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Research Article

Differential Expression of Flowering Genes between Rapid- and Slow-Cycling Brassica rapa

Hayong Song1, Xiangshu Dong1,3, Hankuil Yi1, Ill-Sup Nou2, Yoonkang Hur1,*
Plant Breeding and Biotechnology 2016;4(2):145-157.
Published online: May 31, 2016

1Department of Biological Sciences, College of Biological Science and Biotechnology, Chungnam National University, Daejeon 34134, Korea

2Department of Horticulture, Sunchon National University, Suncheon 57922, Korea

3School of Agriculture, Yunnan University, Kunming, Yunnan 100044, China

*Corresponding author: Yoonkang Hur, ykhur@cnu.ac.kr, Tel: +82-42-821-6279, Fax: +82-42-822-9690
• Received: May 5, 2016   • Revised: May 15, 2016   • Accepted: May 17, 2016

Copyright © 2016 The Korean Society of Breeding Science

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • Flowering time is a very important agronomic trait in Brassica crops and regulation of the time is one of major factor in the breeding program. To understand the control of flowering time in Brassica rapa, we have carried out Br300K microarray with two contrasting Brassica inbred lines, Rapid Cycling B. rapa (RCBr) as rapid cycling type and B. rapa ssp. pekinensis inbred line Chiifu as slow flowering phenotype. Reproductive process-related genes were specifically expressed in RCBr, whereas environmental stimuli-responsive genes in Chiifu. Flowering stimulating genes, such as BrFT and BrSOC1, were preferentially expressed in RCBr, while flowering repressing genes, such as BrFLC and BrMAF4, expressed in Chiifu. Several paralogues present in B. rapa, BrFLCs and BrCOLs, were expressed with paralog-specific pattern depending on flowering phenotypes: i.e., BrFLC1 and BrFLC2, major floral repressors, were expressed in Chiifu, BrFLCL/BrFLC5 in RCBr and BrFLC3 in both plants. The expression of several flowering repressing genes was gradually decreased in RCBr growth, but increased in Chiifu growth. However, the expression of genes involved in photoperiodic flowering was no difference between these two plants under LD and SD conditions, indicating photoperiodic pathway is not major factor to distinguish fast vs. slow flowering in B. rapa. The mechanism underlined in the rapid or fast flowering of RCBr would be further elucidated in association with the controlling mechanism of its short life span.
  • Key words: Br300K microarray, Flowering time, RCBr, Chiifu, Photoperiod
Brassica rapa is an important crop species of the genus Brassica, which is cultivated worldwide and includes a variety of vegetable crops such as Chinese cabbage, Pak-choi, turnip, and turnip green, and oil seed crops including turnip rape and sarson (Gómez-Campo and Prakash 1999). B. rapa ssp. pekinensis (Chinese cabbage) belongs to one of nine subspecies, which is characterized by requirement of long period of cold treatment for flowering. Rapid cycling B. rapa (RCBr), also known as Fast Plant, is a widely used model organism in biology education (Williams and Hill 1986; Musgrave 2000). RCBr was developed by selection of B. rapa for several traits, such as short time to flowering, rapid seed maturation, lack of seed dormancy, petite growth habit, and high female fertility (Williams and Hill 1986). The result is a plant with 10-generations per year. The earliness of flowering is an important trait for crops with higher seed yields (such as oilseed rape), whereas the lateness of flowering is a favourable trait for crops with high leafy head yields (such as Chinese cabbage) (Mao et al. 2014; Wang et al. 2014).
Flowering, a developmental process that transits vegetative stage to reproductive stage, is very important trait in Brassica species. Flowering in Arabidopsis thaliana, a model plant, is controlled by at least five genetically defined regulatory pathways: the photoperiod-dependent, vernalization-dependent, gibberellic acid (GA)-dependent, autonomous promotion, and FRIGIDA (FRI)-dependent pathways (Lee et al. 2000). Flowering time of the Chinese cabbage is largely controlled by vernalization and photoperiod (Kakizaki et al. 2011). Vernalization, exposure to low temperature, rerepresses FLOWERING LOCUS C (FLC) which is a central repressor of flowering (Helliwell et al. 2015). Plants with high FLC activity are late-flowering because FLC directly represses the expression of the floral inducers FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) (Helliwell et al. 2006). Four copies of FLC have been reported in B. rapa, and five copies were identified in B. napus (Lin et al. 2005; Kim et al. 2006; 2007). A genetic-genomics approach revealed that BrFLC2 is a major regulator of flowering time in B. rapa (Xiao et al. 2013; 2014). Recently, Li et al. (2016) reported that mutation in FLC1 and FLC2 leading to production of truncated proteins was associated with shortness of flowering time in B. rapa. In photoperiodic flowering pathway, CONSTAS (CO) induces the day-length specific expression of FT in leaves (Putterill et al. 1995; Samach et al. 2000). Both CO and FT are mainly expressed in the vascular tissue of leaves (Takada and Goto 2003).
Although the regulation of flowering genes are important in Brassica breeding, information on RCBr with respect to gene expression and rapid flowering is still unclear. To get insight on flowering-control mechanism, we have applied Br300K microarray (Dong et al. 2013) using two contrast B. rapa inbred lines, RCBr as rapid cycling B. rapa and B. rapa ssp. pekinesis inbred line Chiifu as slow cycling B. rapa. Differentially expressed genes (DEGs) and major flowering genes were identified and characterized.
Plant materials
Plant materials were rapid cycling Brassica rapa inbred line (RCBr24001) as an extremely early-flowering type and B. rapa ssp. pekinensis, inbred line Chiifu as slow or late flowering type. Plants were grown under either long day (LD; 16 hours light/8 hours dark) or short day (SD; 8 hours light/16 hours dark) conditions with 140±2 μl/m2/s light intensity at 22°C±5°C. For microarray, shoots were sampled at both cotyledon stage and five-leaves stage of RCBr (2 weeks old) and Chiifu (4 weeks old), and stored at −70°C until use. For expression study during RCBr development, RCBr and Chiifu (as control) were sampled until RCBr started flowering. To compare expression of several flowering genes, plants were grown under LD and SD conditions until 21 day after germination (DAG), and sampled at indicated time.
RNA isolation and hybridization to the Br300K microarray GeneChip
Shoot samples from cotyledon stage and five-leave stage were ground in liquid nitrogen, and equal amounts of powders were pooled. Total RNA was isolated from samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and further purified with a NucleoSpin RNA Clean-up Kit (Macherey-Nagel GmbH&Co. KG, Düren, Germany). For biological repeats, RNAs were extracted from two independent samples, and subjected to microarray experiment. Microarray experiments and subsequent analyses were performed as described in our previous report (Dong et al. 2013). To obtain insights regarding the putative biological functions and biochemical pathways of DEGs, we carried out enrichment analyses by searching Gene Ontology (GO) (Ashburner et al. 2000), agriGO (Du et al. 2010), and the Kyoto Encyclopedia of Genes and Genomes (Kanehisa et al. 2008).
Reverse transcription polymerase chain reaction (RT-PCR) analysis
Total RNA was isolated using TRIzol reagent (Invitrogen), and the total RNA (1 μg) from each sample was subjected to the first-strand cDNA synthesis using the Ace-α kit with the Oligo(dT) primers (Toyobo, Osaka, Japan). Complementary DNA was diluted 10-fold and 1 μl of diluted cDNA was used in a 20-μl PCR mixture. RT-PCR primers were listed in Table 1, and primers for BrACT2 (B. rapa Actin 2) used as control are 5′-GAACCGGGTGCTCCTCAGGA-3′ (forward) and 5′-ATGGTACCGGAATGGTCAAGGC-3′ (reverse). To design paralog specific primer, expressed sequence tag (EST)-sequences were subjected to BRAD (http://brassicadb.org/brad/; V1.5), and all sequences were then aligned to figure out polymorphism. A standard PCR was performed with a 5-minute denaturation at 94°C, followed by 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 90 seconds. PCR products were analyzed following electrophoresis through a 1.2% agarose gel.
Overall microarray
Among 47,553 EST clones deposited on Br300K microarray, 40,047 (84%) showed over 500 in probe intensity (PI), which means their transcripts could be detected by regular RT-PCR (Supplementary Table 1). In addition, 8,542 clones were not annotated with Arabidopsis gene locus (18% and expressed as ‘no_hit_found’, HNF). Estimated number of genes in B. rapa ssp. pekinensis inbred line Chiifu and RCBr will be 44,710: 41,173 from BRAD (http://brassicadb.org/brad/; V1.5) (Wang et al. 2011), and 3,537 novel genes recently identified by deep RNA-Seq (Devisetty et al. 2014). Our microarray included more number of EST clones than the estimated numbers, but actual coverage of unigenes will be below 80% due to overlapped EST clones.
Differentially or specifically expressed genes
Genes showing RCBr-specific expression were defined as those genes with PI values greater than 1,000 in RCBr but less than 500 in Chiifu (Supplementary Table 2). Chiifu-specific expression genes were selected with same strategy as RCBr (Supplementary Table 3). RCBr- and Chiifu-specifically expressed genes were 541 and 1,037, respectively. Among them, 178 (33%) and 446 (43%) genes from RCBr and Chiifu, respectively, were NHF: i.e., no Arabidopsis counterpart to be present. These NHFs will be Brassica specific genes (or ophan genes). Only 363 and 591 genes from RCBr and Chiifu, respectively, were annotated with Arabidopsis genome data. RCBr-specific genes included many flowering-stimulating genes and transposable elements. On the other hand, Chiifu-specific genes included several floral repressor genes, such as FLC and MAF4.
To obtain insights regarding the putative biological functions and biochemical pathways of DEGs, we carried out GO enrichment analyses by searching agriGO (Du et al. 2010) using RCBr- (Supplementary Table 2) or Chiifu-induced genes (Supplementary Table 3). With the threshold value (P-value) below 0.05, 51 and 159 GO items were significantly enriched by the RcBr- and Chiifu-induced genes, respectively (Fig. 1). GO items related reproductive processes were only significantly represented by RCBr-induced genes, such as gametophyte development, flower development and cell differentiation. On the other hand, GO items related to stress response or environment stimulus were only significantly represented by Chiifu-induced genes. There are 27 GO Cellular component items were significantly represented in Chiifu-induced genes, and most of them related membrane structure, no such items were represented in RCBr-induced genes.
Transcription factors (TFs)
Since TFs play roles as a master controller for plant growth and development, differentially expressed TF genes were analyzed (Table 2). In this analysis, we omitted TFs classified as flowering genes and then summarized as three catagoris; highly expressed-top 20 genes in both RCBr and Chiifu, RCBr-specific and Chiifu-specific genes. Ethylene-responsive TF RAP2-4 (RAP2.4) was the most highly expressed TF in both plants with over 40,000 in PI values. Genes expressed over two fold in RCBr and Chiifu were 19 and 20, respectively; representative genes were CAULIFLOWER (CAL) and SHINE2 (SHN2) for RCBr, and KNOTTED1-LIKE HOMEOBOX GENE 5 (KNAT5) and CURLY LEAF (CLF) for Chiifu.
Flowering genes
Genes listed in Table 3 were selected from 196 flowering-related genes on Br300K microarray: 26, 9 and 23 genes for up-regulated in RCBr, in Chiifu and both, respectively. Genes (26 genes) up-regulated in RCBr included most flowering-promoting genes, such as FT and SOC1. On the other hand, genes (9) up-regulated in Chiifu included floral repressors, such as FLC and MAF4. Twenty-three genes were highly expressed in both RCBr and Chiifu, which included DWARF AND DELAYED FLOWERING 1 (DDF1) and EARLY FLOWERING 3 (ELF3).
Interestingly, expression of several flowering genes showed paralog-specific pattern: i.e., BrFLC1 (Bra009055; Brapa_ESTC016533) and BrFLC2 (Bra028599; Brapa_ ESTC012276) in Chiifu, BrFLCL/BrFLC5 (FLC-Like, Bra022771; Brapa_ESTC017216 and Brapa_ESTC027067) in RCBr, and BrFLC3 (Bra006051; Brapa_ESTC013303 and Brapa_ESTC021830) in both plants. CONSTNAS-LIKE 9 (COL9) was also paralog specific expression: Brapa_ESTC027877 (Bra029666; BrCOL9-2) and Brapa_ ESTC051650 (Bra001264; BrCOL9-3) for up-regulated ones in RCBr, but rest (most of them are BrCOL9-1; Bra040020) in both plants. These paralog-specific expressions of flowering genes were highlight of this study.
Gibberellin metabolism-related genes
Since gibberellins (GAs) control flowering in concert with cytokinin (Matías-Hernández et al. 2016), the expression of GA-metabolism-related genes were examined (Table 4). Most genes involved in increase of the GA activity were up-regulated in RCBr; GA3OX1/GA4 (GA requiring 4) and GA20OX3 (Gibberellin 20 oxidase 3). Only GA2OX2 (Gibberelin 2-beta-dioxygenase) expression was high in Chiifu, but its function has not been identified besides its involvement in GA catabolism.
Confirmation of selected gene expression
Fig. 2 showed genes whose expression was decreased during the development of RCBr, while increased in Chiifu. These included MADS AFFECTING FLOWERING 4 (FCL4/MAF4), FLC2, FLOWERING LOCUS D (FLD), REDUCED VERNALIZATION RESPONSE 1 (VRN1), REDUCED VERNALIZATION RESPONSE 2 (VRN2, Polycomb group), METHYL-CPG-BINDING DOMAIN 9 (MBD9) and KNAT5. Most of them are known to be floral repressors. The expression of these genes was gradually increased in Chiifu even though this period belongs to be very early developmental stages.
Photoperiod is important for controlling flowering of Brassica species. To elucidate its association, several TF genes and flowering-related genes were selected and their expressions were examined during a day under LD and SD conditions. Plants grown for 21 DAG under LD and SD were sampled at indicated time (Fig. 3A). B. rapa CIRCADIAN CLOCK ASSOCIATED 1 (BrCCA1) was used as a control for the circadian clock. The expression of BrFLC2 was high in Chiifu, but not detected in RCBr. Individual paralogues of BrSEP1 (SEPALLATA1/AGL2) were specific for either plant (Fig. 3B). However, photoperiodic pathway integrator, CO and its relatives (COLs) were expressed with similar patterns in both plants (Fig. 3C). Only BrCOL2-1 was expressed in Chiifu. Taken together, there was no photoperiodic-dependent expression, but the expression of BrFLC2, BrSEP1s and BrCOL2-1 was plant-specific. These daily expression patterns was not agreement with microarray data, due to sampling time difference.
Because of striking difference in periods required for flowering between RCBr and B. rapa ssp. pekinensis, inbred line Chiifu, normalization of developmental stages of these two plants would be almost impossible. Therefore, identification of master gene(s) for controlling flowering time would be also very difficult. However, microarray data in this study showed remarkable difference in the expression of flowering-related genes between two plants; high expression of flowering promoting genes in RCBr, whereas high expression of floral repressor genes in Chiifu. DEGs between RCBr and Chiifu included many NHFs with respect to BlastN against Arabidopsis gene ID, suggesting that these would be Brassica species-specific genes. That can be supported by that most sequences were matched to the public genome data or NCBI data for B. napus and B. oleracea. For example, Brapa_ESTC051261 was the most highly expressed gene (937 fold) in Chiifu compared with RCBr. As result of BlastN in RCBr, this clone was matched to 100% with B. napus uncharacterized LOC106444620, ncRNA and 90% with B. oleracea var. oleracea uncharacterized LOC106327354. Brapa_ ESTC033111 was the most highly expressed gene (301 fold) in RCBr: 93% identical to B. napus uncharacterized LOC106448050 and 92% to B. oleracea var. oleracea uncharacterized LOC106330763. This finding strongly suggests that functional study on these Brassica-specific genes will be very important for understanding evolution of Brassicaceae crops.
Analysis of DEGs
GO enrichment analysis with DEGs showed distinct difference between RCBr, very early flowering phenotype, and Chiifu, a slow or late flowering phenotype (Fig. 1). Flowering process-related genes were specifically induced in RCBr, whereas temperature and other environment stress tolerant-related genes were expressed in Chiifu. Some general biological processes were conserved in both RCBr and Chiifu. This result might imply that Chiifu is not ready to flower yet. However, it was ruled out whether this difference is due to the difference in developmental stages during whole life cycle.
Expression of TF genes
TFs control the expression of a large number genes, thereby exerting big impact on plant morphogenesis. As shown in Table 2, many TFs were highly expressed in both plants, some were specifically expressed in RCBr or in Chiifu. Based on current knowledge, it was hard to find out the specific association with growth and development for highly expressed TFs in both plants; i.e., RAP2.4, the most highly expressed gene, is known to promote leaf senescence (Xu et al. 2010). However, several genes among RCBr-induced TFs seemed to be closely related to early flowering phenotype. TCP family determines in the height of inflorescence shoot (Davière et al. 2014), which might be related to short height of RCBr. SHN2 is known to be related to flower organ (Shi et al. 2011), and CAULIFLOWER (CAL) is closely related to another MADS box gene APETALA1 (AP1) and control floral meristem development (Alvarez-Buylla et al. 2000).
Regarding to genes induced in Chiifu, most TFs appeared to be related to plant growth and development as follows. KNAT5 regulates photomorphogenic responses and represses late steps in gibberellin biosynthesis. (Hay et al. 2002). AP2 encoding a member of the ERF (ethylene response factor) subfamily B-3 of ERF/AP2 TF family may be related to the ethylene-singalling pathway. AtMYB34 regulates indole glucosinolate homeostasis (Celenza et al. 2005). ATHB6, a target of the protein phosphatase ABI1, regulates negatively ABA responses in Arabidopsis (Himmelbach et al. 2002). Only CLF, a repressor of AG function (Goodrich et al. 1997), is related to flowering; it suppresses flowering by inducing misexpression of floral promoting genes (Lopez-Vernaza et al. 2012)
Expression of GA metabolism-related genes
GA content is associated with fertility (Plackett et al. 2012). GA 20-oxidase (GA20ox) regulates GA content (Yamaguchi 2008), and there are five GA20ox genes in Arabidopsis, GA20ox1, GA20ox2, GA20ox3, GA20ox4, and GA20ox5 (Hedden et al. 2002). Among them, GA20ox3 functions almost entirely redundantly with GA20ox1 and GA20ox2 at most developmental stages, including the floral transition, thereby these genes control floral organ growth and anther development (Plackett et al. 2012). In our results (Table 4), GA20OX3 was highly expressed in RCBr, suggesting the possible function in flowering. In addition, several GA responsive genes were up-regulated in RCBr, but their role in flowering have to be solved. Only one gene, GA2OX2 (Gibberellin 2-beta-dioxygenase) was up-regulated in Chiifu, but the function of this gene has not been identified yet.
Expression of flowering-related genes
Regarding to flowering-related genes, several flowering-promoting genes, such as PISTILLATA (PI), SEP1 and AP1, were up-regulated in RCBr, whereas floral repressors, such FLC and MAF4, were up-regulated in Chiifu (Table 3). These results can partially support why these two plants exhibit contrasting flowering phenotypes, rapid vs. slow. Interestingly, several genes contain paralogues; FLC, COL9 and SOC1. Schranz et al. (2002) and Franks et al. (2015) reported presence of four FLC paralogues in B. rapa (BrFLC1, BrFLC2, BrFLC3, and BrFLC5). A genetic-genomics approach revealed that BrFLC2 is a major regulator of flowering time in B. rapa (Wu et al. 2012; Xiao et al. 2013; 2014). Recently, Li et al. (2016) reported that early flowering phenotypes of B. rapa is related to be nonfunctional BrFLCs which are truncated form by premature termination, but slow or late flowering types contain functional or intact forms of BrFLC1, BrFLC2, and BrFLC3. In addition, Song et al. (2015) found out that expression of only one or some of paralogues is closely related to photoperiodic flowering in B. rapa. All these facts might be similar to our study: i.e., BrFLC2 were up-regulated in Chiifu, a slow or late flowering phenotype. However, BrFLC5 was up-regulated in RCBr, suggesting the another possible role of FLC besides flowering control.
RT-PCR results of selected genes demonstrated that the expression of flowering repressor genes was gradually decreased in RCBr, while gradually increased in Chiifu (Fig. 2). These included BrMBD9, BrVRN1, BrVRN2, BrFLC, and BrFLD. MBD9 controls flowering time by modulating gene expression through DNA methylation and histone acetylation (Yaish et al. 2009). VRN1 and VRN2 affect vernalization response of late flowering plants (Chandler et al. 1996). FLD (Flowering Locus D) encodes a plant ortholog of the human Lys-Specific Demethylase 1 (LSD1) protein. FLD functions in histone H3K4 demethylation and H3/H4 deacetylation to repress the expression of FLC (Jiang et al. 2007; Yu et al. 2011). Our data well agree with previous findings.
It was expected that photoperiodic flowering signaling-related genes are differentially expressed between RCBr and Chiifu, but no significant difference was observed (Fig. 3). Special attention paid to COL9 (CONSTNAS-LIKE 9) which delays flowering by down-regulation of expression of CO, FT, and SOC1 (Cheng and Wang 2005). However, three paralogues showed similar expression patterns between two plants.
PBB-4-145_suppl.1.pdf
PBB-4-145_suppl.2.pdf
PBB-4-145_suppl.3.pdf
This work was supported by Research Fund of Chungnam National University (CNU), Daejeon, Korea, to Yoonkang Hur (2015-1115-01).
Fig. 1
Functional categorization of differentially expressed genes between RCBr and Chiifu based on gene ontology dirstribution.
pbb-4-145f1.jpg
Fig. 2
Genes whose expression was decreased in RCBr, while increased in Chiifu. RCBr and Chiifu were grown in long-day condition condition for 19 days and shoot was sampled at 4th hour of light period.
pbb-4-145f2.jpg
Fig. 3
Expression of flowering- and photoperiod pathway-related genes. (A) Photoperiod for plant growth and sampling time a day. (B) Genes showing differential expression in a microarray. (C) Constance (CO) and its paralogs. BrCCA1 was used as control for circadian rhythm.
LD: long-day condition, SD: short-day condition.
pbb-4-145f3.jpg
Table 1
List of primers used in qRT-PCR analysis.
Table 1
Br_IDz) Gene Gene annotation Forward primer Reverse primer
Bra004503 BrCCA1 CIRCADIAN CLOCK-ASSOCIATED 1 5′-CTTATGGACCTTCCGGTGAA 5′-TTCGAGCCACAAGAAGACCT
Bra008669 BrCO CONSTANS 5′-GGCTCCTCAGGAAGTCACTACAA 5′-GCCATATCCTGTGTCATAGTACATTAGC
Bra008668 BrCOL1-1 CONSTANS LIKE-1-1 5′-GCAACAGACCAACTACCTGACC 5′-CGAAGGAACAACTCCATATCCTGTGTTG
Bra023541 BrCOL1-2 CONSTANS LIKE-1-2 5′-CAACGGAGGAATTACCTGAAGCC 5′-TGAAGGAACAATTCCATATCCTGTGTCA
Bra001043 BrCOL2-1 CONSTANS LIKE-2-1 5′-CTGTTGTGCCAGAGTCAACAGAGAG 5′-TTTGCAAAACGTCCCTTGATCCG
Bra021464 BrCOL2-2 CONSTANS LIKE-2-1 5′-CGTTAGTGTTGTGCCAGAGTCATCAAG 5′-CTATCCTCTTTGCGAAACGGCCT
Bra032061 BrCOL3 CONSTANS LIKE-3 5′-ACTTTCCCACACGGATTCAG 5′-TCGCTTAGCAAAACGTCCTT
Bra040020 BrCOL9-1 CONSTANS LIKE-9-1 5′-GTTATGCAGCACATGCAAACTCACC 5′-GCTCTCAGCTGGTTGCACTGAG
Bra029666 BrCOL9-2 CONSTANS LIKE-9-2 5′-AGCCTTTAACACCTCTGGAGAGCTA 5′-ACCGGAGAGCTGCATTGAAGAC
Bra001264 BrCOL9-3 CONSTANS LIKE-9-3 5′-CAGTTCTGCAGCACAAGCAACAA 5′-GCTCCGTTTTAGAACTCATGAACGAGTC
Bra009055 BrFLC1 FLOWERING LOCUS C 1-1 5′-TCGTCAGTCAAAAGCTTTGGACTGT 5′-TCGGAGATTTGTCCTGGTGAGACA
Bra028599 BrFLC2 FLOWERING LOCUS C 1-2 5′-CACACCATGAGCTACTAGAGCTTGTC 5′-GCAGTCGGAGAGTTACCGGAC
Bra006051 BrFLC3 FLOWERING LOCUS C 1-3 5′-CACACCATGAGCTACTAGAGCTTGTC 5′-GCAGTCGGAGAGTTACCGGAC
Bra001357 BrFLD FLOWERING LOCUS D 5′-GGAGTGAAGCTTGTGGGAAG 5′-CACCGGTCCTTTAATCCTGA
Bra023965 BrKNAT5-1 KNOTTED1-LIKE HOMEOBOX GENE 5-1 5′-GCCTGGAGACACAACGTCTGTG 5′-TCACATAACCGAAGGAGAAACCATGTC
Bra011317 BrKNAT5-2 KNOTTED1-LIKE HOMEOBOX GENE 5-2 5′-AGAGCGGGAGAGATCCTTGC 5′-GCAAACCGGTTTCTTCAACCAGTTTC
Bra024351 BrMAF4 MADS AFFECTING FLOWEING 4 5′-CGGRAAACTCTATASCTCCTCC 5′-TTCCGATGACATTGCTCCTTCAC
Bra040518 BrMBD9 METHYL-CPG-BINDING DOMAIN 19 5′-CCTTATTCATTAGCAACTGAAGAAGGC 5′-AGCAGCCGCTGAAAGAGAAGAC
Bra006322 BrSEP1-1 SEPALLATA 1-1 5′-GCCAGCAAAGGAATCTTCTTGGG 5′-GTGATGATGCTCGTACGGAACATTC
Bra008674 BrSEP1-2 SEPALLATA 1-2 5′-CCTCTGAATTCAAAGGAGTTAGAGCAGA 5′-CACCAATCATATCATCCAGCTTCATAGC
Bra021470 BrSEP1-3 SEPALLATA 1-3 5′-CTTGGAGAGGACCTTGGACCC 5′-CTTCATTGACAAAGCACGATTGGCG
Bra039170 BrSEP1-4 SEPALLATA 1-4 5′-GCGTCAACTAGACGGCTCTCTG 5′-GTGATGATGCTCGTACGGAACATTC
Bra037544 BrVRN1 REDUCED VERNALIZATION RESPONSE 1 5′-CCGTTCAAAGTTCTACGAGAGTGC 5′-TCAGACGTACTCGTTGACTCGAWA
Bra021078 BrVRN2 REDUCED VERNALIZATION RESPONSE 2 5′-CAAGAAAGTTATCTGCTGAGCGATC 5′-TATTGCAGTTGTTGATGGTGGC

z)BRAD ID number identified by BlastN with expressed sequence tag sequence at http://brassicadb.org/brad/.

qRT-PCR: quantitative reverse transcription polymerase chain reaction.

Table 2
Transcription factors whose expressions levels were top 20 in both RCBr and Chiifu, and over two-fold in RCBr or Chiifu.
Table 2
Classification At_Locus Gene annotation Br_SEQ_ID Probe intensity Fold change
RCBr Chiifu R/Cz) C/R
Top 20 in both RCBr and Chiifu AT1G78080 RAP2.4 (Ethylene-responsive transcription factor RAP2 4) Brapa_ESTC021276 40264 40257 1.0 1.0
AT3G54810 BME3/BME3-ZF (BLUE MICROPLYLAR END3) Brapa_ESTC043727 38658 39227 1.0 1.0
AT3G15210 ATERF4/ERF4 (ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 4) Brapa_ESTC031149 38808 38636 1.0 1.0
AT1G65440 GTB1 (GLOBAL transcription factor GROUP B1) Brapa_ESTC034584 33003 38201 0.9 1.2
AT5G59820 ZAT12 | RHL41 (RESPONSIVE TO HIGH LIGHT 41) Brapa_ESTC009473 34297 37970 0.9 1.1
AT1G28370 ATERF11/ERF11 (ERF domain protein 11) Brapa_ESTC031565 37200 36359 1.0 1.0
AT1G20696 HMGB3 (HIGH MOBILITY GROUP B 3) Brapa_ESTC008254 34477 35904 1.0 1.0
Brapa_ESTC000857 35055 30406 1.2 0.9
AT1G80840 WRKY40 (WRKY DNA-binding protein 40) Brapa_ESTC014413 35089 33203 1.1 0.9
Brapa_ESTC023350 33999 31364 1.1 0.9
AT4G34410 AP2 domain-containing transcription factor, putative Brapa_ESTC044996 30380 32899 0.9 1.1
AT1G50600 SCL5 (Scarecrow-like protein 5) Brapa_ESTC017171 30623 32226 1.0 1.1
AT1G27730 ZAT10 | STZ (SALT TOLERANCE ZINC FINGER) Brapa_ESTC040097 42218 32094 1.3 0.8
AT4G10710 SPT16 (GLOBAL transcription factor C) Brapa_ESTC033093 29057 31334 0.9 1.1
AT2G37630 MYB91 | AS1 (ASYMMETRIC LEAVES 1) Brapa_ESTC001152 31517 30950 1.0 1.0
AT5G52660 MYB family transcription factor Brapa_ESTC046861 38201 29175 1.3 0.8
AT1G12610 DDF1 (DWARF AND DELAYED FLOWERING 1) Brapa_ESTC039504 30466 29121 1.0 1.0
AT5G48150 PAT1 (PHYTOCHROME A SIGNAL TRANSDUCTION 1) Brapa_ESTC021790 38241 28474 1.3 0.7
AT3G50060 MYB family transcription factor Brapa_ESTC024817 38077 28352 1.3 0.7
AT4G31550 WRKY11 (WRKY DNA-binding protein 11) Brapa_ESTC044727 29815 28222 1.1 0.9
RCBr specific AT5G21120 EIL2 (ETHYLENE-INSENSITIVE3-LIKE 2) Brapa_ESTC037616 1460 116 12.6 .
AT2G45680 TCP family transcription factor, putative Brapa_ESTC042103 3852 377 10.2 .
AT1G26310 CAL (CAULIFLOWER) Brapa_ESTC017036 2570 287 9.0 .
AT1G15050 IAA34 (indoleacetic acid-induced protein 34) Brapa_ESTC033550 2023 250 8.1 .
AT1G76890 GT2 (GT2) Brapa_ESTC003989 1178 199 5.9 .
AT5G25390 SHN2 (Ethylene-responsive transcription factor SHINE3) Brapa_ESTC032163 1763 307 5.7 .
AT1G73830 BEE3 (BR-ENHANCED EXPRESSION 3) Brapa_ESTC019151 2796 496 5.6 .
AT5G22290 ANAC089 (Arabidopsis NAC domain containing protein 89) Brapa_ESTC021310 1579 281 5.6 .
Brapa_ESTC034036 1104 301 3.7 .
AT1G69560 MYB105 (myb domain protein 105) Brapa_ESTC040454 1409 277 5.1 .
AT5G15310 AtMYB16 (myb domain protein 16) Brapa_ESTC025442 1817 419 4.3 .
AT1G20530 Unknown (similar to bzip-related transcription factor -like) Brapa_ESTC000221 1921 497 3.9 .
AT5G60890 AtMYB34 | ATR1 (ALTERED TRYPTOPHAN REGULATION) Brapa_ESTC026773 1226 326 3.8 .
AT1G17590 CCAAT-binding transcription factor (CBF-B/NF-YA) family protein Brapa_ESTC003250 1089 302 3.6 .
AT4G33280 AP2/B3-like transcription factor Brapa_ESTC044866 1607 480 3.3 .
AT2G03060 Similar to AGL102 Brapa_ESTC019197 1325 401 3.3 .
AT3G12720 AtMYB67/AtY53 (myb domain protein 67) Brapa_ESTC042873 1358 467 2.9 .
AT5G06839 bZIP family transcription factor Brapa_ESTC045391 1162 455 2.6 .
AT4G32280 IAA29 (indoleacetic acid-induced protein 29) Brapa_ESTC035154 1126 472 2.4 .
AT1G15360 SHN1/WIN1 (SHINE1/WAX INDUCER 1) Brapa_ESTC017040 1064 464 2.3 .
Chiifu specific AT4G32040 KNAT5 (KNOTTED1-LIKE HOMEOBOX GENE 5) Brapa_ESTC025634 44 18085 . 413.2
AT5G51190 AP2 domain-containing transcription factor, putative Brapa_ESTC013139 145 12449 . 85.7
AT4G01550 ANAC069 (Arabidopsis NAC domain containing protein 69) Brapa_ESTC007789 95 7576 . 80.0
AT3G50260 CEJ1 (COOPERATIVELY REGULATED BY ETHYLENE AND JASMONATE 1) Brapa_ESTC001959 17 1102 . 66.0
AT5G60890 AtMYB34 | ATR1 (ALTERED TRYPTOPHAN REGULATION) Brapa_ESTC009043 408 22870 . 56.1
AT2G22430 ATHB6 (ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 6) Brapa_ESTC004579 323 13848 . 42.9
AT2G46970 PIL1 (PHYTOCHROME INTERACTING FACTOR 3-LIKE 1) Brapa_ESTC042137 108 3570 . 33.1
AT2G42780 similar to Os08g0169600 Brapa_ESTC050862 49 1577 . 32.1
AT1G49950 ATTRB1/TRB1 (TELOMERE REPEAT BINDING FACTOR 1) Brapa_ESTC051029 270 4988 . 18.5
AT4G12020 MAPKKK11 | WRKY19 (WRKY DNA-binding protein 19) Brapa_ESTC030987 149 2540 . 17.1
Brapa_ESTC037364 397 5334 . 13.4
AT5G59820 ZAT12 | RHL41 (RESPONSIVE TO HIGH LIGHT 41) Brapa_ESTC006145 106 1379 . 13.0
AT4G31800 WRKY18 (WRKY DNA-binding protein 18) Brapa_ESTC001172 320 3971 . 12.4
AT3G27810 ATMYB21 (MYB DOMAIN PROTEIN 21) Brapa_ESTC008010 445 4910 . 11.0
AT3G61150 HDG1 (HOMEODOMAIN GLABROUS1) Brapa_ESTC022251 201 1813 . 9.0
AT3G28920 ATHB34 (ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 34) Brapa_ESTC051903 418 3716 . 8.9
AT3G11100 Transcription factor Brapa_ESTC005968 361 2986 . 8.3
AT2G23380 CLF (CURLY LEAF) Brapa_ESTC048306 138 1075 . 7.8
AT1G71030 ATMYBL2 (Arabidopsis myb-like 2) Brapa_ESTC021435 249 1760 . 7.1
AT5G16820 HSFA1B | HSF3 (HEAT SHOCK FACTOR 3) Brapa_ESTC036030 481 1465 . 3.0
AT5G61590 AP2 domain-containing transcription factor family protein Brapa_ESTC021781 465 1249 . 2.7

z)R/C and C/R indicate the expression ratio in RCBr over Chiifu and Chiifu over RCBr, respectively. Flowering-related TF genes were omitted in this Table.

Table 3
Flowering-related genes which were selected from 196 flowering genes on Br300K microarray.
Table 3
Classification At_Locus Gene annotation Br_SEQ_ID Probe intensity Fold change
RCBr Chiifu R/Cz) C/R
Up-regulated genes in RCBr AT5G20240 PI (PISTILLATA) Brapa_ESTC028268 4123 43 95.0 .
Brapa_ESTC008132 6436 365 17.7 .
Brapa_ESTC034308 4875 799 6.1 .
Brapa_ESTC048371 872 163 5.3 .
Brapa_ESTC019275 3390 359 9.4 .
Brapa_ESTC048372 3473 963 3.6 .
AT5G15800 SEP1 (SEPALLATA1)/AGL2 Brapa_ESTC025908 1629 32 50.6 .
Brapa_ESTC011597 2267 94 24.2 .
Brapa_ESTC045715 7932 402 19.7 .
AT1G69120 AP1 (APETALA1)/AGL4 Brapa_ESTC040402 8412 267 31.5 .
Brapa_ESTC017264 4988 242 20.6 .
Brapa_ESTC010543 527 26 20.2 .
Brapa_ESTC008651 5851 313 18.7 .
AT3G02310 SEP2 (SEPALLATA2) Brapa_ESTC008039 5018 200 25.0 .
Brapa_ESTC005886 1654 69 24.0 .
Brapa_ESTC031079 4813 254 19.0 .
Brapa_ESTC049045 1242 197 6.3 .
AT3G54340 AP3 (APETALA 3) Brapa_ESTC010485 2902 138 21.0 .
Brapa_ESTC007881 6834 1754 3.9 .
AT4G18960 AG (AGAMOUS) Brapa_ESTC008198 1927 93 20.6 .
Brapa_ESTC018123 2371 253 9.4 .
AT1G24260 SEP3 (SEPALLATA3)/AGL9 Brapa_ESTC010831 8934 615 14.5 .
AT2G45650 AGL6 (AGAMOUS LIKE-6) Brapa_ESTC018131 2547 932 2.7 .
AT1G26310 CAL (CAULIFLOWER)/AGL10 Brapa_ESTC010942 1086 112 9.7 .
AT1G65480 FT (FLOWERING LOCUS T) Brapa_ESTC017036 2570 287 9.0 .
Brapa_ESTC034579 1985 278 7.1 .
AT3G07650 COL9 (CONSTANS-LIKE 9) Brapa_ESTC019941 2432 462 5.3 .
Brapa_ESTC027877 5149 819 6.3 .
AT2G45660 SOC1/AGL20 (AGAMOUS-LIKE 20)(SOC1-2, SOC1-1, SOC1-3) Brapa_ESTC051650 3495 1501 2.3 .
Brapa_ESTC011095 14204 3600 3.9 .
Brapa_ESTC018989 10579 3022 3.5 .
Brapa_ESTC019044 9961 3960 2.5 .
AT5G60910 FUL/AGL8 (AGAMOUS-LIKE 8) Brapa_ESTC017129 22280 5691 3.9 .
AT5G24860 FPF1 (FLOWERING PROMOTING FACTOR 1) Brapa_ESTC018052 4399 1195 3.7 .
Brapa_ESTC029975 2502 1078 2.3 .
AT5G62165 AGL42 (AGAMOUS LIKE 42) Brapa_ESTC008221 3953 1108 3.6 .
AT5G03840 TFL1 (TERMINAL FLOWER 1) Brapa_ESTC045284 898 253 3.5 .
AT2G03060 Similar to AGL102 Brapa_ESTC019197 1325 401 3.3 .
AT3G21320 Similar to ELF3 (EARLY FLOWERING 3) Brapa_ESTC043198 4038 1494 2.7 .
Brapa_ESTC023599 3673 1782 2.1 .
AT2G43010 PIF4 (PHYTOCHROME INTERACTING FACTOR 4) Brapa_ESTC012017 749 288 2.6 .
Brapa_ESTC018786 4340 1879 2.3 .
AT2G45830 DTA2 (DOWNSTREAM TARGET OF AGL15 2) Brapa_ESTC034818 9026 3696 2.4 .
AT5G15850 COL1 (CONSTANS-LIKE 1) Brapa_ESTC020072 2427 1002 2.4 .
AT3G59060 PIF5/PIL6 (PHYTOCHROME-INTERACTING FACTOR 5) Brapa_ESTC019071 5260 2182 2.4 .
Brapa_ESTC033456 1381 590 2.3 .
AT1G71692 AGL12 (AGAMOUS-LIKE 12) Brapa_ESTC027067 1108 485 2.3 .
AT5G10140 FLC (FLOWERING LOCUS C)/AGL25(FLCL/FLC5) Brapa_ESTC017216 8885 4014 2.2 .
AT5G13790 AGL15 (AGAMOUS-LIKE 15) Brapa_ESTC010865 5636 2491 2.3 .
AT2G33350 Similar to zinc finger CONSTANS-related Brapa_ESTC041607 1624 809 2.0 .
Up-regulated genes in Chiifu AT5G10140 FLC (FLOWERING LOCUS C)/AGL25(FLC1, FLC2) Brapa_ESTC016533 53 1504 . 28.3
Brapa_ESTC012276 6738 13305 . 2.0
AT5G65070 FCL4/MAF4 (MADS AFFECTING FLOWERING 4) Brapa_ESTC013135 393 8499 . 21.6
Brapa_ESTC046597 986 4411 . 4.5
Brapa_ESTC013362 3209 11579 . 3.6
Brapa_ESTC046600 300 1051 . 3.5
Brapa_ESTC046598 3151 9784 . 3.1
AT1G31140 AGL63 (AGAMOUS-LIKE 63) Brapa_ESTC040293 206 2363 . 11.5
AT5G04240 ELF6 (EARLY FLOWERING 6) Brapa_ESTC020902 368 1517 . 4.1
AT5G13790 AGL15 (AGAMOUS-LIKE 15) Brapa_ESTC020193 1290 4174 . 3.2
AT1G30260 AGL79 (AGAMOUS-LIKE 79) Brapa_ESTC014642 276 776 . 2.8
AT1G77080 AGL27/MAF1 (MADS AFFECTING FLOWERING 1) Brapa_ESTC022419 1327 3363 . 2.5
AT1G09530 PIF3 (PHYTOCHROME INTERACTING FACTOR 3) Brapa_ESTC026673 2332 5556 . 2.4
AT3G57390 AGL18 (AGAMOUS-LIKE 18) Brapa_ESTC021222 2876 5626 . 2.0
Highly expressed genes in both RCBr and Chiifu AT1G12610 DDF1 (DWARF AND DELAYED FLOWERING 1) Brapa_ESTC039504 30466 29121 1.0 1.0
Brapa_ESTC002858 19586 15570 1.3 0.8
AT2G25930 ELF3 (EARLY FLOWERING 3) Brapa_ESTC041284 26959 22606 1.2 0.8
Brapa_ESTC041298 22936 16177 1.4 0.7
Brapa_ESTC015252 19828 20034 1.0 1.0
AT1G25540 PFT1 (PHYTOCHROME AND FLOWERING TIME 1) Brapa_ESTC047777 23968 21573 1.1 0.9
AT2G22540 AGL22/SVP (SHORT VEGETATIVE PHASE) Brapa_ESTC014320 23538 18005 1.3 0.8
Brapa_ESTC050176 8869 9304 1.0 1.0
Brapa_ESTC041116 18874 17799 1.1 0.9
AT3G04610 FLK (FLOWERING LOCUS KH DOMAIN) Brapa_ESTC042608 21959 17640 1.2 0.8
AT5G62640.2 ELF5 (EARLY FLOWERING 5) Brapa_ESTC001982 19200 13055 1.5 0.7
AT2G24790.1 COL3 (CONSTANS-LIKE 3) Brapa_ESTC011117 19001 27161 0.7 1.4
Brapa_ESTC014037 15458 24927 0.6 1.6
Brapa_ESTC027269 13996 19872 0.7 1.4
AT5G10140 FLC (FLOWERING LOCUS C)/AGL25 (FLC3) Brapa_ESTC013303 17486 16533 1.1 0.9
Brapa_ESTC021830 13579 14983 0.9 1.1
AT4G24540 AGL24 (AGAMOUS-LIKE 24) Brapa_ESTC036920 17200 17618 1.0 1.0
Brapa_ESTC019065 14515 13686 1.1 0.9
AT1G77300 EFS (EARLY FLOWERING IN SHORT DAYS) Brapa_ESTC040684 17197 13552 1.3 0.8
AT4G22950 AGL19 (AGAMOUS-LIKE 19) Brapa_ESTC044364 16772 15569 1.1 0.9
Brapa_ESTC018998 12476 12321 1.0 1.0
AT3G12810 PIE1 (PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1) Brapa_ESTC042862 16091 12137 1.3 0.8
Brapa_ESTC024641 14327 8484 1.7 0.6
AT2G06210 ELF8 (EARLY FLOWERING 8) Brapa_ESTC004485 15388 12464 1.2 0.8
Brapa_ESTC042284 12694 10565 1.2 0.8
AT1G07050 CONSTANS-like protein-related Brapa_ESTC021115 15190 10154 1.5 0.7
AT1G77080 AGL27, MAF1 (MADS AFFECTING FLOWERING 1) Brapa_ESTC000396 14927 10695 1.4 0.7
AT3G07650 COL9 (CONSTANS-LIKE 9) Brapa_ESTC042798 14472 8979 1.6 0.6
Brapa_ESTC051649 12413 8766 1.4 0.7
Brapa_ESTC021535 12256 7067 1.7 0.6
Brapa_ESTC025316 8692 4979 1.7 0.6
Brapa_ESTC049114 7570 5204 1.5 0.7
Brapa_ESTC049113 7031 5176 1.4 0.7
Brapa_ESTC006144 6478 9542 0.7 1.5
AT4G28190 ULT1 (ULTRAPETALA1) Brapa_ESTC044575 14100 11058 1.3 0.8
AT1G79730 ELF7 (EARLY FLOWERING 7) Brapa_ESTC006396 12278 10830 1.1 0.9
Brapa_ESTC009946 8400 10613 0.8 1.3
AT4G36920 AP2 (APETALA 2) Brapa_ESTC034160 10951 9936 1.1 0.9
Brapa_ESTC007967 7301 8061 0.9 1.1
Brapa_ESTC012323 4120 5899 0.7 1.4
Brapa_ESTC013840 3821 4959 0.8 1.3
Brapa_ESTC023600 2272 1607 1.4 0.7
AT3G04610 FLK (FLOWERING LOCUS KH DOMAIN) Brapa_ESTC021699 10239 6514 1.6 0.6
Brapa_ESTC015151 8990 9444 1.0 1.1
AT2G45660 SOC1/AGL20 (AGAMOUS-LIKE 20)(SOC1-3) Brapa_ESTC034801 9787 7499 1.3 0.8
AT2G40080 ELF4 (EARLY FLOWERING 4) Brapa_ESTC038745 9602 6902 1.4 0.7
Brapa_ESTC023094 8429 13088 0.6 1.6
Brapa_ESTC027935 6642 4190 1.6 0.6
AT5G15850 COL1 (CONSTANS-LIKE 1) Brapa_ESTC013309 9416 16434 0.6 1.7

z)R/C and C/R indicate the expression ratio in RCBr over Chiifu and Chiifu over RCBr, respectively.

Table 4
Genes related to gibberellic acid metabolism.
Table 4
At_Locus Gene annotation Br_SEQ_ID Prove intensity Fold change
RCBr Chiifu R/Cz) C/R
AT1G30040 GA2OX2 (Gibberellin 2-beta-dioxygenase) Brapa_ESTC040202 1319 6104 0.2 4.6
Brapa_ESTC015031 16 1969 0.0 126.4
AT1G02400 GA2OX6/DTA1 (Gibberellin 2-OXIDASE 6) Brapa_ESTC015242 10762 8386 1.3 0.8
AT4G21200 GA2OX8 (Gibberellin 2-OXIDASE 8) Brapa_ESTC024684 431 665 0.6 1.5
Brapa_ESTC015062 423 624 0.7 1.5
AT1G15550 GA3OX1/GA4 (GA REQUIRING 4) Brapa_ESTC027719 554 164 3.4 0.3
AT5G07200 GA20OX3/YAP169 (Gibberellin 20 oxidase 3) Brapa_ESTC045377 1296 155 8.4 0.1
Brapa_ESTC029845 4761 183 26.1 0.0
Brapa_ESTC027440 1114 81 13.7 0.1
AT5G14920 Gibberellin-regulated family protein Brapa_ESTC003065 9782 9954 1.0 1.0
Brapa_ESTC016390 5777 9747 0.6 1.7
Brapa_ESTC042364 10773 8665 1.2 0.8
Brapa_ESTC024407 6879 6685 1.0 1.0
Brapa_ESTC007229 5209 4283 1.2 0.8
Brapa_ESTC010071 5161 3227 1.6 0.6
AT5G59845 Gibberellin-regulated family protein Brapa_ESTC007822 507 20 25.9 0.0
AT1G74670 Gibberellin-responsive protein, putative Brapa_ESTC037994 4417 3177 1.4 0.7
Brapa_ESTC008005 3581 2190 1.6 0.6
AT1G22690 Gibberellin-responsive protein, putative Brapa_ESTC003796 5081 1538 3.3 0.3
AT5G51310 Gibberellin 20-oxidase-related Brapa_ESTC046793 859 1135 0.8 1.3
AT3G10185 Gibberellin-regulated GASA/GAST/Snakin family protein Brapa_ESTC036747 4237 5060 0.8 1.2
AT4G26420 GAMT1 | Gibberellin carboxyl-O-methyltransferase Brapa_ESTC019836 1067 287 3.7 0.3
AT5G56300 GAMT2 | Gibberellin carboxyl-O-methyltransferase Brapa_ESTC009976 773 115 6.7 0.1

z)R/C and C/R indicate the expression ratio in RCBr over Chiifu and Chiifu over RCBr, respectively.

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Differential Expression of Flowering Genes between Rapid- and Slow-Cycling Brassica rapa
Plant Breed. Biotech.. 2016;4(2):145-157.   Published online May 31, 2016
DOI: https://doi.org/10.9787/PBB.2016.4.2.145
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Differential Expression of Flowering Genes between Rapid- and Slow-Cycling Brassica rapa
Plant Breed. Biotech.. 2016;4(2):145-157.   Published online May 31, 2016
DOI: https://doi.org/10.9787/PBB.2016.4.2.145
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Differential Expression of Flowering Genes between Rapid- and Slow-Cycling Brassica rapa
Image Image Image
Fig. 1 Functional categorization of differentially expressed genes between RCBr and Chiifu based on gene ontology dirstribution.
Fig. 2 Genes whose expression was decreased in RCBr, while increased in Chiifu. RCBr and Chiifu were grown in long-day condition condition for 19 days and shoot was sampled at 4th hour of light period.
Fig. 3 Expression of flowering- and photoperiod pathway-related genes. (A) Photoperiod for plant growth and sampling time a day. (B) Genes showing differential expression in a microarray. (C) Constance (CO) and its paralogs. BrCCA1 was used as control for circadian rhythm. LD: long-day condition, SD: short-day condition.

Fig. 1

Fig. 2

Fig. 3

Differential Expression of Flowering Genes between Rapid- and Slow-Cycling Brassica rapa

List of primers used in qRT-PCR analysis.

Br_IDz) Gene Gene annotation Forward primer Reverse primer
Bra004503 BrCCA1 CIRCADIAN CLOCK-ASSOCIATED 1 5′-CTTATGGACCTTCCGGTGAA 5′-TTCGAGCCACAAGAAGACCT
Bra008669 BrCO CONSTANS 5′-GGCTCCTCAGGAAGTCACTACAA 5′-GCCATATCCTGTGTCATAGTACATTAGC
Bra008668 BrCOL1-1 CONSTANS LIKE-1-1 5′-GCAACAGACCAACTACCTGACC 5′-CGAAGGAACAACTCCATATCCTGTGTTG
Bra023541 BrCOL1-2 CONSTANS LIKE-1-2 5′-CAACGGAGGAATTACCTGAAGCC 5′-TGAAGGAACAATTCCATATCCTGTGTCA
Bra001043 BrCOL2-1 CONSTANS LIKE-2-1 5′-CTGTTGTGCCAGAGTCAACAGAGAG 5′-TTTGCAAAACGTCCCTTGATCCG
Bra021464 BrCOL2-2 CONSTANS LIKE-2-1 5′-CGTTAGTGTTGTGCCAGAGTCATCAAG 5′-CTATCCTCTTTGCGAAACGGCCT
Bra032061 BrCOL3 CONSTANS LIKE-3 5′-ACTTTCCCACACGGATTCAG 5′-TCGCTTAGCAAAACGTCCTT
Bra040020 BrCOL9-1 CONSTANS LIKE-9-1 5′-GTTATGCAGCACATGCAAACTCACC 5′-GCTCTCAGCTGGTTGCACTGAG
Bra029666 BrCOL9-2 CONSTANS LIKE-9-2 5′-AGCCTTTAACACCTCTGGAGAGCTA 5′-ACCGGAGAGCTGCATTGAAGAC
Bra001264 BrCOL9-3 CONSTANS LIKE-9-3 5′-CAGTTCTGCAGCACAAGCAACAA 5′-GCTCCGTTTTAGAACTCATGAACGAGTC
Bra009055 BrFLC1 FLOWERING LOCUS C 1-1 5′-TCGTCAGTCAAAAGCTTTGGACTGT 5′-TCGGAGATTTGTCCTGGTGAGACA
Bra028599 BrFLC2 FLOWERING LOCUS C 1-2 5′-CACACCATGAGCTACTAGAGCTTGTC 5′-GCAGTCGGAGAGTTACCGGAC
Bra006051 BrFLC3 FLOWERING LOCUS C 1-3 5′-CACACCATGAGCTACTAGAGCTTGTC 5′-GCAGTCGGAGAGTTACCGGAC
Bra001357 BrFLD FLOWERING LOCUS D 5′-GGAGTGAAGCTTGTGGGAAG 5′-CACCGGTCCTTTAATCCTGA
Bra023965 BrKNAT5-1 KNOTTED1-LIKE HOMEOBOX GENE 5-1 5′-GCCTGGAGACACAACGTCTGTG 5′-TCACATAACCGAAGGAGAAACCATGTC
Bra011317 BrKNAT5-2 KNOTTED1-LIKE HOMEOBOX GENE 5-2 5′-AGAGCGGGAGAGATCCTTGC 5′-GCAAACCGGTTTCTTCAACCAGTTTC
Bra024351 BrMAF4 MADS AFFECTING FLOWEING 4 5′-CGGRAAACTCTATASCTCCTCC 5′-TTCCGATGACATTGCTCCTTCAC
Bra040518 BrMBD9 METHYL-CPG-BINDING DOMAIN 19 5′-CCTTATTCATTAGCAACTGAAGAAGGC 5′-AGCAGCCGCTGAAAGAGAAGAC
Bra006322 BrSEP1-1 SEPALLATA 1-1 5′-GCCAGCAAAGGAATCTTCTTGGG 5′-GTGATGATGCTCGTACGGAACATTC
Bra008674 BrSEP1-2 SEPALLATA 1-2 5′-CCTCTGAATTCAAAGGAGTTAGAGCAGA 5′-CACCAATCATATCATCCAGCTTCATAGC
Bra021470 BrSEP1-3 SEPALLATA 1-3 5′-CTTGGAGAGGACCTTGGACCC 5′-CTTCATTGACAAAGCACGATTGGCG
Bra039170 BrSEP1-4 SEPALLATA 1-4 5′-GCGTCAACTAGACGGCTCTCTG 5′-GTGATGATGCTCGTACGGAACATTC
Bra037544 BrVRN1 REDUCED VERNALIZATION RESPONSE 1 5′-CCGTTCAAAGTTCTACGAGAGTGC 5′-TCAGACGTACTCGTTGACTCGAWA
Bra021078 BrVRN2 REDUCED VERNALIZATION RESPONSE 2 5′-CAAGAAAGTTATCTGCTGAGCGATC 5′-TATTGCAGTTGTTGATGGTGGC

z)BRAD ID number identified by BlastN with expressed sequence tag sequence at http://brassicadb.org/brad/.

qRT-PCR: quantitative reverse transcription polymerase chain reaction.

Transcription factors whose expressions levels were top 20 in both RCBr and Chiifu, and over two-fold in RCBr or Chiifu.

Classification At_Locus Gene annotation Br_SEQ_ID Probe intensity Fold change
RCBr Chiifu R/Cz) C/R
Top 20 in both RCBr and Chiifu AT1G78080 RAP2.4 (Ethylene-responsive transcription factor RAP2 4) Brapa_ESTC021276 40264 40257 1.0 1.0
AT3G54810 BME3/BME3-ZF (BLUE MICROPLYLAR END3) Brapa_ESTC043727 38658 39227 1.0 1.0
AT3G15210 ATERF4/ERF4 (ETHYLENE RESPONSIVE ELEMENT BINDING FACTOR 4) Brapa_ESTC031149 38808 38636 1.0 1.0
AT1G65440 GTB1 (GLOBAL transcription factor GROUP B1) Brapa_ESTC034584 33003 38201 0.9 1.2
AT5G59820 ZAT12 | RHL41 (RESPONSIVE TO HIGH LIGHT 41) Brapa_ESTC009473 34297 37970 0.9 1.1
AT1G28370 ATERF11/ERF11 (ERF domain protein 11) Brapa_ESTC031565 37200 36359 1.0 1.0
AT1G20696 HMGB3 (HIGH MOBILITY GROUP B 3) Brapa_ESTC008254 34477 35904 1.0 1.0
Brapa_ESTC000857 35055 30406 1.2 0.9
AT1G80840 WRKY40 (WRKY DNA-binding protein 40) Brapa_ESTC014413 35089 33203 1.1 0.9
Brapa_ESTC023350 33999 31364 1.1 0.9
AT4G34410 AP2 domain-containing transcription factor, putative Brapa_ESTC044996 30380 32899 0.9 1.1
AT1G50600 SCL5 (Scarecrow-like protein 5) Brapa_ESTC017171 30623 32226 1.0 1.1
AT1G27730 ZAT10 | STZ (SALT TOLERANCE ZINC FINGER) Brapa_ESTC040097 42218 32094 1.3 0.8
AT4G10710 SPT16 (GLOBAL transcription factor C) Brapa_ESTC033093 29057 31334 0.9 1.1
AT2G37630 MYB91 | AS1 (ASYMMETRIC LEAVES 1) Brapa_ESTC001152 31517 30950 1.0 1.0
AT5G52660 MYB family transcription factor Brapa_ESTC046861 38201 29175 1.3 0.8
AT1G12610 DDF1 (DWARF AND DELAYED FLOWERING 1) Brapa_ESTC039504 30466 29121 1.0 1.0
AT5G48150 PAT1 (PHYTOCHROME A SIGNAL TRANSDUCTION 1) Brapa_ESTC021790 38241 28474 1.3 0.7
AT3G50060 MYB family transcription factor Brapa_ESTC024817 38077 28352 1.3 0.7
AT4G31550 WRKY11 (WRKY DNA-binding protein 11) Brapa_ESTC044727 29815 28222 1.1 0.9
RCBr specific AT5G21120 EIL2 (ETHYLENE-INSENSITIVE3-LIKE 2) Brapa_ESTC037616 1460 116 12.6 .
AT2G45680 TCP family transcription factor, putative Brapa_ESTC042103 3852 377 10.2 .
AT1G26310 CAL (CAULIFLOWER) Brapa_ESTC017036 2570 287 9.0 .
AT1G15050 IAA34 (indoleacetic acid-induced protein 34) Brapa_ESTC033550 2023 250 8.1 .
AT1G76890 GT2 (GT2) Brapa_ESTC003989 1178 199 5.9 .
AT5G25390 SHN2 (Ethylene-responsive transcription factor SHINE3) Brapa_ESTC032163 1763 307 5.7 .
AT1G73830 BEE3 (BR-ENHANCED EXPRESSION 3) Brapa_ESTC019151 2796 496 5.6 .
AT5G22290 ANAC089 (Arabidopsis NAC domain containing protein 89) Brapa_ESTC021310 1579 281 5.6 .
Brapa_ESTC034036 1104 301 3.7 .
AT1G69560 MYB105 (myb domain protein 105) Brapa_ESTC040454 1409 277 5.1 .
AT5G15310 AtMYB16 (myb domain protein 16) Brapa_ESTC025442 1817 419 4.3 .
AT1G20530 Unknown (similar to bzip-related transcription factor -like) Brapa_ESTC000221 1921 497 3.9 .
AT5G60890 AtMYB34 | ATR1 (ALTERED TRYPTOPHAN REGULATION) Brapa_ESTC026773 1226 326 3.8 .
AT1G17590 CCAAT-binding transcription factor (CBF-B/NF-YA) family protein Brapa_ESTC003250 1089 302 3.6 .
AT4G33280 AP2/B3-like transcription factor Brapa_ESTC044866 1607 480 3.3 .
AT2G03060 Similar to AGL102 Brapa_ESTC019197 1325 401 3.3 .
AT3G12720 AtMYB67/AtY53 (myb domain protein 67) Brapa_ESTC042873 1358 467 2.9 .
AT5G06839 bZIP family transcription factor Brapa_ESTC045391 1162 455 2.6 .
AT4G32280 IAA29 (indoleacetic acid-induced protein 29) Brapa_ESTC035154 1126 472 2.4 .
AT1G15360 SHN1/WIN1 (SHINE1/WAX INDUCER 1) Brapa_ESTC017040 1064 464 2.3 .
Chiifu specific AT4G32040 KNAT5 (KNOTTED1-LIKE HOMEOBOX GENE 5) Brapa_ESTC025634 44 18085 . 413.2
AT5G51190 AP2 domain-containing transcription factor, putative Brapa_ESTC013139 145 12449 . 85.7
AT4G01550 ANAC069 (Arabidopsis NAC domain containing protein 69) Brapa_ESTC007789 95 7576 . 80.0
AT3G50260 CEJ1 (COOPERATIVELY REGULATED BY ETHYLENE AND JASMONATE 1) Brapa_ESTC001959 17 1102 . 66.0
AT5G60890 AtMYB34 | ATR1 (ALTERED TRYPTOPHAN REGULATION) Brapa_ESTC009043 408 22870 . 56.1
AT2G22430 ATHB6 (ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 6) Brapa_ESTC004579 323 13848 . 42.9
AT2G46970 PIL1 (PHYTOCHROME INTERACTING FACTOR 3-LIKE 1) Brapa_ESTC042137 108 3570 . 33.1
AT2G42780 similar to Os08g0169600 Brapa_ESTC050862 49 1577 . 32.1
AT1G49950 ATTRB1/TRB1 (TELOMERE REPEAT BINDING FACTOR 1) Brapa_ESTC051029 270 4988 . 18.5
AT4G12020 MAPKKK11 | WRKY19 (WRKY DNA-binding protein 19) Brapa_ESTC030987 149 2540 . 17.1
Brapa_ESTC037364 397 5334 . 13.4
AT5G59820 ZAT12 | RHL41 (RESPONSIVE TO HIGH LIGHT 41) Brapa_ESTC006145 106 1379 . 13.0
AT4G31800 WRKY18 (WRKY DNA-binding protein 18) Brapa_ESTC001172 320 3971 . 12.4
AT3G27810 ATMYB21 (MYB DOMAIN PROTEIN 21) Brapa_ESTC008010 445 4910 . 11.0
AT3G61150 HDG1 (HOMEODOMAIN GLABROUS1) Brapa_ESTC022251 201 1813 . 9.0
AT3G28920 ATHB34 (ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 34) Brapa_ESTC051903 418 3716 . 8.9
AT3G11100 Transcription factor Brapa_ESTC005968 361 2986 . 8.3
AT2G23380 CLF (CURLY LEAF) Brapa_ESTC048306 138 1075 . 7.8
AT1G71030 ATMYBL2 (Arabidopsis myb-like 2) Brapa_ESTC021435 249 1760 . 7.1
AT5G16820 HSFA1B | HSF3 (HEAT SHOCK FACTOR 3) Brapa_ESTC036030 481 1465 . 3.0
AT5G61590 AP2 domain-containing transcription factor family protein Brapa_ESTC021781 465 1249 . 2.7

z)R/C and C/R indicate the expression ratio in RCBr over Chiifu and Chiifu over RCBr, respectively. Flowering-related TF genes were omitted in this Table.

Flowering-related genes which were selected from 196 flowering genes on Br300K microarray.

Classification At_Locus Gene annotation Br_SEQ_ID Probe intensity Fold change
RCBr Chiifu R/Cz) C/R
Up-regulated genes in RCBr AT5G20240 PI (PISTILLATA) Brapa_ESTC028268 4123 43 95.0 .
Brapa_ESTC008132 6436 365 17.7 .
Brapa_ESTC034308 4875 799 6.1 .
Brapa_ESTC048371 872 163 5.3 .
Brapa_ESTC019275 3390 359 9.4 .
Brapa_ESTC048372 3473 963 3.6 .
AT5G15800 SEP1 (SEPALLATA1)/AGL2 Brapa_ESTC025908 1629 32 50.6 .
Brapa_ESTC011597 2267 94 24.2 .
Brapa_ESTC045715 7932 402 19.7 .
AT1G69120 AP1 (APETALA1)/AGL4 Brapa_ESTC040402 8412 267 31.5 .
Brapa_ESTC017264 4988 242 20.6 .
Brapa_ESTC010543 527 26 20.2 .
Brapa_ESTC008651 5851 313 18.7 .
AT3G02310 SEP2 (SEPALLATA2) Brapa_ESTC008039 5018 200 25.0 .
Brapa_ESTC005886 1654 69 24.0 .
Brapa_ESTC031079 4813 254 19.0 .
Brapa_ESTC049045 1242 197 6.3 .
AT3G54340 AP3 (APETALA 3) Brapa_ESTC010485 2902 138 21.0 .
Brapa_ESTC007881 6834 1754 3.9 .
AT4G18960 AG (AGAMOUS) Brapa_ESTC008198 1927 93 20.6 .
Brapa_ESTC018123 2371 253 9.4 .
AT1G24260 SEP3 (SEPALLATA3)/AGL9 Brapa_ESTC010831 8934 615 14.5 .
AT2G45650 AGL6 (AGAMOUS LIKE-6) Brapa_ESTC018131 2547 932 2.7 .
AT1G26310 CAL (CAULIFLOWER)/AGL10 Brapa_ESTC010942 1086 112 9.7 .
AT1G65480 FT (FLOWERING LOCUS T) Brapa_ESTC017036 2570 287 9.0 .
Brapa_ESTC034579 1985 278 7.1 .
AT3G07650 COL9 (CONSTANS-LIKE 9) Brapa_ESTC019941 2432 462 5.3 .
Brapa_ESTC027877 5149 819 6.3 .
AT2G45660 SOC1/AGL20 (AGAMOUS-LIKE 20)(SOC1-2, SOC1-1, SOC1-3) Brapa_ESTC051650 3495 1501 2.3 .
Brapa_ESTC011095 14204 3600 3.9 .
Brapa_ESTC018989 10579 3022 3.5 .
Brapa_ESTC019044 9961 3960 2.5 .
AT5G60910 FUL/AGL8 (AGAMOUS-LIKE 8) Brapa_ESTC017129 22280 5691 3.9 .
AT5G24860 FPF1 (FLOWERING PROMOTING FACTOR 1) Brapa_ESTC018052 4399 1195 3.7 .
Brapa_ESTC029975 2502 1078 2.3 .
AT5G62165 AGL42 (AGAMOUS LIKE 42) Brapa_ESTC008221 3953 1108 3.6 .
AT5G03840 TFL1 (TERMINAL FLOWER 1) Brapa_ESTC045284 898 253 3.5 .
AT2G03060 Similar to AGL102 Brapa_ESTC019197 1325 401 3.3 .
AT3G21320 Similar to ELF3 (EARLY FLOWERING 3) Brapa_ESTC043198 4038 1494 2.7 .
Brapa_ESTC023599 3673 1782 2.1 .
AT2G43010 PIF4 (PHYTOCHROME INTERACTING FACTOR 4) Brapa_ESTC012017 749 288 2.6 .
Brapa_ESTC018786 4340 1879 2.3 .
AT2G45830 DTA2 (DOWNSTREAM TARGET OF AGL15 2) Brapa_ESTC034818 9026 3696 2.4 .
AT5G15850 COL1 (CONSTANS-LIKE 1) Brapa_ESTC020072 2427 1002 2.4 .
AT3G59060 PIF5/PIL6 (PHYTOCHROME-INTERACTING FACTOR 5) Brapa_ESTC019071 5260 2182 2.4 .
Brapa_ESTC033456 1381 590 2.3 .
AT1G71692 AGL12 (AGAMOUS-LIKE 12) Brapa_ESTC027067 1108 485 2.3 .
AT5G10140 FLC (FLOWERING LOCUS C)/AGL25(FLCL/FLC5) Brapa_ESTC017216 8885 4014 2.2 .
AT5G13790 AGL15 (AGAMOUS-LIKE 15) Brapa_ESTC010865 5636 2491 2.3 .
AT2G33350 Similar to zinc finger CONSTANS-related Brapa_ESTC041607 1624 809 2.0 .
Up-regulated genes in Chiifu AT5G10140 FLC (FLOWERING LOCUS C)/AGL25(FLC1, FLC2) Brapa_ESTC016533 53 1504 . 28.3
Brapa_ESTC012276 6738 13305 . 2.0
AT5G65070 FCL4/MAF4 (MADS AFFECTING FLOWERING 4) Brapa_ESTC013135 393 8499 . 21.6
Brapa_ESTC046597 986 4411 . 4.5
Brapa_ESTC013362 3209 11579 . 3.6
Brapa_ESTC046600 300 1051 . 3.5
Brapa_ESTC046598 3151 9784 . 3.1
AT1G31140 AGL63 (AGAMOUS-LIKE 63) Brapa_ESTC040293 206 2363 . 11.5
AT5G04240 ELF6 (EARLY FLOWERING 6) Brapa_ESTC020902 368 1517 . 4.1
AT5G13790 AGL15 (AGAMOUS-LIKE 15) Brapa_ESTC020193 1290 4174 . 3.2
AT1G30260 AGL79 (AGAMOUS-LIKE 79) Brapa_ESTC014642 276 776 . 2.8
AT1G77080 AGL27/MAF1 (MADS AFFECTING FLOWERING 1) Brapa_ESTC022419 1327 3363 . 2.5
AT1G09530 PIF3 (PHYTOCHROME INTERACTING FACTOR 3) Brapa_ESTC026673 2332 5556 . 2.4
AT3G57390 AGL18 (AGAMOUS-LIKE 18) Brapa_ESTC021222 2876 5626 . 2.0
Highly expressed genes in both RCBr and Chiifu AT1G12610 DDF1 (DWARF AND DELAYED FLOWERING 1) Brapa_ESTC039504 30466 29121 1.0 1.0
Brapa_ESTC002858 19586 15570 1.3 0.8
AT2G25930 ELF3 (EARLY FLOWERING 3) Brapa_ESTC041284 26959 22606 1.2 0.8
Brapa_ESTC041298 22936 16177 1.4 0.7
Brapa_ESTC015252 19828 20034 1.0 1.0
AT1G25540 PFT1 (PHYTOCHROME AND FLOWERING TIME 1) Brapa_ESTC047777 23968 21573 1.1 0.9
AT2G22540 AGL22/SVP (SHORT VEGETATIVE PHASE) Brapa_ESTC014320 23538 18005 1.3 0.8
Brapa_ESTC050176 8869 9304 1.0 1.0
Brapa_ESTC041116 18874 17799 1.1 0.9
AT3G04610 FLK (FLOWERING LOCUS KH DOMAIN) Brapa_ESTC042608 21959 17640 1.2 0.8
AT5G62640.2 ELF5 (EARLY FLOWERING 5) Brapa_ESTC001982 19200 13055 1.5 0.7
AT2G24790.1 COL3 (CONSTANS-LIKE 3) Brapa_ESTC011117 19001 27161 0.7 1.4
Brapa_ESTC014037 15458 24927 0.6 1.6
Brapa_ESTC027269 13996 19872 0.7 1.4
AT5G10140 FLC (FLOWERING LOCUS C)/AGL25 (FLC3) Brapa_ESTC013303 17486 16533 1.1 0.9
Brapa_ESTC021830 13579 14983 0.9 1.1
AT4G24540 AGL24 (AGAMOUS-LIKE 24) Brapa_ESTC036920 17200 17618 1.0 1.0
Brapa_ESTC019065 14515 13686 1.1 0.9
AT1G77300 EFS (EARLY FLOWERING IN SHORT DAYS) Brapa_ESTC040684 17197 13552 1.3 0.8
AT4G22950 AGL19 (AGAMOUS-LIKE 19) Brapa_ESTC044364 16772 15569 1.1 0.9
Brapa_ESTC018998 12476 12321 1.0 1.0
AT3G12810 PIE1 (PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1) Brapa_ESTC042862 16091 12137 1.3 0.8
Brapa_ESTC024641 14327 8484 1.7 0.6
AT2G06210 ELF8 (EARLY FLOWERING 8) Brapa_ESTC004485 15388 12464 1.2 0.8
Brapa_ESTC042284 12694 10565 1.2 0.8
AT1G07050 CONSTANS-like protein-related Brapa_ESTC021115 15190 10154 1.5 0.7
AT1G77080 AGL27, MAF1 (MADS AFFECTING FLOWERING 1) Brapa_ESTC000396 14927 10695 1.4 0.7
AT3G07650 COL9 (CONSTANS-LIKE 9) Brapa_ESTC042798 14472 8979 1.6 0.6
Brapa_ESTC051649 12413 8766 1.4 0.7
Brapa_ESTC021535 12256 7067 1.7 0.6
Brapa_ESTC025316 8692 4979 1.7 0.6
Brapa_ESTC049114 7570 5204 1.5 0.7
Brapa_ESTC049113 7031 5176 1.4 0.7
Brapa_ESTC006144 6478 9542 0.7 1.5
AT4G28190 ULT1 (ULTRAPETALA1) Brapa_ESTC044575 14100 11058 1.3 0.8
AT1G79730 ELF7 (EARLY FLOWERING 7) Brapa_ESTC006396 12278 10830 1.1 0.9
Brapa_ESTC009946 8400 10613 0.8 1.3
AT4G36920 AP2 (APETALA 2) Brapa_ESTC034160 10951 9936 1.1 0.9
Brapa_ESTC007967 7301 8061 0.9 1.1
Brapa_ESTC012323 4120 5899 0.7 1.4
Brapa_ESTC013840 3821 4959 0.8 1.3
Brapa_ESTC023600 2272 1607 1.4 0.7
AT3G04610 FLK (FLOWERING LOCUS KH DOMAIN) Brapa_ESTC021699 10239 6514 1.6 0.6
Brapa_ESTC015151 8990 9444 1.0 1.1
AT2G45660 SOC1/AGL20 (AGAMOUS-LIKE 20)(SOC1-3) Brapa_ESTC034801 9787 7499 1.3 0.8
AT2G40080 ELF4 (EARLY FLOWERING 4) Brapa_ESTC038745 9602 6902 1.4 0.7
Brapa_ESTC023094 8429 13088 0.6 1.6
Brapa_ESTC027935 6642 4190 1.6 0.6
AT5G15850 COL1 (CONSTANS-LIKE 1) Brapa_ESTC013309 9416 16434 0.6 1.7

z)R/C and C/R indicate the expression ratio in RCBr over Chiifu and Chiifu over RCBr, respectively.

Genes related to gibberellic acid metabolism.

At_Locus Gene annotation Br_SEQ_ID Prove intensity Fold change
RCBr Chiifu R/Cz) C/R
AT1G30040 GA2OX2 (Gibberellin 2-beta-dioxygenase) Brapa_ESTC040202 1319 6104 0.2 4.6
Brapa_ESTC015031 16 1969 0.0 126.4
AT1G02400 GA2OX6/DTA1 (Gibberellin 2-OXIDASE 6) Brapa_ESTC015242 10762 8386 1.3 0.8
AT4G21200 GA2OX8 (Gibberellin 2-OXIDASE 8) Brapa_ESTC024684 431 665 0.6 1.5
Brapa_ESTC015062 423 624 0.7 1.5
AT1G15550 GA3OX1/GA4 (GA REQUIRING 4) Brapa_ESTC027719 554 164 3.4 0.3
AT5G07200 GA20OX3/YAP169 (Gibberellin 20 oxidase 3) Brapa_ESTC045377 1296 155 8.4 0.1
Brapa_ESTC029845 4761 183 26.1 0.0
Brapa_ESTC027440 1114 81 13.7 0.1
AT5G14920 Gibberellin-regulated family protein Brapa_ESTC003065 9782 9954 1.0 1.0
Brapa_ESTC016390 5777 9747 0.6 1.7
Brapa_ESTC042364 10773 8665 1.2 0.8
Brapa_ESTC024407 6879 6685 1.0 1.0
Brapa_ESTC007229 5209 4283 1.2 0.8
Brapa_ESTC010071 5161 3227 1.6 0.6
AT5G59845 Gibberellin-regulated family protein Brapa_ESTC007822 507 20 25.9 0.0
AT1G74670 Gibberellin-responsive protein, putative Brapa_ESTC037994 4417 3177 1.4 0.7
Brapa_ESTC008005 3581 2190 1.6 0.6
AT1G22690 Gibberellin-responsive protein, putative Brapa_ESTC003796 5081 1538 3.3 0.3
AT5G51310 Gibberellin 20-oxidase-related Brapa_ESTC046793 859 1135 0.8 1.3
AT3G10185 Gibberellin-regulated GASA/GAST/Snakin family protein Brapa_ESTC036747 4237 5060 0.8 1.2
AT4G26420 GAMT1 | Gibberellin carboxyl-O-methyltransferase Brapa_ESTC019836 1067 287 3.7 0.3
AT5G56300 GAMT2 | Gibberellin carboxyl-O-methyltransferase Brapa_ESTC009976 773 115 6.7 0.1

z)R/C and C/R indicate the expression ratio in RCBr over Chiifu and Chiifu over RCBr, respectively.

Table 1 List of primers used in qRT-PCR analysis.

BRAD ID number identified by BlastN with expressed sequence tag sequence at http://brassicadb.org/brad/.

qRT-PCR: quantitative reverse transcription polymerase chain reaction.

Table 2 Transcription factors whose expressions levels were top 20 in both RCBr and Chiifu, and over two-fold in RCBr or Chiifu.

R/C and C/R indicate the expression ratio in RCBr over Chiifu and Chiifu over RCBr, respectively. Flowering-related TF genes were omitted in this Table.

Table 3 Flowering-related genes which were selected from 196 flowering genes on Br300K microarray.

R/C and C/R indicate the expression ratio in RCBr over Chiifu and Chiifu over RCBr, respectively.

Table 4 Genes related to gibberellic acid metabolism.

R/C and C/R indicate the expression ratio in RCBr over Chiifu and Chiifu over RCBr, respectively.

Table 1

Table 2

Table 3

Table 4

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