Sequence variation and functional analysis of a FRIGIDA orthologue (BnaA3.FRI) in Brassica napus

Background Allelic variation at the FRIGIDA (FRI) locus is a major contributor to natural variation of flowering time and vernalization requirement in Arabidopsis thaliana. Dominant FRI inhibits flowering by activating the expression of the MADS box transcriptional repressor FLOWERING LOCUS C (FLC), which represses flowering prior to vernalization. Four FRI orthologues had been identified in the domesticated amphidiploid Brassica napus. Linkage and association studies had revealed that one of the FRI orthologues, BnaA3.FRI, contributes to flowering time variation and crop type differentiation. Results Sequence analyses indicated that three out of the four BnaFRI paralogues, BnaA3.FRI, BnaA10.FRI and BnaC3.FRI, contained a large number of polymorphic sites. Haplotype analysis in a panel of 174 B. napus accessions using PCR markers showed that all the three paralogues had a biased distribution of haplotypes in winter type oilseed rape (P < 0.01). Association analysis indicated that only BnaA3.FRI contributes to flowering time variation in B. napus. In addition, transgenic functional complementation demonstrated that mutations in the coding sequence of BnaA3.FRI lead to weak alleles, and subsequently to flowering time variation. Conclusion This study for the first time provides a molecular basis for flowering time control by BnaA3.FRI in B. napus, and will facilitate predictive oilseed rape breeding to select varieties with favorable flowering time and better adaption to latitude and seasonal shifts due to changing climate. Electronic supplementary material The online version of this article (10.1186/s12870-018-1253-1) contains supplementary material, which is available to authorized users.


Background
Timely transition from vegetative to reproductive growth is of great significance for plants, in avoiding adverse environments and ensuring seed set. In the model species Arabidopsis thaliana, a flowering time regulatory gene network has been established [1]. External and internal signals associated with different pathways converge on the common set of key integrators FLOWERING LOCUS T (FT), LEAFY (LFY), and SUPPRESSOR OF OVEREX-PRESSION OF CONSTANS (SOC1), which act on downstream genes to promote floral organ formation [1][2][3][4].
FT is a key flowering integrator gene encoding a florigen protein that moves through the phloem from leaves to the shoot apex, and induces the floral transition in many plant species [5,6].
The photoperiod and vernalization pathways are the two major pathways responding to environmental cues that determine the flowering time of A. thaliana, as well as most other plants [7]. Based on vernalization requirement, genotypes of A. thaliana may be grouped into late flowering (winter-annual) and early flowering ecotypes (summer-annual). FLOWERING LOCUS C (FLC) and FRIGIDA (FRI) are the two key genes in the vernalization pathway [8][9][10]. Dominant FRI represses flowering through activating the expression of FLC [11], which encodes a MADS box transcriptional regulator and represses flowering through directly inhibiting the expression of SOC1 gene [8,12]. Vernalization promotes flowering through proteasome-mediated degradation of FRI protein and epigenetic silencing of FLC [13,14]. Allelic variation at either FRI or FLC locus will lead to a summer-annual life cycle of A. thaliana [10]. Loss-of-function mutations of FRI were demonstrated to be the major determinant of vernalization requirement and explain most flowering time variation of early-flowering ecotypes [15,16].
Brassica napus (AC genome, oilseed rape, canola or rapeseed) is the third most important oil crop in the world. This domesticated amphidiploid species most likely originated from a natural cross between B. rapa (A genome) and B. oleracea (C genome)~7500 years ago [17]. As with Arabidopsis, B. napus can have a vernalization requirement to initiate flowering. Based on vernalization requirement, the three crop types of oilseed rape recognized are spring-type (SOR), semi-winter type (SWOR, rapeseed accessions that initiate flowering with a moderate vernalization condition (0 -4°C for 15-30 d)) and winter-type (WOR). Due to historical duplication events that have occurred since Arabidopsis and Brassica diverged from a common ancestor, the diploid genomes of B. rapa and B. oleracea appear triplicated compared with Arabidopsis, and so the B. napus crop genome is extreme complex [17]. A large number of QTLs and candidate genes that contribute to flowering time variation have been documented in B. napus [18][19][20][21][22][23]. Orthologues of major flowering genes such as CONSTANS (CO), FLC, and FT have been found to be functionally conserved between B. napus and Arabidopsis. For example, the BnaFLC orthologues were proven to confer winter requirement in B. napus and contribute to the major vernalization-responsive flowering time variation in B. napus in a manner similar to that of AtFLC in Arabidopsis [24]. Variations at the BnaFLC orthologous loci that result in loss-of-function or reduced cold sensitivity alleles have been associated with different vernalization requirements and differentiation of WOR and SOR crop types in B. napus [25]. In addition, QTL mapping identified associations between FRI locus at the A3 chromosome (BnaA3.FRI) and vernalization response and flowering time variation in B. napus [18,26,27]. However, the molecular bases towards flowering regulation by BnaA3.FRI in B. napus are still unknown.
More recently, four FRI paralogues (BnaA.FRI.a, BnaX.FRI.b, BnaX.FRI.c and BnaX.FRI.d) were identified in B. napus [28]. In this study, we renamed the four paralogues as BnaA3.FRI, BnaA10.FRI, BnaC3.FRI and BnaC9.FRI, respectively, according to their locations in the 'Darmor-bzh' reference genome [17]. Association study further indicated that one of the FRI orthologues, BnaA3.FRI, was a major regulator of flowering time and vernalization [28]. We analyzed the sequence polymorphisms of the four BnaFRI paralogues in B. napus and performed functional analysis of BnaA3.FRI. By profiling the allelic variation of BnaA3.FRI in a panel of 174 B. napus accessions representing different crop types, we confirmed that the nucleotide polymorphism within this gene was associated with flowering time variation and local adaption. Expression of BnaA3.FRI in wild-type (WT) Arabidopsis Col-0 revealed that sequence variations in the coding region of BnaA3.FRI result in weak alleles, and led to early flowering. This study provides further molecular evidence to support predictive breeding of B. napus to select varieties with favorable flowering time and better adaption to latitude and seasonal shifts due to changing climate.

Plant materials and growth conditions
A collection of 174 B. napus cultivar accessions from across the world, including 17 winter-type oilseed rape (WORs), 39 spring-type oilseed rape (SORs), and 118 semi-winter-type oilseed rape (SWORs), was used for genotype analyses of BnaA3.FRI (Additional file 1). A subset of 30 accessions representing different crop types was used for identification of nucleotide polymorphism and haplotype determination of BnaA3.FRI (Additional file 2). The duration of flowering time (from the date of sowing to the date of half of the plants flowering) of all the accessions was recorded in two successive years (2013-2014) of field trials at spring growth environments (Xining, Qinghai, 36°35′ N, 101°47′ E and Lanzhou, Gansu, 36°02′ N, 103°50′ E) in North China. Seeds were sowed on May 21 and harvested on September 16, 2013 at Xining, and sowed on May 20 and harvested on September 19, 2014 at Lanzhou. At these two spring environments, plants were grown under a day length of 14~14.5 h without vernalization. All these accessions were also grown in semi-winter environment at Wuhan (30°36′ N, 104°18′ E). Seeds were sowed on October 1, 2013 and harvested on May 12, 2014. Plants were grown under a day length of 10~11 h and vernalized in the winter. The temperatures during the whole growth period of oilseed rape in the three field sites were provided in Additional file 3. Each accession was grown in the field under natural rain-fed conditions in two-row plots with 8-10 plants per row. Tissues of roots, hypocotyledonary axis, cotyledons, leaves, stems, floral buds, siliques and seeds from Tapidor (a typical WOR) were collected and used for gene expression pattern analysis. In addition, leaves before and after vernalization, floral buds and flowers from Tapidor, Ningyou7 (a typical SWOR) and Westar (a typical SOR) were collected for analysis of BnaA3.FRI expression. Samples from three individual plants were obtained as biological replicates.
Wild-type A. thaliana ecotype Columbia (Col-0) (fri + FLC) was used for transformation of BnaA3.FRI. Seeds of T1 and T2 (includes both homo-and heterozygous) transgenic lines were screened on half-strength Murashige and Skoog (MS) medium containing 50 mg/L kanamycin under a 16-h-light/8-h-dark photoperiod in greenhouse. All plants were grown under the condition of 16-h-light/8-h-dark photoperiod and 70% humidity at 23°C. Five plants per line were selected (days to flowering was close to the mean of days to flowering of the respective line) as biological repeats for gene expression analysis.
To analyze the expression pattern of BnaA3.FRI, a 1.4 kb promoter fragment from Tapidor (HAP1) was amplified using primers proF-BamHI and proR-HindIII and then cloned into the BamHI-HindIII sites of pCAMBIA2301-GUS, which was generated from pCAMBIA2301 by inserting the β-glucuronidase (GUS) gene into the HindIII-PmlI sites, to construct the pBnaA3.FRI::GUS plasmid. For cellular localization analysis, full-length of BnaA3.FRI cDNA sequence (1729 bp) was isolated from Tapidor (HAP1) using primers BnaA3.FRI-F-PmlI and BnaA3.FRI-R-AsiSI (Additional file 4) and inserted into the PmlI-AsiSI sites of pDOE20 [30] to generate mVenus-BnaA3.FRI (YFP-BnaA3.FRI) fusion protein. Meanwhile, a nuclear marker Ghd7 [31] was introduced into the BamHI-XbaI sites of pDOE20 to generate the mTurquoise2-GHD7 (CFP-GHD7) fusion protein. In this way, two fusion proteins co-express in a single construct.

Plant transformation and phenotyping
For expression pattern determination and functional analysis of BnaA3.FRI, Agrobacterium tumefaciens strain GV3101 harboring plasmid pHAP1::HAP1, pHAP1::HAP2, pHAP1::HAP3, pHAP1::HAP8, pHAP2::HAP2, pHA-P2::HAP1, or pBnaA3.FRI::GUS was used to transform A. thaliana accession Col-0 by a floral dip method [32]. T1 and T2 transgenic plants were screened on half-strength MS plates containing 50 mg/L kanamycin. Flowering time of 15 T2 transgenic plants of each line was recorded. Flowering time was determined by the mean rosette leaf number or the mean days from sowing to bolting time, with the bolting time determined by when the inflorescence stem was 1 cm high.

RT-PCR analysis
Total RNA was isolated using an RNAprep Pure Plant kit (BioTeKe, China) according to the manufacturer's instructions. The concentration of RNA was determined by spectrometry (NanoDrop; Thermo Scientific, USA). RNase-free DNaseI (Thermo Scientific, USA) was used to remove contaminated DNA, and then a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) was used to reverse transcribe RNA samples. Quantitative RT-PCR (qRT-PCR) was performed to analyze the expression levels of BnaA3.FRI in B. napus, and BnaA3.FRI and AtFLC in transgenic plants. Amplification of cDNA was conducted using a SYBR Green master mixture (BioRad, USA) with a LightCycler 96 (Roche, USA). The expression level of Epsin N-terminal homology (BnaENTH) [33] and ACTIN2 gene (AT3G18780) was used as an internal control in B. napus and A. thaliana, respectively. The sequence of primers used for quantitative RT-PCR analyses is listed in Additional file 4.

Histochemical staining and subcellular localization
Tissues including seedlings, leaves, stems, floral buds, and siliques from the pBnaA3.FRI::GUS T1 and T2 transgenic lines were used for GUS staining to determine the expression pattern of BnaA3.FRI. Tissues were incubated in the staining solution containing 1 g/L X-gluc (Sigma-Aldrich, USA) at 37°C overnight followed by decolorization with 70% ethanol [34]. A transient expression experiment was performed in tobacco (Nicotiana benthamiana cv. SR1) leaves to analyze the subcellular localization of BnaA3.FRI according to Voinnet et al. [35]. The Agrobacterium GV3101 cells containing the pDOE20 recombinant plasmid that expresses CFP-GHD7 and YFP-BnaA3.FRI, also expresses the silencing suppressor p19 of Tomato bushy stunt virus, were harvested and re-suspended in the solution of 10 mM MES-KOH, pH 5.6, containing 10 mM MgCl 2 and 150 mM acetosyringone to a final density of 0.8 at 600 nm (OD 600 ). The Agrobacterium suspension was injected into expanded leaves of 6-week-old tobacco plants. Three days after injection, the leaves were observed with a laser scanning confocal imaging system (TCS SP2, Leica, Germany).

Statistical analysis
For flowering time and gene expression comparison between each two data sets, we performed F test to compare the significant level of sample variances (α = 0.05), and then equal variance or unequal variance two-tailed t-test was conducted according to the result of F test (equal variance t-test when the P value of F test equal to or above 0.05, unequal variance t-test when the P value of F test below 0.05).

Sequence variations of BnaFRI paralogues in B. napus
To investigate sequence variations of the four BnaFRIs  (Fig. 1a). Of the 26 SNPs, 14 result in amino acid substitutions (Fig. 1b). In BnaA10.FRI, a total of four INDELs and 26 SNPs were identified (Additional file 5). Three (21 bp, 9 bp and 3 bp) of the INDELs located in exon 1, and the other (1 bp) in intron 1 (Fig. 1a). Eleven of the SNPs result in amino acid substitutions. Six INDELs and 44 SNPs were identified in BnaC3.FRI (Fig. 1a, Additional file 5). Of the six INDELs, two (21 bp and 9 bp) were located in exon 1, three (2 bp, 1 bp and 7 bp) in intron 1, and the other (11 bp) in intron 2 (Fig. 1a). Fourteen of the SNPs cause amino acid substitutions. All the INDELs in exons of the three paralogues result in in-frame amino acid insertion/deletion. Although such variations were found in these three BnaFRI paralogues, no such sequence variation was detected in BnaC9.FRI.
To further explore sequence variations of BnaA3.FRI, the~2.4 kb genomic fragment of BnaA3.FRI mentioned above was amplified from another 20 B. napus accessions representing different crop types, and sequenced (Additional file 2). In addition to the previously identified 30 polymorphic sites (4 INDELs and 26 SNPs) in the 10 B. napus accessions, three more polymorphic sites were identified in exon 1, including one 3-bp INDEL and two SNPs (Fig. 1b). All sequence variations detected across the 30 sequenced accessions could be classified into nine haplotypes (HAP1-HAP9) (Fig. 1b). HAP1 was identified in 10 accessions, HAP2 in eight accessions and HAP8 in five accessions (Additional file 2). All the WOR carried HAP1, which corresponded to the mHAP1 inferred by INDEL markers. HAP2 to HAP7 corresponded to mHAP2, HAP8 to mHAP3, and HAP9 corresponded to mHAP4 (Fig. 1b). To dissect the associations of BnaFRIs and flowering time, the flowering time of different BnaFRI mHAPs within each crop type were compared using t-tests. For BnaA3.FRI, the means of flowering date of SOR accessions with mHAP1, mHAP2, and mHAP3 had no difference in spring environments at 2013XN and 2014LZ (Fig. 2a, b). However, accessions with mHAP1 and mHAP3 displayed a significant difference in flowering time when grown in the semi-winter environment of 2014WH (146.2 ± 17.18 d vs. 160.1 ± 9.7 d, P = 0.0218) (Fig. 2c). In SWOR, accessions with mHAP1 flowered later than those with mHAP3 in all environments (Fig. 2d, e, and f ), and displayed a significant difference in flowering time when grown in the spring environment of 2014LZ (78.1 ± 16.9 d vs. 59.1 ± 8.5 d, P = 0.0113) (Fig. 2e). The SWOR accessions with mHAP2 showed an intermediate flowering time between mHAP1 and mHAP3 (Fig. 2d, e, and f ). The WOR accessions, which only contained mHAP1, flowered extremely late or did not flower when grown in the two spring environments of 2013XN and 2014LZ, and flowered (178.5 ± 5.08 d) much later than all SOR and SWOR accessions when grown in the winter environment of 2014WH (Additional file 1). Different from BnaA3.FRI, the accessions with different BnaA10.FRI or BnaC3.FRI mHAPs within each crop type did not

Expression and subcellular localization of BnaA3.FRI
The expression level of BnaA3.FRI was analyzed in leaves before and after vernalization, floral buds, and flowers of the three crop types represented by Tapidor (WOR with HAP1), Ningyou7 (SWOR with HAP2), and Westar (SOR with HAP2) using quantitative RT-PCR (qRT-PCR). As shown in Fig. 3a, BnaA3.FRI expressed in all the four tissues, and displayed a lowest level in leaves after vernalization and highest in floral buds.
To explore the expression pattern of BnaA3.FRI, we checked the mRNA transcripts of BnaA3.FRI in roots, hypocotyledonary axes, cotyledons, leaves, stems, floral buds, siliques and seeds from the WOR variety Tapidor (Fig. 3b). BnaA3.FRI transcribed in all tissues but showed much higher expression level in roots, leaves, floral buds and seeds (Fig. 3b). Transgenic Arabidopsis lines harboring the construct pBnaA3.FRI::GUS, which contained the 1.5 kb promoter region of BnaA3.FRI from Tapidor (HAP1), were used for GUS staining. Histochemical staining revealed strong GUS activity in roots, leaves, and flowers ( Fig. 3c-g).
Previous studies in Arabidopsis demonstrated that FRI acts as a scaffold protein to recruit several chromatin modifiers in nucleus and epigenetically modify the key flowering regulator FLC [11,13]. BnaA3.FRI is predicted to translate a putative protein containing 596 amino acids, which carries the conserved 'Frigida' domain [36], and shares 58% identity in amino acid sequence with AtFRI (AF228500) [15,28]. To explore the subcellular localization of the BnaA3.FRI protein, the plasmid pDOE20, that co-expresses fusion proteins YFP-BnaA3.FRI and CFP-Ghd7, was introduced into tobacco leaves for transient expression analysis. The results demonstrated that BnaA3.FRI was co-localized with Ghd7 in the nucleus (Fig. 3h).
To confirm whether the later flowering of transgenic lines was caused by elevated AtFLC expression level due to the introduction of a functional BnaA3.FRI gene, qRT-PCR was performed to compare the expression levels of BnaA3.FRI and AtFLC between transgenic lines and WT Col-0. In accordance with the flowering time variations between these lines, the BnaA3.FRI and AtFLC levels in the two HAP2 transgenic lines were significantly lower than those in the two HAP1 transgenic lines (Fig. 4c and e).
The BnaA3.FRI expression levels in pHAP1::HAP3, pHAP2::HAP1, and pHAP1::HAP1 transgenic lines were significantly higher than the transgenic lines harboring pHAP2::HAP2, pHAP1::HAP2, and pHAP1::HAP8, and WT Col-0 (P < 0.01) (Fig. 5b). While driven by the same promoter, the BnaA3.FRI mRNA levels showed significant differences among the transgenic lines of pHAP1::HAP1, pHAP1::HAP2, pHAP1::HAP3, and pHAP1::HAP8, and also between pHAP2::HAP1 and pHAP2::HAP2 (Fig. 5b), suggesting that there might have an important cis-element necessary for transcription within the coding region. The transcription levels of AtFLC were up-regulated in all transgenic lines when compared to WT Col-0 (Fig. 5d). It is worth noting that the expression level of BnaA3.FRI did not correlate with the mRNA level of AtFLC (Fig. 5b, d). However, the expression level of AtFLC was proportional to the flowering time of these transgenic lines (Fig. 5a, d). Taken together, our results demonstrate that HAP1 is stronger than other haplotypes and mutations in the coding sequence of BnaA3.FRI result in weak alleles, which contribute to variation in flowering time.

Discussion
Potential functional divergence of different FRI orthologues in B. napus Four FRI orthologues, BnaA3.FRI, BnaA10.FRI, BnaC3.-FRI and BnaC9.FRI, had previously been identified in B. napus [28,37]. In this study, a large number of sequence variations including INDELs and SNPs were identified within the coding sequences of BnaA3.FRI, BnaA10.FRI and BnaC3.FRI (Additional file 5). In contrast to the high level of polymorphism observed in the other B. napus FRI genes, no sequence variation was identified in BnaC9.FRI. Correspondingly, the orthologue of BnaC9.-FRI in B. oleracea, BolC.FRI.b, was also highly conserved in different crop types of B. oleracea [38]. Moreover, BnaC9.FRI in B. napus displayed a high level of mRNA transcripts in both vernalized and non-vernalized tissues [38]. These results suggest BnaC9.FRI may be functionally important, but requires further investigation.
Marker-based haplotype analysis of BnaFRIs in a panel of 174 B. napus identified four, three, and four mHAPs for BnaA3.FRI, BnaA10.FRI and BnaC3.FRI, respectively. Chi-square analyses showed that the WOR accessions had a biased distribution in mHAPs of all the three Bna-FRIs, and the SOR and SWOR had a biased distribution in mHAPs of BnaA3.FRI and BnaA10.FRI (Table 2). However, of the three BnaFRIs, only BnaA3.FRI variation was associated with flowering time variation in B. napus (Fig. 2), and no association was identified between flowering time and combinations of BnaFRIs alleles (Additional file 8). In accordance with previous studies, our results suggested that BnaA3.FRI plays important roles in vernalization response, crop type differentiation and flowering time variation in B. napus [18,[26][27][28]. However, associations of BnaC3.FRI and BnaA10.FRI with vernalization response and flowering time have not documented. Sequence analyses indicated that the two BnaC3.FRI haplotypes shared extremely high amino acid identity to the two BolC.FRI.a alleles (99.3% between BnaC3.FRI -mHAP1 and BolC.FRI.a-E8, and 99.8% between BnaC3.FRI -mHAP2 and BolC.FRI.a-E1) (Additional file 10), the corresponding orthologues in B. oleracea [38]. Moreover, BnaC3.FRI -mHAP1 and BolC.-FRI.a-E1 were over-represented in both winter type B. napus and B. oleracea [38]. The two BolC.FRI.a alleles have been demonstrated to equally delay flowering time in Arabidopsis [38], indicating that sequence variations in BolC.FRI.a do not affect its function, which is in accordance with our result that BnaFRI.C3 did not associate with flowering time variation in B. napus (Additional file 7). Thus, further study will need to speculate the specific functions of BanC3.FRI and BnaA10.FRI.
Variation of BnaA3.FRI is tightly associated with flowering time variation in B. napus

Previous linkage and association studies indicated that
BnaA3.FRI is an important flowering regulator [28] in B. napus. Extensive sequence variations were identified in both 5' UTR and coding region of BnaA3.FRI (Fig. 1), and could be classified into four mHAPs across the 174 acceessions. The distribution of haplotypes showed strong association with crop type differentiation, as all the WOR accessions contained mHAP1, the majority of SWOR contained mHAP2, while the SOR did not have a major haplotype (Table2). Expression of different BnaA3.FRI haplotypes in Arabidopsis showed that the function of mHAP1 (corresponding to HAP1) is much stronger than mHAP2 (corresponding to HAP2) and mHAP3 (corresponding to HAP8) (Figs. 4 and 5). Thus, our results establish that the BnaA3.FRI mHAP1 is the wild type allele, and confers the vernalization requirement and late flowering of WOR.
Further investigation of the relationship between BnaA3.FRI haplotypes and variations in flowering time in both SWOR and SOR indicated that different BnaA3.-FRI haplotypes were consistent with significant differences in flowering time (Fig. 2). As expected, the SWOR with mHAP1 flowered later than those with mHAP2 or mHAP3, especially in spring environment (Fig. 2e). The later flowering of SWOR with mHAP1 may result from the activated expression of BnaFLCs. In Arabidopsis, FRI is a major determinant of vernalization response and flowering time variation, which is primarily achieved by activating the expression of FLC, and so loss-of-function mutation of FRI could lead to early flowering [15,39].
The vernalization pathway appears to be conserved in B. napus, since QTLs corresponding to BnaA3.FRI and BnaA10.FLC explained the majority of vernalization response and flowering time variations in different populations [18,40]. However, it was interesting to note that the SOR containing mHAP1 flowered earlier than those with mHAP2 or mHAP3 (Fig. 2c). This may because the major FLC locus (BnaA10.FLC) in SORs, which is a key gene in vernalization pathway and acts down-stream of FRI, is mutated (our unpublished data). On the other hand, a similar result has also been documented in Arabidopsis, where summer-annual accessions with functional FRI alleles accelerate flowering relative to those with nonfunctional FRI alleles [16]. Both the SOR and summer-annual Arabidopsis have an early flowering growth type and can initiate flowering without vernalization via the photoperiod pathway. Thus, further investigation will be needed to identify the specific flowering time control mechanism of BnaA3.FRI in SOR, and whether it works independent from FLC or vernalization.

Variation in coding region of BnaA3.FRI results in weak alleles
In this study, we identified 33 polymorphic sites within the 5' UTR and coding region of BnaA3.FRI in 30 B. napus accessions (Fig. 1b). An excess of nonsynonymous mutations were identified within the coding region of BnaA3.FRI, with 14 SNPs and two INDELs predicted to cause amino acid substitution or deletion (Fig. 1b). The majority of these polymorphic sites were common between this and a previous study [28], and no frame shift or stop codons mutations were identified within BnaA3.FRI. This differs from the FRI mutation types in Arabidopsis, where the major two mutant alleles, FRI (Col) and FRI (Ler), are loss-of-function and vernalization insensitive [10,15,39,41]. Functional analysis of four different BnaA3.FRI haplotypes in Arabidopsis (HAP1, HAP2, HAP3, and HAP8) indicated they were all functional (Fig. 5). However, HAP1 showed a much stronger function in delaying flowering time than the other three haplotypes. These results suggest that these sequence variations in the coding region of BnaA3.FRI result in weak alleles, and contribute to subtle flowering time variation in B. napus and appear to be under ongoing active selection since domestication. Candidate gene association analysis by Wang et al. [28] identified several SNPs that associate with flowering time variation. We conclude that further studies will need to identify the key amino acid sites that influence the function of BnaA3.FRI protein. The two INDELs in 5' UTR differentiated two major kinds of promoters. However, transgenic analysis demonstrated they had an equivalent function under the conditions tested (Fig. 5). This indicates that these variations in promoter had no or little effect on the expression of BnaA3.FRI.

Conclusions
In this study we identified a number of sequence variations in the coding region as well as 5' UTR region of