Research. Nadine Ilk 1,2, Jia Ding 1, Anna Ihnatowicz 3, Maarten Koornneef 1,4 and Matthieu Reymond 1,5. Summary. Introduction

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1 Research Natural variation for anthocyanin accumulation under high-light and low-temperature stress is attributable to the ENHANCER OF AG-4 2 (HUA2) locus in combination with PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) and PAP2 Nadine Ilk 1,2, Jia Ding 1, Anna Ihnatowicz 3, Maarten Koornneef 1,4 and Matthieu Reymond 1,5 1 Max Planck Institute for Plant Breeding Research, Cologne, Germany; 2 The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, UK; 3 Laboratory of Plant Protection and Biotechnology, Intercollegiate Faculty of Biotechnology UG & MUG, Gdansk, Poland; 4 Laboratory of Genetics, Wageningen University, Wageningen NL-6708 PE, the Netherlands; 5 Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, Versailles, France Author for correspondence: Maarten Koornneef Tel: koornneef@mpipz.mpg.de Received: 1 July 2014 Accepted: 16 October 2014 doi: /nph Key words: anthocyanin, Arabidopsis thaliana, epistasis, flowering time, HUA2, PAP1, PAP2, quantitative trait loci. Summary Growing conditions combining high light intensities and low temperatures lead to anthocyanin accumulation in plants. This response was contrasted between two Arabidopsis thaliana accessions, which were used to decipher the genetic and molecular bases underlying the variation of this response. Quantitative trait loci (QTLs) for flowering time (FT) and anthocyanin accumulation under a high-light and low-temperature scenario versus a control environment were mapped. Major QTLs were confirmed using near-isogenic lines. Candidate genes were examined using mutants and gene expression studies as well as transgenic complementation. Several QTLs were found for FT and for anthocyanin content, of which one QTL co-located at the ENHANCER OF AG-4 2 (HUA2) locus. That HUA2 is a regulator of both pathways was confirmed by the analysis of loss-of-function mutants. For a strong expression of anthocyanin, additional allelic variation was detected for the PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) and PAP2 genes which control the anthocyanin pathway. The genetic control of variation for anthocyanin content was dissected in A. thaliana and shown to be affected by a common regulator of flowering and anthocyanin biosynthesis together with anthocyanin-specific regulators. Introduction Flavonoids are secondary metabolites involved in a number of mechanisms in plant development, stress and defence (Winkel- Shirley, 2001; Lepiniec et al., 2006). Anthocyanins play a key role as light filters against high light stress in protecting the photosynthetic apparatus against an excess of light and thus preventing photoinhibition (Hatier & Gould, 2009). In high light conditions plants have to deal with oxidative stress as a result of the accumulation of reactive oxygen species (ROS) (Apel & Hirt, 2004) and will produce antioxidants such as ascorbate to avoid ROS damage. At lower temperatures, protection by ascorbate is restricted and additional antioxidants, including anthocyanins, are produced (Lokhande et al., 2003). The biosynthesis of flavonoids and anthocyanin is well established (Winkel-Shirley, 2001). The biosynthesis genes are divided into two subgroups: early biosynthetic genes (EBGs) and late biosynthetic genes (LBGs) (Cominelli et al., 2008; Petroni & Tonelli, 2011). Most knowledge acquired about the genetic control of anthocyanin synthesis in Arabidopsis thaliana has been obtained using induced mutations. Natural variation from accessions collected in nature is increasingly being used for genetic studies (Alonso-Blanco et al., 2009; Weigel, 2012) and allows the identification of specific alleles not highlighted using mutant screens. The genetics of flowering time (FT) has been extensively studied in A. thaliana and nearly 70 genes involved in the variation of this trait have been identified by combining analyses of mutants and natural variation (Simpson & Dean, 2002; Boss, 2004; Turck et al., 2008; Alonso-Blanco et al., 2009). Variation for FT has been analysed in nearly all A. thaliana mapping populations studied to date. Large differences in FT for A. thaliana accessions growing in laboratory conditions are often described as differences between winter (late) and spring (early) annual plants which show strong and weak vernalization responses, respectively (Alonso-Blanco et al., 2009). Most winter annual genotypes carry active alleles of FRIGIDA (FRI) (Johanson et al., 2000) and FLOWERING LOCUS C (FLC) (Michaels & Amasino, 1999) interacting genetically to repress genes promoting FT (Li et al., 2008; Deng et al., 2011). Natural polymorphisms altering FT have been functionally validated in glasshouse studies for 10 FT 422

2 New Phytologist Research 423 genes: CRYTOCHROME2 (CRY2), FRI, FLC, FLOWERING LOCUS M (FLM), ENHANCER OF AG-4 2 (HUA2), PHYTOCHROME A, B, C and D (PHYA, PHYB, PHYC and PHYD), SHORT VEGETATIVE PHASE (SVP) and MADS AFFECTING FLOWERING 2 (MAF2) (Alonso-Blanco et al., 2009; Mendez-Vigo et al., 2011; Rosloski et al., 2013). Only a few studies have been performed on natural variation in flavonoid or anthocyanin production and studied its response to stress (Teng et al., 2005; Diaz et al., 2006; Korn et al., 2010; Saito et al., 2013). Two studies (Teng et al., 2005; Diaz et al., 2006) identified PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) as a candidate gene for anthocyanin production in two different recombinant inbred line (RIL) populations, in which they studied the relationships between anthocyanin accumulation, high carbon stress and nitrogen limitation. In a gene expression screen for cold acclimation in different A. thaliana accessions, PRODUCTION OF ANTHOCYANIN PIGMENT2 (PAP2) was significantly up-regulated in six out of nine accessions (Hannah et al., 2006). Korn et al. (2008) showed that cold acclimation has a major influence on the amount and composition of flavonols, correlating with high expression of PAP2, and that there is considerable natural variation in A. thaliana flavonol composition. In the present study we analysed the genetic variation for anthocyanin accumulation in high-light and low-temperature conditions, in which anthocyanin production is high and which mimic natural conditions for A. thaliana more closely than the commonly used glasshouses or climate chambers. We used an RIL population derived from a cross between the Landsberg erecta (Ler) accession, which does not accumulate anthocyanin in these conditions, and the Eringsboda-1 (Eri-1) accession, which shows strong anthocyanin accumulation in these conditions. The analysis of FT in the same material revealed an unexpected link between one of the quantitative trait loci (QTL) identified for this trait and anthocyanin accumulation. We identified the HUA2 gene as this common regulator which together with allelic variation at the PAP1 and PAP2 genes controls anthocyanin accumulation under these conditions. Materials and Methods Growth conditions and experimental design All Arabidopsis thaliana L. Heyhn. (Brassicaceae) plants were grown on a mixture of commercial soil supplemented with perlite and macronutrients (PRO start; Gebr. Brill Substrate GmbH & Co. KG, Georgsdorf, Germany). To investigate responses to environment, two different growth conditions were set up. In the environment (Supporting Information Fig. S1a) referred to as the control (CT) environment, the plants were subjected to longday photoperiod conditions (16 h : 8 h, light : dark) with overhead lighting in a growth cabinet. Light intensity during the day was on average 100 lmol m 2 s 1. The growth cabinet temperature was maintained at 20 C during the day and 18 C during the night and relative air humidity was maintained at 70%. The other environment was high light at 4 C (HL4). In this environment, plants were exposed to two environmental constraints: low-temperature (4 C) and high-light stress. In addition to the fluorescent tubes (Philips F25T8/TL741) of the cold growth cabinet (Rivacold, Montecchio, Italy), four high-light lamps mounted above the trays (Fig. S1b) were added. All the lamps in the cabinet were set to long-day photoperiod conditions (16 h : 8 h, light/high light : dark). Light intensity during the day was on average 430 lmol m 2 s 1. As a result of the heat generated by the high-light lamps, the temperature at the level of the plants was maintained at C during the day and at 6 7 C during the night. Relative humidity ranged between 60 and 70% during the day and between 45 and 50% during the night. Throughout all experiments, the positions of the pots in the growth cabinet were changed every second day in order to provide equal light distribution. The light intensity in lmol s 1 m 2 was determined from averaged values, measured with a light meter (LI-250A; Li-Cor Biosciences, Lincoln, NE, USA). Before being transferred to HL4, all plants were subjected to CT conditions for 10 d after sowing (DAS). Seeds were stratified for 3 5 d on filter paper moistened with demineralized water at 4 C in the dark before being transferred to soil. All plants were grown on a mixture of commercial soil supplemented with perlite and macronutrients (PRO start; Gebr. Brill Substrate GmbH & Co. KG). Plant material For QTL detection, a population of 110 RILs (CS97359) between the Ler (CS97470) accession and the Eri-1 (CS97359) accession was used (Ghandilyan et al., 2009). Ler is a laboratory strain that harbours a mutation in the erecta gene in the Landsberg genetic background (Redei, 1962). The Ler strain we used carries the hua2-5 nucleotide substitution as described by Doyle et al. (2005) (named Ler-hua2-5 hereafter). The progeny, derived from a cross between Ler-hua2-5 and Eri-1, were grown through eight generations via self-pollinating, single seed descent producing nearly homozygous lines. Seeds from the F 9 progeny (F 10 plants) were grown for QTL experiments. Three plants of each RIL were grown in each environment. A Ler line with the wildtype (WT) HUA2 allele (termed Ler-HUA2) and the Wassilewskija (Ws) line carrying the hua2-6 mutation (Doyle et al., 2005) were provided by Dr Rick Amasino (University of Wisconsin, Madison, WI, USA). For validation of the detected QTL, a near-isogenic line (NIL) with an introgression of the Lerhua2-5 allele at QTL5-2 in an Eri-1 allelic background was generated by backcrossing RIL103 to the parental line Eri-1. The NIL was developed by marker-assisted selection in the progeny of the cross and carries a small Ler-hua2-5 introgression in the QTL5-2 region from 7.71 to 7.82 Mbp. Accessions used as control lines, namely Ws (CS28823), Columbia (Col-0; CS22625) and Nossen (No-0; CS28564), were obtained from the Arabidopsis stock centre ( sis.info). The hua2-4, pap2-10 and transparent testa8 (tt8)-1 mutant lines were obtained from the SALK Institute Genome Analysis Laboratory (SIGnAL) TDNA collection (reference numbers: SALK_032281C, SALK_058359C and SALK_030966) ( The RIKEN line

3 424 Research New Phytologist pst16228 (pap1) carries a loss-of-function allele of PAP1 and is the result of a transposon insertion in the third exon of the PAP1 locus, in the No-0 ecotype background (Kuromori et al., 2004). This allele has been genetically characterized previously by Teng et al. (2005) and was provided by Dr Sheng Teng (Chinese Academy of Sciences, Shanghai, China). The caprice (cpc-2) (KG12704) mutant line isolated from Kazusa T-DNA lines contains a T-DNA insert in the second intron of CPC in the Col-0 background (Kurata et al., 2005) and was provided by Dr Martin H ulskamp (University of Cologne, Cologne, Germany). For identification of T-DNA, a left border T-DNA primer and genespecific primers were used. T-DNA alleles were identified by using two gene-specific primers spanning the T-DNA insertion site. The flowering locus t (ft-10) mutant (CS9869) is a T-DNA insertion line and was described by Yoo et al. (2005). QTL mapping, two-way marker analysis and other statistical analysis Statistical analyses were performed using SPSS 15.0 (IBM Corporation, Armonk, NY, USA) for Windows. Broad sense heritability (H 2 ) of the different FT-related and pigmentationrelated traits was estimated as the proportion of variance explained by between-ril differences based on measurements of three plants per genotype. The correlation between traits was obtained using Pearson correlation in the statistical package SPSS. To determine the significance of differences in the trait values of the parental lines in comparison to the NILs and recombinant lines or the T3 transformants in comparison to the mutant and WT lines, a Student Newman Keuls (SNK) test, the univariate linear model of SPSS and a nested ANOVA in R statistics were used. For each RIL, the mean value was taken for each measured trait in the two different environments for QTL analysis with the software package MAPQTL 5.0 ( MAPQTL â 5.0 was used to detect QTLs on the genetic map using interval mapping (IM) and multiple-qtl model (MQM) mapping methods. For each environment, a separate QTL detection was performed. An epistatic interaction screen for each trait was performed with R statistics (details are given in Methods S1). The significance of both interacting and main effect QTLs were then statistically tested using the general linear model module of SPSS 15.0 (see later Fig. 3c,d). QTL models were composed of all statistically significant (P < 0.05) main and interacting QTLs. The thresholds (10%) of the logarithm of odds (LOD) were determined by permutation test (n = 1000) for each trait. FT and anthocyanin content scoring During the QTL experiments, the F 10 of the 110 RILs were phenotyped in a randomized complete block display in triplicate (three plants observed per line). FT was scored by daily inspection as the number of days from sowing until opening of the first flower bud. The total leaf number (TLN) was also scored when the plants flowered. Approximately d after flowering (43 47 DAS), between three and five (dependent on plant size) rosette leaves of different developmental stages were harvested from each plant for biochemical tests such as determination of anthocyanin content. The effect of the introgression in the NIL or gene mutation in mutant lines on the different traits was tested in comparison to the parental accessions Ler-hua2-5, Eri-1 and WT lines Col-0, No-0 and Ws. The protocol for quantifying the anthocyanin content is based on purification of plant pigments with acetone and subsequent isolation of anthocyanin with chloroform. After extraction, the content was quantified using a photometric test. To calculate the anthocyanin content, the exact weight of the plant samples was recorded. In order to quantify the anthocyanin, samples were dissolved in 80% methanol/1% hydrochloric acid and measured in microplates (96-well flat-bottomed plates; Greiner bio-one GmbH, Frickenhausen, Germany; Fig. S2). Measurement was carried out in a multiscan spectrophotometer at 530 and 657 nm. Relative anthocyanin concentrations were calculated with the formula used in Teng et al. (2005). The relative anthocyanin content was defined as the product of the relative anthocyanin concentration and DW. One anthocyanin unit equals one absorbance unit (A 530 (1/4 9 A 657 )) in 1 ml of extraction solution. Vector construction and complementation tests To confirm that the genes TT8, CPC, PAP1 and PAP2 are also responsible for the anthocyanin content variation in the analysed lines, we created complementation constructs by PCR cloning and transformed these constructs in various genotypes (more details are given in Methods S2). Results Variation in FT and anthocyanin content in the Ler 9 Eri-1 RIL population in both environments tested The RIL population used in this study was derived from the cross between Ler-hua2-5 (from Poland) and Eri-1 (from south Sweden) A. thaliana accessions (Ghandilyan et al., 2009). All the lines were grown in both the HL4 and CT environments. The two parental accessions showed very contrasting phenotypes in HL4: Ler-hua2-5 was early flowering and accumulated low amounts of anthocyanin, whereas Eri-1 was later flowering with a high anthocyanin content. Variation of FT in this population was observed in both growth conditions. The average time to flowering among the RILs lay between the dates observed for Ler-hua2-5 and Eri-1, although some transgression was present in both directions (Table 1). The H 2 values for FT in both environments were similar and high (0.77 and 0.76 for the CT and HL4 environments, respectively). However, no striking differences in anthocyanin content between Ler-hua2-5 and Eri-1 were detected in the CT environment (Table 1), whereas much larger variation (from 1.24 to anthocyanin units 9 g 1 DW) was found in the HL4 environment. Because of the low anthocyanin content and low variation in anthocyanin content in the CT conditions, this trait was not further analysed. In the HL4 environment, a high heritability (0.92) for anthocyanin content

4 New Phytologist Research 425 Table 1 Phenotypic values for the parental lines and the recombinant inbred lines (RILs) in the control (CT) and high-light/low-temperature (HL4) environments Trait Environment Ler-hua2-5 value Eri-1 value Average RILs Range RILs H 2 FT (DAS) CT HL TLN CT HL Anthocyanin (units g 1 DW) CT HL Ler-hua2-5, Landsberg erecta strain carrying the enhancer of ag (hua2-5) nucleotide substitution; Eri-1, Eringsboda-1; H 2, broad sense heritability; FT, flowering time; DAS, days after sowing; TLN, total leaf number. was found and the anthocyanin content of the RILs ranged from just below the value for Ler-hua2-5 to the value for Eri-1 (Table 1). Phenotypic correlations between flowering-related traits and anthocyanin content in the two studied environments Pearson correlation coefficients (R 2 ) between the floweringrelated traits and anthocyanin content in the RILs grown in CT and HL4 are presented in Fig. 1. Within treatments, the highest correlations were observed between FT and TLN. Significant correlations between flowering-related traits and anthocyanin content were found under the HL4 conditions (R ; Fig. 1). QTL detection for FT and anthocyanin content in CT and HL4 conditions QTLs for the variation in FT and anthocyanin content in the two studied environments were detected and are summarized in Table 2 and Fig. 2. QTLs for anthocyanin content could not be detected under the CT conditions because of the absence of variation for this trait. The strong genotype 9 environment (G 9 E) interaction (Table S1), especially for anthocyanin content, explains why specific QTLs were detected only in one environment. In both environments, two QTLs for flowering-related traits (QTL5-1 and QTL5-2) could be mapped to the top of chromosome V, clustering in two separate regions. In both the QTL5-1 and 5-2 regions, QTLs for FT and TLN were detected, with Ler alleles promoting flowering. Anthocyanin content also mapped to QTL5-2 under the HL4 conditions, in which Ler alleles contributed to a decrease in anthocyanin content. Remarkably, this QTL explained 32% of the anthocyanin content variation found in the HL4 conditions. A second QTL for anthocyanin content mapped to chromosome I (referred to as QTL1). Similarly to the major anthocyanin QTL at the QTL5-2 locus, Ler alleles in QTL1 decreased anthocyanin content. The QTL for flowering-related traits mapped to the same position regardless of the environment tested. However, the percentage of explained variance for these QTLs was highly dependent on the environment. All QTLs detected in the CT environment only explained 12.6% and 25.1% of the variation for FT and TLN, respectively, whereas all QTLs for TLN in HL4 explained up to 54% of the variation for this trait. Another minor QTL for all flowering-related traits found in both environments also mapped to chromosome V (referred to as QTL5-3; Table 2; Fig. 2). In addition, a QTL for all flowering-related traits but specific for HL4 was detected at the top of chromosome III (referred to as QTL3; Table 2, Fig. 2), in which the Ler alleles delayed flowering. In the CT environment, two additional minor QTLs for TLN were detected on chromosomes II (referred to as QTL2; Table 2; Fig. 2) and V (referred to as QTL5-4; Table 2, Fig. 2). Fig. 1 Pearson correlations between traits (flowering time (FT), total leaf number (TLN) and anthocyanin content (Antho)) in the control (CT) and high-light/low-temperature (HL4) conditions in the Arabidopsis thaliana Landsberg erecta (Ler)/Eringsboda-1 (Eri-1) recombinant inbred line (RIL) population. The trait values are plotted against each other and the correlation coefficient R 2 of the trait is displayed. Correlations are significant at the 0.01 level (two-tailed). Epistatic interactions For most of the studied traits, the sum of R 2 of the main effect QTLs explained only part of the variation observed in the RIL population, for which the heritabilities were high. This probably means that part of the genetic determinism has not been revealed with the QTLs detected. A further analysis was carried out to detect epistatic interactions on a genome-wide scale for all traits in both CT and HL4 (Fig. 3). A hua2/hua2 marker (at 7.85 mbp) was added to the genetic map as a result of its location in the QTL5-2 region. Significant interactions for all analysed traits between QTL5-2 region at marker hua2/hua2 and other

5 426 Research New Phytologist Table 2 Characteristics of the detected quantitative trait loci (QTLs) explaining flowering time (FT), total leaf number (TLN) and anthocyanin content (Antho) in the Landsberg erecta (Ler)/Eringsboda-1 (Eri-1) recombinant inbred line (RIL) population in control (CT) and high-light/low-temperature (HL4) environments Trait Environment QTL at nearest marker Map position 1 LOD score % of explained variance 2 Additive allele effect 3 FT HL4 CH.318E III:6.1:QTL CT NGA225 V:0.0:QTL CT CIW8/hua2-5 V:15.0:QTL HL4 SO262 V:20.9:QTL CT M5-9 V:41.5:QTL TLN CT F17A22 II:62.7:QTL HL4 F22F7 III:3.2:QTL CT NGA225 V:0.0:QTL HL4 NGA225 V:0.0:QTL HL4 FLC V:4.9:QTL CT CIW8/hua2-5 V:15.0:QTL HL4 CIW8/hua2-5 V:15.0:QTL CT M5-9 V:41.5:QTL HL4 M5-9 V:41.5:QTL CT DF.119L V:60.2:QTL Antho HL4 NF19K23 I:66.0:QTL HL4 CIW8/hua2-5 V:15.0:QTL Chromosome number is given followed by marker position in cm and QTL designation. 2 Percentage of variance explained by main QTL. 3 Additive allele effect = difference of estimated trait means from genotype A (Ler-hua2-5) and genotype B (Eri-1) divided by 2. Positive values indicate that Ler-hua2-5 alleles increased the trait value. Values in bold indicate QTL detected in both environments. LOD, logarithm of odds. Fig. 2 Arabidopsis thaliana Landsberg erecta (Ler)/Eringsboda-1 (Eri-1) linkage map showing the locations of quantitative trait loci (QTLs) for the analysed traits in control (CT) and high-light/low-temperature (HL4) conditions. Chromosomes I, II, III and V of A. thaliana are represented as bars. Marker positions are indicated with lines. The arrows indicate the regions where significant QTLs have been detected (logarithm of odds (LOD) > 2.1; see the Materials and Methods section) with corresponding marker on the left. The direction of the arrows indicates the allelic effect; upwards, Ler-hua2-5 increases and Eri-1 decreases; downwards, Eri-1 increases and Ler-hua2-5 decreases the trait value. QTL regions (QTL1, QTL3-1, QTL3-2, QTL5-1, QTL5-3) were detected (Table 3). For all traits, the LOD value for the additive model versus the single QTL model (QTL1 in Table 3) was always above the thresholds, meaning that adding QTL5-2 to the interaction with other QTLs and regions increased the values. The comparison of the additive versus single QTL models

6 New Phytologist Research 427 (a) (b) (c) (d) Fig. 3 Epistatic interactions between QTL5-2 (ENHANCER OF AG-4 2 (HUA2)) and other quantitative trait loci (QTLs) for (a, c) total leaf number (TLN) and (b, d) anthocyanin content (Antho) in high-light/low-temperature (HL4) conditions. (a, b) Logarithm of odds (LOD) scores for the five chromosomes of Arabidopsis thaliana from a two-dimensional, two-qtl genome scan with TLN HL4 and Antho HL4 phenotypic data. Logarithm of odds - interaction (LODi) is displayed in the upper left triangle and logarithm of odds - full model (LODf) is displayed in the lower right triangle (see Supporting Information Methods S1). In the colour scale on the right, numbers to the left and the right correspond to LODi and LODf, respectively. The most significant interactions are indicated in red and highlighted by a black circle. (c, d) Interaction plots of two-way marker interactions for (c) TLN and (d) anthocyanin content. ANOVA tests between QTL related markers were performed. (QTL3-1 = F22F7, QTL3-2 = CH.96L-Col, QTL5-2 = hua2/hua2, QTL5-1 = FLC, and QTL5-3 = M5-9). Green rectangles, Ler allele at marker position 1; red triangles, Eri-1. Asterisks indicate significant interactions:, P 0.1; *, P 0.01; **, P Error bars indicate SD between samples. Table 3 Characteristics of the detected epistatic interactions explaining flowering time (FT), total leaf number (TLN) and anthocyanin content (Antho) in the Landsberg erecta (Ler)/Eringsboda-1 (Eri-1) recombinant inbred line (RIL) population in control (CT) and high-light/low-temperature (HL4) environments Environment trait QTL1 1 QTL2 1 LOD score interaction % of explained variance LOD score full model 2 LOD score additive 3 CT FT QTL5-2 QTL * 5.58* QTL5-2 G * 2.18* QTL5-2 QTL * 2.35* QTL5-2 QTL * 3.76* HL4 FT QTL5-2 QTL * 9.55* QTL5-2 QTL * * 1.07 CT TLN QTL5-2 QTL * 5.42* QTL5-2 QTL * * 3.54* QTL5-2 QTL * 1.88 HL4 TLN QTL5-2 QTL * 5.42* QTL5-2 QTL * * 3.54* QTL5-2 QTL * * 1.88 HL4 Antho QTL5-2 QTL * 2.32* QTL5-2 QTL * 1.70 QTL5-1 QTL * * Quantitative trait locus (QTL) region or closest marker. 2 Logarithm of odds (LOD) full model (QTL1 + QTL2 + QTL1 9 QTL2). 3 LOD additive model (QTL1 + QTL2 versus QTL1). *Above the thresholds (10%). Thresholds are determined by permutation test (n = 1000) for each trait and model. (Fig. 3a,b) and interaction plots between QTLs (Fig. 3c,d) supports the occurrence of such interactions and therefore epistasis between the hua2/hua2 marker and other loci for FT, TLN (QTL3-1 and QTL5-1) and anthocyanin content (QTL1 and QTL3-2). It is worth mentioning that the anthocyanin QTL3-2 was only detected in the interaction with QTL5-2 (hua2/hua2

7 428 Research New Phytologist marker), not as a main effect QTL. Another significant interaction for TLN was observed between QTL5-3 and QTL5-2. Additionally, an interaction between QTL5-3 and QTL5-1 for anthocyanin content was detected. Validation and characterization of QTL5-2 We focussed on the validation and characterization of QTL5-2 as this was a major QTL for both FT and the anthocyanin accumulation trait. Additionally, this QTL5-2 showed genetic interactions with several other regions, of which some were specific for anthocyanin content and others for FT traits. To confirm the presence and the effects of detected QTLs, the parental accessions and NILs that had introgression of alleles from one parental accession at QTL5-2 in a genetic background that otherwise originated from the other parental accession were phenotyped and compared in the HL4 environment. An Eri NIL QTL5-2 with a small Ler-hua2-5 introgression in the QTL5-2 region from marker ICE5 (7.71 Mbp) to marker CDPK9 (7.82 Mbp) was obtained (Fig. 4) and grown in the HL4 conditions, and its TLN and anthocyanin content were quantified and compared with those of Eri-1. The NIL showed a decrease in TLN ( 11 leaves) compared with Eri-1 (Fig. 4), making it to flower at a similar time as the Ler-hua2-5 parental line (TLN = 9). In addition, the anthocyanin content of Eri NIL QTL5-2 grown in HL4 was also reduced when compared with Eri-1 (20 and 104 units g 1 DW for the Eri NIL and Eri-1, respectively). These results provided validation of the effects of QTL5-2 on FT and anthocyanin content. According to the reference sequence of the Col-0 accession, the 110-kb region between the positions of the flanking markers contains only 26 genes with 33 transcripts in total (Table S2). A candidate gene for flowering-related traits was present in the sequence of Col-0 in this region: HUA2. Single hua mutants are early flowering and have reduced levels of FLC mrna (Doyle et al., 2005). HUA2 activates FLC expression and enhances AGAMOUS (AG) function (Chen & Meyerowitz, 1999). The Ler parent used to generate the Ler/Eri-1 RIL population carries a point mutation described as the hua2-5 allele (Doyle et al., 2005), which was confirmed by sequencing the HUA2 gene in our parental Ler strain (Ler-hua2-5). By contrast, the Eri-1 parent carries a functional HUA2 Eri-1 allele determined by resequencing the Eri-1 accession (see Figs S3a, S4a,b). Further confirmation of HUA2 as a candidate gene for QTL5-2 for TLN as well as for anthocyanin content in HL4 was obtained by phenotyping the Ler strain carrying the WT HUA2 Ler (Ler-HUA2) and comparing it with the parental Ler strain (Ler-hua2-5). Other hua2 mutants in, respectively, the Ws and Col genetic backgrounds (the hua2-6 and hua2-4 mutants, respectively) were also compared with their respective WT in HL4. It was found that HUA2 in Ler and Ws consistently had increased TLN and anthocyanin content values when compared with the mutants hua2-5 and hua2-6 (Fig. 4). The effect of HUA2 in Ler and Ws was minor compared with HUA2 in Col-0, which showed a stronger increase of TLN and anthocyanin content values when compared with the corresponding mutant line hua2-4. In Col-0 and Eri-1, HUA2 seems to be a major regulator, which shows pleiotropic effects on flowering-related traits and anthocyanin accumulation under specific environmental conditions. To determine whether the effect of high anthocyanin content is a consequence of late flowering per se, we analysed the ft-10 (Col-0 background) mutant in HL4. The ft-10 mutant is late flowering as a result of a T-DNA insertion in FLOWERING LOCUS T, which encodes an integrator of various flowering pathways. In the mutants, TLN had c. 12 more leaves than the WT Col-0. However, the mutant accumulated less (15 units g 1 DW) anthocyanin in the HL4 conditions than Col-0 plants (88 units g 1 DW). This result indicated that late flowering and anthocyanin accumulation under HL4 are traits that can be physiologically uncoupled. Expression patterns of HUA2 and FLC in the lines Lerhua2-5,Ler-HUA2, Eri NIL QTL5-2 and Eri-1 grown in CT and HL4 conditions HUA2 was previously shown to be expressed in diverse tissues and at different developmental stages (Chen & Meyerowitz, 1999; Poduska et al., 2003; Wang et al., 2007). To induce flowering, the down-regulation of FLC is required (Michaels et al., 2003). In order to investigate the role of HUA2 after transition to flowering and under different environmental conditions, the transcript levels of HUA2 and FLC before and after flowering in CT and HL4 conditions were determined (Methods S3). In the CT conditions, the expression of HUA2 was examined in Lerhua2-5, Ler-HUA2, Eri NIL QTL5-2 and Eri-1 at day 14 after germination (before flowering for all the lines) at day 26 (after flowering for the lines Ler-hua2-5, Ler-HUA2 and Eri NIL QTL5-2) or at 31 d (after flowering for Eri-1) (Fig. 5). No significant differences in the expression of HUA2 could be found between these lines. However, when the same genotypes were grown in the HL4 environment, significant differences in HUA2 expression were detected between the lines before and after flowering. Lines with a functional HUA2 allele (Ler-HUA2 and Eri- 1) showed higher expression levels of HUA2 compared with the lines carrying the mutant hua2-5 allele (Ler-hua2-5). Eri-1 belongs, like Col-0, to the functional FLC A haplotype group (Caicedo et al., 2004) (determined by resequencing the Eri-1 accession). The transcript levels of FLC are higher in the Eri-1 line compared with Ler-HUA2, which carries a weak allele of FLC (Gazzani et al., 2003; Michaels et al., 2003), with an even lower level of FLC expression in the Ler-hua2-5 line. Transcript levels of genes involved in the biosynthesis and regulation of anthocyanin production in the lines Ler-hua2-5,Ler-HUA2, Eri NIL QTL5-2 and Eri-1 grown in CT and HL4 conditions The mechanism by which HUA2 contributes to the regulation of anthocyanin production has not yet been described. For an indication of the level on which it could act, the expression levels of different genes (Table S3) involved in the regulation of anthocyanin accumulation, including biosynthesis genes, were determined using quantitative real-time PCR (qrt-pcr). Out of 17 tested genes, five genes, TRANSPARENT TESTA8 (TT8), CAPRICE

8 New Phytologist Research 429 (a) (b) (c) Fig. 4 (a) Total leaf number and (b) anthocyanin content of different Arabidopsis thaliana enhancer of AG-4 2 (hua2) mutant lines and corresponding HUA2 wild-type (WT) lines grown in high-light/lowtemperature (HL4) conditions. (a, b) The boxplots represent the median with the lower and upper quartiles and the lowest and highest trait values (indicated by error bars). n = 8 plants per line. The colours of the boxes represent the background lines (green boxes, Landsberg erecta (Ler); red boxes, Eringsboda-1 (Eri-1); yellow boxes, Wassilewskija (Ws); violet boxes, Columbia (Col-0)). The five bars above the boxplots indicate the five chromosomes of A. thaliana;ler alleles in green and Eri-1 alleles in red. The Eri near-isogenic line (NIL) QTL5-2 carries a very small introgression in the Eri-1 background at the position of the green arrow. (c) Photograph of Ler-hua2-5,Ler-HUA2, Eri NIL QTL5-2 and Eri-1 plants grown for 45 d in the HL4 environment. (CPC), PAP1, PAP2 and DIHYDROFLAVANOL 4-REDUCT ASE (DFR), showed significant differences in transcript levels between the four lines (Fig. 6). All genes except DFR are regulators of anthocyanin biosynthesis; their proteins form a tertiary transcription factor complex (the myeloblastosis (MYB)-basic helix-loop-helix (bhlh)-tryptophan-aspartate (WD) 40 (MBW) complex), binding to promoters of late anthocyanin biosynthesis genes and leading to the biosynthesis of anthocyanin. In HL4, the expression of the five differentially expressed genes was highest in Eri-1, whereas their expression in Ler-hua2-5 was always low, and in Ler-HUA2 and the Eri NIL QTL5-2 their expression was between the levels of Ler-hua2-5 and Eri-1. An exception was the expression of CPC, for which Ler-HUA2 and Eri-1 showed higher expression, whereas Ler-hua2-5 and Eri-NIL QTL5-2 plants had lower expression of this gene. The highest expression level differences between the lines were found for PAP1 and PAP2. The Ler-hua2-5 line showed almost no expression of PAP1 and PAP2, whereas Ler-HUA2 showed low levels of expression. The Eri NIL QTL5-2 had higher PAP1 expression levels but lower levels for PAP2 in comparison with Ler-HUA2, Fig. 5 Gene expression analysis of Arabidopsis thaliana ENHANCER OF AG-4 2 (HUA2) and FLOWERING LOCUS C (FLC) before flowering (before flowering time (FT) at day 14 in control (CT) conditions and day 20 in high-light/low-temperature (HL4) conditions) and after flowering (after FT at day 26/31 in CT conditions and day 42 in HL4 conditions) occurred in CT (light grey bars) and HL4 (dark grey bars) conditions. Values were obtained by quantitative real-time PCR and are the mean SD of three biological replicates with three technical replicates, normalized to Actin2. whereas Eri-1 had very high expression of both genes. A similar tendency in expression levels in the four lines, but with lower values, was found for expression of TT8 (Fig. 6). CPC, PAP1 and PAP2 had higher expression in the Ler-HUA2 line and Eri-1 line compared with Ler-hua2-5, which indicates interplay between HUA2 and the genes PAP1, PAP2 and CPC. The DFR gene showed expression differences in the four lines during this screen, which is consistent with DFR being transcriptionally regulated by the regulators encoded by HUA2, PAP1, PAP2 and CPC described above. The relative expression values of TT8, PAP1, PAP2 and DFR were very low in the CT conditions. Only CPC, a negative regulator of anthocyanin content, showed higher expression in the CT conditions compared with the HL4 environment. Structural differences in HUA2, TT8, CPC, PAP1 and PAP2 between Ler-HUA2 and Eri-1 accessions The observation that TT8, PAP1 and PAP2 showed much higher expression levels in Eri-1 compared with Ler-HUA2 (Fig. 6), which contains a WT (HUA2) allele, suggests that either Eri-1 has a more effective HUA2 allele than Ler-HUA2 or that other alleles from Eri-1 elsewhere in the genome (TT8, PAP1 and PAP2) promote anthocyanin biosynthesis. In HUA2, two nonsynonymous single nucleotide polymorphisms (SNPs) between

9 430 Research New Phytologist Fig. 6 Gene expression analysis of regulatory and biosynthesis Arabidopsis thaliana anthocyanin genes. Transcript levels of TRANSPARENT TESTA 8 (TT8), CAPRICE (CPC), PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1), PRODUCTION OF ANTHOCYANIN PIGMENT2 (PAP2) and DIHYDROFLAVANOL 4-REDUCTASE (DFR) were determined in Landsberg erecta (Ler)-hua2-5, Ler-HUA2, Eringsboda (Eri) near-isogenic line (NIL) QTL5-2 and Eri-1 grown in control (CT) conditions (light grey bars) and high-light/low-temperature (HL4) conditions (dark grey bars). Values were obtained by quantitative real-time PCR and are the mean SD of four biological replicates with three technical replicates, normalized to Actin2. the Eri-1 and Ler-HUA2 alleles (Figs S3a, S4a,b) are present: the first SNP is a substitution from T to A (position ) in the third exon resulting in an amino acid change from serine to threonine between the Eri-1 and Ler-HUA2 alleles, respectively. The second SNP (T to C, in the fourth exon, position ) has phenylalanine in Eri-1 compared with serine in Ler-HUA2 (Fig. S4a,b). To determine whether expression level differences in TT8, CPC, PAP1 and PAP2 are attributable to structural differences (SNPs and insertions-deletions (INDELs)) between the Eri-1 and Ler-HUA2 alleles, we also used the resequencing data for Eri-1 and the publicly available data for Ler-HUA2 to visualize the sequences of the alleles of the highlighted genes in the Integrative Genomics Viewer (IGV, (Fig. S3). Ler-HUA2 carries several SNPs in the coding region of TT8 which could lead to functional differences in TT8 in Ler HUA2 compared with Eri-1. The sequence of Eri-1 in CPC shows a deletion in the first exon, as well as several SNP substitutions in the big second intron and third exon. In Eri-1, the expression of CPC is high, but shows no negative regulation of anthocyanin production, which is consistent with a loss of function for this gene. Despite the apparent loss-of-function genotype, no effect was observed on the formation of root hairs which is known to be defective when the function of CPC is lost, in the Col background (Fig. S5). The expression differences and the structural differences in the sequences of PAP1 and PAP2 between Ler-HUA2 and Eri-1 (Figs S3d,e, S6) indicate that PAP1 and PAP2 are obvious candidates for the QTL1 effects on anthocyanin content in HL4. The sequence of PAP1 in Ler- HUA2 shows deletions in the first and second introns as well as deletions in the 3 0 untranslated region (UTR) and, as shown above, the transcription of PAP1 is also affected. SNPs in the promoter region of PAP2 are also present between Ler-HUA2 and Eri-1, as well as in the first intron and third exons of this gene. These SNPs could be an explanation for the differences in transcript levels of PAP2 between Ler-HUA2 and Eri-1. QTL1: natural variation of PAP1 and PAP2 is involved in anthocyanin accumulation in the Ler/Eri-1 RIL population To confirm that the genes TT8 and CPC and the candidate genes for QTL1, PAP1 and PAP2, are involved in the regulation of anthocyanin production in the Ler/Eri-1 population, we performed complementation experiments. For each gene, a genomic DNA fragment from Ler-HUA2, Eri-1 and Col-0 (as a positive control) encompassing the entire coding region, including upstream and downstream intergenic regions, was cloned and transformed into loss-of-function mutants of the respective genes. T2 plants transformed with the At4g09820 (TT8) and At2g46410 (CPC) alleles of Ler-HUA2, Eri-1 and Col-0 showed no significant differences in anthocyanin content between three independent transformants when grown under the HL4 conditions. However, significant differences in anthocyanin content were found using nested ANOVA (Table S4) among pap1 plants transformed with the alleles of PAP1 from Ler-HUA2, Eri-1 or Col-0 (Fig. 7b). Because the pap1 mutant is in a Nossen background and the WT of Nossen shows low anthocyanin accumulation, Nossen WT plants were also transformed with PAP1 from Ler-HUA2, Eri-1 or Col-0

10 New Phytologist Research 431 (a) (b) (c) Fig. 7 Complementation test: anthocyanin content of Arabidopsis thaliana T3. (a) Nossen wild-type (WT) plants transformed with the PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) allele of Landsberg erecta (PAP1 Ler ; green bars), with the PAP1 allele of Eringsboda-1 (PAP1 Eri 1 ; red bars) and with the PAP1 allele of Columbia-0 (PAP1 Col 0, violet bars). (b) pap1 (pst16228 = T-DNA insertion at PAP1 in Nossen WT; see the Materials and Methods section) lines transformed with PAP1 Ler (green bars), PAP1 Eri 1 (red bars) and PAP1 Col 0 (violet bars). (c) pap2-10 (T-DNA insertion at PAP2 in Col-0 WT; see the Materials and Methods section) lines transformed with PAP2 alleles of Landsberg erecta (PAP2 Ler ; green bars), with PAP2 alleles of Eringsboda-1 (PAP2 Eri 1 ; red bars) and with PAP2 alleles of Columbia-0 (PAP2 Col 0 ; violet bars). T3 (n = 11 plants per line) of three independent transformants (1, 2 and 3) per PAP1 allele and PAP2 allele are displayed. The error bars indicate the SD of the 11 plants per line. As a control, the corresponding mutant line and Col-0 and Nossen WT were also analysed (grey bars). The letters (a, b, c) on the x-axis indicate the different groups identified by Student Newman Keuls test (95% confidence interval). (Fig. 7a). All three alleles of PAP1 (from Ler-HUA2, Eri-1 and Col-0) were able to complement the anthocyanin phenotype of the pap1 mutant. However, the different alleles of PAP1 resulted in different increases in anthocyanin content compared with the low levels of the mutant line. The PAP1 alleles from Ler-HUA2 resulted in an increased anthocyanin content of 39 units g 1 DW, PAP1 from Eri-1 an increase of 54 units g 1 DW and PAP1 from Col-0 an increase of 37 units g 1 DW compared with 20 units g 1 DW in the pap1 mutant line. In the Nossen WT lines transformed with PAP1 from Eri-1 and Col-0, the anthocyanin content was even higher than (almost double) that in the Nossen WT itself and that in Nossen plants transformed with PAP1 from Ler-HUA2. The higher amount of anthocyanin produced by these lines could also be observed in the accumulation of anthocyanin in leaves (Fig. S7a,b) and stems, especially in Eri-1 (Fig. S8). Indeed, plants transformed with PAP1 alleles from Eri-1 showed dark violet stems, whereas plants transformed with the other alleles or Nossen WT showed green stems. Also, by transforming the pap2-10 mutant (in the Col- 0 background) with PAP2 alleles from Eri-1 or from Col-0, we could complement the nonaccumulating anthocyanin mutant phenotype to produce high anthocyanin accumulation. Transformants of both alleles showed higher accumulation of anthocyanin than the transformants with the PAP2 allele from Ler-HUA2 (Figs 7c, 5c). Discussion The effect of environmental variation on FT and anthocyanin content was striking in our study in different light and temperature conditions, with a delay in FT and an increased number of leaves being observed in the high-light and cold environment (HL4). The correlation between FT and leaf number found under long day control conditions in our study is in agreement with the findings of previous studies (Koornneef et al., 1991). In addition, new correlations were found in this strong-light, cold environment. In HL4, the delay of FT and increase in leaf number were correlated with anthocyanin accumulation. Phenotypes of the ft-10 mutant (late flowering without anthocyanin accumulation) growing under these conditions indicated that late flowering in these conditions can occur without anthocyanin accumulation. FT-related QTLs It has been demonstrated that most of the late flowering accessions in A. thaliana carry active FRI and FLC loci, which act together genetically to delay FT (Andres & Coupland, 2012). Several accessions, such as the laboratory strains Col and Ler (both Ler-hua2-5 and Ler-HUA2, in this context referred to as Ler), carry nonfunctional or less functional alleles of FRI and exhibit an early flowering phenotype (Johanson et al., 2000; Shindo et al., 2005). Ler carries a weak allele caused by a 375-bp deletion in the promoter region and a 31-bp insertion in the first exon region (Johanson et al., 2000), which both were also found in Eri-1 (Fig. S3c). The latter is consistent with not finding a FT QTL at FRI (top of chromosome IV). At the QTL level, the correlations between FT and TLN are strongly supported by three co-locating QTLs on chromosome V (QTL5-1, QTL5-2 and QTL5-3) detected in both environments. Another QTL for flowering-related traits was mapped to chromosome III (QTL3-1) under HL4 solely. QTL3-1, involved in flowering variation, has already been detected in previous studies (El-Lithy et al., 2006; Tisne et al., 2010; Mendez-Vigo et al., 2011). To date, this QTL has only been detected when Ler- HUA2 and Ler-hua2-5 are used as parental lines in the mapping populations studied, suggesting that Ler carries a rare allele of the

11 432 Research New Phytologist gene(s) underlying QTL3-1. In the region of FLC at the top of chromosome V, co-locating QTLs for FT-related traits in the Ler/Eri-1 population were detected in both environments (QTL5-1). As a result of a 1233-bp insertion of a transposable element in the first intron, Ler carries a less effective FLC allele (Gazzani et al., 2003; Michaels et al., 2003). Eri-1 belongs to the FLC A haplotype group, similar to Col-0, which is functional (Caicedo et al., 2004). Therefore, FLC is probably the gene underlying QTL5-1, being responsible for over 40% of the variation in FT between Ler and Eri-1 in both environmental conditions tested. QTL5-2 is another major FT QTL explaining 25% and 54% of variation in the CT and HL4 conditions, respectively. This QTL co-located with the HUA2 locus. Interactions suggested that the QTL5-2 region (hua2/hua2 marker) is interacting with other regions on chromosome V. The main interaction was found between HUA2 and QTL5-1, where the HUA2-Ler allele is epistatic to FLC. This is also suggested by similar map positions in the Ler 9 Cvi RIL population (Alonso-Blanco et al., 1998). HUA2 is a known repressor of flowering through enhancement of the expression of FLC, AG and MAF2-MAF5 (Chen & Meyerowitz, 1999; Doyle et al., 2005). HUA2 contains an arginine- and proline-rich (RPR:SMART 00582) domain (SMART 00582), a motif found in proteins that function in RNA metabolism, pre-mrna processing and splicing. Poduska et al. (2003) pointed out that HUA2 (called ART1 in their study and shown to be HUA2 by Wang et al., 2007) activates FLC independently of FRI, but with synergistic effects when an active FRI allele is present. The Ler accession used for the development of the Ler/Eri-1 population contains a premature stop codon in HUA2 leading to a loss-of-function mutant hua2-5 allele (Doyle et al., 2005). By contrast, Eri-1 carries a functional allele of HUA2. The early flowering phenotype of the monogenic FLC lines (functional FLC Sy-0 in a Ler background) created by Poduska et al. (2003) is comparable to the early flowering phenotype of Eri-NIL QTL5-2 in comparison with the Eri-1 parent. This NIL carries hua2-5 and functional FLC alleles, confirming the epistasis of hua2-5 in relation to FLC, consistent with the effect of hua2 mutations in both Col and Ws. The third group of FT QTLs (QTL5-4) in Ler/Eri-1 co-locating with already known genes involved in naturally occurring flowering variation was detected at the bottom of chromosome V. The MAF2-MAF5 cluster has been implicated in FT variation and has been shown to be very polymorphic between accessions (Caicedo et al., 2009; Rosloski et al., 2010). Doyle et al. (2005) previously mentioned that HUA2 also interacts with MAF2-MAF5 and showed that the expression of MAF2 is also reduced by hua2 mutations, similar to FLC. When Ler was used as a parent of the RILs, as in the present work, Ler alleles at MAF2-MAF5 always delayed FT, indicating that Ler carries functional MAFs and Eri-1 possibly carries lossof-function or less functional MAF alleles. Sequence analysis of the Eri-1 accession (this work) revealed that Eri-1 carries deletions in the promoter, 5 0 UTR and 3 0 UTR of MAF2 compared with Ler and Col-0. Although we detected three QTLs for which strong candidate genes have already been described for variation of FT (FLC, HUA2 and MAF2-MAF5), we also identified additional, not yet cloned QTLs (QTL2, 3-1 and 5-3) using the Ler/Eri-1 population. Anthocyanin content QTLs The pleiotropic nature of HUA2 revealed in this study indicates that this gene is involved not only in FT but also in anthocyanin content variation, affecting the expression level of anthocyanin regulators in specific environments. Pleiotropic effects of HUA2 have previously been shown by Wang et al. (2007), suggesting that a particular allele of HUA2 (HUA2 Sy-0 ) affects FT and plant morphology. Similar changes in side-shoot morphology were not observed in our experiments, probably because of the different genetic materials and conditions used. Other studies on natural variants in A. thaliana have connected genes to unexpected effects, based on co-location of different QTLs and subsequent confirmation in experiments with mutants and transgenic plants. For example, CRY2 has been shown to affect FT as well as ovule number per fruit (El-Assal et al., 2004) and FRI has been shown to have an effect on dehydration avoidance in addition to its large effect on FT (Lovell et al., 2013). Van Zanten et al. (2009) reviewed the many pleiotropic effects of ERECTA. Our results illustrate a direct link between flowering and anthocyanin production attributable to a common regulator. That the effect of HUA2 is via its effect on FLC is unlikely, as neither a significant QTL for anthocyanin at this position nor an interaction effect between the two loci for this trait was detected. The different expression of HUA2 in mutant and WT lines in the HL4 environment indicates a specific function of HUA2 correlating with a higher expression level in that environment. Two-way interactions revealed interactions across the A. thaliana genome for anthocyanin content variation. Most interactions mapped to chromosome V, where many of the structural genes of the anthocyanin pathway and the main interactor HUA2 are located. For anthocyanin content, possible interactions with the HUA2 region and other regions on chromosome III and chromosome I have also been detected. The epistatic effect of hua2 mutants on anthocyanin accumulation in HL4 is explained by the important regulatory role of the HUA2 gene in these conditions. Anthocyanin QTL1 highlights the G 9 E interaction observed for this trait, because there were QTLs only detected in HL4 conditions. Between locus positions T27K12 (16.1 Mbp) and NF19K23 on chromosome I, PAP1, a positive regulator of anthocyanin pigment production (Borevitz et al., 2000), is present. PAP1 encodes an R2R3 MYB protein. PAP1 together with a bhlh protein, GLABRA 3 (GL3)/(ENHANCER OF GLABRA3 (EGL3)/TT8), and a WD40 protein, TRANSPARENT TESTA GLABRA 1 (TTG1), acts in the MBW complex which controls late genes of the anthocyanin biosynthesis pathway (Gonzalez et al., 2008; Hichri et al., 2011). The MBW complex binds the promoter of one of the late key genes (DFR) to start the biosynthesis of anthocyanin (Zimmermann et al., 2004). The QTL previously detected in high-carbon or low-nitrogen environments (Teng et al., 2005; Diaz et al., 2006) co-locates with the QTL1 detected in this work. The Ler allele at QTL1 decreased the anthocyanin content