Van x Col Rotation Project

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I started my pHD at the department of Ecology and Evolution here, at the University of Chicago, in the fall 2007. My research interests and list

of publications can be found on my web page, http://home.uchicago.edu/~nbloch.

I am currently doing a rotation in the Borevitz lab and I am working on the Van x Col Seasonal Flowering time Project. Below is a description of the project:

Here is a link to the talk I gave at the end of my rotation (Lab meeting 04/15/2008)

QTL MAPPING OF FLOWERING TIME UNDER GEOGRAPHICAL AND SEASONAL CONDITIONS IN Arabidopsis

SNP Markers

Old Set
New Set

Phenotypes

Experiment 2 (Spring, Spain & Sweden, 2006)
Experiment 3 (Spring, Spain & Sweden, 2007)

QTL mapping

This study is very similar to Li et al.

Introduction

Flowering time is a major life history transition essential for plant's reproductive success and hence, fitness. Many environmental cues, such as vernalization, photoperiod, or drought stress, will influence flowering time. Arabidopsis thaliana is widely distributed over a wide latitudinal range, and hence inhabits latitudes that vary systematically in the environmental factors responsible for flowering time variation. The latitudinal cline in flowering time in wild A. thaliana suggest there are precise mechanisms regulating flowering time which are responsible for local adaptation.
Previous studies have already identified genes regulating flowering time in response to environmental cues:

Modified from Simpson & Dean 2002. Model representing the different pathways controlling flowering time and their predominance in different seasons. In late summer FRI prevents flowering stimulation by long days, through the photoperiod pathway. In winter, vernalization in responsible for the reducing FLC levels, and finally, in the spring the photoperiod pathway is predominant upregulating FPI.

Flowering time adaptations to local environment are thus a consequence of genetic variation and the corresponding response of each genotype to environmental signals (G X E)
Quantitative trait locus (QTL) mapping provides a unique opportunity to test for genotype x environment interactions allowing us, not only to determine which QTLs are responsible for variation in flowering time, but also to understand QTL x environment interactions.

Latest technology allows us to replicate field conditions in walk in growth chambers at different latitudes without the unpredictability of field studies. These chambers allow us to simulate environmental conditions at a particular latitude/longitude by controlling sunrise and sunset times, light spectrum, temperature and relative humidity.

Methods

Growth chambers

The two walk-in chambers were programed to simulate the seasonal conditions of Stockholm, Sweden and Madrid, Spain. 30 years averages of humidity, day length (sunrise and sunset), temperature and light spectrum were used to set up chambers during the experiment. The same method as Li et al. were used to program the chambers . Mathematical models were generated based on latitude, longitude, day of the year and time zone. The resulting fluctuations in temperature and light conditions are summarized in figures 3 and 4 respectevely.

Figure 2: Simulated temperature fluctuations in Sweden and Spain during the course of the experiment.

Figure 4: Simulated light quality spectrum in Sweden and Spain during the course of the experiment.

Two experiments were done, in 2006 and 2007. Form this point we will refer to the 2006 experiment as experiment 2, and to the 2007 experiment as experiment 3.

RIL set

Set up a graph explaining how RIL are produced

Van stock center number CS6884

Protocol:

To increase the number of recombination events in each set, we have performed for most sets several generations of random intercrossing before beginning with single-seed descent (see table). Briefly, beginning with the F2, 75 (or 150 VanC) random, non-overlapping pairs of individuals were selected for crosses. Two plants from each cross were kept for the next round of random intercrossing.

To generate recombinant inbred lines, each set of F2s or advanced intercross lines (AILs) is being taken through six additional generations of random single-seed descent, which should yield lines that are more than 98% homozygous across the genome. For lines with relatively uniform flowering times (not requiring vernalization), 20 seeds from each plant are imbibed in water for 4 days at 4°C. After stratification, seeds are placed on soil in 12-pot flats, with 4 lines per pot (one line in each corner of the pot). For each line, only the seedling closest to the corner of the pot is kept, thereby randomly propagating seedlings with respect to growth characteristics to avoid selection bias. For lines requiring vernalization, 20 seeds per line are sterilized, soaked overnight in water, and then plated on 1/2 MS phytagar plates; plates with gridlines are being used, and one line is plated per gridded square. Seeds are then stratified for an additional 4 days in the dark at 4°C, moved to 23°C light for 3 days to induce germination, and placed at 4°C in a short-day incubator for 4 to 6 weeks. After vernalization, one seedling per line is chosen (the upper-leftmost seedling to prevent selection bias) and transplanted to soil.

After the sixth round of selfing, the RILs

will be genome-wide genotyped. When the lines are finished, we will deposit them in the stock center. At the present time, we cannot give a firm estimate when this will happen, but likely sometime during 2004 for the first set of lines, if not sooner. We will provide an update at this site regarding submission status of the lines on this site.

SNP Markers

For these experiments two sets of SNP markers were used. After carefull analysis and elimination of bad markers we had a total of 164 SNP markers.

Experimental Design/Layout

How were the blocks set up???

All the phenotypic raw data as well as the genotype raw data can be found at http://natural.uchicago.edu/naturalvariation/VanC/.

We processed all of the files to have them in the proper format for genotype, phenotype and QTL analyses (data organization scripts). The processed data files can be found here.

All the scripts used to process and analyze the data can be found here.

Results

Phenotype Description

To go to the scripts use for phenotype descriptions click here

Figure 5: A) Flowering time in each block for experiment 2. Blocks 1,2 & 3, on the left, correspond to Spain seasonal conditions. Blocks 4, 5 & 6, on the right correspond to Sweden.
B) Flowering time in each block for experiment 2. Blocks purple, red & white, on the right, correspond to Spain seasonal conditions. Blocks blue, green & orange, on the left correspond to Sweden.

Genotype Description

We began by controlling the quality of the genotypes for each RIL, and eliminate the ones that got systematically bad readings or for which the new and old markers were inconsistent (possibly because of mislabeling of samples). Here is a link to the corresponding figures. IN PROGRESS...

Figure 6: A) Histogram of the proportion of col genotypes for each RIL. We chose to keep only lines were the proportion of col genotypes was between 0.3 and 0.7 (between the two vertical lines)

B) Markers positions along the 5 chromosomes. C) Distribution of intermaker distances.