Naturally Occurring Indel Variation in the Brassica nigra COL1 Gene Is Associated With Variation in Flowering Time

Corresponding author: Ulf Lagercrantz, Swedish University of Agricultural Sciences, Box 7080, S-750 07 Uppsala, Sweden., ulf.lagercrantz{at}vbiol.slu.se (E-mail)

Communicating editor: O. SAVOLAINEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Previous QTL mapping identified a Brassica nigra homolog to Arabidopsis thaliana CO as a candidate gene affecting flowering time in B. nigra. Transformation of an A. thaliana co mutant with two different alleles of the B. nigra CO (Bni COa) homolog, one from an early-flowering B. nigra plant and one from a late one, did not show any differential effect of the two alleles on flowering time. The DNA sequence of the coding region of the two alleles was also identical, showing that nucleotide variation influencing flowering time must reside outside the coding region of Bni COa. In contrast, the nucleotide sequence of the B. nigra COL1 (Bni COL1) gene located 3.5 kb upstream of Bni COa was highly diverged between the alleles from early and late plants. One indel polymorphism in the Bni COL1 coding region, present in several natural populations of B. nigra, displayed a significant association with flowering time within a majority of these populations. These data indicate that a quantitative trait nucleotide (QTN) affecting flowering time is located within or close to the Bni COL1 gene. The intergenic sequence between Bni COL1 and Bni COa displayed a prominent peak of divergence 1 kb downstream of the Bni COL1 coding region. This region could contain regulatory elements for the downstream Bni COa gene. Our data suggest that a naturally occurring QTN for flowering time affects the function or expression of either Bni COL1 or Bni COa.

ADAPTIVE traits typically show quantitative phenotypic variation, and it is often assumed that this variation is shaped largely by natural selection. While there is mounting evidence that natural selection was, at least in part, involved in the evolution of adaptive traits (KREITMAN and AKASHI 1995 ), the relative importance and nature of its contribution is still a matter of debate. Because most quantitative traits are controlled by a large number of interacting loci, it is conceivable that selection intensity at each of these loci is limited, most of the variation observed within and among natural populations coming from variation among loci rather than at loci themselves (e.g., LATTA 1998 ). Polymorphism in natural populations could have also resulted mainly from selection at a few regulatory genes, variation at structural genes being predominantly neutral (PURUGGANAN 2000 ). Unfortunately, there are still limited data linking the phenotypic variation of adaptively important quantitative traits to variation of underlying genes, because most studies of molecular variation so far have concentrated on a few genes.

Clearly, flowering time has a high adaptive value as reproductive success in plants closely depends on the timing of flower initiation. A correct timing ensures that flowers develop at a most favorable time for fertilization and consequently leads to a sufficient production of seeds. The process probably also includes trade-offs between timing of flowering and importance of seed production (DORN and MITCHELL-OLDS 1991 ). Flowering time exhibits considerable variation between, but also within, natural populations and a significant part of this variation has a genetic basis (for variation between populations, see KOWALSKI et al. 1994 ; CLARKE et al. 1995 ; MITCHELL-OLDS 1996 ; KUITTINEN et al. 1997 ; ALONSO-BLANCO et al. 1998 ; for variation within populations, see PORS and WERNER 1989 ; FU and RITLAND 1994 ).

Much progress has been made lately in understanding the genetic control of flowering time in the cruciferous model species Arabidopsis thaliana (COUPLAND 1995 ; SIMPSON et al. 1999 ; REEVES and COUPLAND 2000 ). A substantial number of genes affecting flowering time have been identified through mutagenesis studies and utilization of natural variation among ecotypes. The identification of a pathway and its underlying genes through mutagenesis studies does not in itself show that phenotypic variation in the wild is due to variation at these loci, although the nucleotide variation at genes involved in the response to environmental cues is a likely target for adaptive selection. The information on A. thaliana provides a large number of candidate genes for the control of flowering time in the wild and it would be of great interest to see which of the many possible alternatives has been targeted by natural selection in different species and/or populations within species. Recently, the A. thaliana FRIGIDA (FRI) gene was shown to be a major determinant of flowering time variation in natural populations (JOHANSON et al. 2000 ). Without vernalization, functional FRI alleles confer late flowering, and most early-flowering ecotypes carry loss-of-function mutations at FRI.

We have taken a comparative mapping approach to try to identify quantitative trait nucleotides (QTNs) affecting flowering time (FT) in natural populations of Brassica species. Brassica species belong to the same family as A. thaliana and the induction of flowering shows great similarities with A. thaliana (TOMMEY and EVANS 1991 ). Species in both genera are induced to flower by long days and respond in a similar way to vernalization. The first step in our approach is to map quantitative trait loci (QTL) for flowering time in Brassica species. A QTL is simply a chromosome location, and to identify candidate genes within this area, comparative mapping is then used to identify homologous chromosome segments in A. thaliana. These segments are searched for candidate genes on the basis of map position and knowledge about gene function in Arabidopsis. Finally, the effects on flowering time of natural variation in the Brassica homologs are specifically tested.

In the present case, previous QTL mapping in Brassica nigra identified two main genomic areas influencing flowering time (LAGERCRANTZ et al. 1996 ). On the basis of comparative mapping, a B. nigra homolog to the Arabidopsis gene CONSTANS (CO) was suggested as a likely candidate gene for at least one of the QTL. CO acts in the pathway that accelerates flowering in response to long photoperiods (PUTTERILL et al. 1995 ). The gene activates at least four early target genes with diverse biochemical function that act to promote flowering, making CO a key component in the regulation of flowering time in response to the environment (SAMACH et al. 2000 ).

As a major QTL for flowering time in B. nigra maps close to a CO homolog, we tested whether natural variation at this homolog affected flowering time. In the present study, we cloned alleles of the B. nigra CO homolog at the major QTL (Bni COa) from early- and late-flowering plants. To test any differential effect of the two alleles on flowering time, they were transformed into the A. thaliana co mutant. As no such effect was detected, we examined allelic variation around Bni COa. Notably, we analyzed variation at the B. nigra COL1 gene, which is located 3.5 kb upstream of Bni COa. COL1 displayed highly diverged alleles from early- and late-flowering plants. Studies of association between this allelic variation and flowering time were conducted in natural populations. These studies detected a significant association in several B. nigra populations, indicating that nucleotide variation within or close to COL1 affects flowering time.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of B. nigra CO and COL1 alleles:
A cDNA of the A. thaliana CO gene (PUTTERILL et al. 1995 ) was used to screen a B. nigra genomic library in EMBL3. The library was prepared from the rapid cycling line used as the early-flowering parent in our previous QTL mapping experiments (LAGERCRANTZ et al. 1996 ). Hybridizations were performed at moderate stringency. More than 80 clones were isolated and characterized. Restriction mapping and hybridization experiments identified four different genes. One representative of each gene was subcloned and sequenced using cycle sequencing and an automatic sequencer (ABI377). The four genes were positioned on the B. nigra linkage map using a previously described mapping population (LAGERCRANTZ and LYDIATE 1995 ). A genomic library in Zap was then prepared from the late-flowering parent used to construct our mapping populations (LAGERCRANTZ et al. 1996 ). This library was screened with the previously isolated B. nigra genes. Subcloned alleles were sequenced as above.

Introduction of B. nigra CO alleles into Arabidopsis:
Alleles of Bni COa were cloned in pPZP211 (HAJDUKIEWICZ et al. 1994 ). The constructs contained genomic clones comprising the coding region with a 2-kb upstream sequence and a 1-kb downstream sequence. The plasmids were transformed into Agrobacterium strain GV3101 that was used to vacuum infiltrate an A. thaliana co mutant (co-2; Nottingham stock center no. N175). Offspring from homozygous transformants were grown in controlled-environment rooms with a 16-hr photoperiod. Flowering time was measured as the number of days from sowing to the opening of the first flower, and as the number of rosette leaves after bolting. The means were calculated from 17 to 25 plants. Five independent transformants with a Bni COa allele from an early-flowering plant and six with an allele from a late-flowering plant were tested.

Population samples:
Seed samples (population samples) originating from Ethiopia (accession no. BRA1163), Spain (accession no. BRA101), Portugal (accession no. BRA153), Italy (samples 1, 2, and 3: accession nos. BRA1045, BRA26, and BRA27), Greece (accession no. BRA185), and Germany (accession no. BRA1045) (Fig 1) were obtained from the Institute für Plantzengenetik und Kultur (Gatersleben, Germany).

View larger version (26K): In this window In a new window Download PPT slide   Figure 1. Geographic locations of the original seed collections of B. nigra and distributions of flowering time from one common garden experiment.

Flowering time experiments:
The seeds were germinated 24 hr in petri dishes and planted in pots. In the first experiment conducted with the two Italian populations (Ital 2 and Ital 3), the plants were grown under artificial light with a 16-hr photoperiod. In the second experiment plants were grown in the greenhouse with supplementary 16 hr light. Flowering time was scored as the number of days from planting to the opening of the first flower.

Marker analysis:
Genomic DNA was prepared from leaf samples as described by LISCUM and OELLER 1997 . Two indels (Ind1 and Ind2) in COL1, identified comparing alleles from the early- and late-flowering parents in our mapping cross, were amplified separately using PCR. The primers were for Ind1 CO34 (5' AGA AGA TGA AGC AGA GGC) and CO56 (5' ACT GTA ATC GAC AAG GTC CAG) and for Ind2 CO57 (5' CTG GAC CTT GTC GAT TAC AGT) and CO58 (5' GAC TCT GGC ACA AGA CTA ACC). Primers CO34 and CO57 were labeled with 6-FAM and HEX, respectively, and run on an ABI377 automatic sequencer.

Estimates and tests of population genetics parameters were performed using Arlequin (SCHNEIDER et al. 2000 ). Linkage disequilibrium was tested using a likelihood-ratio test, whose empirical distribution is obtained by a permutation procedure (SLATKIN and EXCOFFIER 1996 ). The association between flowering time and marker genotype was tested within each population using analysis of variance in Statview 4.0 (Abacus Concepts, Berkeley, CA). The analyses were performed within each population as well as with all populations in a nested model with country of origin and genotype with country as factors.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To test the hypothesis that allelic variation in a B. nigra homolog to CO influences flowering time in natural populations, CO homologs were isolated from B. nigra. Our screens identified two CO homologs, Bni COa and Bni COb, corresponding to the two CO loci previously mapped to QTL on linkage groups 2 (LG2) and 8 (LG8), respectively (LAGERCRANTZ et al. 1996 ). In addition, one homolog each of A. thaliana COL1 (Bni COL1) and COL2 (Bni COL2) was identified in our screens.

We concentrated on Bni COa at the major QTL on LG2 and isolated alleles from the late- and early-flowering parents in our mapping cross. The DNA sequences of the coding region and 600 bp of the 5' untranslated region of the two alleles of Bni COa were virtually identical in the two alleles. In total, two nucleotide substitutions were detected at positions -268 and -466 from the translation start site. To test if variation in the flanking regions or the intron might affect flowering time, we introduced the two alleles into an Arabidopsis co mutant (co-2). Both alleles were functional and contained enough regulatory sequence to restore early flowering in the co mutant (Table 1). However, no significant difference in flowering time was detected between alleles from early- and late-flowering B. nigra plants.

Thus, if diversity between the two B. nigra CO alleles affects flowering time variation, important nucleotide variation is likely to reside farther away from the coding region. A gene with high sequence similarity to Bni COa was found 3.5 kb upstream of COa. This gene, Bni COL1, is a homolog to A. thaliana CONSTANS LIKE 1 (COL1; PUTTERILL et al. 1997 ), which is located 3.5 kb upstream of A. thaliana CO. Considering the near-identical alleles at Bni COa, alleles of Bni COL1 from early- and late-flowering plants exhibited surprising sequence divergence (Fig 2). The coding region in the two alleles differed by 16 nucleotide substitutions and two in-frame indels (Ind1 and Ind2) separated by 235 bp. Ind1 is part of trinucleotide AAC repeat coding for a run of asparagine residues, and the allele from the early plant contained six additional repeat units. The 18-nucleotides-longer Ind1 was associated with a deletion of 18 nucleotides at Ind2 in the "early" allele. Most of the nucleotide substitutions (9) were nonsynonymous, resulting in amino acid substitutions that were concentrated around the indels and the C-terminal end of the protein (Fig 2). The deduced amino acid variation was thus located exclusively outside the two highly conserved motifs present in COL1 genes (Fig 2; LAGERCRANTZ and AXELSSON 2000 ).

View larger version (8K): In this window In a new window Download PPT slide   Figure 2. A schematic view of the B. nigra COL1 gene indicating nucleotide substitutions between alleles from an early- and a late-flowering B. nigra plant. N indicates nonsynonymous substitutions, and S denotes synonymous ones. The zinc-finger and basic regions are two highly conserved motifs in COL1 genes (LAGERCRANTZ and AXELSSON 2000 ).

Sequence analysis of COL1 in A. thaliana and other Brassica species indicates that the Ind2 polymorphism present in B. nigra predates the divergence of the lineage leading to B. nigra from the one leading to B. rapa and B. oleracea. The insertion was present in A. thaliana but absent in alleles sampled from B. oleracea, B. rapa, and B. juncea (Fig 3). A deletion of the extra 18 bp present in A. thaliana and some B. nigra alleles could have occurred independently in the lineages leading to B. nigra, B. rapa, and B. oleracea, but a single deletion event before the split of the B. nigra and B. rapa/B. oleracea lineages seems a more parsimonious explanation.

View larger version (17K): In this window In a new window Download PPT slide   Figure 3. Alignment of a part of the COL1 gene around the B. nigra Ind2 indel from A. thaliana, B. juncea, B. rapa, and B. oleracea.

Polymorphism at Ind1 and Ind2:
The two indels in COL1 were genotyped in seven B. nigra populations originating from Europe and Africa. At Ind2, only the two previously identified alleles (L and S) were segregating, while in total six alleles were found at the microsatellite locus Ind1 (Table 2). The populations were highly differentiated, in particular at Ind1, which displayed an F_ST of 45%, while the F_ST estimate for Ind2 was 20%. Within populations, no significant departure from Hardy-Weinberg equilibrium was detected (data not shown). Linkage disequilibrium was detected between Ind1 and Ind2 in four of the five populations where both loci were polymorphic (Table 2).

Flowering time variation:
Flowering time for the plants in the seven populations was measured in two separate experiments with different environmental conditions. Thus, flowering time cannot be compared between the experiments. However, plants from six populations grown under identical long-day conditions showed significant differences in flowering time (Fig 1 and Table 2). Plants from the most southern population, Ethiopia, flowered the earliest while plants from the most northern one, Germany, flowered the latest. Plants from Spain, Portugal, Italy, and Greece displayed intermediate flowering times.

Association between flowering time and indel polymorphisms in COL1:
Due to strong population structure, both for indel genotypes and FT, all associations between genotype and FT were tested within populations. Allelic variation at Ind1 was observed in six populations; however, no significant association between the Ind1 genotype FT was seen in any of these populations nor in the nested model (Table 3). At Ind2, variation was detected in five populations, and in four of those, FT was significantly different among genotypes (Fig 4 and Table 3). The overall effect of genotype within country was also highly significant in the nested model. In all cases, the S (short) allele was associated with earlier flowering. In accordance with these data, the two populations fixed for the S allele (Ethiopia and Portugal) flowered earlier than any of the other populations (Fig 1 and Table 2).

View larger version (13K): In this window In a new window Download PPT slide   Figure 4. Average flowering times for LL, LS, and SS genotypes at Ind2 in different B. nigra populations. Vertical lines indicate standard errors. Experiments I and II were conducted under different environmental conditions, and the flowering times are not comparable between experiments.

Genetic diversity in the intergenic region between COL1 and COa:
The alleles of COL1 from early- and late-flowering plants were highly diverged, in particular toward the C-terminal part, while the corresponding alleles of COa 3.5 kb downstream were virtually identical. We thus wanted to check if the allelic diversity also extended into the noncoding region between the two genes. A comparison of the 3.5-kb intergenic region identified 42 indels. Most of those were smaller than 10 bp, but three indels were larger than 30 bp (98, 147, and 400 bp; Fig 5). The two alleles also differed by a large number of nucleotide substitutions, with a peak of divergence 1 kb downstream of the COL1 coding region (Fig 5).

View larger version (15K): In this window In a new window Download PPT slide   Figure 5. Distribution of silent/synonymous substitutions along the COL1–COa region between two alleles from early- and late-flowering B. nigra plants. A sliding window of 100 sites was used. The organization of the B. nigra COL1–COa region is given below the plot. Boxes indicate genes, lines depict intergenic regions, and solid triangles denote large indels.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our previous QTL mapping identified a CO homolog as a candidate for a gene affecting naturally occurring flowering time variation in B. nigra. In the present study we could not detect any effect of allelic variation in Bni COa on flowering time. Introduction of allelic variants from early- and late-flowering B. nigra plants into a A. thaliana co mutant resulted in plants flowering earlier than the wild type, although transformants with either of the two alleles displayed similar flowering times.

In contrast to the near sequence identity found between Bni COa alleles from early- and late-flowering plants, we detected a surprising sequence divergence between the corresponding Bni COL1 alleles located only 3.5 kb upstream of Bni COa. Furthermore, the genotype at Ind2 located within the Bni COL1 coding region showed a strong association with flowering time in several populations. In these populations the S allele was consistently associated with early flowering. These data indicate that flowering time is affected by some nucleotide variation (QTN) close to Ind2, possibly within the COL1 gene. From the present data it is not possible to say how close the QTN really is. When a mutation occurs, it has a strong positive disequilibrium with the carrier haplotype and a negative disequilibrium with other haplotypes in the population. Recombination in each generation will then decrease linkage disequilibrium. For old mutations in large equilibrium populations, strong linkage disequilibrium is expected only over distances as short as a few kilobases or less (KRUGLYAK 1999 ; LANGLEY et al. 2000 ; NORDBORG 2000 ; THORNSBERRY et al. 2001 ).

However, we do not know the age of mutations at Ind2 and the QTN, or the number of meioses that have occurred, and cannot therefore estimate the distance between the two loci. There is some indication that the Ind2 polymorphism is old because the insertion was present in Arabidopsis but absent in samples of more closely related Brassica species. These observations suggest that the Ind2 polymorphism in B. nigra was present before the split of the lineage leading to B. nigra and the one leading to B. oleracea and B. rapa. This split most likely occurred several million years ago (R. PRICE, personal communication). Furthermore, the Ind1 polymorphism located only 250 bp from Ind2 did not show any significant association with the QTN for flowering time. Either recombination has been frequent enough to break the association between the QTN and Ind1, or the lack of association is due to the accumulation of new mutations at Ind1. Even if the mutation rate at the simple sequence repeat Ind1 locus could be relatively high, the data indicate that a considerable number of generations have passed since the occurrence of the QTN mutation. In conclusion, our data suggest that recombination in multiple generations of meiosis should have broken disequilibria over larger distances, indicating that the QTN is probably close to Ind2.

Is the QTN for flowering time likely to reside within COL1?
Alleles of COL1 from early- and late-flowering plants showed considerable sequence divergence with a total of nine amino acid substitutions in addition to the two indels. Most of the substitutions were radical and occurred in regions of the protein characterized by a high evolutionary rate (Fig 2; LAGERCRANTZ and AXELSSON 2000 ). However, studies of COL1 in Arabidopsis have not suggested a role for COL1 in the control of flowering time. LEDGER et al. 2001 did not detect any effect on flowering time when over- or underexpressing COL1 in A. thaliana, but overexpression of COL1 did shorten the period of two circadian rhythms. Although these data do not support the location of a QTN for flowering time in Bni COL1, further studies are needed to test this hypothesis.

The Bni COa gene is still an attractive candidate for the QTN:
CO has been shown to be essential for the induction of flowering in Arabidopsis (PUTTERILL et al. 1995 ). One of the pathways for induction of flowering promotes flowering in response to day length and includes the CO gene. The co mutant flowers considerably later than wild type under long days, but not under short days, indicating that the CO gene is required to promote flowering under long-day conditions. The CO mRNA is also more abundant under long days than under short days, indicating that transcriptional regulation of CO, at least in part, is likely to regulate flowering time (SUÁREZ-LÓPEZ et al. 2001). Flowering time in long days is also correlated with amount of CO expression (PUTTERILL et al. 1995 ). One possibility is that the B. nigra QTN for flowering time resided in the intergenic region between COL1 and CO and that it affects flowering time through regulation of COa expression levels. Our transformation experiments with CO alleles from early- and late-flowering B. nigra plants into Arabidopsis co did not show any differences in flowering time. These constructs included 2 kb of DNA upstream of the CO coding region, which is likely to contain the most important promoter elements in Arabidopsis. However, we cannot exclude that cis-acting elements are located farther away. The intergenic region between COL1 and CO is highly diverged between alleles from early- and late-flowering B. nigra plants (Fig 5). Polymorphic enhancer elements for COa could potentially reside close to COL1. An obvious peak of divergence between early and late alleles was observed 2.5 kb upstream of the COa coding region. The very high level of sequence diversity at this position could be due to the action of balancing selection, because theoretical models predict a peak of polymorphism near a site that is under balancing selection (HUDSON and KAPLAN 1988 ).

Data from Drosophila suggest that variation affecting quantitative traits can be complex at the nucleotide level, with multiple interacting sites. A minimum of three polymorphic sites in one 2.3-kb region of the ADH gene contributes to naturally occurring variation in the concentration of ADH protein (STAM and LAURIE 1996 ). To locate the QTN/QTNs for flowering time in the COL1/COa region we need to extend the association studies with more markers to establish the extent of linkage disequilibrium in the region and more individuals to increase the statistical power of the tests. We also need to conduct additional transformation experiments, where specific parts of the COL1–COa region from early and late alleles are introduced into a common genetic background (e.g., an Arabidopsis col1, co double mutant).

	ACKNOWLEDGMENTS

We thank Agneta Ottosson for help with Arabidopsis transformation experiments. This work was supported by the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, and by the Swedish Research Council. M.K.Ö. was supported by a fellowship from the Lawski foundation.

Manuscript received September 24, 2001; Accepted for publication February 1, 2002.

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				M. Riihimaki and O. Savolainen Environmental and genetic effects on flowering differences between northern and southern populations of Arabidopsis lyrata (Brassicaceae) Am. J. Botany, July 1, 2004; 91(7): 1036 - 1045. [Abstract] [Full Text] [PDF]

				O. Podlaha and J. Zhang Positive selection on protein-length in the evolution of a primate sperm ion channel PNAS, October 14, 2003; 100(21): 12241 - 12246. [Abstract] [Full Text] [PDF]

				M. E. Schranz, P. Quijada, S.-B. Sung, L. Lukens, R. Amasino, and T. C. Osborn Characterization and Effects of the Replicated Flowering Time Gene FLC in Brassica rapa Genetics, November 1, 2002; 162(3): 1457 - 1468. [Abstract] [Full Text] [PDF]

				U. Lagercrantz, M. Kruskopf Osterberg, and M. Lascoux Sequence Variation and Haplotype Structure at the Putative Flowering-Time Locus COL1 of Brassica nigra Mol. Biol. Evol., September 1, 2002; 19(9): 1474 - 1482. [Abstract] [Full Text] [PDF]