Progress 07/01/02 to 06/30/05

Outputs
Most plant species have multiple copies of genes that arose during evolution mainly by rounds of chromosome multiplication. This genetic redundancy appears to confer a selective advantage, and it probably has contributed to the diversification of agricultural plants. The mechanisms by which genetic redundancy contribute to variation are not known, but potentially this could occur through the function of key regulatory genes that behave in a dosage dependent manner. One such gene is FLC. This gene controls flowering time in the model organism Arabidopsis, and multiple copies of FLC also regulate flowering time in Brassica species, and important group of crop plants including vegetables (cabbage, broccoli, cauliflower, turnip and kales) and oilseeds (canola). We plan to study the function of multiple FLC genes in Brassica crops by analyzing diverse forms for variation in gene expression and for the individual effects of these genes on flowering time. These data, in addition to DNA sequence analyses, will tell us if multiple copies of FLC contribute to the large variation in flowering time observed among Brassica crops. Our finding may establish an important mechanism that could be used to create useful variation in many different crop plants.

Impacts
This project is providing new insight on the effects of duplicate gene function in organisms with complex genomes.

Publications

• No publications reported this period

Progress 01/01/04 to 12/31/04

Outputs
The purpose of this study was to determine if different alleles at replicated loci of FLOWERING LOCUS C (FLC) have different expression levels and effects on flowering time in Brassica rapa. The specific goals of this project are to: 1. Develop genetic stocks that segregate for single alleles at each of the four FLC paralog loci using three B. rapa genotypes as donors and an early-flowering genotype (R500) as the recipient. 2. Test the effects of each allele on flowering time in the common R500 background. 3. Sequence each allele and determine how sequence divergence is related to differences in phenotypic effects. 4. Determine expression levels of each FLC allele in the parents and backcross lines. At the suggestion of the proposal reviewers, work was initiated on goal 4. This goal was achieved by doing two related experiments using SYBR green I real-time PCR. First an RNA expression assay was conducted using as genetic materials the genetic stocks developed with the alleles coming from the donor winter turnip rape cultivar Per. We found higher levels of expression for Per alleles than R500 alleles at every locus. The largest expression differences were for alleles that differentially affect flowering time. The transcript levels of both alleles at each locus were decreased by vernalization, but the decrease was greater for loci that had larger phenotypic responses to vernalization. Second, based on these results we decided to assess the expression levels at each FLC locus of 22 cultivars from diverse morphological groups, along with R500 and Per. Results from this analysis showed the same trend observed in the first experiment. In both experiments BrFLC3 was the locus showing the highest expression levels for unvernalized treatments but its higher expression was not associated with delay in flowering. However, in the vernalized treatment, it was the gene showing the strongest association with flowering time and it explained 48% of the observed phenotypic variation. The highest association between flowering time and FLC alleles was detected for FLC2, which explained 16% of the observed phenotypic variation. Progress toward goal 1 is one generation from completion. The BC3S1 progenies derived from two diverse B. rapa morphotypes, the Chinese cabbage cv Michihili and the Japanese green salad cv Mizuna, will be planted and screened this spring. Selected homozygous genotypes will be bagged to produce BC3S2 lines that will be subjected to flowering time analysis this summer. Goal 2 will be performed, upon completion of goal 1, in a controlled environment (Biotron) during the Summer 2005 (mid-July). This experiment will comprise the BC3S2 lines of each FLC locus derived from three diverse B. rapa morphotypes (Per, Michihili and Mizuna) along with R500, the recurrent parent used in their development. We are in the process of sequencing alleles at the 4 FLC loci in 5 genotypes (R500, Per, Michihli, Mizuna and IMB218). Based on these results, we will design specific primers for each gene and sequence the other genotypes that were used in the expression experiment described above.

Impacts
This project is providing new insight on the effects of duplicate gene function in organisms with complex genomes.

Publications

• Schranz ME, Osborn TC (2004) De novo variation in life-history traits and responses to growth conditions of resynthesized polyploid Brassica napus (Brassicaceae). Amer J Bot 91:174-183
• Lukens, LN, Quijada PA, Udall JA, Pires JC, Schranz ME, Osborn TC (2004) Genome redundancy and plasticity within ancient and recent Brassica crop species. Biol J Linnaen Soc 82:665-674
• Osborn TC (2004) The contribution of polyploidy to variation in Brassica species. Physiol Plant 121:531-536
• Pires JC, Zhao J, Schranz ME, Leon EJ, Quijada PA, Lukens LN, Osborn TC (2004) Flowering time divergence and genomic rearrangements in resynthesized Brassica polyploids (Brassicaceae). Biol J Linnaen Soc 82:675-688

Progress 01/01/03 to 12/31/03

Outputs
The purpose of this study is to determine if different alleles at replicated copies of FLOWERING LOCUS C (FLC) have different expression levels and effects on flowering time in Brassica rapa. FLC is an inhibitor of flowering and exists as a single locus in Arabidopsis. We previously showed that B. rapa has four FLC loci, and that alleles from winter turnip rape (Per) at three loci delay flowering to varying degrees compared to alleles from a spring sarson (R500). We now have assayed these alleles for FLC transcript levels and found higher levels for Per alleles than R500 alleles at every locus. The largest expression differences were for alleles that differentially affect flowering time. The transcript levels of both alleles at each locus were decreased by vernalization, but the decrease was greater for loci that had larger phenotypic responses to vernalization. We are now analyzing expression levels of alleles from other morphotypes within B. rapa, and backcrossing these alleles into the R500 background. Since most of the other B. rapa morphotypes used in the expression analysis showed significant heterogeneity for flowering time, we screened individual plants with PCR primers specific for each FLC locus and self-pollinated selected plants to create S1 lines. We are now analyzing the S1 lines for FLC expression using real-time PCR. During the last year, we also backcrossed late-flowering FLC alleles from two morphotypes (Chinese cabbage cv. Michihli and Japanese green salad cv. Mizuna) into the same spring sarson genotype (R500) used in our previous work. We have made two backcrosses (BC) with these two donors (Michihli and Mizuna) and developed families segregating at each FLC locus. We now are screening the FLC BC2 families to select heterozygous plants that will be backcrossed to the recurrent parent (R500) to create BC3 families. The BC3 families will be selfed and homozygous plants for each FLC allele at each locus will be selected. The lines with contrasting FLC alleles will be assayed for phenotypic variation and FLC expression levels.

Impacts
This project is providing new insight on the effects of duplicate gene function in organisms with complex genomes.

Publications

• Osborn TC, Lukens L (2003) The molecular genetic basis of flowering time variation in Brassica species. In: Nagata T, Lorz H, Widholm JM (eds) Biotechnology in Agriculture and Forestry, Nagata T, Tabata S (eds) Vol. 52: Brassicas and Legumes, from Genome Structure to Breeding. Springer-Verlag, Berlin, Heidelberg, pp. 69-86

Progress 01/01/02 to 12/31/02

Outputs
The purpose of this study is to determine if different alleles at replicated copies of FLOWERING LOCUS C (FLC) have different expression levels and effects on flowering time in Brassica rapa. FLC is an inhibitor of flowering and exists as a single locus in Arabidopsis. We previously showed that B. rapa has four FLC loci, and that alleles from winter turnip rape (Per) at three loci delay flowering to varying degrees compared to alleles from a spring season (R500). We now have assayed these alleles for FLC transcript levels and found higher levels for Per alleles than R500 alleles at every locus. The largest expression differences were for alleles that differentially affect flowering time. The transcript levels of both alleles at each locus were decreased by vernalization, but the decrease was greater for loci that had larger phenotypic responses to vernalization. We are now analyzing expression levels of alleles from other morphotypes within B. rapa, and backcrossing these alleles into the R500 background.

Impacts
The project will provide basic information on the contribution of replicated genes to phenotypic variation in agricultural plants.

Publications

• Schranz ME, Quijada P, Sung S-B, Lukens L, Amasino R, Osborn TC (2002) Characterization and effects of the replicated flowering time gene FLC in Brassica rapa. Genetics 162:1457-1468
• Schranz ME, Quijada PQ, Amasino R, Osborn TC (2002)The Flowering Rheostat: Multiple FLC loci regulate flowering time in Brassica rapa. 6th Gatersleben Research Conference, Abstract P62