Characterization of FLOWERING LOCUS C 5 in Brassica rapa L.

doi:10.21203/rs.3.rs-2490319/v1

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Characterization of FLOWERING LOCUS C 5 in Brassica rapa L.

https://doi.org/10.21203/rs.3.rs-2490319/v1

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published 19 Jul, 2023

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Brassica rapa L., which includes Chinese cabbage, turnip, and pak choi, has more complex flowering time regulation than does Arabidopsis thaliana due to the presence of multiple paralogous flowering time genes. FLOWERING LOCUS C (FLC) is one of the key genes regulating flowering time. Although there is a single gene in A. thaliana, there are four FLC paralogs in B. rapa. BrFLC1, BrFLC2, and BrFLC3 are involved in regulating flowering time, whereas BrFLC5 is deemed a pseudogene because of a mutation (from G to A) in the splice site of the third intron. However, some accessions do not have this mutation, and a flowering-time quantitative trait locus (QTL) covering BrFLC5 associated with late flowering has been identified, suggesting that some accessions in B. rapa have a functional BrFLC5 gene. However, it has not been shown whether BrFLC5 acts as a floral repressor. To examine the allelic variation of BrFLC5, we genotyped 310 B. rapa accessions and found 19 had homozygous and 81 had heterozygous putative functional BrFLC5 alleles. BrFLC5 acts as a floral repressor when overexpressed in A. thaliana. Variation in the BrFLC5 expression level in pre-vernalized plants was identified and this transcriptional variation was not associated with the G/A polymorphism in the third intron. However, higher BrFLC5 expression in pre-vernalized plants is associated with 584 bp insertion in the promoter region. The possibility that a high expression of a functional BrFLC5 could contribute to producing premature bolting-resistant lines in B. rapa vegetables is discussed.

FLOWERING LOCUS C

vernalization

floral repressor

transcription

Brassica

Brassica rapa L. comprises a large number of morphological variants and includes commercially important leafy vegetables such as Chinese cabbage (var. pekinensis), pak choi (var. chinensis), komatsuna (var. perviridis), root vegetables such as turnip (var. rapa) and oil seed/mustard (var. trilocularis, var. dichotoma, var. oleifera) (Lv et al. 2020). B. rapa leafy vegetables are grown mainly in Asian countries, and root vegetables are grown in Europe countries and the USA as well as in Asian countries. In India, oil seeds and mustard are the main crops (Qi et al. 2017). The whole-genome sequence of B. rapa was the first to be determined in the genus Brassica using the Chinese cabbage doubled haploid line, Chiifu-401-42 (Wang et al. 2011), followed by the whole genome sequence in yellow sarson (Belser et al. 2018) and pak choi (Li et al. 2020; 2021). The reference genome sequence of Chiifu-401-42 was updated by long-read sequencing technology and high-through chromosome conformation capture (Hi-C) technology (Zhang et al. 2018).

Flowering time is one of the major influences on the quality of the harvested product and seed in the genus Brassica. Brassica species originally required prolonged cold exposure to induce flowering, and this phenomenon is called vernalization (Itabashi et al. 2018; Akter et al. 2018; 2021). B. rapa has two main flowering types, one requires little or no vernalization, and the other is vernalization requiring (Su et al. 2018). Even within B. rapa accessions requiring vernalization, there is a diversity in the degree of vernalization required, a variation of the length of the period of cold exposure for flowering. For oilseed or mustard, where seed is the product and crops are grown in warm climates, accessions with little or no vernalization requirement are used. Most leafy or root vegetables have a high vernalization requirement because early bolting leads to a decrease in quality, especially in spring Chinese cabbage that is sown in late winter or early spring and requires a long period of vernalization (Akter et al. 2021).

The floral repressor, FLOWERING LOCUS C (FLC), a MADS-box transcription factor, mediates vernalization in B. rapa (Shea et al. 2019; Takada et al. 2019; Akter et al. 2021). Due to the whole genome triplication (WGT) in B. rapa, there are four copies of BrFLC (BrFLC1, BrFLC2, BrFLC3, and BrFLC5) (Shea et al. 2018; Akter et al. 2019; 2021). All four BrFLC paralogs are expressed in pre-vernalized plants and have active histone marks, histone H3 lysine 4 trimethylation (H3K4me3) and H3K36me3, in their coding regions (Mehraj et al. 2021). The expression of all four BrFLC paralogs gradually decreases during prolonged cold exposure (Akter et al. 2020). During this period, the repressive histone mark, H3K27me3, accumulates around the transcription start site of BrFLCs (Akter et al. 2019). After returning to a warm environment, H3K27me3 spreads across the entire encoding regions of all BrFLC paralogs, and the repression of their expression is maintained (Kawanabe et al. 2016; Akter et al. 2019; 2021). There are variations in expression levels in pre-vernalized plants and in the degree of suppression of expression following vernalization among the four BrFLC paralogs in the same plant (Takada et al. 2019). Furthermore, there are variations in expression levels of each BrFLC gene between accessions in pre-vernalized plants, and in the degree of suppression of expression following vernalization in each BrFLC gene between accessions (Takada et al. 2019). The total expression level of the four BrFLC paralogs in pre-vernalized and the degree of suppression of expression in each BrFLC following prolonged cold treatment are important factors for the inter-specific variation of the vernalization requirement (Takada et al. 2019; Akter et al. 2021).

BrFLC5 is classified as a pseudogene due to the deletion of two exons in the reference genome (Schranz et al. 2002; Wang et al. 2011). Later, a polymorphism in the sequence of the splicing donor site of the third intron (G/A) was found among B. rapa accessions, and the presence of accessions encoding the complete BrFLC5 gene was reported (Xi et al. 2018). Quantitative trait loci (QTLs) affecting flowering time in different populations of B. rapa have been identified (Shea et al. 2018; Akter et al. 2021), and some QTLs cover regions including BrFLC1, BrFLC2, or VERNALIZATION INSENSITIVE3.1 (BrVIN3.1) (Zhao et al. 2010; Su et al. 2018). A flowering time QTL covering BrFLC5 was identified (Kakizaki et al. 2011), and the possibility that BrFLC5 is involved in flowering time variation has been shown (Kakizaki et al. 2011; Xi et al. 2018).

An accession encoding a complete BrFLC5 has been identified, and there is a flowering time difference between plants having a functional (G) and non-functional (A) polymorphism at the 5′ splice site in the third intron of BrFLC5 in the F₂ population (Xi et al. 2018). These results suggest that a functional BrFLC5 contributes to the flowering time difference, however, it does not clarify whether BrFLC5 acts as a floral repressor. Here, we identified accessions having a functional (G) or non-functional (A) polymorphism at the 5′ splice site in the third intron of BrFLC5 from over 300 B. rapa accessions. We confirmed that BrFLC5 acts as a floral repressor by overexpressing BrFLC5 in Arabidopsis thaliana. The variation of expression levels of BrFLC5 in pre-vernalized plants among B. rapa accessions and the variation in the repression rate of BrFLC5 expression following prolonged cold treatment were examined. Finally, we discuss whether BrFLC5 can be used as a breeding resource.

Plant materials and growth conditions

310 B. rapa accessions from four different varieties were used to examine a polymorphism in the sequence of the splicing donor site of the third intron (G/A). Ten B. rapa accessions [Chinese cabbage; Chukanbohon Nou 6 gou (Nou 6) (Kakizaki et al. 2011), ‘Eishun’, ‘Nozaki Hakusai No. 2’ (NOZAKI SAISHUJO LTD., Japan), ‘CR Kisaku-80’, ‘Raiou-90’ (MARUTANE CO., LTD, Japan), ‘CR-Okiniiri’, ‘Homarenokiwami’, ‘Shoshun’ (TAKII & CO., LTD, Japan), Turnip; ‘Aishinku No. 3’, ‘CR Takamaru’ (Musashino Seed Co.,Ltd, Japan)] were used to examine the expression of BrFLC5 in pre-vernalized condition. After surface sterilization, the seeds were sown in agar solidified Murashige and Skoog (MS) plates with 1% (w/v) sucrose under long day (LD) conditions (16 h light) at 22°C. Fourteen-day seedlings on MS plate were treated for four weeks at 4° C under LD conditions.

Sequencing DNA fragments of BrFLC5 gene

Genomic DNAs were isolated from 14-day first and second leaves by the Cetyl trimethyl ammonium bromide (CTAB) method (Murray and Thompson 1980). The regions including the third intron were amplified by PCR using a primer set, BrFLC5-F/R (Fig. S1; Table S1). Amplified PCR products were treated by illustra ExoProStar (GE Healthcare Life Sciences) and were directly sequenced using ABI Prism 3130 (Applied Biosystems).

The 1,355 or 771 bp upstream regions from the transcription start site (TSS) were amplified by PCR using a primer set, BrFLC5_pro-F1/R1 (Fig. S1; Table S1). Amplified PCR fragments (771 bp) of seven accessions (‘Nozaki Hakusai No. 2’, ‘CR Kisaku-80’, ‘Raiou-90’, ‘Eishun’, ‘Shoshun’, ‘Homarenokiwami’, and ‘CR-Okiniiri’) were directly sequenced. Amplified PCR products (1,355 bp) of three accessions (Nou 6, ‘Aishinku No. 3’, and ‘CR Takamaru’) were cloned into pGEM®-T Easy vector (Promega, Madison, WI, USA). Nucleotide sequences of three clones of PCR products were determined with the ABI Prism 3130 (Applied Biosystems). Primers, M13F/R and BrFLC5_pro F2/R2, were used for sequencing (Fig. S1; Table S1). The data were analyzed using Sequencher (Gene Codes Corporation, MI, USA). PCR was performed using the following conditions; 1 cycle of 94°C for 2 min, 35 cycles of 94°C for 30 s, 58°C for 30 s, and 68°C for 2 min. Primer sequences and their position used for sequencing are shown in Fig. S1 and Table S1.

RNA extraction and RT-qPCR

Total RNA was isolated from 14-day first and second leaves or from first and second leaves following four weeks of cold treatments to 14-day seedling using the SV Total RNA Isolation System (Promega). The leaves from three individual plants in each condition were harvested as biological replicates. The cDNA was synthesized from 500 ng total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Ltd., Osaka, JAPAN). RT-qPCR was performed using LightCycler 96 (Roche Diagnostics International Ltd., Switzerland). The cDNA was amplified using FastStart Essential DNA Green Master (Roche). PCR conditions were 95°C for 10 min followed by 40 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 15 s, and the Melting program (60°C to 95°C at 0.1°C/s). After amplification cycles, each reaction was subjected to melt temperature analysis to confirm single amplified products. The expression level of each gene relative to BrACTIN was automatically calculated using automatic CQ calling according to the manufacturer’s instructions (Roche) (Fujimoto et al. 2006). Data presented are the average and standard error (s.e.) calculated from three biological and experimental replications. Primer sequences used for RT-qPCR are shown in Table S1.

RNA-sequencing

For RNA-sequencing (RNA-seq), total RNA from 14-day first and second leaves of pre-vernalized in ‘Aishinku No. 3’, ‘CR Takamaru’, Nou 6, and ‘Homarenokiwami’ were used. A library was prepared using NEBNext® Ultra™II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA), and sequencing was performed using NovaSeq 6000 (Illumina Inc., San Diego, CA) (paired-end, 150 bp). Low quality reads were filtered using FaQCs ver 2.10 (Lo and Chain 2014), and HISAT2 ver 2.2.1 (Kim et al. 2015) was used to align the filtered reads to Brassica rapa reference sequence v1.5 (http://brassicadb.org/cn/). The levels of gene expression were scored by fragments per kilo-base per million (FPKM) using cuffdiff v2.2.1 (Trapnell et al. 2010). The RNA-seq data was deposited in the DNA Data Bank of Japan (DDBJ; accession no. DRA00929)

Constructs and plant transformation

The cDNA from leaves of Nou 6 was used for RT-PCR. PCR fragments of a coding sequence (CDS) of BrFLC5 were amplified by RT-PCR using a primer pair, BrFLC5_5UTR_Sal and BrFLC5_3UTR_XbaI-2 (Table S1). PCR was performed using the following conditions; 1 cycle of 94°C for 2 min, 35 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 30 sec. Amplified PCR products were then cloned into the pGEM®-T Easy vector (Promega). The Sal I and Xba I fragment including BrFLC5 CDS was inserted between the 35S promoter and Nos terminator in the overexpression vector prepared by modifying the pCAMBIA1301 binary vector (Itabashi et al. 2019). Ligation was carried out using In-Fusion HD Cloning Kit (TaKaRa Bio) and a primer pair, BrFLC5_CDS-fusF3 and BrFLC5_CDS-fusR3, was used (Table S1). This construct was transformed into Agrobacterium tumefaciens strain EHA105, and the transformation of Columbia-0 (Col) accession in A. thaliana was carried out by the floral dip procedure (Clough and Bent 1998). Transgenic seedlings were selected, which showed hygromycin resistance on a selection medium. T₂ plants seeds were sown on MS medium and grown under LD conditions (16 h light) at 22°C. After growing plants on MS medium, they were transferred to soil and grown under the conditions described above. The flowering time observed in A. thaliana was expressed as the number of rosette leaves at the time of flowering. Total RNA was isolated from mature leaves of T₂ plants with and without transgene, and RT-PCR was performed to confirm the expression of transformed BrFLC5 gene. PCR conditions were 1 cycle of 94°C for 2 min, 30 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 30 sec. Primer sequences used for RT-PCR are shown in Table S1.

Identification of functional and non-functional alleles in B. rapa germplasm

In the reference genome sequence of B. rapa, Chiifu-401-42, BrFLC5 (Bra022771/BraA03g015950.3C) is annotated with five exons, while the other three BrFLC paralogs (BrFLC1, BrFLC 2, and BrFLC3) are annotated with seven exons (Wang et al. 2011; Zhang et al. 2022), implying that BrFLC5 is a pseudogene in B. rapa. The reason for the decrease in the number of exons is the presence of a mutation from G to A in the splicing donor site (GT) in the third intron (Fig. S1). However, some lines of B. rapa do not have this mutation; there are seven exons in BrFLC5 (Xi et al. 2018). In this study, we examined the presence of this mutation in 310 accessions of B. rapa that were mainly collected from Japanese commercial cultivars. Direct sequencing of the 673 bp sequence harboring this mutation site was performed in 310 B. rapa accessions (Fig. S1). Among the 310 accessions, 19 accessions (6.1%) had a homozygous G/G allele, 81 accessions (26.1%) had a heterozygous G/A allele, and 210 accessions (67.7%) had a homozygous A/A allele (Table 1). Among four varieties of B. rapa, the frequency of homozygous G/G alleles was 31.3% in turnip, 5.0% in komatsuna, 4.8% in Chinese cabbage, and none in pak choi. In all varieties, the frequency of a homozygous G/G allele is lower than that of a homozygous A/A allele (Table 1).

Table 1

Frequency of G/A mutation in the splicing donor site of the third intron of *BrFLC5* in *B. rapa* vegetables
		G/G		G/A		A/A		Total
		Number	(%)	Number	(%)	Number	(%)	Number
Chinese Cabbage	var. pekinensis	11	4.8%	67	29.4%	150	65.8%	228
Turnip	var. rapa	5	31.3%	2	12.5%	9	56.3%	16
Komatsuna	var. perviridis	2	5.0%	11	27.5%	27	67.5%	40
Pak choi	var. chinensis	0	0.0%	0	0.0%	17	100.0%	17
Others		1	11.1%	1	11.1%	7	77.8%	9
Total		19	6.1%	81	26.1%	210	67.7%	310

BrFLC5 functions as a floral repressor

BrFLC1, BrFLC2, and BrFLC3 but not BrFLC5 have been shown to act as floral repressors (Kim et al. 2007; Takada et al. 2019). The nucleotide sequence of the coding region of BrFLC5 in Nou 6 was determined. The similarities of the predicted amino acid sequence of BrFLC5 to BrFLC1, BrFLC2, and BrFLC3 were 81%, 84%, and 80%, respectively (Fig. 1). The MADS-box domain responsible for binding to target DNA was conserved in BrFLC5 (Fig. 1).

A 35 S promoter::BrFLC5 CDS was transformed into the Columbia-0 accession of A. thaliana that has no AtFLC expression because of the loss of function of FRIGIDA (AtFRI) (Johanson et al. 2000). More than twenty T₁ plants were obtained. Some T₁ plants did not flower and offspring were not obtained. Three T₂ populations were examined for flowering time, and the flowering time of plants transgenic for BrFLC5 transgene (TG+) was later than plants without the transgene (TG-) (Fig. 2a, b). Expression of BrFLC5 in transgenic plants was confirmed by RT-PCR (Fig. 2c).

Expression levels of BrFLC5 varied among B. rapa accessions

We have suggested that the level of FLC expression prior to prolonged cold treatment and the rate of suppression of FLC expression following prolonged cold treatment are important in the vernalization requirement in B. rapa (Takada et al. 2019; Akter et al. 2021). The expression level of BrFLC5 was examined by qPCR using a BrFLC5 specific primer set in ten accessions of B. rapa of which seven had homozygous G/G alleles and three had homozygous A/A alleles. ‘Aishinku No. 3’ showed the highest expression levels followed by ‘CR Takamaru’ and Nou 6, which all have a homozygous G/G allele (Fig. 3). The expression levels of ‘Homarenokiwami’ and ‘CR-Okiniiri’, which have the homozygous A/A allele, were similar to that in ‘Raiou-90’ and higher than those in ‘Nozaki Hakusai No. 2’, ‘CR Kisaku-80’, and ‘Eishun’ that all have a homozygous G/G allele (Fig. 3), indicating that the expression level of BrFLC5 in pre-vernalized plants was independent of a G/A mutation in its third intron.

The promoter sequences of ten accessions of B. rapa were determined. Among these ten accessions, there were two types of fragment sizes of promoter regions, 1,335 bp (Type C) and 771 bp (Type A; A/A allele, B; G/G allele) (Figs. 3, 4). There were no sequence differences among accessions having the same fragment sizes, and the 771 bp sequence was identical to the sequences in the reference genome. The 1,335 bp fragment has 584 bp insertion (Fig. 4) and all accessions having this insertion tended to have higher expression levels of BrFLC5 before vernalization (Figs. 3, 4). Blast searches using this 584 bp sequence as a query revealed a homologous sequence in the promoter region of FLC.A03b of Brassica napus (Identity 583/584, 99.8%) that is an ortholog of BrFLC5 (Akter et al. 2021), and there were many regions homologous to this 584 bp sequence in the genome of B. rapa (Fig. S2).

In response to four weeks of cold treatment, BrFLC5 expression levels decreased to between 6.2–39.4% of the non-vernalized level, and the average repression rate in accessions having G/G homozygous (25.1%) was similar to those having A/A homozygous (24.3%) (Fig. 3).

BrFLC5 showed the lowest expression level among four BrFLC paralogs

We performed RNA-seq in the 14-day first and second leaves in four accessions of B. rapa (Nou 6, ‘Aishinku No. 3’, ‘CR Takamaru’, and ‘Homarenokiwami’); three of the four accessions (Nou 6, ‘Aishinku No. 3’, and ‘CR Takamaru’) have a homozygous G/G allele with 584-bp insertion in the BrFLC5 promoter region. From 82.2%-92.1% of total filtered sequence reads (15.5 M to 21.2 M reads) were mapped to the reference genome (Table S2). Expression levels of BrFLC1, BrFLC2, BrFLC3, and BrFLC5 were identified using the FPKM value. BrFLC3 had the highest expression levels in all four accessions, from 40.1–56.9% of the total BrFLC expression level, and BrFLC5 had the lowest expression levels, from 2.4–6.2% of the total BrFLC expression level (Fig. 5).

Flowering time is an important trait for B. rapa vegetables, and FLC plays an important role in controlling flowering time in B. rapa (Akter et al. 2021). Leafy and root B. rapa vegetables need a high requirement for vernalization to prevent premature bolting, which reduces yield and quality (Akter et al. 2021). To achieve a high dependence on vernalization for flowering, a high level of the total amount of BrFLC expression in pre-vernalized plants is required together with a low repression rate of the total amount of BrFLC expression following prolonged cold treatment (Takada et al. 2019; Akter et al. 2021). These two characteristics also contribute to the difference in vernalization requirements in canola (B. napus); winter types, which have high vernalization requirements, showed a high level of total BnFLC expression or residual BnFLC expression level in specific paralogs following prolonged cold treatment (Calderwood et al. 2021). Since total BrFLC expression level in pre-vernalized plants is important, loss of function of one BrFLC paralog will result in a reduction in the total expression level of functional BrFLCs, and a reduction in the vernalization requirement. For example, the early flowering accession, yellow sarson, has a loss of function of BrFLC1 and BrFLC2 (Li et al. 2009; Yuan et al. 2009). Similarly, if one BrFLC paralog is not reduced in expression following prolonged cold treatment, the repression rate of total BrFLC expression will be reduced, resulting in a higher vernalization requirement. For example, in Tsukena No. 2, the expression levels of BrFLC2 and BrFLC3 are less reduced following prolonged cold treatment (Kitamoto et al. 2014), and the expression level of BrFLC1 is less reduced in BRA2209 (Takada et al. 2019); these two accessions both have a high vernalization requirement.

BrFLC5 originated from the α replication of the common ancestor of A. thaliana and B. rapa, but it was not localized in the syntenic region where AtFLC is located (Yang et al. 2006; Akter et al. 2019). In the reference genome of B. rapa, BrFLC5 was considered to be a pseudogene because it lacks two exons (Schranz et al. 2002; Wang et al. 2011). Furthermore, the expression level of BrFLC5 is the lowest among the four paralogs (Xi et al. 2018). BoFLC5 can also be a pseudogene and has the lowest expression levels among the paralogs in cabbage (Itabashi et al. 2019). The expression levels of BnaFLC.A03b and BnaFLC.C03b, which are orthologs of BrFLC5 and BoFLC5, respectively, tended to be lower than the other BnFLC paralogs in B. napus (Calderwood et al. 2021), suggesting that low expression levels of FLC5 were fixed early after the speciation of Brassica species. Therefore, there are a few studies examining the relationship between BrFLC5 and flowering time/vernalization requirement. However, some accessions having a full BrFLC5 amino acid sequence have been identified (Xi et al. 2018). We also identified some accessions with full annotation of the BrFLC5 amino acid sequence among 310 B. rapa accessions. In this study, the frequency of the A allele (non-functional) is higher than the G allele (putative functional), and turnip showed the highest ratio having a homozygous G allele (31.3%). These results are similar to a previous report using different accessions (Xi et al. 2018), indicating that these results could reflect the overall B. rapa accessions. Furthermore, we showed that BrFLC5 acts as a floral repressor, allowing us to discuss the contribution of BrFLC5 to flowering time/vernalization requirement.

QTL analysis using Nou 6 and A9709, which are high and low vernalization requirements, respectively, were performed in field conditions, sowing seeds in the winter season in Japan (January or February) without any cold treatment. Two QTLs were identified and one of them covered BrFLC5 (Kakizaki et al. 2011). In this study, we confirmed that A9709 has a homozygous A-allele and Nou 6 has a homozygous G-allele, suggesting that the flowering time difference of these two accessions might be due to a difference in BrFLC5 function. The contribution of functional BrFLC5 to flowering time variation was also shown using the F₂ population derived from accessions having A homozygous and G homozygous alleles (Xi et al. 2018). Xi et al. (2018) showed that BrFLC5 functions weakly to control flowering time. As above mentioned, the BrFLC5 expression level in pre-vernalized material is important for a vernalization requirements (Takada et al. 2019; Akter et al. 2021). A variation of BrFLC5 expression levels among accessions in pre-vernalized plants was observed in this study. In B. rapa, BrFLC5 expression levels were the lowest among the four paralogs (Xi et al. 2018). The expression levels of BrFLC5 between accessions used for creating F₂ population were not shown (Xi et al. 2018), but if the accession having a functional BrFLC5 had a low expression level in pre-vernalized material, the contribution of a functional BrFLC5 to flowering time would be little. Kakizaki et al. (2011) showed that expression levels in Nou 6 were higher than A9709 in pre-vernalized material and during cold treatments, thus a QTL would have been detected in the region covering BrFLC5. In this study, we also confirmed that the BrFLC5 expression level in Nou6 was higher than in other accessions before vernalization, although the expression level of BrFLC5 was the lowest among the four BrFLC paralogs. These results suggest that the BrFLC5 allele of Nou 6 could be utilized late-bolting breeding.

Transposable elements (TEs) were found in the genomic regions in FLC in B. rapa and B. napus, and sometimes affect the FLC expression levels before vernalization or the repression rate of FLC expression following prolonged cold treatments (Akter et al. 2021). We found that 584 bp insertion in the promoter region led to increased BrFLC5 expression before vernalization, but did not affect the repression of BrFLC5 expression following prolonged cold treatments. A higher expression level before vernalization is one important characteristic of a high vernalization requirement, developing a DNA marker harboring this insertion will be useful for marker-assisted selection.

There is variation of BrFLC5 expression levels between accessions prior to vernalization, while the variation of rate of repression of BrFLC5 following prolonged cold treatments was small. Higher expression levels of BrFLC5 expression in unvernalized material could be due to the 584 bp insertion in the promoter region and could contribute to increased vernalization requirements. Thus, a DNA marker developed for the 584 bp insertion region could be useful for developing high bolting-resistant breeding in B. rapa vegetables.

Acknowledgments: We thank Ms. Tomoko Kusumi and Ms. Yumiko Arai for their technical assistance.

Author contribution: AA, TK, EI, KK, MAA, and HM performed experiments, and AA, EI, and MS performed data analysis. AA, ESD, and RF drafted the manuscript. AA, TK, KO, and RF participated in the design of the study. ESD and RF revised the manuscript. All of the authors have read and approved the final version of the manuscript.

Funding: This work was supported by Kobe University Strategic International Collaborative Research Grant (Type B Fostering Joint Re-search), Fund for the Promotion of Joint Research (16KK0171), and Grant-in-Aid for Scientific Re-search (B) (18H02173, 21H02163) of Japan Society for the Promotion of Science (JSPS) granted to R.F.

Data availability: DDBJ; accession no. DRA00929

Ethics approval: Not applicable.

Consent to participate: Not applicable.

Consent for publication: Not applicable.

Conflicts of interest: The authors declare no conflicts of interest.

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Characterization of FLOWERING LOCUS C 5 in Brassica rapa L.

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