A naturally occurring InDel variation in BraA.FLC.b (BrFLC2) associated with flowering time variation in Brassica rapa

Abstract

Background: Flowering time is an important trait in Brassica rapa crops. FLOWERING LOCUS C (FLC) is a MADS-box transcription factor that acts as a potent repressor of flowering. Expression of FLC is silenced when plants are exposed to low temperature, which activates flowering. There are four copies of FLC in B. rapa. Analyses of different segregating populations have suggested that BraA.FLC.a (BrFLC1) and BraA.FLC.b (BrFLC2) play major roles in controlling flowering time in B. rapa.

Results: We analyzed the BrFLC2 sequence in nine B. rapa accessions, and identified a 57-bp insertion/deletion (InDel) across exon 4 and intron 4 resulting in a non-functional allele. In total, three types of transcripts were identified for this mutated BrFLC2 allele. The InDel was used to develop a PCR-based marker, which was used to screen a collection of 159 B. rapa accessions. The deletion genotype was present only in oil-type B. rapa, including ssp. oleifera and ssp. tricolaris, and not in other subspecies. The deletion genotype was significantly correlated with variation in flowering time. In contrast, the reported splicing site variation in BrFLC1, which also leads to a non-functional locus, was detected but not correlated with variation in flowering time in oil-type B. rapa, although it was correlated with variation in flowering time in vegetable-type B. rapa.

Conclusions: Our results suggest that the naturally occurring deletion mutation across exon 4 and intron 4 in BrFLC2 gene contributes greatly to variation in flowering time in oil-type B. rapa. The observed different relationship between BrFLC1 or BrFLC2 and flowering time variation indicates that the control of flowering time has evolved separately between oil-type and vegetable-type B. rapa groups.

Figure 1

FLC2primers used in this study. (a) Genomic structure of A. thaliana FLC gene (from [12]). E, exon; exon sizes shown by white boxes. (b) FLC2IndelF/R: FLC2-specific primers used to screen for 57-bp InDel polymorphisms across exon 4 and intron 4. (c) FLC2F8/FLC2R6: FLC2-specific primers in exon 4 and exon 7. (d) FLC012F1/FLC5: FLC2-specific primers in exon 2 and exon 7. (e) BrCFLC2F/R: FLC2-specific primers in 5′UTR and 3′UTR.

Figure 2

Schematic model of development of InDel marker used to detect 57-bp deletion mutation across exon 4 and intron 4 ofBrFLC2. (a) Insertion and deletion identified in B. rapa ssp. olerferious. Del, deletion; In, insertion. Amplified region used to detect deletion mutation covered part of exon 4, intron 4, and part of exon 5. (b) Amplified fragments. In and Del represent insertion allele and deletion allele, respectively, followed by fragment size (in base pairs). Nine accessions (1–9) are listed in same order as in Table 2 .

Figure 3

Sequence variation affects RNA splicing. (a) PCR fragments amplified from 11 E. coli clones transformed with BrFLC2 RT-PCR products from yellow sarson accession L143. (b) Three alternative splicing patterns identified in accession L143. SPI: constitutive splicing pattern [11]; SPD1–3: alternative splicing patterns. White boxes indicate exons; shaded box, missing parts of exon 4; dotted lines, intron parts missing from transcripts; solid lines, retained parts of introns; black bars, the 5 bp insertion (TAAAT).