2025 Volume 94 Issue 2 Pages 232-242
Broccoli produces green florets under normal growth conditions, but produces darkish-colored florets after low-temperature exposure due to the accumulation of anthocyanin, which often reduces its commercial value. Selecting anthocyanin-free genotypes requires exposing the florets to low temperatures, which is laborious and time-consuming. We identified a genomic region on chromosome C09, which was associated with anthocyanin pigments in florets at low temperatures, through quantitative trait locus sequencing (QTL-seq) analysis using the phenotype data obtained from field evaluation of anthocyanin pigments of F2 plants over two years. Among nine differentially expressed genes in the QTL region, one gene encoding flavonoid 3′-hydroxylase (F3′H) was selected as a candidate. Sequence analysis of Brassica oleracea F3′H (BoF3′H) of parental lines revealed the presence of three alleles, with one derived from the anthocyanin-free parents, Bof3′h-2, exhibiting a 43-bp deletion in the second exon, causing a frame-shift mutation. Designing DNA markers capable of distinguishing this mutation, we demonstrated that, among 35 F1 cultivars released in Japan, eight homozygous for the Bof3′h-2 allele showed no anthocyanin pigments and lower anthocyanin contents in florets in the autumn- to over-winter harvesting. The expression level the BoF3′H gene was not necessarily related to the difference in anthocyanin contents. Furthermore, seedlings of the Arabidopsis f3′h mutants exhibited no pigments and a significantly reduced anthocyanin content under low-temperature treatment compared to that in the wild type. These results suggested that the 43-bp deletion in BoF3′H is responsible for the anthocyanin-free trait in broccoli genetic resources, and the developed marker was deemed useful for marker-assisted breeding.
Broccoli (Brassica oleracea var. italica) is one of the most important vegetable crops worldwide and is grown to produce edible flower buds (florets) and stalks. This vegetable originated in the Eastern Mediterranean, and its early forms were highly esteemed by ancient Romans; it spread from Italy to northern Europe and the USA (Biggs et al., 2006), and arrived in Japan at the end of the 19th century. While the production of many leafy vegetables is decreasing, the demand for broccoli has increased in recent years because it contains high amounts of dietary fiber, vitamins, minerals, and antioxidant compounds such as sulforaphane (Fahey et al., 2015; Yuan et al., 2010). There are two types of cultivation in Japan, i.e., summer and spring sowing (Shinohara, 2006). Summer sowing and autumn-to-over-winter harvesting are the general patterns in temperate areas. However, plants are exposed to low temperatures close to harvesting time in autumn- to over-winter harvesting, and purple coloring often occurs on the surface of the bud. This purple coloring is due to anthocyanin accumulation (Liu et al., 2020).
Anthocyanins, a class of water-soluble flavonoids synthesized in the cytosol, are localized in the vacuoles and confer color to pigments. The six types of major anthocyanins in plants are pelargonidin, cyanidin, delphinidin, peonidin, malvidin, and petunidin (Zhu et al., 2020), which give a range of colors, such as red, orange, purple, and blue, in vegetables and fruits (Sunil and Shetty, 2022; Tanaka et al., 2008). In Brassica crops, purple cauliflower and kohlrabi accumulate cyanidin as their main pigment (Chiu et al., 2010; Rahim et al., 2018; Zhang et al., 2015), and the purple color of broccoli florets is mainly attributable to the accumulation of cyanidin (Yu et al., 2023). Antioxidant activities of anthocyanins have attracted growing attention owing to their health benefits to humans. Anthocyanins also play a role in protecting plants from various biotic and abiotic stresses and accumulate under various environmental conditions, including insect invasion (Park et al., 2015), strong light, salt stress (Lotkowska et al., 2015), exposure to reactive oxygen species (Xu et al., 2017), and low temperatures (He et al., 2020). Anthocyanin biosynthetic pathway genes are well-characterized and conserved in many plant species (Liu et al., 2020; Sunil and Shetty, 2022). Anthocyanin biosynthesis consists of three steps (He et al., 2020; Liu et al., 2020). The first phenylpropanoid metabolic pathway converts phenylalanine to 4-Coumaroyl-CoA using phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL). The next step is the early biosynthesis pathway, converting 4-Coumaroyl-CoA to dihydroflavonols using chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxyl enzyme (F3H), flavonoid 3′-hydroxylase (F3′H), and flavonoid 3′,5′-hydroxylase (F3′5′H). The final pathway is the late biosynthesis pathway, which catalyzes the conversion of dihydroflavonols to anthocyanins using dihydroflavonol reductase (DFR) and anthocyanin synthase (ANS) or leucoanthocyanidin dioxygenase (LDOX). These structural genes associated with the anthocyanin biosynthesis pathway are transcriptionally controlled by the MBW complex composed of R2R3-MYB, basic helix-loop-helix (bHLH), and WD40 repeats (WDRs), and these transcription factors function as positive regulators to increase anthocyanin accumulation or as negative regulators to suppress it.
Various environmental factors, including low temperatures, activate a series of downstream transcription factors that influence the expression of anthocyanin biosynthetic genes. BrMYB8 and BrTT8 genes were upregulated after cold treatment in purple-head Chinese cabbage (He et al., 2020). Zhang et al. (2012) reported that the expression of BoPAP1 and BoTT8 genes increased under cold conditions in kale and that anthocyanin accumulation was 50-fold higher than that under normal conditions. Purple pigments gradually and strongly accumulated in purplish cauliflower curd in winter, in which BoPAP2, BoEGL3, BoANS, BoDFR, and BoUGT79B1 were upregulated (Chen et al., 2022). Accumulation of anthocyanins plays a role in environmental stress tolerance (Saigo et al., 2020). The purple coloring of broccoli florets due to anthocyanin accumulation induced by low temperatures leads to darkish-colored florets and often reduces their commercial value in Japan. Therefore, the lack of purple coloring is one of the important traits in broccoli breeding, and some F1 cultivars with no purple coloring or less coloring, called anthocyanin-free or anthocyanin-less cultivars, are cultivated. However, because anthocyanin accumulation is believed to be affected by floret development and environmental conditions, the precise selection of anthocyanin-free genotypes in field trials is difficult and time-consuming.
In this study, we evaluated the degree of anthocyanin accumulation in the florets of the F2 population and attempted to identify loci that control anthocyanin pigments using quantitative trait locus sequencing (QTL-seq) (Takagi et al., 2013) and transcriptome analysis to develop DNA markers to select anthocyanin-free individuals in our breeding materials. We demonstrated the sequence variation of three alleles of the candidate gene and developed a DNA marker to identify anthocyanin-free genotypes. Furthermore, we performed a functional analysis of the candidate gene using Arabidopsis mutants and validated the usefulness of the marker using F1 broccoli cultivars released in Japan.
Four broccoli inbred lines, 11-1, 14-1-2, 14-3, and 3-2, were used as the parental lines to generate two segregating F2 populations. The 11-1 and 14-1-2 accumulate anthocyanins under low-temperature conditions, whereas 14-3 and 3-2 are anthocyanin-free genotypes. Two F2 populations, F2_01 and F2_02, were generated by the self-pollination of F1 plants derived from crosses between 11-1 and 14-3 and 14-1-2 and 3-2, respectively, and grown from mid-August to early December in the experimental field at Iwate University for two years, i.e., 2022 and 2021, respectively. Thirty-five commercial F1 broccoli cultivars (Table S1), which included 11 cultivars released as anthocyanin-free or reduced anthocyanin, were grown in the same experimental field in the autumn–winter season in 2023.
Arabidopsis thaliana Col-0 and two mutant lines, tt7-6 (CS2105577) and tt7-7 (CS2105578), harboring T-DNA in exon 4 and intron 1 of the F3′H gene (At5g07990), respectively, were used for total anthocyanin analysis. Homozygosity of the mutant allele was confirmed using primers described by Appelhagen et al. (2014).
Evaluation of anthocyanin accumulationIn two field experiments, the degree of anthocyanin accumulation in florets was evaluated visually at three grades: 0 (0%), 1 (< 50%), and 2 (> 50%), based on the ratio of areas of the florets containing anthocyanin pigment (Fig. 1). The presence or absence of anthocyanins in the leaves was also visually determined. F2 individuals with florets < 4 cm in diameter were excluded from the analysis.
Phenotypic variations in anthocyanin pigments in broccoli florets. A, Three grades of anthocyanin pigments in broccoli florets. The white bars indicate 5 cm. B, Frequency distribution of F2_01 (left) and F2_02 (right) for the degree of anthocyanin pigmentation based on visual evaluation.
The total anthocyanin content of broccoli florets and Arabidopsis seedlings was calculated according to previous reports (Goswami et al., 2018; Mehrtens et al., 2005; Rahim et al., 2019) with slight modifications. Briefly, 100 mg of florets and 30–40 mg of Arabidopsis seedlings (approximately 7–12 seedlings) were sampled, frozen with liquid N2, and crushed with stainless-steel beads. Acidic methanol (1 mL; 1% HCl, w/v) was added to fresh plant material. The samples were incubated in the dark at room temperature at 50 rpm with shaking for 18 h. Plant material was sedimented by centrifugation and 450 μL of the supernatant was added to 450 μL of acidic methanol. The absorbances of the extracts at wavelengths of 530 and 657 nm were determined using a UV-Vis spectrophotometer (U-5100; Hitachi High-Tech, Tokyo, Japan). Three replicates were used for each plant.
QTL-seq analysisTotal DNA was extracted from fresh leaves using a NucleoSpin Plant II (TaKaRa Bio, Shiga, Japan). From the two F2 populations, 10 to 20 individuals with green and purple florets with anthocyanin, respectively, were selected, and an equal amount of DNA was pooled to construct the anthocyanin (A)-bulk and no anthocyanin (NP)-bulk samples. These DNA bulk and the two parental DNA samples were used for whole-genome resequencing. High-quality reads from the DNA bulks and parental DNAs were aligned and mapped to the B. oleracea reference genome, HDEM (https://plantgardTen.jp/ja/index). After precise single nucleotide polymorphisms (SNPs) were obtained, the SNP indices in the A-bulk and NA-bulk were calculated, and their subtraction (delta-SNP index) was mapped to show their distribution on the chromosomes.
RNA-seq analysisThe buds of lines 11-1 and 14-3 were sampled when the anthocyanin pigments appeared in the florets of 14-3. Total RNA was isolated from the buds of lines 11-1 and 14-3 with a Monarch Total RNA Isolation Kit (New England Biolabs, Ipswich, MA, USA) at room temperature according to the manufacturer’s instructions. The RNA quality was measured using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Library construction and sequencing were performed by Genewiz-Azenta (Leipzig, Germany). Low-quality reads and adaptors were removed using Trimmomatic ver. 0.36 (Bolger et al., 2014) and Cutadapt ver. 1.17 (Martin, 2011), and the resulting reads were aligned to the reference sequence (HDEM) using Hisat2 ver. 2.1.0 (Kim et al., 2015). Read counts were performed using FeatureCounts ver. 1.4.6 (Liao et al., 2014) were normalized by transcripts per kilobase million (TPM) measure, and genes with a false discovery rate (FDR) < 0.01 were extracted as differentially expressed genes (DEGs).
For RT-PCR analysis, total RNA was extracted from buds of F1 cultivars when anthocyanin pigments appeared in the florets of anthocyanin accumulating genotypes with a Monarch Total RNA Isolation Kit (New England Biolabs) and converted to first-strand cDNA using the SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Scientific). PCR was performed using Quick Taq HS DyeMix (Toyobo, Osaka, Japan) according to the following program: 2 min at 94°C; 35 cycles of 10 s at 94°C, 30 s at 57°C, 2 min at 68°C; and a final extension of 3 min at 72°C. Primers are listed in Table S2. The BoActin gene (Rahim et al., 2018) was used as a control.
Candidate gene analysis and DNA marker genotypingSeveral primer pairs were designed to amplify the genomic DNA fragments of the promoter and coding regions of the candidate genes (Table S2). Amplification was carried out using QuickTaq HS DyeMix (Toyobo) and the cycling conditions were 2 min at 94°C and 30 cycles of 10 sec at 94°C, 30 sec at 57°C, and 3 min at 68°C. For amplification of the intron 1 sequence, the extension time and PCR cycles were changed to 10 min and 35 cycles, respectively. PCR products were purified on a PCR96 plate (Merck KGaA, Darmstadt, Germany) and used for Sanger sequencing. If direct sequencing failed, the PCR products were directly cloned into the pCR2.1 vector (Thermo Scientific) and used for Sanger sequencing. Sequences were assembled using CodonCode Aligner (CodonCode Corporation, Dedham, MA, USA) and SnapGene software (www.snapgene.com).
Total DNA was isolated from frozen leaves of F2 individuals and F1 broccoli cultivars using the cetyltrimethylammonium bromide (CTAB) method. Amplification for genotyping was carried out using QuickTaq HS DyeMix (Toyobo) and consisted of an initial 2 min at 94°C and 30 cycles of 10 sec at 94°C, 30 sec at 57°C, and 1 min at 68°C. PCR fragments were separated on 2% agarose gels (Merck KGaA) or 2.5% agarose XP (Nippon Gene, Tokyo, Japan).
Cold treatment of Arabidopsis seedlingsCold treatment was performed according to the method described by Zhang et al. (2011) with slight modifications. Seeds were surface sterilized with 1% hypochlorous acid solution and sown on 0.8% agar containing 1/2 MS medium. Seedlings were incubated at 22°C with a photoperiod of 14 h for 5 d. Seedlings were then incubated at 4°C with a photoperiod of 14 h for 4 d as the cold condition. For the control condition, seedlings were incubated at 22°C with a photoperiod of 14 h for 4 d.
Visible evaluation of anthocyanin pigments in broccoli florets during autumn–winter cultivation revealed a continuous distribution in F2 plants in both F2 populations (F2_01 and F2_02). Variations in the levels of anthocyanin pigments were observed, and the ratio of plants categorized into three grades differed between the two F2 populations (Fig. 1). When plants with Grades 1 and 2 were assigned to anthocyanin pigments and Grade 0 was assigned to no pigments, the segregation of the plants with anthocyanin and no pigments fitted the 3:1 ratio in both populations (0.6 > P > 0.2). These results indicated that anthocyanin pigmentation is controlled by a single dominant gene.
Identification of loci associated with the anthocyanin-free trait under the cold conditionThe QTL-seq analysis revealed a significant delta-SNP index in the genomic region of 59.2–61.7 Mb on chromosome 9 in F2_01 and 59.2–61.5 MB in F2_02, which was associated with the anthocyanin pigmentation of broccoli florets in two populations, F2_01 and F2_02 (Figs. 2, S1, and S2). We then used RNA-seq data to narrow down the candidate genes located in the QTL region. We identified 9 DEGs located in the 59.2–61.7 Mb region on B. oleracea HDEM chromosome 9 (Table 1). Of these, four genes were upregulated in the anthocyanin line 11-1 and five and were upregulated in the anthocyanin-free line 14-3. Among the 9 DEGs, we focused on one gene (BolC9t59639H) encoding F3′H (flavonoid 3′-hydroxylase), which was rarely expressed in florets of 14-3 and was involved in the anthocyanin biosynthesis pathway (hereafter referred to as BoF3′H). The BoF3′H is a single copy gene in the B. oleracea genome (Han et al., 2021). Then, we determined the sequence of genomic fragments of the BoF3′H genes, including ~1.2 kb of 5ʹ upstream and ~1.0 kb of downstream regions. We found that BoF3′H derived from 11-1 was identical to the reference genome HDEM and that there were three alleles, which included two non-synonymous SNPs, six synonymous SNPs, and a 43-bp insertion/deletion (Indel) in the coding sequence among the parental lines (Fig. 3A). There were 26 SNPs and four Indels in the 5′ upstream region and 23 SNPs and seven Indels in the 3′ downstream region. In addition, an ~3.5-kb Indel was found in intron 1 of 14-1-2, 14-3, and 3-2. Among these polymorphisms, only a 43-bp Indel in the second exon of BoF3′H was identified in the anthocyanin-free parental lines, 14-3 and 3-2. This 43-bp deletion caused a frame-shift mutation, resulting in the truncated protein lacking highly conserved motifs important for function, such as oxygen- and heme-binding motifs, among cytochrome P450 and F3′H proteins (Huang et al., 2012; Vikhorev et al., 2019; Werck-Reichhart and Feyereisen, 2000; Xu et al., 2007; Zhou et al., 2016; Fig. 3B). Based on these polymorphisms, the alleles derived from 11-1 and 14-1-2 were denoted as BoF3′H-1 and BoF3′H-2, respectively, and the allele of 14-3 and 3-2 containing the 43-bp deletion as Bof3′h-2 (Fig. 3).
The delta-SNP index values on B. oleracea chromosome C09 for floret colors at low temperature. Upper: the results of F2_01, lower: the results of F2_02.
List of DEGs located in the QTL region.
The genomic structure and deduced amino acid sequences of the BoF3′H alleles. A, Schematic representation of the genomic structure of the BoF3′H alleles. Black boxes indicate exons, and solid lines between exons indicate introns. The white box indicates the 43-bp deletion found in exon 2 of the Bof3′h-2 allele. Arrowheads indicate primers that detected the 43-bp deletion. White triangles and asterisks indicate the positions of > 10 bp and < 10 bp indels compared with the BoF3′H-1 allele, respectively. The sequences of the three BoF3′H alleles have been deposited in the GenBank database (accession nos. LC816091, LC816092, and LC816093). B, Alignment of deduced amino acid sequences of the three BoF3′H alleles. Identical amino acids are indicated by gray letters. Highly conserved regions in cytochrome P450 and F3′H proteins are indicated in solid and dotted boxes, respectively.
We designed primer pairs to detect the 43-bp indel, which amplified 265 bp or 262 bp in the anthocyanin parents, 219 bp fragments in the anthocyanin-free parent, and two bands from the anthocyanin parents and anthocyanin-free parents in F1 plants (Fig. 4). Then, the genotypes of the BoF3′H-Indel marker were validated in two F2 populations. The results showed co-segregation of 100% between genotypes and visible phenotypic data in F2_01; the genotype Bof3′h-2/Bof3′h-2 showed no visible anthocyanin pigments in florets, but there were over 92% in F2_02 (Table 2). Some exceptions between appearance and genotype observed in F2_02 were possibly because diseases occurred due to prolonged rain, leading to disease spots on the flower buds being recorded as anthocyanin pigments. Furthermore, we investigated the relationship between marker genotype and anthocyanin content in the florets of the F2_01 population. The anthocyanin content in florets at low temperatures was significantly different among three genotypes, BoF3′H-1/BoF3′H-1, BoF3′H-1/Bof3′h-2, and Bof3′h-2/Bof3′h-2, and we confirmed that the anthocyanin contents of plants with BoF3′H-1/BoF3′H-1 and BoF3′H-1/Bof3′h-2 were higher than those of the plants with genotype Bof3′h-2/Bof3′h-2, while no differences were detected between the BoF3′H-1/BoF3′H-1 and BoF3′H-1/Bof3′h-2 genotypes (Fig. 5).
Genotyping of BoF3′H alleles in parental lines and F1 plants. PCR analysis using primers, BoF3′H_Indel_F and BoF3′H_Indel_R, amplifies 265 bp, 262 bp, and 219 bp, for BoF3′H-1, BoF3′H-2, and Bof3′h-2, respectively. F1 was derived from a cross between 11-1 and 14-3.
Relationships between anthocyanin pigments and the Indel marker genotype.
Differences in floret anthocyanin contents among three genotypes categorized using the BoF3′H marker. Each plot indicates the average value of three replicates in the individual.
Further, we investigated the relationship between anthocyanin accumulation and marker genotypes in 35 F1 broccoli cultivars released in Japan after autumn–winter cultivation at Iwate University. Purple pigments were observed on the florets of 24 cultivars, whereas no pigments were observed in 11 cultivars, all of which were released as anthocyanin-free cultivars (Table 2). However, 10 cultivars exhibited purple pigments in both the florets and leaves, while 14 cultivars had purple pigments in the florets, but no pigments in the leaves. Large variations in anthocyanin contents in broccoli florets were observed among F1 cultivars: the anthocyanin contents of the anthocyanin-free cultivars were low, while those of ‘Grandome’, ‘Satomidori’, ‘Pixel’, and ‘Keirin’ were significantly higher (Table 3).
The F3′H genotype, anthocyanin pigments, and anthocyanin contents in 35 F1 broccoli cultivars.
Genotyping of the Indel marker revealed that three genotypes were detected in most of the F1 cultivars, as expected. However, an additional band was detected in the upper part of the 265/262-bp band corresponding to the wild type allele of BoF3′H in some cultivars (Fig. 6). Sequencing analysis revealed that this additional band was generated due to heteroduplex of BoF3′H-1 and BoF3′H-2 alleles. Six genotypes were identified based on the band patterns (Table 3). Of 35 cultivars, genotyping analysis of the indel markers revealed that 27 cultivars harbored BoF3′H-1 or BoF3′H-2 alleles, and eight were homozygous for the Bof3′h-2 allele. None of the eight cultivars showed anthocyanin pigments in either leaves or florets and accumulated low levels of total anthocyanin after autumn–winter cultivation. These results indicated that the recessive allele of BoF3′H gene harboring the 43-bp deletion is associated with the lack of anthocyanin pigment accumulation under low-temperature cultivation. Of 27 cultivars with wild type alleles, BoF3′H-1 or BoF3′H-2 alleles, 24 showed anthocyanin pigments, while 3 cultivars (‘Konnichiwa’, ‘Castle’, and ‘Kairyo-ryokuen’) showed no anthocyanin pigments.
Genotypes of the BoF3′H-Indel marker in 35 F1 broccoli cultivars. PCR analysis using primers, BoF3′H_Indel_F and BoF3′H_Indel_R, amplifies 265 bp, 262 bp, and 219 bp, for BoF3′H-1, BoF3′H-2, and Bof3′h-2, respectively. 1: ‘Ohayo’, 2: ‘Grandome’, 3: ‘Pixel’, 4: ‘Ryokurei’, 5: ‘Konnichiwa’, 6: ‘SK0-099’, 7: ‘Speed dome 052’, 8: ‘Delicious dome’, 9: ‘Tokuminori’, 10: ‘Sachiyoshi’, 11: ‘Brocken’, 12: ‘Brocken W’, 13: ‘Blanca’, 14: ‘YQQ422’, 15: ‘Shuster’, 16: ‘Heights SP’, 17: ‘Castle’, 18: ‘Subaru’, 19: ‘Fighter’, 20: ‘Kairyo-ryokuen’, 21: ‘Reirin’, 22: ‘Keirin’, 23: ‘Satomidori’, 24: ‘N-85’, 25: ‘Windbell’, 26: ‘Bellstar’, 27: ‘Yumeataru’, 28: ‘Yumehibiki’, 29: ‘Top star’, 30: ‘Dream sky’, 31: ‘Super dome’, 32: ‘Salinas early’, 33: ‘Ryokuzan’, 34: ‘One cut’, 35: ‘Neo green’. Details of the cultivars used are indicated in Table S1. M: 100 bp DNA Ladder.
The expression of BoF3′H was analyzed using RT-PCR with buds from four anthocyanin-accumulating and four anthocyanin-free cultivars (Fig. 7). The expression level the BoF3′H gene in the four anthocyanin-free cultivars was lower compared to three anthocyanin-accumulating cultivars, whereas the expression in ‘Reirin’ exhibiting high anthocyanin contents was as low as that of the anthocyanin-free cultivars.
Expression analysis of the BoF3′H gene in F1 broccoli cultivars. RT-PCR analysis was performed using Total RNA isolated from buds of anthocyanin-accumulating cultivars (‘Delicious dome’, ‘Ryokurei’, ‘Shuster’, and ‘Reirin’) and anthocyanin-free cultivars (‘Blanca’, ‘Fighter’, ‘Yumeataru’, and ‘Dream sky’) collected in early December. The BoActin gene was used as a control.
Anthocyanin pigments and contents of two f3′h mutants, tt7-6 and tt7-7, treated with low temperature conditions were compared with those of the wild type to investigate the effect of F3′H gene deficiency on anthocyanin accumulation induced by low temperatures. Wild-type seedlings grown at 22°C exhibited light purple pigments in the upper parts of the hypostyle (Fig. 8A). After 4°C treatment, anthocyanin pigments were observed in wild-type seedlings, but not in tt7-6 and tt7-7 mutant seedlings. The total anthocyanin content in f3′h mutant seedlings was approximately half that of wild-type seedlings, which was approximately three times higher than that of mutant seedlings after 4°C treatment (Fig. 8B). Unexpectedly, anthocyanin contents of the mutants increased at the same level as the wild type at 22°C. These results indicated that a disruption in the F3′H gene in Arabidopsis repressed the induction of anthocyanin accumulation with or without low-temperature treatment.
Anthocyanin accumulation of Arabidopsis wild type (Col-0) and f3′h mutants after cold treatment. A, Phenotypes of wild-type and mutant seedlings after cold treatment. Arrows indicate anthocyanin pigments. The black bar indicates 5 mm. B, Anthocyanin content of wild type and mutant seedlings grown at 22°C (white bars) and 4°C (black bars). The P-value of the Wilcoxon’s rank sum test was corrected using the Bonferroni method, and different letters indicate significant differences at the 5% level.
In the present study, we identified a genomic region on chromosome C09 of B. oleracea using QTL-seq to develop DNA markers for anthocyanin-free traits. Based on the RNA-seq data, we focused on one gene encoding the anthocyanin biosynthesis-related gene BoF3′H in the QTL region, which was rarely expressed in florets of the anthocyanin-free 14-3. We determined the full sequences of the BoF3′H genes in the parental lines. One of the three alleles, Bof3′h-2, harbored in the anthocyanin-free parents exhibited a 43-bp deletion in the second exon, encoding a truncated protein lacking highly conserved motifs important for function, such as oxygen- and heme-binding motifs, among cytochrome P450 and F3′H proteins (Huang et al., 2012; Vikhorev et al., 2019; Werck-Reichhart and Feyereisen, 2000; Xu et al., 2007; Zhou et al., 2016). F3′H belongs to the subfamily CYP75B of cytochrome P450-dependent monooxygenase and is necessary for the formation of cyanidin-type anthocyanin (Nitarska et al., 2021; Tanaka and Brugliera, 2013; Vikhorev et al., 2019). This enzyme is associated with purple and/or red coloration in Chinese cabbage, turnips, and radishes (Park et al., 2021; Segawa et al., 2021; Tao et al., 2022). Genotyping analysis of the BoF3′H-Indel marker capable of distinguishing the 43-bp deletion revealed that the genotype Bof3′h-2/Bof3′h-2 showed no visible anthocyanin pigments and significantly lower anthocyanin contents in florets in F2 and commercial F1 broccoli cultivars. Recently, the same Bof3′h-2 allele has been reported to be responsible for keeping florets green at low temperatures using broccoli germplasms in China (Liu et al., 2022; Yu et al., 2023). In this study, we demonstrated that the expression level of the BoF3′H gene is not necessarily related to the difference in anthocyanin contents of F1 cultivars. This result suggested that the anthocyanin-free trait of our broccoli breeding materials at low temperatures is caused not by the expression level, but by a 43-bp deletion of BoF3′H. The high anthocyanin content of ‘Reirin’ with low expression of the BoF3′H gene may be due to upregulation of the regulatory genes under cold conditions (Zhang et al., 2012).
To date, the anthocyanin accumulation in Arabidopsis f3′h mutants subjected to low-temperature treatment has not been investigated. We found that seedlings of two Arabidopsis Atf3′h mutants had very low levels of anthocyanin under normal conditions and did not show purple pigments under low-temperature conditions, while those of the wild type visible purple pigments. These results further supported the idea that a defect in the BoF3′H gene confers the anthocyanin-free trait in broccoli florets at low temperatures. However, the total anthocyanin content of mutant seedlings after low-temperature treatment increased to the same level as that of the wild type under normal conditions. Because Arabidopsis does not harbor F3′5′H (Bak et al., 2011), two pathways are considered for anthocyanin biosynthesis: one via cyanidin and the other via pelargonidin (Appelhagen et al., 2014); the production of pelargonigin derivatives is enhanced in f3′h mutants at low temperature.
Genotyping of the BoF3′H-Indel marker in 35 commercial F1 cultivars revealed that six genotypes were recognizable in commercial F1 broccoli cultivars. Although a large variation in anthocyanin accumulation was found among F1 cultivars, eight cultivars homozygous for the Bof3′h-2 allele did not show anthocyanin pigments after autumn–winter cultivation. This result indicated that the Bof3′h-2 allele has been introgressed into the anthocyanin-free F1 cultivars in Japan. Of 27 cultivars having wild-type alleles, BoF3′H-1 or BoF3′H-2 alleles, 24 showed anthocyanin pigments, while three cultivars showed no anthocyanin pigments. This may be due to mutations in other structural or regulatory genes involved in anthocyanin biosynthesis, as reported by Tang et al. (2017), in which a 1-bp insertion in the second exon of BoDFR caused a frame-shift mutation, resulting in green leaves in Chinese kale.
The Bof3′h-2 allele has also been found in broccoli germplasms in China (Liu et al., 2022; Yu et al., 2023). Therefore, it is considered that the Bof3′h-2 is widely distributed in broccoli genetic resources through the world. This could be because a certain anthocyanin-free cultivar harboring the Bof3′h-2 allele was frequently used as a genetic resource in broccoli breeding. Another possibility is that the presence of BoF3′H gene near genes important for broccoli characteristics is advantageous for selection over other anthocyanin biosynthesis genes. The Indel marker we developed can select anthocyanin-free genotype and F1 cultivars harboring the Bof3′h-2 allele and they could be used as genetic resources for marker-assisted breeding of new anthocyanin-free cultivars without field evaluation.
In conclusion, we identified a candidate gene, BoF3′H, that conferred no anthocyanin pigmentation on broccoli florets at low temperatures. Sequence analysis revealed that one of the three alleles, Bof3′h-2, harbored in the anthocyanin-free parents, exhibited a 43-bp deletion in the second exon. We developed a DNA marker to distinguish the mutant allele and demonstrated its effectiveness in detecting distribution of the mutant allele in F1 broccoli cultivars in Japan. Expression analysis of the BoF3′H gene suggests that low anthocyanin contents observed in broccoli cultivars is caused not by the expression level of the BoF3′H gene, but by a 43-bp deletion found within this gene. Furthermore, the Arabidopsis f3′h mutant line exhibited significantly reduced anthocyanin accumulation compared to the wild type with or without the low-temperature treatment. These results provide genetic insights for the development of new broccoli varieties with anthocyanin-free traits.
The authors thank Prof. F. Tatsuzawa, Iwate University, for helpful comments on this manuscript and M. Tsukazaki for her technical assistance. We thank the Arabidopsis Biological Resource Center for providing the Arabidopsis mutant seeds and Editage (https://www.editage.com) for English language editing.
