BACKGROUND
brassica rapa is a genus comprising a variety of vegetables such as chinese cabbage , pak choi , and turnip  as well as oil crops including turnip rape  and sarson .
flowering time is an important trait in brassica vegetables because early flowering often leads to low yield and low quality.
it is also important for oilseed rape varieties as they are divided into “winter” and “spring” types according to their different flowering times and responsiveness to vernalization.
winter types must be exposed to cold to transition from the vegetative growth stage to the reproductive stage, while this is not necessary for the spring types, which are generally grown in shorter-season areas.
in arabidopsis, studies of natural variation demonstrated that the vernalization requirement is largely conferred by two dominant genes, fri and flc  <cit> .
fri acts upstream of flc to positively regulate flc expression  <cit> .
flc encodes a mads-box transcription factor that functions as a repressor of flowering by inhibiting downstream floral integrator genes  <cit> .
vernalization represses the expression of flc and induces flowering.
the promoter and first exon of flc are sufficient to initiate the repression of flc during vernalization, while the maintenance of repression requires additional regions of the gene body  <cit> .
there are four copies of flc in b. rapa <cit> .
they are located on chromosomes a <dig> , a <dig> , and a <dig>  owing to polyploidy evolution  <cit> .
colinearity analysis indicated that brflc <dig>  brflc <dig>  and brflc <dig> are located in three r blocks , which is consistent with the three flc copies that would be expected after a triplication event  <cit> .
brflc <dig> is located between blocks i and j  <cit> .
multiple gene copies are thought to be responsible for dose-regulated expression, and the mechanism appears to affect variations in flowering time in brassica crops  <cit> .
these replicated genes may have additive effects.
depending on the specific cross studied, different alleles of the various flc paralogs may exert different effects on flowering time.
in a backcross population derived from two recombinant inbred lines, created from a cross between per and r <dig>  a quantitative trait locus co-located with brflc <dig> explained more of the flowering time variation than brflc <dig> <cit> .
it was also reported that a naturally occurring splicing variation in brflc <dig> was associated with variation in flowering time in b. rapa, and this locus contributed most of its effect to late flowering  <cit> .
however, zhao et al.
<cit>  studied a doubled haploid  population derived from a cross between pak choi and yellow sarson, and reported brflc <dig> as a candidate gene for a major qtl for flowering time and the vernalization response in b. rapa.
the decreased transcript level of brflc <dig> upon cold treatment provided further evidence for this hypothesis.
since there are apparent contradictions in the proposed roles of brflc <dig> and brflc <dig>  we analyzed sequence variation of brflc <dig> and brflc <dig> in a large collection of b. rapa accessions, and the relationship between sequence variations and flowering time.
our results indicate that among the various b. rapa crop types, there are different genetic controls of flowering time.
RESULTS
flowering time variation
we determined flowering time for the germplasm collection of  <dig> b. rapa accessions in two separate experiments; one in an open field in kunming, south china, and one in a heated greenhouse in beijing, north china.
the data from the two experiments were significantly correlated with each other .
the flowering time varied from  <dig> to 155 days from sowing to the opening of the first flower  in the open field experiment, and  <dig> to  <dig> dtf in the greenhouse experiment .
flowering time varied more within turnip rape than within the other subspecies, with a variation range of 87 days in the open field to 104 days in the greenhouse.
the three yellow sarson accessions showed the narrowest variation .
the pak choi accessions showed the greatest difference in flowering time between the two experiments, indicating that day length likely affected the timing of flowering more than vernalization, since the average day length was more than 200 h per month during december to april in kunming, compared with 170–190 h per month in beijing.
in the open field experiment, among the vegetable-type b. rapa accessions, caixin and zicaitai flowered earlier, with an average flowering time of  <dig> dtf and  <dig> dtf, respectively, while both chinese cabbage and pak choi flowered at  <dig> dtf on average.
turnips flowered latest,  <dig> dtf on average, followed by wutacai .
the results from the greenhouse experiments were largely consistent with those obtained from the open field experiments .
acc., accessions; ave. dtf, average days to flowering; var., variation.
nucleotide polymorphisms at the brflc <dig> gene
nine accessions were selected to sequence brflc <dig> .
these nine accessions represented seven b. rapa subspecies, and showed a wide range of variation in flowering times.
for six of the nine accessions, flowering time varied from  <dig> to  <dig> dtf in the greenhouse in  <dig>  the other three accessions  had not flowered by 90 days after sowing.
to investigate the allelic variation in brflc <dig>  a 1400-bp fragment was amplified from b. rapa genomic dna with the primer combination of flc2f <dig> in exon  <dig> and flc2r <dig> in exon  <dig> .
sequencing of the amplified fragments from the nine selected accessions revealed that the amplified region had multiple sequence variations among the nine accessions.
all of these polymorphisms were single nucleotide substitutions, with two additional insertion/deletions  in line l <dig>  a yellow sarson accession.
one was a deletion of 57 bp, started at 1851 bp of brflc <dig>  and ended at 1914 bp, across exon  <dig> and intron  <dig>  the deletion was interrupted by  <dig> nucleotides  that could not be mapped to a certain position of the reference sequence .
the other one was an insertion of 29 bp at the position of 2430 bp located in intron  <dig> of brflc <dig>  all the single nucleotide substitutions were synonymous.
an indel marker designated as brflc2indel was developed to distinguish the 57 bp deletion across exon  <dig> and intron  <dig> and validated in the nine sequenced accessions .
a <cit> .
.
relationship between flowering time and nucleotide polymorphisms in brflc2
the brflc <dig> indel was screened over the germplasm collection consisting of  <dig> accessions.
the deletion allele was absent from all of the vegetable-type subspecies, but it was present as a homozygous allele in nine out of  <dig> b. rapa ssp.
oleifera accessions and in all three of the ssp.
tricolaris accessions, and as a heterozygous allele in  <dig> b. rapa ssp.
oleifera accessions .
the correlation analysis showed that the indel polymorphism was significantly associated with variation in flowering time among the oil-type b. rapa accessions .
in all cases, accessions with the deletion allele flowered early, with an average flowering time measured as days to flowering  of  <dig> in the open field experiment and  <dig> dtf in the greenhouse experiment.
in contrast, the accessions without this deletion  flowered significantly later  with an average dtf of  <dig> in the open field experiment and  <dig> in the greenhouse experiment.
the flowering time of the accessions with the heterozygous brflc <dig> locus were similar to those of accessions with the homozygously mutated locus, indicating that early flowering was dominant over late flowering.
dtf  data are presented as means ± se.
significant differences  between lines are indicated by different letters.
*includes ssp.
oleifera and ssp.
tricolaris.
we further analyzed the  <dig> accessions using the previously reported brflc1mvai caps marker, which can distinguish a/g alleles located at the splicing site of exon  <dig> and intron  <dig> of brflc <dig>  the a allele results in alternative splicing, which is correlated with early flowering in b. rapa <cit> .
brflc <dig> allelic variation was observed in both vegetable-types and oil-types, showing a, g, and heterozygous alleles .
the flowering time in vegetable-type b. rapa was significantly correlated with the a/g genotype; accessions with the a genotype had an average flowering time of  <dig> dtf in the open field experiment and  <dig> dtf in the greenhouse experiment.
accessions with g genotype flowered at  <dig> dtf in the open field experiment and  <dig> dtf in the greenhouse experiment.
however, there was no significant relationship between the brflc <dig> a/g genotype and variations in flowering time among oil-type b. rapa accessions.
the mean dtf of the  <dig> oil-type accessions with the a allele was  <dig> in the open field experiment and  <dig> in the greenhouse experiment.
in contrast, the average flowering time of  <dig> accessions with the g allele was  <dig> dtf in the open field experiment and  <dig> dtf in the greenhouse experiment.
similar to brflc <dig>  early flowering controlled by brflc <dig> was dominant over late flowering .
the association between flowering time and the brflc <dig> indel alleles was also analyzed for dh progenies from a bc <dig> population, using a chinese cabbage accession z <dig> as the recurrent parent and yellow sarson accession l <dig> as the donor.
both parents had “a” alleles for brflc <dig>  while z <dig> had a brflc <dig> allele without deletions that was possibly functional, and l <dig> had the deletion allele of brflc <dig>  of the  <dig> screened dh lines,  <dig> had the deletion allele.
we investigated the flowering phenotype of these three lines, five randomly selected lines with the functional allele, and the two parental lines.
the lines with the deletion allele showed significantly shorter flowering times than those of lines with the functional allele .
the flowering times of dh lines with the functional brflc <dig> allele ranged from  <dig> dtf to  <dig> dtf, while those of lines with the mutated deletion allele ranged from  <dig> dtf to  <dig> dtf.
dtf  data are presented as means ± se.
significant differences  between lines are indicated by different letters.
alternative splicing of brflc2
to identify any alternative splicing of the mutated brflc <dig> allele, rt-pcr was conducted with brflc2-specific primers  using the yellow sarson accession l <dig>  which had a homozygous deletion allele.
three fragments of different lengths were amplified from the cdna of l <dig> .
sequencing of these fragments revealed three alternative splicing patterns.
the  <dig> nucleotides  which interrupted the  <dig> bp deletion were found in transcripts from all three splicing patterns.
the three patterns were as follows: spd1) 600-bp transcript— <dig> bp skipped at the end of exon  <dig> following the  insertion and  <dig> bp retained at the end of intron 4; spd2) 551-bp transcript— <dig> bp skipped at the beginning and  <dig> bp skipped at the end of exon  <dig>  the retained  <dig> bp of exon  <dig> followed by the  insertion and  <dig> bp retained at the end of intron 4; and spd3) 686-bp transcript—same as spd <dig> besides intron  <dig> retained.
of the  <dig> sequenced cdna clones,  <dig> showed spd <dig>  six showed spd <dig>  and two showed spd <dig>  this indicated that the spd <dig> was the major splicing pattern for the deletion allele of brflc <dig>  while spd <dig> and spd <dig> were derived from spd <dig>
discussion
an indel polymorphism across exon  <dig> and intron  <dig> of brflc <dig> 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 <cit> .
an indel variation in the col <dig> gene was reported to be correlated with variation in flowering time in b. nigra <cit> .
in a. thaliana, flc encodes a mads-box transcription factor that acts as a dose-dependent flowering repressor  <cit> .
four copies of the flc gene in b. rapa increase the potential variation in flowering time  <cit> .
in our previous study, we identified a splicing site polymorphism pi6 +  <dig> in brflc <dig> that was significantly associated with the naturally occurring variation in flowering time in b. rapa <cit> .
in that study, we examined  <dig> 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 brflc <dig> and flowering time variation.
in the present study, we could not detect any effect of allelic variation in brflc <dig> pi6 +  <dig> 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  <dig> and intron  <dig> of brflc <dig> 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.
<cit>  suggested that brflc <dig> was a major determinant of flowering time variation in b. rapa.
however, there were no firm conclusions from several studies on brflc <dig> <cit>  and brflc <dig> <cit> .
since brflc <dig> and brflc <dig> 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  <dig> accessions .
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 brflc <dig> 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 brflc <dig> 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 <cit>  and capsella bursa-pastoris <cit> , and for brflc <dig> in b. rapa <cit> .
alternative splicing variants were also reported for brflc <dig> in a biennial oilseed cultivar, although their relationship with flowering time was not addressed  <cit> .
in the present study, we detected three alternative splicing patterns for brflc <dig> in the yellow sarson accession l <dig>  which has a homozygous deletion allele of brflc <dig>  all three alternative splicing variations led to the insertion of premature stop codons in the transcripts.
the alternative splicing pattern iii of brflc <dig> has been reported by zhao et al.
<cit>  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 brflc <dig> 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 brflc <dig>  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  <cit> .
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  <cit> , 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  <cit> .
after the hexaploid process, the three sub-genomes of the ancestor were partitioned into lf, mf <dig>  and mf <dig>  <cit> .
brflc <dig> is located in lf, brflc <dig> in mf <dig>  and brflc <dig> in mf <dig>  <cit> , while brflc <dig> is located in the homologous region generated from an α-duplication event that occurred before the diversification of arabidopsis-brassica <cit> .
the fact that a non-functional brflc <dig> mutation introduces early flowering time variation in vegetable-type b. rapa, while the non-functional brflc <dig> 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  <cit> .
we have shown that the deletion in brflc <dig> also promoted flowering in a genetic background of chinese cabbage line z <dig>  however, it remains unknown why different alleles of brflc <dig> show no difference in flowering time in oil-type b. rapa accessions.
we need to sequence all of the brflc <dig> 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 brflc <dig> or brflc <dig>  we sequenced brflc <dig> or brflc <dig> using primers designed from sequences in exon  <dig> and exon  <dig>  respectively, and we did not identify any functional sequence variation for the nine accessions .
however, we can not exclude the possibility that there are sequence variations located in other regions that might affect their functions.
further research on brflc <dig> in sub-genome mf <dig> and brflc <dig> 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, brflc <dig> and brflc <dig>
CONCLUSIONS
our results suggest that the naturally occurring deletion mutation across exon  <dig> and intron  <dig> in brflc <dig> contributes greatly to the variation in flowering time in oil-type b. rapa.
the different relationships between brflc <dig> or brflc <dig> and the variation in flowering time of vegetable-type and oil-type b. rapa indicate that control of flowering time undergone separate evolution between these two groups.

 brassica rapa is a genus comprising a variety of vegetables such as chinese cabbage, pak choi, and turnip as well as oil crops including turnip rape and sarson. flowering time is an important trait in brassica vegetables because early flowering often leads to low yield and low quality. it is also important for oilseed rape varieties as they are divided into “winter” and “spring” types according to their different flowering times and responsiveness to vernalization. winter types must be exposed to cold to transition from the vegetative growth stage to the reproductive stage, while this is not necessary for the spring types, which are generally grown in shorter-season areas. in arabidopsis, studies of natural variation demonstrated that the vernalization requirement is largely conferred by two dominant genes, fri and flc. fri acts upstream of flc to positively regulate flc expression. flc encodes a mads-box transcription factor that functions as a repressor of flowering by inhibiting downstream floral integrator genes. vernalization represses the expression of flc and induces flowering. the promoter and first exon of flc are sufficient to initiate the repression of flc during vernalization, while the maintenance of repression requires additional regions of the gene body. there are four copies of flc in b. rapa. they are located on chromosomes a, a, and a owing to polyploidy evolution. colinearity analysis indicated that brflc brflc and brflc are located in three r blocks, which is consistent with the three flc copies that would be expected after a triplication event. brflc is located between blocks i and j. multiple gene copies are thought to be responsible for dose-regulated expression, and the mechanism appears to affect variations in flowering time in brassica crops. these replicated genes may have additive effects. depending on the specific cross studied, different alleles of the various flc paralogs may exert different effects on flowering time. in a backcross population derived from two recombinant inbred lines, created from a cross between per and r a quantitative trait locus co-located with brflc explained more of the flowering time variation than brflc. it was also reported that a naturally occurring splicing variation in brflc was associated with variation in flowering time in b. rapa, and this locus contributed most of its effect to late flowering. however, zhao et al. studied a doubled haploid population derived from a cross between pak choi and yellow sarson, and reported brflc as a candidate gene for a major qtl for flowering time and the vernalization response in b. rapa. the decreased transcript level of brflc upon cold treatment provided further evidence for this hypothesis. since there are apparent contradictions in the proposed roles of brflc and brflc we analyzed sequence variation of brflc and brflc in a large collection of b. rapa accessions, and the relationship between sequence variations and flowering time. our results indicate that among the various b. rapa crop types, there are different genetic controls of flowering time.  flowering time variation we determined flowering time for the germplasm collection of b. rapa accessions in two separate experiments; one in an open field in kunming, south china, and one in a heated greenhouse in beijing, north china. the data from the two experiments were significantly correlated with each other. the flowering time varied from to 155 days from sowing to the opening of the first flower in the open field experiment, and to dtf in the greenhouse experiment. flowering time varied more within turnip rape than within the other subspecies, with a variation range of 87 days in the open field to 104 days in the greenhouse. the three yellow sarson accessions showed the narrowest variation. the pak choi accessions showed the greatest difference in flowering time between the two experiments, indicating that day length likely affected the timing of flowering more than vernalization, since the average day length was more than 200 h per month during december to april in kunming, compared with 170–190 h per month in beijing. in the open field experiment, among the vegetable-type b. rapa accessions, caixin and zicaitai flowered earlier, with an average flowering time of dtf and dtf, respectively, while both chinese cabbage and pak choi flowered at dtf on average. turnips flowered latest, dtf on average, followed by wutacai. the results from the greenhouse experiments were largely consistent with those obtained from the open field experiments. acc., accessions; ave. dtf, average days to flowering; var., variation. nucleotide polymorphisms at the brflc gene nine accessions were selected to sequence brflc. these nine accessions represented seven b. rapa subspecies, and showed a wide range of variation in flowering times. for six of the nine accessions, flowering time varied from to dtf in the greenhouse in the other three accessions had not flowered by 90 days after sowing. to investigate the allelic variation in brflc a 1400-bp fragment was amplified from b. rapa genomic dna with the primer combination of flc2f in exon and flc2r in exon. sequencing of the amplified fragments from the nine selected accessions revealed that the amplified region had multiple sequence variations among the nine accessions. all of these polymorphisms were single nucleotide substitutions, with two additional insertion/deletions in line l a yellow sarson accession. one was a deletion of 57 bp, started at 1851 bp of brflc and ended at 1914 bp, across exon and intron the deletion was interrupted by nucleotides that could not be mapped to a certain position of the reference sequence. the other one was an insertion of 29 bp at the position of 2430 bp located in intron of brflc all the single nucleotide substitutions were synonymous. an indel marker designated as brflc2indel was developed to distinguish the 57 bp deletion across exon and intron and validated in the nine sequenced accessions. a.. relationship between flowering time and nucleotide polymorphisms in brflc2 the brflc indel was screened over the germplasm collection consisting of accessions. the deletion allele was absent from all of the vegetable-type subspecies, but it was present as a homozygous allele in nine out of b. rapa ssp. oleifera accessions and in all three of the ssp. tricolaris accessions, and as a heterozygous allele in b. rapa ssp. oleifera accessions. the correlation analysis showed that the indel polymorphism was significantly associated with variation in flowering time among the oil-type b. rapa accessions. in all cases, accessions with the deletion allele flowered early, with an average flowering time measured as days to flowering of in the open field experiment and dtf in the greenhouse experiment. in contrast, the accessions without this deletion flowered significantly later with an average dtf of in the open field experiment and in the greenhouse experiment. the flowering time of the accessions with the heterozygous brflc locus were similar to those of accessions with the homozygously mutated locus, indicating that early flowering was dominant over late flowering. dtf data are presented as means ± se. significant differences between lines are indicated by different letters. *includes ssp. oleifera and ssp. tricolaris. we further analyzed the accessions using the previously reported brflc1mvai caps marker, which can distinguish a/g alleles located at the splicing site of exon and intron of brflc the a allele results in alternative splicing, which is correlated with early flowering in b. rapa. brflc allelic variation was observed in both vegetable-types and oil-types, showing a, g, and heterozygous alleles. the flowering time in vegetable-type b. rapa was significantly correlated with the a/g genotype; accessions with the a genotype had an average flowering time of dtf in the open field experiment and dtf in the greenhouse experiment. accessions with g genotype flowered at dtf in the open field experiment and dtf in the greenhouse experiment. however, there was no significant relationship between the brflc a/g genotype and variations in flowering time among oil-type b. rapa accessions. the mean dtf of the oil-type accessions with the a allele was in the open field experiment and in the greenhouse experiment. in contrast, the average flowering time of accessions with the g allele was dtf in the open field experiment and dtf in the greenhouse experiment. similar to brflc early flowering controlled by brflc was dominant over late flowering. the association between flowering time and the brflc indel alleles was also analyzed for dh progenies from a bc population, using a chinese cabbage accession z as the recurrent parent and yellow sarson accession l as the donor. both parents had “a” alleles for brflc while z had a brflc allele without deletions that was possibly functional, and l had the deletion allele of brflc of the screened dh lines, had the deletion allele. we investigated the flowering phenotype of these three lines, five randomly selected lines with the functional allele, and the two parental lines. the lines with the deletion allele showed significantly shorter flowering times than those of lines with the functional allele. the flowering times of dh lines with the functional brflc allele ranged from dtf to dtf, while those of lines with the mutated deletion allele ranged from dtf to dtf. dtf data are presented as means ± se. significant differences between lines are indicated by different letters. alternative splicing of brflc2 to identify any alternative splicing of the mutated brflc allele, rt-pcr was conducted with brflc2-specific primers using the yellow sarson accession l which had a homozygous deletion allele. three fragments of different lengths were amplified from the cdna of l. sequencing of these fragments revealed three alternative splicing patterns. the nucleotides which interrupted the bp deletion were found in transcripts from all three splicing patterns. the three patterns were as follows: spd1) 600-bp transcript— bp skipped at the end of exon following the insertion and bp retained at the end of intron 4; spd2) 551-bp transcript— bp skipped at the beginning and bp skipped at the end of exon the retained bp of exon followed by the insertion and bp retained at the end of intron 4; and spd3) 686-bp transcript—same as spd besides intron retained. of the sequenced cdna clones, showed spd six showed spd and two showed spd this indicated that the spd was the major splicing pattern for the deletion allele of brflc while spd and spd were derived from spd discussion an indel polymorphism across exon and intron of brflc 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. an indel variation in the col 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. 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 +  in brflc that was significantly associated with the naturally occurring variation in flowering time in b. rapa. in that study, we examined 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 brflc and flowering time variation. in the present study, we could not detect any effect of allelic variation in brflc pi6 +  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 and intron of brflc 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 brflc was a major determinant of flowering time variation in b. rapa. however, there were no firm conclusions from several studies on brflc and brflc. since brflc and brflc 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 accessions. 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 brflc 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 brflc 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 brflc in b. rapa. alternative splicing variants were also reported for brflc 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 brflc in the yellow sarson accession l which has a homozygous deletion allele of brflc all three alternative splicing variations led to the insertion of premature stop codons in the transcripts. the alternative splicing pattern iii of brflc 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 brflc 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 brflc 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, mf and mf. brflc is located in lf, brflc in mf and brflc in mf, while brflc 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 brflc mutation introduces early flowering time variation in vegetable-type b. rapa, while the non-functional brflc 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 brflc also promoted flowering in a genetic background of chinese cabbage line z however, it remains unknown why different alleles of brflc show no difference in flowering time in oil-type b. rapa accessions. we need to sequence all of the brflc 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 brflc or brflc we sequenced brflc or brflc using primers designed from sequences in exon and exon respectively, and we did not identify any functional sequence variation for the nine accessions. however, we can not exclude the possibility that there are sequence variations located in other regions that might affect their functions. further research on brflc in sub-genome mf and brflc 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, brflc and brflc  our results suggest that the naturally occurring deletion mutation across exon and intron in brflc contributes greatly to the variation in flowering time in oil-type b. rapa. the different relationships between brflc or brflc and the variation in flowering time of vegetable-type and oil-type b. rapa indicate that control of flowering time undergone separate evolution between these two groups. 
in contrast, the reported splicing site variation in brflc 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. the observed different relationship between brflc or brflc and flowering time variation indicates that the control of flowering time has evolved separately between oil-type and vegetable-type b. rapa groups. flowering locus c is a mads-box transcription factor that acts as a potent repressor of flowering. our results suggest that the naturally occurring deletion mutation across exon and intron in brflc gene contributes greatly to variation in flowering time in oil-type b. rapa. there are four copies of flc in b. rapa. analyses of different segregating populations have suggested that braa.flc.a and braa.flc.b play major roles in controlling flowering time in b. rapa. we analyzed the brflc sequence in nine b. rapa accessions, and identified a 57-bp insertion/deletion across exon and intron resulting in a non-functional allele. the indel was used to develop a pcr-based marker, which was used to screen a collection of b. rapa accessions. the deletion genotype was present only in oil-type b. rapa, including ssp. flowering time is an important trait in brassica rapa crops.
 flowering time is an important trait in brassica rapa crops. flowering locus c 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 and braa.flc.b play major roles in controlling flowering time in b. rapa.  we analyzed the brflc sequence in nine b. rapa accessions, and identified a 57-bp insertion/deletion across exon and intron resulting in a non-functional allele. in total, three types of transcripts were identified for this mutated brflc allele. the indel was used to develop a pcr-based marker, which was used to screen a collection of 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 brflc 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.  our results suggest that the naturally occurring deletion mutation across exon and intron in brflc gene contributes greatly to variation in flowering time in oil-type b. rapa. the observed different relationship between brflc or brflc and flowering time variation indicates that the control of flowering time has evolved separately between oil-type and vegetable-type b. rapa groups. 