A naturally occurring splicing site mutation in the Brassica rapa FLC1 gene is associated with variation in flowering time

Abstract

FLOWERING LOCUS C (FLC), encoding a MADS-domain transcription factor in Arabidopsis, is a repressor of flowering involved in the vernalization pathway. This provides a good reference for Brassica species. Genomes of Brassica species contain several FLC homologues and several of these colocalize with flowering-time QTL. Here the analysis of sequence variation of BrFLC1 in Brassica rapa and its association with the flowering-time phenotype is reported. The analysis revealed that a G-->A polymorphism at the 5' splice site in intron 6 of BrFLC1 is associated with flowering phenotype. Three BrFLC1 alleles with alternative splicing patterns, including two with different parts of intron 6 retained and one with the entire exon 6 excluded from the transcript, were identified in addition to alleles with normal splicing. It was inferred that aberrant splicing of the pre-mRNA leads to loss-of-function of BrFLC1. A CAPS marker was developed for this locus to distinguish Pi6+1(G) and Pi6+1(A). The polymorphism detected with this marker was significantly associated with flowering time in a collection of 121 B. rapa accessions and in a segregating Chinese cabbage doubled-haploid population. These findings suggest that a naturally occurring splicing mutation in the BrFLC1 gene contributes greatly to flowering-time variation in B. rapa.

Fig. 1.

Frequency distribution of flowering-time phenotype in the collection of 121 B. rapa accessions. The plants were grown in the open field (OF) or in the growth chamber (GC) after 25 d of vernalization of germinated seeds. 60NF indicates that the plants did not show flower buds after 60 d.

Fig. 2.

Seven sites of nucleotide polymorphism among 30 sequenced B. rapa accessions in the BrFLC1 gene. Sites of base variation are indicated with a vertical arrow according to their relative position in BrFLC1 and their names are given above the arrow.

Fig. 3.

RT-PCR amplification of BrFLC1. Leaf samples were collected from plants grown for 42 d in the growth chamber after cold treatment at 4 °C in the dark for 25 d. The constitutive transcript and alternative transcripts are indicated by arrows.

Fig. 4.

Discovery of five splicing patterns. SpG represents the constitutive splicing pattern; SpA1–SpA3 indicate alternative splicing patterns; solid boxes, exons; the dotted box, a missing exon; dotted lines, introns that are missing completely or partly compared with the constitutive spliced transcripts; continuous lines, retention of intron parts; the numbers 25 and 55 indicate the number of nucleotides retained in intron 6; the shaded region, the transcribed region from exon 7 for different splicing patterns. Premature stop codons (12–14nt for SpA1 and SpA2) are indicated by an arrow. In SpA3, an alternative splice acceptor site in the last exon is used, leading to a deletion of exon 6 sequences.

Fig. 5.

Schematic model of the development of the CAPs marker used to detect the splicing site mutation of BrFLC1. (A) MvaI restriction site in the amplified fragment of BrFLC1 covering the region from exon 4 to exon 7 (E4–E7). MvaI recognition sites are indicated by a solid arrow; the splicing site is shown by a dotted arrow; and the corresponding fragment size is indicated. (B) MvaI digestion of PCR products. A and G represent Pi6 + 1(A) and Pi6 + 1(G) alleles, respectively, followed by fragment size (in base pairs).

Fig. 6.

Distribution of flowering time and CAPS marker genotype in a collection of B. rapa germplasm (n=121). (A) Plants were grown in the open field; (B) plants were grown in the growth chamber. Black and white columns represent accessions with G and A alleles, respectively, at the Pi6+1 site of BrFLC1. Arrows indicate mean values of lines with an A or G allele. NF indicates lines that did not flower during the experiment.