Purple Brassica oleracea var. capitata F. rubra is due to the loss of BoMYBL2–1 expression (2024)

Plant materials

Cabbages used for BoMYBL2–1 cloning and marker validation were B. oleracea var. capitata f, alba or rubra plants from six green inbred lines, 32 green F₁ cultivars, two purple inbred lines, two near-isogenic lines (NILs), 16 purple F₁ cultivars, and 11 recombinants, as shown in Table1. Inbred lines of green cabbages and purple cabbages, supplied by Asia Seed Co. (Korea), were selected to distinguish the genetic differences responsible for anthocyanin content.

Cabbages used for sequence analyses were as follows: inbred lines 337 and 154, selected for high anthocyanin content at low temperature; inbred lines 2437 and 09WH-45, selected for low anthocyanin content; inbred lines 2409 and 842, selected for no anthocyanin content; and the US cultivars Green A and B. The purple cabbages selected were the inbred lines 7S4–51 and 7S4–63, the NILs B90 and B98, and Purple A and B. Additional seeds not listed in Table1 were purchased at local markets. Most plants were cultivated in a greenhouse at Chungnam National University, Daejeon, Korea, although some leaf samples were obtained from Korean Seed companies.

Cloning of genomic DNA and sequence analysis for BoMYBL2–1

Genomic DNA was isolated from leaf samples using the DNeasy Plant Mini kit (QIAGEN Gmbh, Germany). The DNA sequences around BoMYBL2–1 (Bol016164 = Bo6g112670) available in two databases (http://brassicadb.org/brad/ and http://plants.ensembl.org/Brassica_oleracea/Info/Index) did not match each other. Bo6g112680 of TO1000 [56] was annotated as Bol016161, Bol016162, and Bol016163 of O2–12 [57] (Additionalfile1: Table S1). Therefore, DNA sequences from three cultivars, kale, broccoli, and cauliflower, were reanalyzed using four primer sets (Additional file1: Table S1; Additionalfile2: Figure S2A). After that, DNA sequences of heading-type cabbages (B. oleracea var. capitate) were cloned and analyzed.

Genomic PCR was performed under the following conditions: denaturation for 5min at 94°C, 30cycles of amplification (30s at 94°C, 30s at 58°C, and 2–5min at 72°C), and a final extension period of 7min at 72°C. PCR products were purified using a LaboPass Gel Extraction kit (Cosmogenetech, Korea) and cloned into the TA-vector using the T&A cloning kit (RBC Bioscience Co., Taiwan). Escherichia coli (DH5α) cells were transformed with plasmid DNA carrying the desired insert. Plasmid DNA was purified using DNA-Spin (Intron Biotech. Inc., Korea) before sequencing. As cabbages might contain multiple MYBL2–1 alleles, at least ten clones from each line were sequenced and analyzed. Any possible PCR and/or sequencing errors were eliminated by aligning independent sequences.

Inverse PCR (iPCR)

To clone DNA sequences from purple cabbages that could not be amplified using any of the primer sets (Additional file1: Table S1), inverse PCR (iPCR) was performed on Bol010163 gene sequences using the Universal GenomeWalker™ 2.0 (Clonetech, USA). DNA from purple cabbages and control DNA from the kit were digested with DraI, EcoRV, PvuII, and StuI for 16–18h at 37°C, and purified using the NuceloSpin Gel kit and PCR Clean-Up kit (Macherey-Nagel GmbH & Co, Germany). After ligation with the GenomeWalker adaptor, primary PCR was performed with a Bol010163 gene-specific primer (5’-AGACGTTGATGAGATCAACGGTTGTGA), followed by secondary PCR with an adaptor primer (5’-CATCCAATAAAGGCGAGCAAGAAAGGA). PCR products were electrophoresed on 1% agarose gels, and the resulting bands were excised, purified, and cloned using the T&A cloning kit (RBC Bioscience Co., Taiwan).

Three sizes of DNA fragments were obtained: 700bp from the PvuII library, 2.1 kbp from the DraI library, and 4.0 kbp from the StuI library. To minimize sequence error, at least five clones from each library were sequenced and analyzed.

RT-PCR

Leaf samples were collected from at least three individual plants, and total RNA was isolated from liquid nitrogen-ground samples using TRIzol reagent (Invitrogen, USA), and further purified using the NucleoSpin RNA Clean-up Kit (Macherey-Nagel GmbH & Co., Germany). Total RNA (1μg) was treated with RQ1 RNase-free DNAase (Promega, USA), and cDNA was synthesized using the Ace-α kit with oligo(dT) primers (Toyobo, Japan). Complementary DNA was diluted 10-fold, and 1μl of diluted cDNA was used in a 20μl PCR mixture. RT-PCR primers are listed in Additionalfile3: Table S2; primers for ACTIN 2 (BoACT2) were used as a control. A standard PCR was performed with a 5min denaturation at 94°C, followed by 20–30cycles of amplification (30s at 94°C, 30s at 60°C, and 30s at 72°C), and a final extension time of 7min at 72°C. PCR products were analyzed following electrophoresis through 1.2% agarose gels.

Marker development and validation

To distinguish green and purple cabbages based on their BoMYBL2–1 sequences, three primer sets were designed following sequence alignment using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) (Table2). The PCR mix consisted of 10–30ng of genomic DNA, 5pmol of each forward and reverse primer, 10pmol of BoACT2 primers, and 1× HiPi Plus PCR premix buffer in 20μl. To optimize PCR conditions for each primer pair (BoMYBL2–1w-F and BoMYBL2–1w-R; BoMYBL2–1v-F and BoMYBL2–1v-R; BoMYBL2–1sub-F and BoMYBL2–1sub-R), several annealing temperatures, extension times, and cycle numbers were tested and set. For the primer pair BoMYBL2–1w-F and BoMYBL2–1w-R, used to identify the presence of BoMYBL2–1, the PCR conditions were 5min at 94°C, followed by 28cycles of 30s at 94°C, 30s at 65°C, and 2min at 72°C, with a final extension phase of 5min at 72°C. For the primer pair BoMYBL2–1v-F and BoMYBL2–1v-R, used to identify promoter-substituted BoMYBL2–1, the reaction conditions were 5min at 94°C, followed by 30cycles of 30s at 94°C, 30s at 60°C, and 1min at 72°C, and a final extension of 5min at 72°C. For the primer pair BoMYBL2–1sub-F and BoMYBL2–1sub-R, used to identify DNA with a substitution of the whole BoMYBL2–1 gene, the reaction conditions were 5min at 94°C, followed by 30cycles of 30s at 94°C, 30s at 65°C, and 2min at 72°C, and a final extension of 5min at 72°C.

Presence of genes associated with anthocyanin biosynthesis

To examine whether additional genes were defective in purple cabbages, the presence of genes associated with anthocyanin biosynthesis was studied using genomic PCR. The genes selected were transcriptional activators and repressors (Additional file3: Table S2). PCR conditions were 5min at 94°C, followed by 20–30cycles of 30s at 94°C, 30s at 60°C, and 30s at 72°C, and a final extension time of 5min at 72°C.

Promoter analysis

To confirm whether the substituted promoter found in purple cabbages retained promoter activity, the wild-type and substituted versions of the BoMYBL2–1 promoter were fused to the GUS reporter gene. Arabidopsis plants were transformed with the reporter constructs, and examined for GUS activity and expression. The MYBL2–1 promoter regions from green and purple B. oleracea (1926bp upstream from the ATG start codon for green B. oleracea and 1804bp upstream for purple B. oleracea) were amplified by PCR using the specific forward primers (5’-GATCAGGATCCAAGAACACATGAACTT for green cabbage and 5’-GATCAGGATCCAAGAACCAGTGTTC for purple cabbage) and the same reverse primer (5’-GTGAGCCATGGTACGAGAAGCA). These primers contain the BamHI and NcoI restriction sites (underlined).

The amplified fragments were inserted into the T&A cloning vector (Real Biotech Co., Taiwan), and the presence of the MYBL2–1 promoter sequence was confirmed by sequencing. The fragments were liberated by digestion with BamHI and NcoI, and subcloned into the pCambina3301-GUS binary vector, digested with the same enzymes. The resulting constructs were transformed into Arabidopsis plants using the Agrobacterium tumefaciens-mediated floral dip procedure [58]. Transformed plants were selected using 0.1% BASTA herbicide, and their identity was confirmed by PCR analysis of genomic DNA. T₃ hom*ozygous lines containing a T-DNA insertion at a single locus, determined by a 3:1 ratio of segregation of basta-resistance: sensitivity, were selected for the assay of promoter activity.

Arabidopsis thaliana wild-type (Col-0) and transgenic plants were grown in a growth chamber under 16h light/8h dark photoperiods at 22°C and a light intensity of 100μmolm^− 2s^− 1. For plate culture, seeds were surface-sterilized with 30% bleach containing 0.1% Triton X-100, stratified for 3days at 4 °C, and plated onto solidified half-strength Murashige and Skoog (MS) medium, plus or minus 90mM sucrose. Three plants were sampled for RT-PCR study of GUS expression, and another three plants were used to analyze GUS expression.

For GUS expression studies, plants were incubated in GUS staining solution (1mM X-GlucA in 100mM sodium phosphate, pH7.0, containing 5mM Na₂EDTA, 0.5mM potassium ferrocyanide, 0.5mM potassium ferricyanide, and 0.1% Triton X-100) for 16h at 37 °C. After staining, the plants were washed in 95% ethanol at room temperature until wild-type (Col-0) Arabidopsis appeared clear.

Quantification of total anthocyanin content

Total anthocyanin content was determined in cabbage leaves using methanol containing 1% HCl [59]; three independent biological replicates from three individual plants were used in each experiment. The tissues were ground under liquid nitrogen, and the powder was resuspended in a 1.5ml tube containing methanol (1% HCl) at room temperature and centrifuged at 14,000rpm for 10min at 4 °C. The absorbance of the supernatants was determined spectrophotometrically at 530nm and 657nm. Total anthocyanin content was quantified as

where Q = total anthocyanins; A530 = absorption at 530nm; A657 = absorption at 657nm; FW = fresh weight of tissues (g).

Selection of BoMYBL2–1 gene

To dissect the association of BoMYBL2 with anthocyanin biosynthesis, expression of selected key genes in the pathway was examined in six inbred lines of green cabbage (337, 154, 2477, 09WH-45, 2409, and 842) and two inbred lines of purple cabbage (7S4–51 and 7S4–63). Two-month-old cabbage plants were grown for a month, either under greenhouse (G) conditions, which were non-inductive for anthocyanin accumulation, or outside (C), which was inductive for anthocyanin accumulation because it exposed the plants to low temperature (Fig.1).

Expression of BoMYB1 (Bol042409 = Bo3g081880), BoMYB2 (Bol012528 = Bo6g100940), BoDFR1 (Bol035269 = Bo9g058630), BoMYBL2–1 (Bol016164 = Bo6g112670), and BoMYBL2–2 (Bol034966 = Bo2g070770) was analyzed. Two BoMYBL2 genes (one on chromosome 6 and the other on chromosome 2) matched Arabidopsis MYBL2 (At1G71030), and BoMYBL2 designations were given according to the level of amino acid sequence identity with the Arabidopsis counterpart. As shown in Fig.1, the expression of BoDFR1 correlated with anthocyanin accumulation (Additionalfile4: Figure S1). BoMYB1 and 2 were highly expressed in purple cabbages, whereas BoMYBL2–2 expression was detected in all samples. BoMYBL2–1 expression was not detected in purple cabbages, and attempts to amplify the gene by PCR from the genomic DNA of purple cabbages failed, suggesting either a high level of sequence variation or deletion of BoMYBL2–1.

To investigate whether BoMYBL2–1 had been deleted from the genome of purple cabbages, we examined the presence and expression of the gene in additional cultivars (Fig.2). Both the BoMYBL2–1 gene and its transcripts were detected in all the green cabbages tested. Two different types of result were obtained from two purple cabbages: (1) no amplification product from either genomic DNA or cDNA templates; and (2) amplification product from genomic DNA but not from cDNA. More specifically, the BoMYBL2–1 transcript could not be detected in the purple B cultivar, although presence of the gene was confirmed from analysis of genomic DNA. These data suggest that two distinct mechanisms for the loss of BoMYBL2–1 expression were responsible for the color of the two purple cabbages tested.

Cloning and sequence analysis of BoMYBL2–1 from various B. oleracea species

Genomic cloning of BoMYBL2–1 was undertaken using six primer pairs (Additional file2: Figure S2; Additional file1: Table S1) and eight inbred lines of cabbages obtained from the Asia Seed Co. (Korea). An initial comparison of PCR results revealed that high levels of sequence variation were present around BoMYBL2–1, as observed in the distinct reference sequences previously obtained from two different varieties of B. oleracea [56, 57] (Additional file1: Table S1). We therefore re-established a sequence assembly for kale (B. oleracea var. sabellica), broccoli (B. oleracea var. italica), and cauliflower (B. oleracea var. botrytis) cultivars using one allele from each variety (Additionalfile5). Based on this new sequence information, sequencing and analysis were extended to inbred lines and cultivars of green cabbages, as well as purple cabbages (B. oleracea var. capitata) (Additional file5). Nonetheless, no PCR product could be amplified from several lines of purple cabbages, including 7S4–51, 7S4–63, and B98. We used iPCR with primers designed around Bol016163 to obtain sequence information for these plants.

The simplified structure of the BoMYBL2–1 genes and upstream nucleotide sequences from all of the lines used in our analysis (Additional file5), which confirmed high levels of sequence variation in this region, are shown in Fig.3. Kale and cauliflower were both heterozygous for Bol016163 alleles, one with complete and the other with deleted versions, while broccoli appeared to have hom*ozygous alleles. All heading-type cabbages and two purple cabbages (Purple B and B90) contained the deleted version of Bol016163; the other purple cabbages contained a complete version of Bol016163.

As shown in Fig.3, all heading-type green cabbages carried BoMYBL2–1 (1208bp) plus approximately 1900bp of upstream sequences. By contrast, two types of DNA sequence were observed in purple cabbages: BoMYBL2–1 with a substituted promoter containing 1190bp of sequence, also found on chromosome 1, 347bp upstream of the start codon (called BoMYBL2–1 variant or BoMYBL2–1v), and a substitution (or deletion) type of the entire BoMYBL2–1 plus regulatory regions with putative chromosome 3 and chromosome 7 (called BoMYBL2–1 substitution or BoMYBL2–1sub). Most purple cabbages contained BoMYBL2–1sub. To distinguish the wild-type or intact BoMYBL2–1 sequence from BoMYBL2–1v, the wild-type version was designated BoMYBL2–1w. Interestingly, both BoMYBL2–1w and BoMYBL2–1v contained 159bp repeat sequences upstream of the ATG start codon, which included seven ACCCGA repeats, 11 CGAA repeats, seven AAAT repeats, and so on. The MYB core motif GGATA [55] was detected in this repeat.

Promoter analysis

To test whether the promoter region of BoMYBL2–1v had any transcriptional activity, 1926bp from BoMYBL2–1w and 1804bp from BoMYBL2–1v upstream of the ATG start codon were fused to the GUS reporter gene and the constructs were used to transform Arabidopsis plants. ß-glucuronidase (GUS) activity in Arabidopsis transgenic plants was evaluated using both histochemical staining and levels of expression of GUS transcript (Figs.4 and5). Two independent pCambia3301 transgenic plants were used as positive controls. Comparable levels of GUS staining (Fig.4a) and GUS transcript expression (Fig.4b) were shown by seven independent pMYBL2–1w::GUS transgenic plants. On the other hand, neither GUS staining nor GUS transcripts were detected in wild-type Arabidopsis (Col-0) or in seven independent pMYBL2–1v::GUS transgenic plants. These results indicated that the pMYBL2–1v promoter was non-functional under normal growth conditions.

Since anthocyanin biosynthesis is induced by various environmental factors, GUS expression in transgenic Arabidopsis plants was also tested under conditions of sucrose and temperature (low and high temperature) stress. As shown in Fig.6, GUS staining was not detectible in pMYBL2–1v::GUS transgenic plants, even when the plants were subjected to the stress of 90mM sucrose or exposure to high or low temperature. Taken together, these data strongly suggest that the promoter substitution found in BoMYBL2–1v resulted in the loss of promoter activity and BoMYBL2–1 expression in some types of purple cabbages. In others, the change of color from green to purple may be explained by the complete deletion of the BoMYBL2–1 coding sequence.

Confirmation of a unique deletion of BoMYBL2–1 in purple cabbages

To determine whether the color change to purple in B. oleracea var. capitata f rubra is attributable to the loss of other important genes regulating anthocyanin biosynthesis, we investigated the presence of regulatory genes, including BoMYBL2–1, associated with anthocyanin biosynthesis. BoMYBD, BoSPL9, BoMYBH, and BoMYBL2–1 are negative regulators of anthocyanin biosynthesis, while BoMYB1–4, BoMYB11, and BoMYB12 are transcriptional activators. BoANL (Bo9g002480/Bol011495) is a homeobox-leucine zipper gene equivalent to ANTHOCYANINLESS 2 (AT4G00730).

As shown in Additionalfile6: Figure S3, all these genes other than BoMYBL2–1, which was lost in several lines of purple cabbage, were present in various varieties of B. oleracea. This strongly suggested that, at least for some purple cabbages (B. oleracea var. capitata f, rubra), the deletion of BoMYBL2–1 was responsible for the development of purple coloration.

Since the presence of a gene does not guarantee its expression, expression of key biosynthetic (BoCHS1, BoCHS2, BoF3H, BoF3’H, and BoDFR1) and regulatory genes affecting anthocyanin accumulation was examined in 20 different purple cabbage (Additional file6: Figure S3). Transcript levels of most genes did not differ between these purple cabbages, but expression levels of several genes (BoCHS1, BoF3H, BoDFR1, and BoMYB2) were higher in those plants than in the green cabbage Daebakna. All the genes tested, other than BoMYB4, were expressed at relatively high levels, possibly as a result of defective BoMYBL2–1 activity.

And validation of molecular markers to distinguish purple cabbage

Markers that are associated with a particular horticultural trait, such as purple coloration, are very important in developing new B. oleracea varieties with the desired trait from crosses of different subspecies. To develop molecular markers able to identify purple B. oleracea var. capitata carrying defective BoMYBL2–1, we designed three sets of primer pairs: (1) to amplify the BoMYBL2–1 coding sequence; (2) to amplify the promoter-substituted variant BoMYBL2–1 (MYBL2v); and (3) to amplify the DNA sequence that replaced the entire BoMYBL2–1 gene (BoMYBL2–1sub) (Table2; Fig.3). The optimal PCR conditions for each pair are described above (Methods). The validity of these primer pairs was tested using the 35 green and 33 purple cabbages listed in Table1.

As shown in Fig.6, the band corresponding to BoMYBL2–1 coding sequence was amplified by the F1 and R1 primers (BoMYBL2w-F and BoMYBL2w-R) from all the green cabbages tested, as well as from four purple cabbages (B90, Purple B, NP-J-156, and-047). All of the purple cabbages tested, however, other than B90, contained DNA that could be amplified using the F3 and R3 primers (BoMYBL2sub-F and BoMYBL2sub-R) (Fig.7), implying that these plants contained the substituted version of the entire BoMYBL2–1 gene, including the promoter and coding regions. In addition, only four purple cabbages contained DNA that could be amplified using the F2 and R2 primers (BoMYBL2v-F and BoMYBL2v-R), which were specific for the promoter-substituted BoMYBL2–1 variant (BoMYBL2–1v) (Fig.8). Taken together, we concluded that purple cabbages contain either BoMYBL2–1v or BoMYBL2–1sub or both. Our data indicated that most of the purple cabbages in this study were hom*ozygous for BoMYBL2–1sub, although B90 was hom*ozygous for BoMYBL2–1v. Purple B, NP-J-0156, and HK-047 were heterozygous, carrying both the BoMYBL2–1v and BoMYBL2–1sub alleles.

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Competing interests

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