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ANALYZING SEQUENCE POLYMORPHISMS AND HvFT1 EXPRESSION IN A BIPARENTAL BARLEY POPULATION Trabajo Fin de Grado en Biotecnología. CARLOTA RUIZ GAZULLA Directores: Dr. ERNESTO IGARTUA Dra. ANA M. CASAS Departamento de Genética y Producción Vegetal. Estación Experimental de Aula Dei. Consejo Superior de Investigaciones Científicas. 2014 1 SUMMARY: Time of flowering in barley has a large impact on yield, and many researches have focus on finding polymorphisms in genes underlying the adaptation of different cultivars to changing environmental conditions. Previous studies have identified three vernalization and two photoperiod genes, which have a major role in the determination of flowering. Other genes (HvFT1, HvCEN, HvELF3 or HvLUX) also contribute to fine tune the time to heading within spring or winter barley cultivars. In this study we use a doubled haploid population, derived from the cross of two spring two-row barley cultivars, Beka and Logan. Previous QTL analyses had identified three major QTL controlling flowering time in this population. The aim of this study was to analyze the expression of three genes, HvELF3, HvCEN and HvFT1, candidates for the major QTLs segregating in this population. Gene expression was analyzed in eight doubled haploid lines with contrasting alleles for these genes. Plants with the Logan allele in the three genes flowered the earliest. Expression of HvFT1 was dependent on the genetic background. Higher HvFT1 expression, and earlier flowering, was detected in plants with the Logan allele in the three genes. Most of the results were coherent with the previous literature. 2 RESUMEN: La fecha de floración en la cebada tiene gran impacto en la producción de las cosechas y por tanto, existe un interés por conocer los polimorfismos de los genes que están detrás de la adaptación de diferentes cultivares a ambientes con condiciones ambientales muy diversas. Estudios previos han identificado tres genes de vernalización y dos de fotoperiodo, que juegan un papel importante en la determinación de la fecha de floración. Otros genes, (HvFT1, HvCEN, HvELF3 o HvLUX) también contribuyen a este fenómeno, tanto en variedades de primavera, como en las de verano. En este estudio hemos usado una población biparental, que deriva del cruce entre dos variedades de primavera, Beka y Logan. A partir de análisis de QTLs se han identificado tres QTL que controlan la fecha de floración en esta población. El objetivo de este estudio era analizar la expresión en los tres genes, HvELF, HvCEN y HvFT1; principales candidatos de los QTLs identificados. Se analizó la expresión en las ocho líneas de la población, las cuales, tenían diferente combinación de alelos cada una. Por un lado se vio como las plantas con alelos de Logan florecían antes. Además, las plantas que tenían alelos de Logan para los tres genes, presentaban mayor expresión de HvFT1 que el resto. La mayoría de los resultados que se obtuvieron fueron coherentes con los observados en otros estudios similares. 3 INDEX: 1. INTRODUCTION 2. BACKGROUND 2.1 Domestication of Barley 2.2 Flowering of barley 2.3 Vernalization genes 2.4 Interactions between Vrn-H genes 2.5 Photoperiod response 2.6 Importance of early flowering 3. PREVIOUS RESULTS 4. OBJECTIVES 5. MATERIALS AND METHODS 5.1 Plant material 5.2 Plants growth conditions 5.3 RNA isolation 5.4 cDNA synthesis 5.5 PCR quantification 5.6 Statistical analysis 6. RESULTS 6.1 Phenotypic observations 6.2 Expression of HvFT1 6.3 Expression of HvCEN 6.4 Expression of HvELF3 7. DISCUSSION 8. BIBLIOGRAPHY 4 1. INTRODUCTION: Barley (Hordeum vulgare L.) is a major cereal grain known from ancient times. It was one of the first grains in the Fertile Crescent, in Western Asia and near the Nile River. 10,000 years ago the crop was domesticated from its wild relative H.vulgare ssp. spontaneum. (1) Nowadays, barley ranks fourth in importance among cereals after wheat, maize and rice. The main producing countries worldwide are Russia, Ukraine and France. Spain has an important production on a worldwide scale. In 2012, the area harvested was 2.676.200 ha and the production was 5,976,900 tons. (2) The reason for its importance is due to its wide ecological adaptation, including extreme latitude and altitude. This is the result of a long domestication process started centuries ago. The crosses between efficient cultivars led to new varieties that could be adapted to new regions with diverse climatic conditions. At the beginning, the improvement of barley cultivars was made by observing the phenotype and selecting the best seeds. Nowadays, the use of molecular tools allows to associate physical traits with the genotype, and facilitates the obtaining of varieties with desirable characteristics for specific regions and assures bumper crops under very different conditions. In this project we studied the relation between some recently described QTLs and the date of flowering in a barley population. By quantitative PCR we were able to follow the expression of some genes which are part of the flowering process. 5 2. BACKGROUND Barley is diploid species with 14 chromosomes. It has been considered as an important model organism for genetic studies. Its diploid genome has facilitated the study of other crops such as wheat or rye. 2.1. Domestication of barley: During domestication of barley cultivars, farmers selected seeds with favorable traits, and over time, they converted wild species in bountiful crops. This transition started around 10 000 years before present and has led to an accumulation of characteristics that facilitate agricultural production. The most important traits acquired are non-brittle rachis, an increase in the size and number of seeds, changes in plant architecture, improved seed fertility, loss of seed shattering, and adaptation of flowering time to different areas. (1) Barley can be classified in different groups: - Two-rowed and six rowed barley: Depending on the development of lateral kernels. Two-row barley has lower protein content, so it is usually used for malting, while six-row barley, with a higher protein content is best suited for animal feed. - Hulled and “naked” seeds: Based on the seed covers. Naked barley has an outer hull easier to remove and generally falls of during harvesting. - Spring and winter barley: Depending on the growth habit. Winter barley is planted in autumn because it requires a cold treatment before being competent to flower. Spring barley is planted in different times of the year, depending on the region. All these diverse characteristics appeared due to the unconscious selection of mutations during the domestication process. 2.2. Flowering of barley: Flowering time, as explained in the introduction, depends on the vernalization requirement of a cultivar, and its photoperiod sensitivity. As seen before, barley can be classified in two types: spring and winter barley. In winter varieties, prolonged exposure to cold accelerate flowering. It occurs during winter, when temperatures do not exceed 10ºC. This phenomenon is called vernalization. These cereals are planted in autumn, whereas spring varieties are planted in different times of the year and do not have vernalization requirement. (3) The spring growth habit appeared after domestication of winter varieties. Independent mutations led to phenotypes that did not require vernalization, this modification converted flowering time in a highly variable phenotype (4). Remains of the first barley crops were found in the Fertile Crescent, and over time, varieties without the need for an extended period of cold were extended to regions with severe winters, leading to a great variety of cultivars that are 6 suitable for different geographic regions and climatic conditions. Cultivars from regions with mild winters, have an intermediate vernalization requirement, alleles from these cultivars might offer advantages in areas like the Mediterranean region, were winter is not so cold (5). 2.3. Vernalization genes: The genes underlying the flowering process are:  Vrn-H1 gene encodes a MAD-box transcription factor which regulates the transition of the vegetative shoot apical meristem to the reproductive phase. It is induced by vernalization, and it depends on the length of the cold treatment, the longer is the exposure, the greater is the effect. (6, 7)  Vrn-H2 is a floral repressor. It encodes a protein with zinc-finger motif, which mediates DNA binding. The main role of Vrn-2 is to block flowering in long days, by repressing Vrn-3. (6). It is under the control of circadianclock. (7)  Vrn-H3 seems to be an orthologous of the Arabidopsis thaliana FLOWERING LOCUS T gene (FT). (8, 9) Vrn-3, or HvFT1, is a promoter of flowering that encodes a phosphatidylethanolamine-binding protein (PEBP) (8). It is induced by long days and interacts with the PHOTOPERIOD1 gene (Ppd-H1). Ppd-H1 is a pseudo-response regulator gene expressed with a diurnal pattern; it moderates day-length induction of HvFT1 by controlling CONSTANS (CO) activity. (3) Different alleles of the HvFT1 gene have polymorphisms in the promoter and first intron. In the promoter, the haplotypes differ in nine linked SNPs and two indels (8, 10). Two SNPs in the first intron, TC or AG, are associated with differences in flowering time (8, 10). These regions of the gene are presumably important for the repression of HvFT1, and might contain a binding site for the Vrn-2 protein. (3) In spring cultivars, HvFT1 can carry more than one copy of the gene (11, 12). Increased copy number has been associated with early flowering (11). Spring barley has evolved from the ancestral winter growth habit. In addition to allelic variation in HvFT1, deletions at Vrn-2 and, different alleles at Vrn-1, resulting from deletions of a cis-regulatory motif in the first intron of this gene confer spring behavior. (13). 2.4. Interactions between Vrn-H genes: Flowering is explained by a model that integrates vernalization and photoperiod pathways. In general, barley planted in late summer or fall does not flower before winter because both vernalization and day length response pathways are inactive. Vrn-1 expression is low and Vrn-2 activity represses the possible 7 induction of HvFT1. Vrn-1 is slightly up-regulated during winter, due to low temperatures, and then represses Vrn-2. Low levels of Vrn-2 transcripts, add up to the increase of the day-length, cause high levels of CO protein and favor the activation of HvFT1. This promotes consecutive stages of reproductive development. Flowering occurs in spring or early summer (Figure 1). (3) Figure 1. Expression levels of the Vrn-H genes during time. The red line represents the seasonal variation in temperature. Rectangles on top indicate the duration of light (white) and dark (grey) in each season. Taken from Distelfeld et al. (2009) 2.5. Photoperiod response: The photoperiod pathway is also involved in the flowering process. The circadian clock provides diurnal and seasonal control of flowering. Two genes have been identified and characterized:  Ppd-H1, mentioned above, up-regulates FT1 expression, which is mediated by the activity of CO. Both genes are regulated by transcriptional outputs from the circadian clock (Figure 2). The recessive allele of this gene, ppd-H1, delays flowering under long days and shows reduce expression of FT1 (14).  Ppd-H2, encodes a distinct PEBP, HvFT3, and promotes flowering under short day conditions (5). 8 Figure 2. Model of flowering that integrates vernalization requirement (VrnH1, Vrn-H2) and photoperiod response (Ppd-H1, CO). Vernalization induces VrnH1 under both long or short days, which repress Vrn-H2. Vrn-H3 responds to photoperiod signals by interacting with trimeric HAP2/3/5 complexes. Taken from Distelfeld et al. (2009) Other components of the circadian clock have been related with the photoperiod response and its effect in flowering. The gene EAM8 was identified as a barley orthologous of the A. thaliana circadian clock regulator EARLY FLOWERING 3 (15, 16). There is strong evidence that mutations on this gene cause early flowering phenotype in plants, by increasing the expression of FT1, but this process is independent of allelic variation at Ppd-H1. 2.6. Importance of early flowering: The development of DNA markers facilitates the characterization of quantitative trait loci (QTL), regions within genome that contain genes related with a particular quantitative trait. DNA markers have an enormous potential in plant breeding, and have been used to determine regions underlying time to flowering. Other studies identified the location of the genetically assigned Earliness per se locus Eps2 (or Eam6) on chromosome 2H, one of the major determinants of flowering time under Mediterranean conditions (17, 19). The region predicted contains more than 300 genes, and by screening all genes within the interval, candidates for EPS2 were identified. One of these is HvCEN, a homolog of Antirrhinum CENTRORADIALIS (18). Two main alleles related with a winter and spring growth habit, have been identified. The difference between the alleles consists in a non-synonymous substitution. The haplotype Pro135 is found in cultivars with winter growth habit while haplotype Ala135 is linked with the spring growth habit. This gene is considered as a contributor to environmental adaptation (18). 15 Table 4. qPCR protocol. Table 5. Primer efficiencies cDNA (250 ng/ µL) 1 µL H2O 4 µL 2x Syber Green 10 µL Mix Primers F+R, 2 µM each 5 µL Total volume 20 µL The threshold cycle (Ct), at the beginning of the exponential phase, was recorded. We used the average of the two technical repeats for calculations. Results were treated with the ΔCt method, taking into account the efficiency (E) of the primers. We used the next equation, were the “Ct” is the average Ct for the gene, and the “gene” can be any of the three genes in the study: HvFT1, HvCEN and HvELF3. 5.6. Statistical analysis Results were compared in Excel. Analysis of variance for differences among marker alleles and their interactions in field and greenhouse experiments were performed using mixed models procedures implemented with REML (residual maximum likelihood), in package Genstat 15.0 (23). Markers were considered fixed effects, environments in the field experiments and replicates in the greenhouse experiments were consider random. Actin 0,97 HvFT1 0,86 HvCEN 0,96 HvELF3 0,95 16 6. RESULTS: HvFT1 has been described as floral integrator of photoperiod and vernalization. The expression of the gene has been related with early flowering, so cultivars that flower earlier are expected to have higher expression of HvFT1. The first association of HvFT1 haplotype with differences in flowering time was reported by Yan et al (6), predicting that cis regulatory sites on the promoter and first intron might be important for transcriptional regulation. Recent studies describe however, incoherent results for haplotype-phenotype relation. They suggest that not only the haplotype is important for the growth habit, but also the copy number of HvFT1. (9) 6.1 Phenotypic observations: Logan is earlier than Beka, so plants with Logan alleles are supposed to flower before those with Beka alleles (19). The time to flag leaf expansion and awn emergence, support the above affirmation. Logan’s flag leaf was expanded two days before that of Beka, and also the awns appeared earlier (Figure 5). There was a relation between the genetic constitution of the doubled haploid lines and their phenotypes; those with a majority of Logan alleles flowered before those with a majority of Beka alleles Besides, all lines with the Logan allele in Ctig4047 (2775, 2779, 2771, 2820) were the earliest to flower. Figure 5. Days to flag leaf expansion and awns appearance. 12-nov 17-nov 22-nov 27-nov 02-dic 07-dic 12-dic 17-dic Beka Logan 2814 2785 2796 2799 2775 2779 2771 2820 Date Genotype 50% flag leaf expanded 50% awn appearance 17 6.2 Expression of HvFT1 Figure 6. Expression of HvFT1 normalized to Actin. Analyzing the expression of HvFT1 (figure 6) in the second sampling, plants with higher expression of HvFT1 flowered earlier, in particular DH lines 2771 and 2820. Those plants that flowered later showed lower HvFT1 expression. There is no apparent relation between the expression of HvFT1 and the allele they carry. 6.3 Expression of HvCEN Figure 7. Expression of HvCEN normalized to Actin. The expression of HvCEN was consistent considering the parents. Logan had more expression in the second sampling than Beka, and it also flowered earlier. 17-nov 22-nov 27-nov 02-dic 07-dic 12-dic 17-dic 0 0,2 0,4 0,6 0,8 1 Beka Logan 2814 2785 2796 2799 2775 2779 2771 2820 Expression levels relative to Actin Genotypes HvFT1 07-nov 22-nov Awn appearance 0 0,2 0,4 0,6 0,8 1 Beka Logan 2814 2785 2796 2799 2775 2779 2771 2820 Expression levels relative to Actin Genotypes HvCEN 07-nov 22-nov 18 However, looking at the doubled haploid lines, expression pattern has no apparent link with the time to flowering. In the second sampling date, lines with more HvCEN mRNA levels do not always coincide with earlier plants. 6.4 Expression of HvELF3 Figure 8. Expression of HvELF3 normalized to Actin. The expression of HvELF3 was very similar in all plants. No apparent difference can be suggested based on the Ctig4047 allele they carry. We also tested possible interactions between the expressions of the different genes by analysis of variance. No differences were detected for any of the analyzed genes in the first sampling date. In the second sampling date, there were differences for the expression of HvFT1, according to the HvELF3 and HvCEN. Plants with Logan alleles in those markers showed higher HvFT1 expression. Also, two significant interactions were detected, one between the alleles of HvELF3 and HvCEN (P=0.028) and another one between the alleles of HvCEN and HvFT1 (P=0.004). In both cases, plants with the Logan alleles in those markers showed higher HvFT1 expression. 0 0,2 0,4 0,6 0,8 1 Beka Logan 2814 2785 2796 2799 2775 2779 2771 2820 Expression levels relative to Actin Genotypes HvELF3 07-nov 22-nov 19 7. DISCUSSION: The three analyzed genes in the project have been proposed to be good candidates for the QTLs detected and described before. In different researches, they have been reported to influence in the time to flower. Nowadays, it can be assumed that the gene HvFT1 responds to environmental signals and they affect flowering time. Polymorphisms affecting both the promoter and the intron have wide range of effects in flowering time; those that confer earliness development usually have higher levels of expression. However, as reported in Loscos et al. (12) we cannot consider this gene as an individual factor affecting flowering time, but we also have to bear in mind the genetic background. Beka and Logan have different haplotypes, 139-142-TC y 135-146-TC and number of copies (2 vs 1), respectively. Plants with the Logan allele flower earlier than those with the Beka allele. Logan has a different promoter, similar to that of SBCC145 (22). This promoter clearly results in earlier flowering, as we saw in some landraces from the Spanish barley core collection (10) and also in the SBCC145 x Beatrix population (22). Beka also contributes something to earlier flowering on chromosome 5H, marker HvM30. What we see in the expression analysis is that the highest expression of the gene HvFT1 is found in the earliest plants (2771 and 2820). Nevertheless, the HvFT1 alleles of these plants are one from Beka and the other one from Logan. If HvFT1 was the main determinant of flowering, genotypes with the Logan allele for HvFT1 gene were expected to flower before, these are: 2779, 2785, 2799 and 2820. As it can be seen on Figure 6, this does not occur, 2820 is the genotype that flowered the earliest, but the three others mentioned flowered later than genotypes with the HvFT1 Beka allele. As we said before, it is important to consider the genetic background. Besides that, it has been reported copy number variation in HvFT1, suggesting that can lead to higher expression in those genotypes that have more copies. Beka has two copies of the gene and Logan one. In the Beka x Mogador population, (12) there was a QTL and a slight higher HvFT1 expression in plants with the Beka allele, with 2 copies of the gene, whereas Mogador has only one copy. In this study, the allele from Logan, with just one copy, but a different promoter, was associated with earlier flowering. Apparently, there is no evidence that copy number variation affects the expression of HvFT1 in the Beka x Logan population. Apart from HvFT1, the expression of two other genes is also analyzed. HvELF3 and HvCEN have been considered the best candidates for two QTLs described in the previous results: Ctig4047 is on the long arm of chromosome 1H, very 20 close to Mat-a, or HvELF3 (16). Ctig1158 maps near the centromere on 2H, very close to the position of Eam6, HvCEN (18). For the gene HvCEN, fourteen SNPs were identified that defined thirteen haplotypes. Logan carries haplotype I and Beka haplotype III of the gene (18). They differ in two SNPs within an intron and, both have the same amino acid in position 135, Ala. Apparently; the difference in nucleotide sequence does not explain the different flowering behavior. Logan carries the same HvCEN allele as cultivar Morex. In the Steptoe x Morex population a QTL was detected on 2H, in the region of Eam6. It showed QTL x environment interaction, but the early allele seemed to be the one from Morex (21). In the Beka x Mogador population, Eam6 was the major determinant for flowering time in autumn sowings (17). The parents of that population carry the two contrasting haplotypes III (Beka) and II (Mogador), as reported by Comadran et al. (18). The Mogador allele of HvCEN (haplotype II) was associated with earlier flowering (12). The earliest plants in that study were those with this haplotype. A comparison of selected DH lines from this population showed a slight difference in flowering time and higher expression in plants that contained alleles from Beka in HvFT1 and HvCEN. The alleles were different from those in Beka x Logan, so no comparison can be made. In any case, the genotypes in our study that carry Beka allele for both genes are 2814 and 2775, and they are not earlier than the others (Figure 5). On the other hand, in the second sampling date, an analysis of variance showed a significant interaction between HvFT1 and HvCEN also in this population. Plants with the Logan allele in HvCEN had higher HvFT1 expression than those with the Beka allele (2796 and 2799 vs 2814 and 2785; or 2771 and 2820 vs 2775 or 2779). Our results also suggest that HvCEN, or a closely linked gene, is the responsible of the 2H QTL. The most evident relation between genotype and phenotype is when we compared the expression of HvELF3 and the effect of both alleles in the time to flower. Many alleles has been described for HvELF3, the variety Mari has a 4bp deletion in exon 2 (mat-a.8) and Early Russian (eam8.w) a C-to-T point mutation in exon 2, both of which would encode a truncated protein (15); a large rearrangement of the gene was reported in eam8-k (16) The mutations in this gene give early flowering and photoperiod insensitivity. Several authors have reported that mutant alleles showed higher HvFT1 expression (7, 15, 16, and 20). Logan has an allele that differs from those reported. What we saw in the results (Figure 8) is that those plants that flowered first; 2771, 2775, 2779 and 2820 are those with the Logan allele for the gene HvELF3 (marker Ctig4047). Those plants also have higher expression for HvFT1. These results suggest that the alleles of HvELF3 may have influence on the expression of HvFT1 and therefore in the time to flower, as it has been previously reported. 21 Expression of HvELF3 is very similar in all plants, independently on the date they flowered. Maybe the expression levels do not have big influence in flowering, and the effects are due to the change in the protein sequence that affects the pathway somehow. HvELF3 is a circadian clock gene that contributes to photoperiod-dependent flowering in plants. Wild type ELF3 represses the activity of core circadian clock genes and it is essential for maintaining correct diurnal expression patterns. A significant interaction was found between HvELF3 and HvCEN. Future studies could investigate whether the proteins encoded by HvELF3 and HvCEN are part of the same pathway. In summary, what we can conclude is: - We verified, as in previous studies, that HvFT1 expression increase in plants that have an earlier development, although we could not demonstrate any connection between the alleles of this gene and the phenotype. - Logan alleles for Ctig4047 and Ctig1158 have big influence in the time to flower, compared with the other alleles for the other genes, even if the expression pattern does not differ among plants. - There are two significant interactions found between Ctig4047 x Ctig1158 and Ctig1158 x HvFT1, which have to be investigated in more detail. - Our results suggest that HvCEN might be the gene responsible for the QTL identified close to Ctig1158, and that HvELF3 is the gene under Ctig4047. The work reported here is a small example of research in how some genes can affect time to flowering in barley, which has a big impact on yield. These researches are useful as long as they discover important breeding targets on barley that could be used by plant breeders to assure efficient crops in regions with very different environmental conditions. 22 8. BIBLIOGRAPHY. 1. Pourkheirandish M. and T. Komatsuda (2007). The importance of barley genetics and domestication in a global perspective. Ann Bot 100:999-1008. 2. FAOSTAT 2012 http://faostat.fao.org/ 3. Trevaskis B., M. Hemming, E.S. Dennis and W.J. Peacock (2007). The molecular basis of vernalization-induced flowering in cereals. Trends Plant Sci 112:352-357. 4. Saisho D., M. Ishii, K. Hori and K. Sato (2011) Natural variation of barley vernalization requirements: implication of quantitative variation of winter growth habit as an adaptive trait in East Asia. Plant Cell Physiol 52: 775–784 5. Casao M.C., I. Karsai, E. Igartua, M.P. Gracia, O. Veisz and A.M. Casas (2011) Adaptation of barley to mild winters: A role for PPDH2. BMC Plant Biol 11:164 6. Diestelfeld A., C. Li and J. Dubcovsky (2009) Regulation of flowering in temperate cereals. Curr Opin Plant Biol 12:178-184. 7. Turner A.S., S. Faure, Y. Zhang and D.A. Laurie (2013) The effect of day neutral mutations in barley and wheat on the interaction between photoperiod and vernalization. Theor Appl Genet 126:22672277 8. Yan L., D. Fu, C. Li, A. Blech, G. Tranquilli, M. Bonafede, A. Sanchez, M. Valarik and J. Dubcovsky (2006) The wheat and barley vernalization gene VNR3 is an orthologue of FT. Proc. Natl. Acad. Sci. U.S.A. 103:19851–19856. 9. Hemming M.N., W.J. Peacock, E.S. Dennis and B. Trevaskis (2008) Low-temperature and daylength cues are integrated to regulate FLOWERING LOCUS T in barley. Plant Physiol 147:355–366. 10. Casas A.M., A. Djemel, F.J. Ciudad, S. Yahiaoui, L.J. Ponce, B. Contreras-Moreira, M.P. Gracia, J.M. Lasa and E. Igartua (2011) HvFT1 (VrnH3) drives latitudinal adaptation in Spanish barleys. Theor Appl Genet 122:1293–1304. 11. Nitcher R., A. Distelfeld, C. Tan, L. Yan and J. Dubcovsky (2013) Increased copy number at the HvFT1 locus is associated with accelerated flowering time in barley. Mol Genet Genomics 288:261– 275. 12. Loscos J., E. Igartua, B. Contreras-Moreira, M.P. Gracia and A.M. Casas (2014) HvFT1 polymorphism and effect - survey of barley germplasm and expression analysis. Frontiers Plant Sci 5:251. 13. Hemming M.N., S. Fieg, W.J. Peacock, E.S. Dennis and B. Trevaskis (2009) Regions associated with repression of the barley (Hordeum vulgare) VERNALIZATION1 gene are not required for cold induction. Mol Genet Genomics 282:107–117. 23 14. Turner A., J. Beales, S. Faure, R.P. Dunford and D.A. Laurie (2005) The pseudo-response regulator Ppd-H1 provides adaptation to photoperiod in barley. Science 310, 1031–1034 15. Faure S., A.S. Turner, D. Gruszka, V. Christodoulou, S.J. Davis, M. von Korff and D.A. Laurie (2012). Mutations at the circadian clock gene EARLY MATURITY 8 adapts domesticated barley (Hordeum vulgare) to short growing seasons. Proc Natl Acad Sci USA 109:8328-8333. 16. Zakhrabekova S., S.P. Gough, I. Braumann, A.H. Müller, J. Lundqvist, K. Ahmann, C. Dockter, I. Matyszczak, M. Kurowska, A. Druka, R. Waugh, A. Graner, N. Stein, B. Steuernagel, U. Lundqvist and M. Hansson (2012) Induced mutations in circadian clock regulator Mat-a facilitate short season adaptation and range expansion in cultivated barley. Proc Natl Acad Sci USA 109:4326-4331 17. Cuesta-Marcos A., E. Igartua, F.J. Ciudad, P. Codesal, J.R. Russell, J.L. Molina-Cano, M. Moralejo, P. Szűcs, M.P. Gracia, J.M. Lasa and A.M. Casas (2008) Heading date QTL in a spring x winter barley cross evaluated in Mediterranean environments. Mol Breeding 21:455–471. 18. Comadran J., B. Kilian, J. Russell, L. Ramsay, N. Stein, M. Ganal, P. Shaw, M. Bayer, W. Thomas, D. Marshall, P. Hedley, A. Tondelli, N. Pecchioni, E. Francia, V. Korzun, A. Walther and R. Waugh (2012). Natural variation in a homolog of Antirrhinum CENTRORADIALIS contributed to spring growth habit and environmental adaptation in cultivated barley. Nat Genet 44:1388–1392. 19. Moralejo M., J.S. Swanston, P. Muñoz, D. Prada, M. Elia, J.R. Russell, L. Ramsay, L. Cistue, P. Codesal, A.M. Casas, I. Romagosa, W. Powell and J.L. Molina-Cano (2004) Use of new EST markers to elucidate the genetic differences in grain protein content between European and North American two-rowed barleys. Theor Appl Genet 110:116-125 20. Boden S.A., D. Weiss, J.J. Ross, N.W. Davies, B. Trevaskis, P.M. Chandler and S.M. Swain (2014) EARLYFLOWERING3 regulates flowering in spring barley by mediating gibberellins production and FLOWERING LOCUS T expression. Plant Cell 26:1557-1569. 21. Borràs-Gelonch G., M. Denti, W.T.B. Thomas and I. Romagosa (2012) Genetic control of pre-heading phases in the Steptoe x Morex barley population under different conditions of photoperiod and temperature. Euphytica 183, 303–321. 22. Ponce-Molina L.J., A.M. Casas, M.P. Gracia, C. Silvar, E. Mansour, W.B.T. Thomas, G. Schweizer, M. Herz and E. Igartua (2012). Quantitative trait loci and candidate loci for heading date in a large population of wide barley cross. Crop Sci. 52, 2469-2480. 23. Payne R.W., D.A. Murray, S.A. Harding, D.B. Baird and D.M. Soutar (2009). GenStat for Windows (12th Edition) Introduction. Hemel Hempstead: VSN International.