An InDel polymorphism across exon 4 and intron 4 of BrFLC2 was discovered in a subset of oil-type B. rapa accessions, including ssp. oleifera and ssp. tricolaris. In this study, we investigated its relationship with flowering time both in a collection of B. rapa natural accessions and in a BC2DH population.
Plants need to sense their environment and initiate flowering at the appropriate time to ensure successful fertilization and production of abundant seeds. There is considerable variation in the flowering time among, but also within, natural populations, as we observed in the present study of a B. rapa germplasm collection. In A. thaliana, the FRI gene was shown to be a major determinant of flowering time variation in the natural population through its effects on the expression of FLC[21, 22]. An InDel variation in the COL1 gene was reported to be correlated with variation in flowering time in B. nigra. In A. thaliana, FLC encodes a MADS-box transcription factor that acts as a dose-dependent flowering repressor [9, 22]. Four copies of the FLC gene in B. rapa increase the potential variation in flowering time . In our previous study, we identified a splicing site polymorphism Pi6 + 1(G/A) in BrFLC1 that was significantly associated with the naturally occurring variation in flowering time in B. rapa. In that study, we examined 96 lines, six of which were oil-types. In contrast, half of the lines examined in the present study were oil-types. Because there were so few oil-types in our previous study, we did not identify that oil-type and vegetable-type B. rapa showed different relationships between alleles of BrFLC1 and flowering time variation. In the present study, we could not detect any effect of allelic variation in BrFLC1 Pi6 + 1(G/A) on the variation in flowering time for oil-type B. rapa, including ssp. oleifera and ssp. tricolaris. However, we detected an InDel polymorphism across exon 4 and intron 4 of BrFLC2 among the accessions of ssp. oleifera and ssp. tricolaris. This sequence polymorphism was not detected in any of the other vegetable-type B. rapa subspecies. Furthermore, this allelic variation was strongly associated with variations in flowering time in oil-type B. rapa. Zhao et al.  suggested that BrFLC2 was a major determinant of flowering time variation in B. rapa. However, there were no firm conclusions from several studies on BrFLC1[12, 18] and BrFLC2. Since BrFLC1 and BrFLC2 have specific roles in controlling flowering time in different B. rapa groups, we deduced that there was an independent evolution of the control of flowering time, at least the control of the vernalization pathway, between oil-type B. rapa including ssp. oleifera and ssp. tricolaris and the other vegetable-type and turnip B. rapa subspecies. The oil-type B. rapa formed an evolutionary branch that was independent of other B. rapa species in an analysis of molecular phylogeny based on whole genome re-sequencing data generated from 108 accessions (Dr. Xiaowu Wang, unpublished data). This indicates that the evolutionary history of oil-type B. rapa is isolated from that of the vegetable-type subspecies. The fact that the deletion mutation of BrFLC2 was absent from vegetable-type B. rapa indicates that this mutation may have arisen after the division of oil-type from vegetable-type B. rapa, while the splicing site mutation of BrFLC1 may have arisen before this division and been maintained during their respective evolutions.
Relationships between naturally occurring alternative splicing variants and flowering time variation have been reported for the FLC gene in A. thaliana and Capsella bursa-pastoris, and for BrFLC1 in B. rapa. Alternative splicing variants were also reported for BrFLC5 in a biennial oilseed cultivar, although their relationship with flowering time was not addressed . In the present study, we detected three alternative splicing patterns for BrFLC2 in the yellow sarson accession L143, which has a homozygous deletion allele of BrFLC2. All three alternative splicing variations led to the insertion of premature stop codons in the transcripts. The alternative splicing pattern iii of BrFLC2 has been reported by Zhao et al.  using DH lines derived from a cross between the same yellow sarson accession and a pak choi accession, and was deduced to be a regulatory mechanism for the differential expression of BrFLC2 in response to vernalization. In the present study, the transcripts from splicing pattern iii were the minor fraction of transcripts from the deletion allele of BrFLC2. This could be due to the different cultivation conditions in the two studies, as the plants were not cold-treated in this study. A possible reason for the differential expression in response to cold treatment might be that alternative splicing transcripts were eliminated by the mRNA surveillance system. Eukaryotes have an mRNA surveillance system to eliminate the transcripts that are deliberately spliced to contain premature stop codons as a part of their intricate autoregulatory system .
B. rapa is a mesohexaploid that has undergone whole genome triplication after divergence from a common ancestor of A. thaliana. During the diploidization process afterwards, which involved considerable gene loss, some gene family showed preferential retention such as circadian clock genes , and also many of flowering time genes. It has been speculated that polyploidy and lost of the duplicated genes may have contributed to the evolution of variations in flowering time, a key component of morphological diversity . After the hexaploid process, the three sub-genomes of the ancestor were partitioned into LF, MF1, and MF2 . BrFLC1 is located in LF, BrFLC2 in MF2, and BrFLC3 in MF1 , while BrFLC5 is located in the homologous region generated from an α-duplication event that occurred before the diversification of Arabidopsis-Brassica. The fact that a non-functional BrFLC1 mutation introduces early flowering time variation in vegetable-type B. rapa, while the non-functional BrFLC2 introduces early flowering time variation in oil-type B. rapa, indicates that these two loci of FLC in B. rapa play different roles in different groups. It has been proposed that non-functionalization of duplicate genes could provide an important source of phenotypic variation . We have shown that the deletion in BrFLC2 also promoted flowering in a genetic background of Chinese cabbage line Z16. However, it remains unknown why different alleles of BrFLC1 show no difference in flowering time in oil-type B. rapa accessions. We need to sequence all of the BrFLC1 alleles in these accessions to determine whether they contain additional mutations. Genetic redundancy provides flexibility for plants growing in changeable environments. It is also possible that the other two homologs of FLC might function to compensate for the loss of function of BrFLC1 or BrFLC2. We sequenced BrFLC3 or BrFLC5 using primers designed from sequences in exon 4 and exon 7, respectively, and we did not identify any functional sequence variation for the nine accessions (unpublished data). However, we can not exclude the possibility that there are sequence variations located in other regions that might affect their functions. Further research on BrFLC3 in sub-genome MF1 and BrFLC5 as a relic of the α-duplication event and their influence on flowering is underway. We anticipate that they may have differentiated from, and are functionally different from, BrFLC1 and BrFLC2.