1. Cynthia Weinig and Matthew Rubin
Phenotypic Variation in Circadian Near Isogenic Lines of Arabidopsis thaliana
Sarah Cheeney
Advisors:
Cynthia Weinig and Matthew Rubin

2. Circadian Rhythms The Earth rotates on its axis once every 24 hours
Providing predictable cycles of light and temperature
Most organisms exhibit circadian rhythms
Circadian rhythms are endogenous repeating rhythms
Circadian rhythms are “set” by external cues
Many physiological and behavioral characters are controlled by circadian rhythms
Best example in plants is photosynthesis 2

3. Arabidopsis thaliana: The Real-Live Plant
Member of the mustard family
Related to cabbage, cauliflower, kohlrabi, broccoli, and kale
Useful for environmental studies:
Primarily self-fertilizing
Naturally highly inbred families
Grow replicates of each genotype in each environment.
Short-lived annual
Fitness estimated from fruit number

4. Research Questions
Do previously characterized circadian NILs show patterns in other non-circadian traits?
Can physiological parameters be measured at both the whole plant and leaf level?
Are any of the measured traits adaptive?

5. Recombinant Inbred Lines
Parent 1
Parent 2 X
Each RIL is a novel combination of parental alleles F1
(8 more generations)

6. Temperature Compensation
Temperature compensation in Ler, Col, Cvi, and the CvL and CoL RILs. (A) Mean leaf-movement period plotted against temperature for the Ler (open circles), Col (solid triangles), and Cvi (solid circles) accessions.
Edwards K D et al. Genetics 2005;170: 6

7. QTL Mapping
QTL mapping tests for an association between a genetic marker state and phenotype
marker M M m M m m m m M
QTL Q Q q Q q q q q Q
M = Marker allele 1
m = Marker allele 2
Q = Allele for branching
q = Allele for fewer branches

8. QTL Mapping Results
Genetic mapping of circadian period QTL in the CvL and CoL RILs. QTL likelihood (LOD) maps across Arabidopsis chromosomes 1 and 5 in the CvL (A) and CoL (B) RILs. Chromosome numbers are indicated in the upper right corner of each graph. Names on the x-axis correspond to molecular markers. QTL were mapped independently at 12° (open diamonds), 22° (shaded diamonds), and 27° (solid diamonds). Dashed line represents 2.7 LOD significance threshold of P < 0.05 (as calculated from Van Ooijen 1999). Solid boxes on x-axis span the 2-LOD support interval of mapped QTL. Putative QTL designations are indicated in italics. Selected markers used as cofactors in mapping are identified above the x-axis with diamonds shown as open (12°), shaded (22°), and solid (27°) according to temperature.
Edwards K D et al. Genetics 2005;170: 8

9. NILs test the effect of a single introgression
PerCv1a, PerCv1b, and PerCv5d NILs. Mean leaf-movement period of Ler is compared to NILs for PerCv1a and PerCv1b (A and B) and for PerCv5d (D and E) at 12°, 22°, and 27°. See insets for line identification. Error bars represent SEM of period estimates. NIL genotypes across the five Arabidopsis chromosomes are shown to the right of each graph. Open regions represent the Ler genotype and solid regions represent the Cvi genotype. Detailed view of NIL genotypes around PerCv1a and PerCv1b (C) and PerCv5d (F) regions shows the position of mapping markers (solid diamonds) and candidate genes (open diamonds). Horizontal lines correspond to mapping markers and solid (Cvi) and open (Ler) regions of vertical bars represent the genotype of NILs at the markers. Breakpoints are estimated as the midpoint between mapping markers.
Edwards K D et al. Genetics 2005;170: 9

10. Greenhouse Project Genotypes: Cvi x Ler NILs 16 replicates of each NIL
Grew at warm temperature (from Edwards et al)
As a result we have NILs that are either short, WT, or long period
Cold-treated seeds for 4 days
Measure Physiology traits
Days to Germination
Days to Flowering
Leaf number
Leaf size

11. IRGA (infra-red gas analyzer) gas exchange system
Parameters measured or calculated:
photosynthetic carbon assimilation (Photo),
stomatal conductance (Cond),
intercellular CO2 concentration (Ci),
fluorescence (Fs),
leaf transpiration,
vapor pressure deficit calculated from measure leaf temperature (VpdL),
measured air temperature (Tair),
measured leaf temperature (Tleaf),

12. Days to Germination and Bolting Advanced in Short and Long Period NILs

13. Short Period NILs Have Reduced Size and Leaf Number

14. Net photosynthesis and quantum yield tend to be higher in WT plants

15. Difficulties in Physiological Measurements
Whole plant IRGA requires plants to be at an earlier developmental stage than other studies making it difficult to compare across experiments
Soil respiration is confounded with plant measurements
Overcome by “capping soil” with plastic or clay

16. Circadian Clock is Adaptive
Earth rotates every 24 hours providing predictable cycles of light and temperature
WT and WT-like NILs have increased leaf number and size. These two traits are often positively correlated with fruit set.
WT plants tended towards higher net photosythesis and more effiecient photosytem II activity.

17. Future Research Increase replication
Lead to better sampling of NILs and period classes

18. Acknowledgements
Funding NSF EPSCoR Research Fellowships for Undergraduates Thanks to: Advisor: Cynthia Weinig and Matthew Rubin Planting/Data Collection : Kyle Anderson, Meredith Pratt and Yulia Yarkhunova