A Single Amino Acid Change In Histone Methyltransferase CURLY LEAF Results In Premature Bolting In Chinese Cabbage (Brassica Rapa L. Ssp. Pekinensis)

Background: Flowering is an important inection point in the transformation from vegetative to reproductive growth, and premature bolting severely decreases crop yield and quality. Results: In this study, a stable early-bolting mutant, ebm3, was identied in an ethyl methanesulfonate (EMS)-mutagenized population of a Chinese cabbage doubled haploid (DH) line ‘FT’. Compared with ‘FT’, ebm3 showed early bolting under natural cultivation in autumn, and curled leaves. Genetic analysis showed that the early-bolting phenotype was controlled by a single recessive nuclear gene. Modied MutMap and genotyping analyses revealed that Brebm3 (BraA04g017190.3C), encoding the histone methyltransferase CURLY LEAF (CLF), was the causal gene of the emb3. A C to T base substitution in the 14th exon of Brebm3 resulted in an amino acid change (S to F) and the early-bolting phenotype of emb3. The mutation occurred in the SET domain (Suppressor of protein-effect variegation 3-9, Enhancer-of-zeste, Trithorax), which catalyzes site- and state-specic lysine methylation in histones. Tissue-specic expression analysis showed that Brebm3 was highly expressed in the ower and bud. Promoter activity assay conrmed that Brebm3 promoter was active in inorescences. Subcellular localization analysis revealed that Brebm3 localized in the nucleus. Transcriptomic studies supported that Brebm3 mutation might repress H3K27me3 deposition and activate expression of the AGAMOUS (AG) and AGAMOUS-like (AGL) loci, resulting in early owering. Conclusions: Our study revealed that an EMS-induced early-bolting mutant ebm3 in Chinese cabbage was caused by a nonsynonymous mutation in BraA04g017190.3C, encoding the histone methyltransferase CLF.These results improve our knowledge of the genetic and genomic resources of bolting and owering, and may be benecial to the genetic improvement of Chinese cabbage.

The photoperiod and vernalization pathways control owering in response to seasonal changes in day length and temperature [5]. In the photoperiod pathway, CONSTANS (CO) is the main positive regulator of FT/TSF. CYCLING DOF FACTORs (CDFs) are transcriptional repressors of CO. FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) and GIGANTEA (GI) form a stable complex that releases repression of CO by inducing degradation of CDF1 [6]. At the posttranscriptional level, CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and SUPPRESSOR OF PHYTOCHROME A (SPA1) form a ubiquitin ligase complex that facilitates CO degradation in the dark [5]. In the vernalization pathway, FLOWERING LOCUS C (FLC), which encodes a MADS-box transcription factor, acts as a central oral repressor by directly repressing the transcription of the oral promoting genes FT, SOC1, and SQUAMOSA PROMOTER-BINDNG PROTEIN-LIKE 15 (SPL15) [7]. FRIGIDA (FRI), which encodes a coiled-coil protein, positively regulates FLC by affecting its chromatin structure [8]. VERNALIZATION INSENSITIVE 3 (VIN3), which encodes a PHD-nger protein, is necessary for epigenetic silencing of FLC [9]. Two long noncoding RNAs (lncRNAs), cold-induced long antisense RNA (COOLAIR) and cold-assisted intronic noncoding RNA (COLDAIR), are responsible for transcriptional shutdown of FLC [10][11].
The ambient temperature pathway controls owering in response to the daily growth temperature. SHORT VEGETATIVE PHASE (SVP) plays a key role in this pathway by reducing FT transcription at lower temperatures [3,5].
Chinese cabbage is the most leafy B. rapa crop in East-Asian countries, composed of a large number of tightly wrapped heading leaves. Flowering time is an important agronomic trait for Chinese cabbage, and premature bolting can severely reduce crop yield and quality. In the present study, we characterized an earlybolting Chinese cabbage mutant identi ed from an EMS-mutagenized population. By performing MutMap and kompetitive allele-speci c polymerase chain reaction (KASP) genotyping analyses, a nonsynonymous base substitution in Brebm3 was identi ed to cause the mutant phenotype. The expression pattern of the causal gene Brebm3 was comprehensively analyzed by evaluating spatiotemporal expression, promoter activity, and subcellular localization. Transcriptome pro ling was conducted to identify potential Brebm3regulated genes responsible for owering time in Chinese cabbage. We expected our ndings to be of great signi cance for further study of the molecular mechanism of bolting and owering in Chinese cabbage.

Results
Morphological characteristics and genetic analysis of the mutant ebm3 Following EMS treatment, 528 M 0 lines were obtained. By continuous identi cation and further screening for generations, the mutant ebm3 exhibiting obvious early-bolting characteristics in spring and autumn cultivation was selected as the study material. Except for curled leaves, the mutant emb3 showed no other pleiotropic effects when compared with the wild-type line 'FT' (Fig. 1a).
Under normal cultivation conditions in autumn, the wild-type line 'FT' will not premature bolting without exposure to a prolonged cold period (vernalization); however, but the mutant emb3 exhibited obvious bolting under these conditions. To more intuitively assess the characteristics of the mutant, three indices, squaring period (SP), owering time (FT), and days to reaching a 10 cm-high elongated oral stalk (DE), were measured in 30 individuals and the average values are presented. SP, FD, and DE of the mutant ebm3 were 40, 43, and 46 days, respectively.
The reciprocal cross F 1 generation had the same phenotype as the wild-type line 'FT', indicating that the earlybolting phenotype of the mutant ebm3 was recessive and controlled by nuclear gene. In the F 2 generation, 1,225 and 401 individuals exhibited the wild-type line 'FT' and mutant ebm3 phenotype, respectively. This segregation ratio was consistent with the Mendelian ratio of 3:1 segregation (χ 2 = 0.08 < χ 2 0.05 = 3.84). In addition, all 518 BC 1 (F 1 × 'FT') generation individuals exhibited the phenotype of the wild-type line 'FT'. For the BC 1 (F 1 × ebm3) generation, 264 and 272 individuals exhibited the wild-type line 'FT' and mutant ebm3 phenotype, respectively. This 1:1 segregation ratio was consistent with the expectations (χ 2 = 0.09 < χ 2 0.05 = 3.84). These data indicated that the phenotype of the mutant ebm3 was controlled by a single, recessive nuclear gene (Table 1).  To verify the reliability of these six candidate SNPs, the sequences surrounding them were ampli ed from DNA from the mutant ebm3 and wild-type line 'FT'. Sequence alignment results showed that all SNPs were real (data not shown), and the sequencing peak of the C/T allele of SNP 13,129,878 was displayed in Fig. 1c.
We conducted genotyping analysis of 200 F 2 individuals to con rm the causal SNP for the early-bolting mutant phenotype. A KASP assay showed that SNP 13,129,878 of BraA04g017190.3C co-segregated with the mutant phenotype in the F 2 individuals. All F 2 individuals exhibited a T:T genotype, whereas the wild-type line 'FT' was C:C genotype (Fig. 1d). For the other ve SNPs, recombinants (C:T genotype) were detected in the F 2 individuals, indicating these SNPs did not co-segregate with the mutant phenotype (data not shown). These results con rmed that BraA04g017190.3C, harboring SNP 13,129,878, was the causal gene of the mutant ebm3. Gene annotation con rmed that BraA04g017190.3C encoded an important histone methyltransferase, CLF. Loss-of-function of A. thaliana homologous CLF (At2g23380) causes an early owering phenotype and upwardly curled leaves [30]. In this study, the causal gene of the mutant ebm3 is referred to as Bremb3.
The full-length gene sequence of Bremb3 was found to be 4,406 bp, and Bremb3 consists of 17 exons and 16 introns (Fig. 2a). Sequence alignment of the mutant ebm3 and wild-type line 'FT' showed that besides SNP 13,129,878 in the 14th exon, there was no variation. The coding sequence of Bremb3, 2,715 bp in length, encodes a protein of 904 amino acids with a molecular weight of 1000 kDa and a theoretical pI of 90.5. The SNP 13,129,878 (C→T) of Brebm3 causes an amino acid substitution from serine (S) to phenylalanine (F) at residue 766 (Fig. 2b). The amino acid substitution is localized in a typical SET domain that is highly conserved among diverse species ( Fig. 2c; The original gure refers to Additional le 1: Figure S1).

Spatiotemporal expression of Brebm3
To study the relative expression levels of Brebm3 in different tissues, RNA from root, stem, leaf, bud, ower, and pod of the wild-type line 'FT' was used as a template for qRT-PCR. The data showed that Brebm3 expression was the highest in the ower, followed by the bud, leaf, and pod, with extremely low expression in the stem (Fig. 3a).

Brebm3 promoter activity
We analyzed Brebm3 promoter activity in A. thaliana tissues by using the fusion vector Brebm3 pro:GUS. Following screening based on hygromycin resistance and the GUS reporter gene, 32 transgenic plants were obtained (Fig. 3b, c; The original gure of Fig. 3c refers to Additional le 1: Figure S2). Tissues (root, stem, leaf, in orescence, and pod) of homozygous T 2 generation transgenic plants were stained in a GUS histochemical assay. Analysis of the transformed plants showed that Brebm3 transcriptional activity was the highest in the in orescence, followed by leaf and pod (Fig. 3d). These results were in line with those of spatiotemporal expression analysis, indicating that ebm3 expression shows a tissue-speci c pattern.
Brem3 is located to the nucleus To detect the subcellular localization of Brebm3, we constructed recombinant 35S:GFP-Brebm3 plasmid for transiently expression. Co-localization analysis of GFP and mKate uorescent signals in the transformed Arabidopsis mesophyll cell protoplasts indicated that 35S:GFP-Brebm3 vector was exclusively located in the nucleus, suggesting that Brem3 is a nucleoprotein.Whereas the 35S:GFP control vector was detected within both the nucleus and cytoplasm (Fig. 4).
Transcriptome pro ling of the mutant ebm3 We further conducted RNA-Seq to analyze the molecular mechanism of Brebm3 in regulating early bolting in Chinese cabbage. After ltering and quality control, 22 Figure S3). The most signi cantly enriched GO terms were "transcription, DNA-templated" in biological process, "plasma membrane" in cellular component, and "transcription factor activity, sequence-speci c DNA binding" in molecular function. 944 DEGs were assigned to 19 signi cantly enriched KEGG pathways (p vaule ≤ 0.03), including starch and sucrose metabolism, phenylalanine metabolism, and circadian rhythm-plant (Additional le 1: Figure S4).
Flowering is an essential stage in the life cycle of higher plants and is tightly controlled by complex molecular pathways. To further explore the molecular mechanism underlying the early-bolting phenotype of the mutant ebm3, we conducted an in-depth analysis of the transcriptome data. The causal gene Brebm3 (BraA04g017190.3C) was not signi cantly differentially expressed between the mutant ebm3 and wild-type line'FT' (Additional le 2: Table S5; Fig. 5a). The oral integrator genes FT, TSF, TFL1, and SOC1, the vernalization pathway-related genes FLC and FRI, the ambient temperature-related gene SVP, the photoperiod pathway-related gene GI, age pathway-involved genes SPL3, SPL9 and SPL15, gibberellin pathway-involved genes GA20OX1-4, oral homeotic genes AG, AGL19, SEP1/AGL2, SEP2/AGL4 and FUL/AGL8 were detected in our data. The SOC1 genes (BraA04g031640.3C, BraA05g005370.3C, and BraA03g023790.3C), two AG genes (BraA03g048590.3C and BraA01g010430.3C), AGL19 (BraA01g013570.3C), SEP1/AGL2 (BraA10g023780.3C), SEP2/AGL4 (BraA01g044460.3C) were signi cantly upregulated in the mutant ebm3 as compared to wild-type line 'FT' (Additional le 2: Table S5). We assessed Brebm3, SOC1, and FLC gene expression by RT-qPCR to verify the reliability of the RNA-seq data. As shown in Fig. 5, the expression patterns of the eight genes were generally consistent with the transcriptome pro le.

Discussion
Chinese cabbage is generally grown in the autumn and spring seasons. The harvest period of spring varieties coincides with the off-season supply, accommodating for the annual demand. Unlike autumn varieties, the seedlings of spring varieties require exposure to a prolonged cold period. Premature bolting, which mostly occurs in spring varieties and is caused by low-temperature and long-day conditions, severely reduces crop yield and quality. To overcome restrictions related to planting season and geographic distribution, diverse spring varieties with high bolting resistance have been selected through the incorporation of genetic resources in the past few years [31].
A good understanding of the molecular mechanism of owering time can accelerate the breeding of boltingresistant varieties [20]. To adapt to the diverse agro-environments, vegetable crops have employed a complex and elaborate network that tightly controls owering time. Mutants are important materials for plant functional genomics studies. The genetic basis of natural variation in owering time has been extensively evaluated in quantitative trait loci (QTL) studies [32][33][34][35]. However, there is insu cient natural variation for effective research due to the low probability. Here, we characterized an EMS-induced early-bolting mutant, ebm3, with curled leaves, which was derived from a Chinese cabbage DH line 'FT' (Fig. 1a). The genetic background of the mutant ebm3 was relatively homozygous, and highly consistent with that of the wild-type line 'FT', which was conducive to highlight the bolting phenotype caused by the causal gene. Genetic analysis showed that the mutant trait was quality character, controlled by a single recessive nuclear gene (Table 1).
Multi-season planting indicated that the early-bolting trait was genetically stable and not affected by external factors. Therefore, the mutant ebm3 is an ideal material to study important node genes in the owering regulatory pathways in Chinese cabbage.
EMS mutagenesis has multiple advantages, such as high mutation frequency, easy screening, and stable inheritance, which is why EMS is the most widely used chemical mutagen in plants [36]. The combination of high-throughput sequencing with bulk segregant analysis (BSA) has laid the foundation for rapid mining of new genes using mutants, which has greatly facilitated functional genome studies. In our study, we used a modi ed MutMap method and KASP genotyping to map the causal gene. Brebm3 (BraA04g017190.3C), encoding histone methyltransferases CLF, was found to be responsible for the early-bolting trait (Fig. 1b-d). A nonsynonymous SNP in the 14th exon of Brebm3 caused an amino acid substitution from S to F (Fig. 2a). Unlike loss-of-function of Arabidopsis CLF, a single amino acid change of in the Enhancer of zeste (E(z)) ortholog CLF, clf-59 retained FLC repression by promoting histone H3 lysine 27 trimethylation (H3K27me3) deposition in FLC chromatin, causing early owering [37]. Sequence comparison of CLF of various species revealed that the protein has a highly conserved SET domain, and the nonsynonymous SNP was located in this domain (Fig. 2b, c). The SET domain is a 130-140-amino acid evolutionarily conserved sequence motif [38]. SET domain proteins have been characterized in diverse plant species, including Arabidopsis, rice, maize, barley, grapevine, and poplar [39][40][41][42][43][44].  [45]. Moreover, SDG proteins have been suggested to affect owering time. Mutations in ve Arabidopsis SDG genes, including ASHR3/SDG2 [46], ASHH2/SDG8/EFS [47], ATX1/SDG27 [48], ATXR7/SDG25 [49], and CLF/SDG1 [30], cause an early-owering phenotype, and mutations in Arabidopsis ASHH1/SDG26 [50], and three rice genes, including SDG708 [51], SDG724 [52] and SDG725 [53], confer a lateowering phenotype. The present and previous studies indicate that an amino acid substitution in the SET domain of Brebm3 is expected to cause the early-bolting phenotype in Chinese cabbage.
Epigenetic factors play crucial roles in owering regulation by activating or repressing the transcription of owering genes. Two functionally distinct multiprotein complexes of the Polycomb Group (PcG), PcG Repressive Complex 1 (PRC1) and PRC2, are the core epigenetic factors in eukaryotes [54]. PRC2 is a key repressive epigenetic mark, which maintains the repressed state of a target gene by catalyzing H3K27me3 [55]. In A. thaliana, PRC2 acts on various growth and developmental processes, including leaf morphology, oral organogenesis, cell pluripotency, vegetative-to-reproductive phase transition, and embryonic development [58][59][60][61]. In A. thaliana, CLF is the main component of the E(z) subunit of PRC2 [62]. Extensive evidence supports that CLF maintains suppressed expression of FLC and FT, as well as that of several oral homeotic genes, including AG, AGL19, and SEP3 [30,60,[63][64][65][66][67]. As a typical example of reprogramming of epigenetic states in plants, H3K27me3 repressive marks on FLC can be erased by ELF6 histone demethylases during seed development [68]. A noncoding RNA transcribed from the second intron of AG associated with CLF can silence AG expression by mediating H3K27m3 deposition to form repressive chromatin [69]. The temporal-speci c interaction of NF-YC and CLF mediates epigenetic regulation by derepressing FT expression in photoperiod-induced owering [4]. Loss-of-function of ASHH1/SDG26 retains SOC1/AGL20 repression by reducing H3K4me3 and H3K36me3 deposition in SOC1/AGL20 chromatin, resulting in the late-owering phenotype [50]. A tilling mutant of B. rapa, braA.clf-1 (Gln615Stop), displayed small plant size, altered oral development, and curled leaves due to reduced H3K27me3 and high expression levels of oral homeotic genes such as AG and AGL loci [70]. In the present study, transcriptome pro ling revealed that AG and AGL loci, e.g., SOC1/AGL20, AGL19, SEP1/AGL2, SEP2/AGL4, were signi cantly upregulated in the mutant ebm3 (Additional le 2: Table S5). Therefore, it is reasonable to speculate that mutation in Brebm3 mediates reduced H2K27me3 deposition and high expression of the AG and AGL loci in Chinese cabbage.

Conclusions
The transition to owering is an essential developmental stage in the plant life cycle. Plants need to owering in the most favorable conditions to ensure maximal reproductive success. Timely owering is conducive to crop production, harvesting, and marketing. In this study, based on MutMap and KASP genotyping analyses, Brebm3, encoding the histone methyltransferase CLF, was determined to control the early-bolting trait in Chinese cabbage. Brebm3 was highly expressed in the oral organs, and the translation product localized in the nucleus. Transcriptome pro ling was conducted to identify potential CLF-repressed genes in mutant ebm3. Collectively, our ndings will be invaluable for understanding the molecular mechanism of owering time in Chinese cabbage.

Plant materials and genetic analysis
The Chinese cabbage DH line 'FT' was used as a wild-type line in this study propagated from Chinese cabbage variety 'Fukuda 50', which was screened by Shenyang greenstar Chinese cabbage research institute (Shenyang, China) [71]. An early-bolting mutant with stable inheritance was obtained from 'FT' by multigenerational screening after EMS mutagenesis, and was designated ebm3. The mutant generation method has been described in detail in Fu et al. [72]. To study the inheritance characteristics, the mutant ebm3 and wild-type line 'FT' were used as parents. An F 1 generation obtained by a reciprocal cross was selfcrossed to obtain an F 2 segregating generation. The F 1 generation was backcrossed with both parents to obtain a BC 1 population. The segregation ratios of the F 2 and BC 1 populations were analyzed using the chisquare test. The F 2 population was also used for mutant gene identi cation and genotyping. Individual plants were grown in a greenhouse at Shenyang Agricultural University.
A. thaliana ecotype Columbia-0 (Col-0) was obtained from the Arabidopsis Biological Resource Center (ABRC; http://abrc.osu.edu) and preserved by the Liaoning Key Laboratory of Genetics and Breeding for Cruciferous Vegetable Crops at Shenyang Agricultural University. All Arabidopsis plants were grown in a growth chamber at Shenyang Agricultural University. Culture conditions were as described by Wang et al. [73].

Evaluation of bolting characteristics
Three bolting characteristics were measured, i.e., SP, DE, and FT, as previously reported by Yu et al. [74]. Candidate SNP identi cation by the MutMap method A modi ed MutMap method was used to identify the candidate gene for the mutant ebm3 [75]. DNA was extracted from 15 F 2 individuals with the early-bolting phenotype and the parental lines using a DNAsecure Plant Kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. Equal amounts of each DNA from the 15 F 2 individuals were mixed to construct an offspring pool. Sequencing libraries of the mutant ebm3 (ebm3), wild-type line 'FT' ('FT'), and offspring pool (F 2 _ebm3) were generated using a TruSeq Nano DNA HT Sample preparation Kit (Illumina, San Diego, CA, USA). The libraries were sequenced using Illumina HiSeq TM PE150 (Novogene Co., Ltd., Beijing, China). After quality control and ltration, the clean reads of each sample were aligned to the B. rapa reference genome (http://brassicadb.org/brad/, v3.0) using Burrows-Wheeler Alignment tool (BWA) [76]. Alignment les were converted to BAM les using the SAMtools software [77]. SNP calling was performed using GATK [78] and annotated using ANNOVAR [79]. The screened SNPs between the M and W library were used to calculate the SNP index in offspring-pool library. The sliding window method was used to determine the SNP index of the whole genome in offspring pool library.

SNP genotyping by KASP
To verify the real existence of the candidate SNP, a sequence surrounding the locus was ampli ed using DNA from the mutant ebm3 and wild-type line 'FT' and the primer pair 5′-ATACTTTGCTTTGGTTGACTCTAC-3′ and 5′-TCGTGTTTACTTACACTGTTCTGT-3′. Puri ed PCR product was ligated into the PMD 18-T Vector (Takara Biotech Co., Ltd., Dalian, China), and transformed into TOP10 competent cells (ComWin Biotech Co., Ltd., Beijing, China). The recombinant plasmid was sequenced by Sanger sequencing (Genewiz lnc., Tianjin, China). Sequence alignment was performed using the SeqMan software.
The candidate SNP was con rmed using a KASP assay to detect whether the locus co-segregated with the mutant phenotype. For KASP genotyping, DNA from 200 F 2 individuals with the early-bolting phenotype was used. Two allele-speci c primers carrying the uorescence probes FAM and HEX and the candidate SNP at the 3′ end (Primer_AlleleFAM: AGGTTTTACTTGGAATATCTGATGTATC; Primer_AlleleHEX: CAGGTTTTACTTGGAATATCTGATGTATT), and a common genome-speci c primer (Primer_Common: GTTACGCATCTACTATACCTTTAGGAAAG), were designed following standard KASP guidelines of the laboratory of the Government Chemist (LGC http://www.lgcgenomics.com/). The primer mixture was prepared as recommended by LGC Genomics. PCR mixture preparation and cycling were conducted as described by Xi et al. (2018). Fluorescence data were read using a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA , USA).

Quantitative reverse transcription-PCR (qRT-PCR)
Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using FastKing gDNA Dispelling RT SuperMix (Tiangen Biotech Co., Ltd., Beijing, China). The reaction system was performed with UltraSYBR Mixture (ComWin Biotech Co., Ltd., Beijing, China). PCR ampli cation was run in a QuantStudio TM 6 Flex Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The Actin gene was selected as an internal control. Relative gene expression data were calculated by the 2 −△△Ct method [80]. The data were analyzed using the QuantStudio TM 6 Flex Manager software. Three technical and biological replications were included for each sample. The qRT-PCR primer pairs were listed in Additional le 2: Table S6.

Promoter activity assay
The promoter sequence (2,000 bp upstream of the initiation codon) of Brebm3 was ampli ed from DNA of the wild-type line 'FT', using the primer pair 5′-ccgggatccTCTAGAgcgaagccaagtagtaagcact-3′ and 5′-gcaggtcgacTCTAGAtgtcgaggagccagatcgga-3′ (uppercase letters indicate an XbaI site). The ampli cation product was digested with XbaI and ligated into the pC1301IgT vector containing fused GUS reporter gene. The recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3103. A. tumefaciensmediated transformation was used to transfer the Brebm3 pro:GUS vector into A. thaliana Col-0 by the oral dip method. Transgenic plants were screened on 0.5× Murashige and Skoog (MS) medium containing 0.25 mg L -1 hygromycin. The GUS reporter gene was ampli ed from DNA of all hygromycin-resistant plants, using the primer pair 5′-AACCACAAACCGTTCTACTTTACTG-3′ and 5′-TACATTACAAGACGCTGCGAGT-3′. A GUS histochemical assay was performed on various tissues (root, stem, leaf, in orescence and pod) of the transgenic plants [81].

Subcellular localization
The full-length Brebm3 coding sequence without the stop codon was ampli ed from cDNA of the wild-type line 'FT', using the primer pair 5′-cgatCACCTGCaaaacaacatggcgtcgggagcttcgcc-3′ and 5′-cagtCACCTGCaaaatacaagcaaccttcttgggtctac-3′ (uppercase letters indicate an AarI site). The ampli cation product was digested with AatI and inserted into the pBWA(V)HS-ccdb-GLosgfp vector, resulting in an Nterminal fusion vector with GFP under the control of the CaMV35S promoter (35S:GFP-Brebm3). The 35S:GFP vector was used as a control. The constructs were respectively transiently transformed into A. thaliana mesophyll cell protoplasts, as described by Wang et al. [70]. The pBWA(V)HS-NLS-mKATE vector was served as a nucleus marker. Fluorescence data were obtained by confocal laser-scanning microscope (Leica TCS SP8, Wetzlar, Germany).Excitation wavelengths used were 488nm for GFP and 561nm for mKate. Emission wavelengths were 507nm for GFP and 580nm for mKate.

Transcriptome pro ling
When the mutant ebm3 reached the critical point of bolting, total RNA was extracted from the SAM of mutant and wild-type line 'FT', with three biological replications (designated emb3-1, emb3-2, emb3-3, 'FT'-1, 'FT'-2, and 'FT'-3). RNA quantity and purity was analyzed using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent Technologies, SantaAdditional le 2: Clara, CA, USA). Following puri cation and fragmentation, the cleaved RNA fragments were reverse-transcribed to create cDNA libraries using a mRNASeqsample preparation kit (Illumina, San Diego, CA, USA). The libraries were paired-end sequenced using an Illumina HiSeq 4000 platform (LC-Bio Technology Co., Ltd., Hangzhou, China). Following quality control and ltration, the clean reads were aligned to the B. rapa reference genome (v3.0) using HISAT. StringTie was used to assemble the alignments into transcripts and to compute transcript abundance by calculating Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Differentially expressed genes (DEGs) were de ned based on |log 2 (fold change)| ≥ 1 and p <0.05, using the R package Ballgown [82]. Functional analysis of the DEGs included Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses [83][84].

Availability of data and material
The datasets supporting the conclusions of this article are included within the article and its additional les.
Any other datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
CT and JR analyzed the data and drafted the manuscript. LW, WF, JZ, and MQ participated in the creation of materials and performed the experiments. XY, HF and ZL directed the whole study including designing experiments and revising the manuscript. All authors have read and approved the nal manuscript. Figure 1 Identi cation of the mutant ebm3 and candidate SNPs. a Phenotypic characterization of the wild-type line 'FT' (left) and the mutant ebms3 (right). b The distribution of SNP index in offspring pool on chromosome A04 generated by MutMap analysis. b Sequencing peak of the C/T allele of SNP 13,129,878 generated by Sanger sequencing. d KASP genotyping results of SNP 13,129,878 in F2 individuals.

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