Functional alleles of the flowering time regulator FRIGIDA in the Brassica oleracea genome

Abstract

Background: Plants adopt different reproductive strategies as an adaptation to growth in a range of climates. In Arabidopsis thaliana FRIGIDA (FRI) confers a vernalization requirement and thus winter annual habit by increasing the expression of the MADS box transcriptional repressor FLOWERING LOCUS C (FLC). Variation at FRI plays a major role in A. thaliana life history strategy, as independent loss-of-function alleles that result in a rapid-cycling habit in different accessions, appear to have evolved many times. The aim of this study was to identify and characterize orthologues of FRI in Brassica oleracea.

Results: We describe the characterization of FRI from Brassica oleracea and identify the two B. oleracea FRI orthologues (BolC.FRI.a and BolC.FRI.b). These show extensive amino acid conservation in the central and C-terminal regions to FRI from other Brassicaceae, including A. thaliana, but have a diverged N-terminus. The genes map to two of the three regions of B. oleracea chromosomes syntenic to part of A. thaliana chromosome 5 suggesting that one of the FRI copies has been lost since the ancient triplication event that formed the B. oleracea genome. This genomic position is not syntenic with FRI in A. thaliana and comparative analysis revealed a recombination event within the A. thaliana FRI promoter. This relocated A. thaliana FRI to chromosome 4, very close to the nucleolar organizer region, leaving a fragment of FRI in the syntenic location on A. thaliana chromosome 5. Our data show this rearrangement occurred after the divergence from A. lyrata. We explored the allelic variation at BolC.FRI.a within cultivated B. oleracea germplasm and identified two major alleles, which appear equally functional both to each other and A. thaliana FRI, when expressed as fusions in A. thaliana.

Conclusions: We identify the two Brassica oleracea FRI genes, one of which we show through A. thaliana complementation experiments is functional, and show their genomic location is not syntenic with A. thaliana FRI due to an ancient recombination event. This has complicated previous association analyses of FRI with variation in life history strategy in the Brassica genus.

Figure 1

Cloning BoFRIa and BoFRIb. (a) Southern analysis of A12 BACs identified by colony hybridisation and probed with AtFRI. Lanes 1, 4, 5, 8, 11, 12 and 13 contain clones that show homology to FRIGIDA. BACs in lanes 2, 3, 6, 7, and 10 do not cross hybridise (b) HindIII digest of six BACs probed with conserved region from exons 2 and 3 of BoFRIa. Lane 5 contains JBo72I23 from which BoFRIa was sequenced. Lane 7 contains JBo88G16. Lanes 1, 2, 3 and 6 contain four further BACs showing the same hybridization pattern as JBo88G16. Note the intensity of the hybrization is indicative of the sequence divergence between BoFRIa and BoFRIb (See 1c) (c) Comparison of the protein sequences of BoFRIa and BoFRIb with other members of the FRI sub-family. From top to bottom they are Brassica oleracea BoFRIa, Brassica oleracea BoFRIb, Thelliungiella halophila ThFRI, Arabidopsis thaliana AtFRI Arabidopsis lyrata ssp lyrata AllFRI and Arabidopsis lyrata ssp petraea AlpFRI. The N-terminal domain containing the conserved region of 37 amino acids (indicated by solid bar) that defines copies of FRIGIDA from other members of the FRI superfamily [17]. The coiled-coil domains are indicated by the red lines.

Figure 2

Mapping BoFRIa and BoFRIb. (a) A12DHdxGDDH33 mapping population probed with conserved region from exons 2 and 3 of BoFRIa. Two loci are identified; one monomorphic (upper band) and a second segregating with the two parental alleles (lower two bands). (b) Meiotic pachytene spread with JBo72I23 (BoFRIa, green, white arrow) hybridizing to C3 between the telomere and JBo62M08 (red, yellow arrow). (c) and (d) JBo88G16 (BoFRIb) hybridizes to the short arm of C9: (c) mitotic metaphase with JBo88G16, (green, arrows), BoB061G14 and 45S rDNA (red), (d) reprobe of (c) with JBo32J18 (green, arrows), a marker for C9.

Figure 3

BoFRIa and BoFRIb map to regions of the B. oleracea genome that are non-syntenic with A. thaliana. (a) Comparison of BoFRIa genomic clone with annotated regions of A. thaliana chromosomes 4 and 5 represented in the 5' to 3' orientation. Solid black rectangles correspond to the three exons that make up FRI. Arrows indicate the orientation of other genes. (i) BoFRIa, genomic clone. (ii) Region of A. thaliana chromosome 5 containing an iron superoxide dismutase (FSD2, At5g51100) and At51090 showing homology to FRI intron 1 and exon 3. (iii) Region of A. thaliana chromosome 4 containing AtFRI and At4g400651 showing homology to exon 9 of FSD2. (b) Proposed derivation of A. thaliana and B. oleracea linkage groups from ancestral karyotype (AK6 and AK8) as suggested in [53] showing the position of FRI and FLC. The rearrangement of ancestral blocks W and R results in orthologues of FRI and FLC being brought into close proximity in the ancestral Brassica genome. This region is present on chromosomes C2, C3 and C9 of B. oleracea and is located in regions of the B. oleracea genome showing synteny to A. thaliana chromosome 5. BoFRIa is located on C3 and BoFRIb on C9. The copy of FRI on C2 has been lost.

Figure 4

Comparison of first 160 amino acids of BoFRIa from 55 BolDFS genotypes. Protein comparison of BoFRIa from cultivated genotypes of B. oleracea listed by crop type. The plate co-ordinates refer to those listed in Table 1. The conserved region of 37 amino acids that defines FRI from other members of the FRI superfamily is delineated by the horizontal black bar.

Figure 5

Distribution of flowering times in T1 transformants carrying BoFRIa-1 and BoFRIa-4 alleles. Histogram of the flowering time of T1 lines transformed with BoFRIa-1 and BoFRIa-4 measured as days to flower. The flowering time of wild type Col-0 plants and Col-0 transformed with AtFRI are indicated.

Figure 6

Functional analysis of the two most common BoFRIa alleles. Average flowering time of T2 families transformed with the two major BoFRIa alleles BoFRIa-1 and BoFRIa-4 and compared to Col-0 transformed with the AtFRI allele and Col-0 wild type. (a) Flowering time measured as days-to-flower. The error bars show 95% confidence intervals. (b) Flowering time measured as final leaf number. The error bars show 95% confidence intervals. Segregating progeny with and without the transgene are indicated by + and - respectively.