Sherrie L. Sanda and Richard M. Amasino*
Department of Biochemistry, University of Wisconsin-Madison,
Madison, WI 53706-1569
*To whom correspondence should be addressed at
420 Henry Mall
University of Wisconsin
Madison, WI 53706-1569
The length of time required for a particular genotype of Arabidopsis to initiate flowering can be an important consideration in the choice of strain for molecular and genetic studies. Genes involved in the regulation of flowering time have been identified by examining naturally occurring variation. In crosses of early-flowering ecotypes to late-flowering ecotypes, the dominant FRIGIDA (FRI) gene was identified as a major regulator of flowering time (Napp-Zinn, 1985; Burn et al., 1993; Lee et al., 1993; Clarke and Dean, 1994). Lee et al. (1994) and Koornneef et al. (1994) reported that Landsberg erecta (Ler) contains an allele of another flowering gene, FLC, that suppresses the late-flowering phenotype of FRI. FLC alleles from other ecotypes (e.g., FLC alleles from Columbia (Col) and Sf-2) act in a semidominant manner with FRI to cause late flowering. The suppressor allele of FLC had only been found in Ler.
In this study, the flowering behavior of C24 was genetically analyzed. C24 contains the unique combination of a FRI allele that delays flowering and an FLC allele that suppresses the late-flowering phenotype of FRI. Although C24 has been reported to have been derived from the Col ecotype, microsatellite analysis indicates that C24 and Col are polymorphic, and thus do not share a common heritage.
Figure 1: The phenotype of C24 and of the F1 progeny of crosses of C24 to Columbia (Col) and Landsberg erecta (Ler). A)C24. B) F1 of C24 crossed to Ler. C) F1 of C24 crossed to Col. D) F1 of C24 crossed to the Ler line containing the FLC allele from Col.
In the F2 population resulting from self-pollination, more than 1/4 of the plants were later flowering than the latest parent (C24) (Figure 2) indicating that at least two genes are segregating for flowering time.
Figure 2. Frequency distribution of rosette leaf number in an F2 population derived from a cross between C24 and Columbia (Col). The genotypes at FLC and FRI of the plants flowering similar to the parental lines were not determined (black columns with white dots). The genotypes of plants that were homozygous for both FRI from C24 (FRI-C24) and FLC from Col (FLC-Col) are shown as filled columns. The genotypes of plants that were homozygous for FRI-C24 and heterozygous for FLC-Col are shown as open columns. The genotypes of plants that were heterozygous for FRI-C24 and homozygous for FLC-Col are shown as dotted columns. The genotypes of plants that were heterozygous for both FRI-C24 and FLC-Col are shown as stripped columns. The horizontal bars represent the leaf number distributions of the C24 line (filled bar), Col (open bar) and F1 (striped bar).
Microsatellite analysis was used to determine which loci contributed to late flowering in the F2 population. Late-flowering co-segregated with Col DNA at microsatellite locus nga249 in a homozygous or heterozygous state and with C24 DNA at microsatellite locus nga8 in a homozygous or heterozygous state. These two loci are located in the region of two previously identified genes that regulate flowering time, FLC and FRI (Lee et al. 1994). These data indicate that one copy of the FRI allele from C24 (FRI-C24) and one copy of the FLC allele from Col (FLC-Col) are required to cause the late flowering observed in the F2 plants. The C24 strain therefore appears to contain a late-flowering allele of FRI and an allele of FLC which suppresses the late-flowering effect of FRI similar to the Ler allele of FLC.
Figure 3. Frequency distribution of rosette leaf number in F2 populations derived from crosses between A) C24 and Landsberg erecta (Ler) and B) C24 and a Ler line containing the FLC allele from Columbia (FLC-Col). The horizontal bars represent the leaf number distributions of the C24 line (filled bar), Ler (open bar), the FLC-Col line (dotted bar) and F1 (striped bar).
Figure 4. Average rosette leaf number of the C24 line and crosses of C24 to Columbia (Col) and Landsberg erecta (Ler) under different environmental conditions. C24 (open columns), the F1 of C24 crossed to Col (thin striped columns), the F1 of C24 crossed to Ler (thick striped columns), Col (dotted columns), and Ler (filled columns) were grown under long-day photoperiods with varying amounts of red/far-red light, short-day photoperiods, and short-day photoperiods with cold treatment. Long-day photoperiods (LD) consisted of 20 hrs light with a red/far-red of 4.6 and 0.8 (Lee et al. 1995). The short-day photoperiod (SD) consisted of 8 hrs light with a red/far-red ratio of 1.3. Plants were cold treated for 40 days in short days as described by Lee et al. (1994) and then grown under short-day photoperiod (SDV) with a red/far-red light ratio of 1.3. The averages and standard deviations of the values from 10 plants are presented.
All genotypes exhibit a photoperiod response. C24 is late flowering under long-day photoperiods and even later flowering under short-day photoperiods. The time to flowering of C24 and F1 plants derived from C24 is greatly reduced by low red/far-red light ratios and cold treatment. F1 plants from the cross of C24 to Col are later flowering than the C24 parent under all conditions due to the presence of FLC-Col. These results are similar to the interactions of the FRI allele from Sf-2 with the FLC alleles from Col and Ler (Lee et al., 1994; Lee and Amasino, 1995).
The origin of the C24 strain is not known. Microsatellite analysis demonstrates that C24 is not genetically similar to any of the commonly used Col lines. C24 contains a late-flowering FRI allele similar to many late-flowering ecotypes (Napp-Zinn, 1985; Burn et al., 1993; Lee et al., 1993; Clarke and Dean, 1994). The relatively early-flowering C24 strain may have been derived from a late-flowering ecotype by acquisition of an allele of FLC that suppresses the late-flowering phenotype of FRI. FLC suppressor alleles can be readily derived through mutagenesis of late-flowering FLC alleles (Michaels et al. unpublished). The recessive glabrous trait of C24 indicates that this line may have been subjected to mutagenesis. This mutagenesis may have created the suppressor allele of FLC present in C24, or this allele may have originated naturally.
The presence of a late-flowering allele of FRI and suppressor allele of FLC in C24 should be considered in the choice of ecotypes with which C24 will be crossed. Crosses of C24 to plants which contain a late-flowering allele of FLC (like Col) will result in late-flowering plants. Crosses of C24 to plants derived from Landsberg, which contain a suppressor allele of FLC, will result in plants which flower in a reasonable amount of time.
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