplasm will facilitate the planning and implementation of molecular markers in wheat breeding programs. This information will be helpful to develop elite wheat cultivars carrying suitable vernalization and photoperiod alleles with higher grain yield potential and better quality suitable for different production environments through marker assisted selection.


Introduction
Bread wheat was originated in the Fertile Crescent about 8000 years ago.Today this crop is widely grown in various geographical regions ranging from 67°North to 45°South (Kilian et al. 2009).For adaptation to such diverse areas, a wide range of genetic variations at specific loci is indispensable.Among these, the regulation of flowering time is most important criterion for adaptation to such a wide range of growing conditions.Growth and developmental phases (i.e.tillering, stem elongation, heading, anthesis and maturity) of wheat are primarily determined by vernalization genes (Vrn), genes that control the photoperiod response (Ppd) and earliness per se genes (Eps) (Dubcovsky et al. 1998).The adaptability of bread wheat to a wide range of environments has been favored by allelic diversity in genes regulating growth habit (Vrn genes) and photoperiod response (Ppd genes) (Distelfeld et al. 2009).Differences in Vrn genes divide wheat into spring, winter and facultative types, whereas differences in the Ppd genes divide the germplasm into day length-sensitive and day length-insensitive classes.
Increasing knowledge about the genetics of growth habit will contribute to a better understanding about the adaptation of wheat and promote their breeding for specific environments (Goncharov 2003).In bread wheat, vernalization requirement is primarily controlled by alleles at three orthologous loci, Vrn-A1, Vrn-B1 and Vrn-D1 (collectively these three alleles are designated as 'Vrn-1' loci, Santra et al. 2009), which are located on the long arms of chromosomes 5A, 5B and 5D, respectively (Yan et al. 2003;2004;Zhang et al. 2008).Santra et al. (2009) reported that the presence of a dominant Vrn-1 allele in any genome confers spring growth habit, whereas the presence of recessive alleles in the homozygous state across Vrn-1 loci confers winter growth habit.According to Santra et al. (2009), Vrn-A1 is most effect allele providing complete insensitivity to vernalization, and is epistatic to Vrn-B1 and Vrn-D1, whereas, Vrn-B1 and Vrn-D1 are slightly vernalization sensitive alleles, having small residual vernalization requirements.In addition to these alleles, other groups of vernalization genes have been detected in wheat such as the Vrn-2 series located on chromosomes 4B, 4D and 5A; Vrn-B3 (located on chromosome 7BS) and Vrn-D5 (located on chromosome 5D) (Goncharov 2003;Yan et al. 2006;Santra et al. 2009).
In recent years, the Vrn-1 alleles were cloned, sequenced and, diagnostic molecular marker for Vrn1 alleles have been developed (Yan et al. 2003(Yan et al. , 2004;;Fu et al. 2005).Diagnostic molecular markers provide a unique opportunity to screen large collections of wheat germplasm for allelic diversity, because detection of vernalization and photoperiod genes by traditional genetic methods is time consuming.Yan et al. (2003Yan et al. ( , 2004) ) reported that different dominant Vrn-A1 alleles have been found as follow (i) Vrn-A1a resulted due duplication in the promoter region, (ii) Vrn-A1b developed due to deletion in the promoter region and (iii) Vrn-A1c developed due to large deletion in the Intron1.Zhang et al. (2008) studied allelic variation at the vernalization genes Vrn-A1, Vrn-B1, Vrn-D1 and Vrn-B3 in Chinese wheat cultivars using diagnostic marker developed by Fu et al. (2005) and Yan et al. (2004).These authors reported that the dominant Vrn-D1 allele showed highest frequency in Chinese wheat cultivars (37.8%), followed by the dominant Vrn-A1, Vrn-B1 and Vrn-B3 alleles.Ninety-two winter cultivars carried recessive alleles for all four vernalization loci, whereas 172 spring genotypes contained at least one dominant Vrn allele.
Wheat are classified as 'photoperiod sensitive', which require long days for flowering, or 'photoperiod insensitive', which flower under long or short day environments (Dyck et al. 2004).Scarth and Law (1984) reported that three genes control photoperiod response in wheat including Ppd-D1 located on the long arm of chromosome 2D, Ppd-B1 on the short arm of 2B and Ppd-A1 located on the long arm of 2A.Insensitivity of wheat to day length is conferred by dominant alleles represented as Ppd-D1a, Ppd-B1a and Ppd-A1a, whereas recessive forms of these alleles (Ppd-D1b, Ppd-B1b and Ppd-A1b) result in photoperiod sensitivity.Worland (1998) reported that the potency of the group 2 photoperiod genes for insensitivity has been ranked in the order Ppd-D1>Ppd-B1>Ppd-A1. Recently, significant progress has been achieved in molecular characterization of photoperiod responsive genes in bread wheat.Beales et al. (2007) developed diagnostic markers for Ppd-D1 that enables to detect allelic variation within Ppd-D1 loci.Using these DNA markers, Yang et al. (2009) studied the distribution of the photoperiod insensitive allele Ppd-D1a in Chinese wheat cultivars.They screened 926 Chinese wheat landraces and improved cultivars collected from nine wheat growing zones for Ppd-D1 locus using diagnostic markers.They observed that 66% of Chinese wheat landraces and improved varieties carried photoperiod-insensitive allele.
Modern breeding in Turkey started in 1925 with the goal to select well-adapted lines from local landraces for wheat improvement.In 1967, the national wheat release and training project was established with contribution of international organizations, by introducing several cultivars from foreign countries (Braun et al. 2001).The national wheat breeding program, meanwhile, developed more than 100 wheat cultivars, most of which has significant impact on the economy of country.However, Turkish wheat cultivars released since 1935, have not been screened systematically for vernalization and photoperiod alleles.Therefore, we aimed to characterize a collection of Turkish bread wheat cultivars and landraces for allelic variations at four vernalization and one photoperiod gene in order to select parental lines for marker assisted breeding programs.

Plant material
In total, seventy bread wheat genotypes, including sixty-three bread wheat cultivars (Triticum aestivum L.) most of them released in Turkey during the last 70 years and seven bread wheat landraces from Turkey were included in this study (Table 1).Three wheat genotypes, namely Chinese Spring (vrn-A1), Thatcher (Vrn-A1a) and Hope (Vrn-B3) were used as controls, in order to confirm the presence or absence of specific vernalization alleles.

DNA extraction and PCR analysis for vernalization and photoperiod alleles
Three plants of each cultivar and landrace were grown in the greenhouse.Young leaf samples were collected from 10 days old seedlings, frozen in liquid nitrogen and stored at -80ºC until use.Genomic DNA was then extracted by the CTAB method (Doyle and Doyle 1987) with minor modifications (Ozkan et al. 2005).For the characterization of vernalization alleles, eleven diagnostic markers were used to detect dominant or recessive alleles of Vrn-A1, Vrn-B1, Vrn-D1 and Vrn-B3 (Yan et al. 2004;Fu et al. 2005).In order to identify dominant and recessive photoperiod alleles, two primer combinations as described by Yang et al. (2009), were used.All PCR amplifications were performed in a gradient thermal cycler (Eppendorf, Hamburg, Germany).Details about PCR based markers for vernalization and photoperiod genes, their sequences and amplification conditions are briefly presented in Table 2.All amplification reactions were carried out in a 25 μl volume containing 75 mM Tris-HCl, pH 8.8, 20 mM (NH 4 ) 2 SO 4 , 2.0 mM MgCl 2 , 0.1% Tween 20, 0.2 μM primer pair, 100 μM each of dATP, dGTP, dCTP and dTTP, 1 unit of Taq DNA polymerase and 20 ng of genomic DNA.All amplified products were analyzed by gel electrophoresis on 2% agarose gels in 0.5 × TBE buffer, stained with ethidium bromide and photographed under ultraviolet light.The experiments were replicated three times in order to confirm the reproducibility of results.

Allelic frequencies at Ppd-D1
To study allelic diversity at Ppd-D1 locus, two specific primer combinations were used (Yang et al. 2009; Table 2).Out of 70 cultivars and landraces, 25 cultivars and 4 landraces yielded a 418 bp PCR fragment, indicating the presence of the photoperiod sensitive Ppd-D1b allele, and 38 cultivars and 3 landraces gave a 290 bp fragment, indicative of the photoperiod-insensitive Ppd-D1a allele.The overall frequency of the dominant Ppd-D1a allele in Turkish cultivars and landraces were 59%, with frequencies of 43% and 60% in landraces and cultivars, respectively.
Allelic frequencies at Vrn-A1, Vrn-B1, Vrn-D1 and Vrn-B3 Diagnostic markers VRN1AF and VRB1-INT1R as described by Yan et al. (2004) were used in all Turkish bread wheat cultivars and landraces, in order to identify the variation in the promoter region of the dominant spring allele Vrn-A1 (Table 2).We found that only seven cultivars and one landrace yielded two fragments similar to Thatcher used as control (970 bp and 870 bp).This indicated that these genotypes carry the dominant Vrn-A1a allele at the Vrn-A1 locus.A 720 bp PCR product was observed only in two cultivars, namely Ak-702 and Yayla, which represents the presence of Vrn-A1b allele (Table 2).The remaining 60 cultivars and landraces including the control Chinese Spring gave a 740 bp PCR product, which indicated that these genotypes carry either the dominant Vrn-A1c allele or the recessive vrn-A1 allele, as reported by Zhang et al. (2008).In order to differentiate between these two alleles; we used two primers pairs Intr1/A/F2-Intr1/A/R3 and  Using primer pair Intr1/D/F-Intr1/D/R3, nineteen wheat cultivars generated a 1670 bp PCR product, demonstrating that these varieties carried the dominant Vrn-D1 allele.A 991 bp fragment was generated from 44 wheat cultivars and seven landraces using the primer pair Intr1/D/F-Intr1/D/R4, indicating the presence of the recessive vrn-D1 allele.None of the bread wheat cultivars or landraces gave any PCR product using the primer pair VRN4-B-INS-F -VRN4-B-INS-R, which demonstrated the lacking of the dominant Vrn-B3 allele.For vrn-B3, a 1150 bp fragment was generated from 63 bread wheat cultivars and seven landraces using the primer pair VRN4-B-NOINS-F -VRN4-B-NOINS-R, indicating presence of the recessive vrn-B3 allele

Discussion
The productivity of wheat and their adaptation to climatic conditions are associated with their growth habits and heading dates, which are controlled to great extent by vernalization requirement and sensitivity to photoperiod.Vernalization and photoperiod genes play a significant role in the geographical adaptation, agronomic performance, quality and yield potential of wheat cultivars (Santra et al. 2009).Vernalization and photoperiod genes cause flowering and maturity time variations to escape drought or heat stresses during grain filling and damage from early frost in fall in some regions (Iqbal et al. 2007).
Different dominant Vrn and Ppd alleles in wheat were found to have different affects on vernalization requirement, flowering time and response to day length.Various reports have been published describing allelic variation of vernalization and photoperiod genes and, their effect on grain yield (Santra et al. 2005;Iqbal et al. 2007;Zhang et al. 2008;Yang et al. 2009).Stelmakh (1993) discovered that average grain yield per plant across environments was highest for spring wheat genotypes with Vrn-A1 or Vrn-B1, whereas cultivars with all three dominant alleles at homoeologous Vrn-1 genes gave the lowest yield.Recently, Santra et al. (2009) concluded that if temperature and drought stress occurred during grain filling, the highest grain yield was reported for photoperiod insensitive wheat genotypes with the dominant allele at chromosome 5D (Vrn-D1), in combination with either Vrn-A1 or Vrn-B1.Therefore, optimizing the allelic composition at Vrn-1 loci with photoperiod insensitivity may offer possibilities for developing wheat cultivars with higher yield potential and adaptation to a broader range of different climates.
Several physiological and morphological methods are available to detect vernalization and photoperiod genes in the background of wheat and other cereals, however, some of them are time consuming, labor intensive and influenced by environmental conditions, and thereby difficult to use in breeding programs.Recent advances in molecular genetics have facilitated the cloning of vernalization and photoperiod genes of wheat, which resulted in the development of gene and allele specific markers (diagnostic marker or functional markers) (Yan et al. 2004;Fu et al. 2005;Zhang et al. 2008).The development of diagnostic marker for Vrn-A1, Vrn-B1, Vrn-D1 and Ppd-D1 allows the detection of alleles of these genes in large wheat collections and is important for any wheat breeding programs.
In this study, sixty-three bread wheat cultivars and seven bread wheat landraces from Turkey were analyzed using eleven diagnostic primer combinations in order to identify the allelic status at four vernalization and one photoperiod genes.Our results revealed that 14% of Turkish wheat cultivars (8 cultivars) studied, carried dominant alleles for Vrn-A1, 52% (37) for  for Vrn-D1.The dominant Vrn-A1a allele was present in the genotypes Kirkpinar, Çukurova-86, Doçankent-1, Gönen-98, Tahirova-2000, Nurkent, Yüreçir and Apure, whereas the dominant allele Vrn-A1b was found in only 2 cultivars .No Turkish wheat cultivar or landrace carried the Vrn-A1c allele.The Vrn-A1a frequency in Turkish bread wheat collection is much lower than in wheat collections when compared with different parts of the world.For instance, Fu et al. (2005) screened 117 spring wheat genotypes from Argentina and California and found that Vrn-A1a and Vrn-A1b were the predominant alleles, either alone or in combination with Vrn-B1 and Vrn-D1.The same authors and Iqbal et al. (2007) did not find any Vrn-A1c allele in their wheat varieties collections.Vrn-A1a was found only in the spring hexaploid wheat landraces from Afghanistan (Zhang et al. 2008).Iqbal et al. (2007) identified the Vrn-A1a allele in 85% of the Canadian spring wheat genotypes, Vrn-A1b in turn was found in only one genotype.Yan et al. (2004) reported that more than half of the spring wheat cultivars released between 1970-2004 in the USA and Argentina, carried the Vrn-A1a allele.The increase in the frequency of Vrn-A1a allele in spring wheat varieties in Argentina and USA might be related to the introduction of semi-dwarf germplasm from CIMMYT (International Wheat and Maize Improvement Center).We thought that the situation in Turkey could be similar; many wheat cultivars have been introduced from CIMMYT.However, we observed that only few cultivars introduced from CIMMYT carried the Vrn-A1a allele (Çukurova-86, Doçankent-1, Gönen-98, Yüreçir-89).Several other cultivars like Balatilla, Genç-99 and Adana-99 etc., were also introduced from CIMMYT (Yücel et al. 2009), but these genotypes did not carry the Vrn-A1a allele.This is in contrast to the above-mentioned studies.Vrn-B1 is most frequent allele in bread wheat varieties from Turkey.Iqbal et al. (2007) reported that 50% of their lines carried the Vrn-B1 allele.However, the frequency of the dominant Vrn-B1 allele was less in Chinese cultivars (26%) studied by Zhang et al. (2008).The dominant Vrn-B3 allele was not observed in our Turkish bread wheat collection.These finding are consistent with Zhang et al. (2008), that reported only two out of 278 cultivars from China carried the dominant Vrn-B3 allele.Vrn-D1 was the second most frequent allele (27%) among the screened Turkish wheat varieties.This might be also due to the use of parental lines from CIMMYT germplasm.Yediay et al. (2010) reported that nearly all Turkish wheat cultivars in their study were bred directly and/or indirectly from CIMMYT germplasm.Eagles et al. (2009) reported that Vrn-D1 is the most frequent allele in Australian wheat cultivars developed from CIMMYT materials.This is also consistent with the situation in China (Zhang et al. 2008).Vrn-D1 was not found in any bread wheat landrace from Turkey, which is in contrast to the results of Iwaki et al. (2001) that reported Vrn-D1 is common in Indian bread wheat landraces.
Twenty-four bread wheat cultivars and one landrace carried recessive alleles at all four vernalization loci studied.Vrn-A1a was found in combination with the dominant Vrn-B1 allele in five bread wheat cultivars, namely Çukurova-86, Doçankent-1, Gönen-98, Nurkent and Yüreçir.The cultivars, Çukurova-86, Doçankent-1, Gönen-98, Nurkent and Yüreçir, carry both dominant vernalization alleles Vrn-A1a and Vrn-B1 in combination with Ppd-D1a.These cultivars were originated from CIMMYT parental lines.We observed that 11 cultivars contained both Vrn-B1 and Vrn-D1 alleles, and only the cultivar Ppd-D1a genes can be used to accelerate the development of wheat cultivars showing wide adaptation with good yield potential for specific environments.
In Turkey, the majority of wheat is sown in autumn (in October and early November) such as in central and southeastern Anatolian region or early winter (in November and early December) as in Mediterranean region, and harvested in the following summer.Therefore these varieties should flower late enough to avoid frost damage but sufficiently early enough to mature before summer drought and high temperature occur.The photoperiod insensitive allele Ppd-D1 in combination with specific vernalization genes is required for different geographical regions.This would be highly desirable in Turkish wheat breeding programs.Based on our results presented here, Turkish wheat breeders can now modify the flowering time behavior suitable to a particular geographic region.This information will be helpful to wheat breeders trying to develop elite wheat cultivars carrying suitable vernalization and photoperiod alleles with higher grain yield potential and better quality suitable for different production environments through marker assisted selection.
/F-Intr1/AB/R specific for Intron1 deletion (Vrn-A1c) and the recessive vrn-A1 allele, respectively.We found the absence of the Vrn-A1c allele in all tested Turkish wheats and in the control 'Chinese Spring'.On the other hand, all genotypes gave 1060 PCR products, indicating that they carry the recessive vrn-A1 allele.Thirty-two bread wheat cultivars and 5 landraces including the control Chinese Spring harbor the dominant Vrn-B1 allele because a 712 bp band was amplified in these lines using the primer combination Intr1/B/F-Intr1/B/R3.The absence of a PCR product in the remaining wheat genotypes suggests that they carry the recessive allele.The primer combination Intr1/B/F-Intr1/B/R4 was used to confirm the presence of the recessive allele.Thirty-one bread wheat cultivars and two landraces amplified 1160 bp PCR product, showing the presence of the recessive vrn-B1 allele.

Table 2 .
Specific DNA molecular markers for vernalization and photoperiod genes considered in this study