PBG/MCB 622 – Class Exercise 1 Friday Nov 14th 2014

PBG/MCB 622 – Class Exercise 1 Friday Nov 14th 2014 Today’s lab will focus on QTL mapping use WinQTL, a windows version of QTL Cartographer. We will focus upon loading genotypic, phenotypic and map data into WinQTL so that you can run Simple Interval Mapping, Composite Interval Mapping and Multiple Interval Mapping and compare the results from the three approaches. The data set is based upon the Steptoe x Morex mapping population of 150 random DH lines. We scored the population for 3 quantitative traits over 4 seasons at JHI in Scotland and, for the purposes of this class, have taken simple arithmetic means of each trait so there are 3 available to map but we will just try to detect QTL for PSpot (Physiologic Leaf Spotting) in this exercise. You should have the following files available to create a master file in ‘MCD’ format that WinQTL requires to analyse data: SxM-‐chlab-‐mknum.txt, SxM-‐mklab.txt, SxM-‐mkpos.txt, and SxM Means-‐
mkttot-‐lab.txt. The first file contains chromosome labels with the number of markers for each, the second contains the marker names for with those for chromosome listed on one line, the third has the marker positions for each chromosome listed in the same way, and the fourth has the population genotypic and phenotypic data for individuals with all the data for one individual listed on one line. The first line of the last file also contains some header information, with the last 3 values being the trait names. Task 1. Import your data into WinQTL. 1.
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Find WinQTL and double click on the icon to load the program. Click on New to create a new ‘MCD’ file for WinQTL This opens a ‘Create New Source File’ dialog box. Step 1 of 6: Adjust the Chromosome number to 7, leave the Traits at 3, put the other trait at 0, Invdividual Number at 150, Symbol for missing vale as ‘*’, Cross Type as ‘Ri0’ (equivalent to doubled haploid). Choose a filename to store your data in so that you can go back to the file later if you want then leave the Marker Genotype Table alone as the data is formatted that way and then click ‘Next’ Step 2 of 6: Change the Map Function to Kosambi, and check that ‘Position’ is selected as Position Type and ‘cent Morgan’ as the Position Units. Click ‘Browse’ and go to the ‘Exercise 1’ directory and then select SxM-‐chlab-‐mknum.txt to load in the chromosome labels and numbers. Make sure the ‘Include label’ box is checked and then click ‘Send Data’. This should transfer the data into the box above. Click ‘Next’ Step 3 of 6. Load in Marker Labels and Positions. Browse to SxM-‐mklab.txt, ensure that the ‘Labels’ button is on, and click ‘Send Data’. Now switch the ‘Positions’ button on and Browse to SxM-‐mkpos.txt and click Send Data. This should populate the Marker Labels and Marker Positions in the table above and make the ‘Next’ button active. Step 4 of 6. Make sure the top radio button is on ‘All data in one file….’ and click ‘Next’ Step 5 of 6. Under ‘Cross Information Filename’ Browse to SxM Means-‐mkttot-‐lab.txt and click Send Data. This should populate the table above with marker genotypes and trait data. Click Next. Click ‘Finish’ and then ‘OK’ on the message box. 12. This will create a Source File in the tree on the LHS and a listing of the map in the main window. Task 2. Simple Interval Mapping of Physiological Leaf Spotting 1. Ensure that the node with your MCD file is highlighted and then click the IM icon (8th from Left) 2. This opens a dialog box for the input settings for SIM. Accept all the default values but change ‘Trait Selection’ from ‘All Traits’ to ‘Trait 1 – Pspot_Mn’, switch the ‘Manual Input’ radio button of ‘Threshold Value Setting’ on and enter 14 (just greater than LOD=3) into the ‘Threshold (LR)’ box. 3. Press ‘Start’ to begin Interval mapping and changing values in the middle of the box indicate that the process is running – should take <1min. 4. When it has run, a graph window will open up showing the results of the LOD profile and estimated effects for chromosome 1. 5. Select ‘All Chromosomes’ from the ‘Chrom’ menu in the chromosome window to display the results from all chromosomes QUESTION 1. Click on the Tools Menu of the chromosome menu and choose the ‘Show QTL Information’ Option and then Automatic locating QTLs with a minimum of 15cM between QTL. Then press OK and the graph window is modified. How many QTL are detected by SIM and on what chromosomes are they located? Question 2. How many Steptoe alleles at the detected QTL decrease the character, and if there are any, on what chromosomes are they located? Task 3. Composite Interval Mapping of Physiological Leaf Spotting 1. Ensure that the node with your MCD file is highlighted and then click the CIM icon (9th from Left) 2. This opens a dialog box for CIM. Adjust the default values as in Task 2.2 3. Press ‘Start’ to begin Interval mapping and changing values in the middle of the box indicate that the process is running – should take <2min. 4. When it has run, a graph window will open up showing the results of the LOD profile and estimated effects for chromosome 1. 5. Select ‘All Chromosomes’ from the ‘Chrom’ menu in the chromosome window to display the results from all chromosomes QUESTION 3. Click on the Tools Menu of the chromosome menu and choose the ‘Show QTL Information’ Option and then Automatic locating QTLs with a minimum of 15cM between QTL. Then press OK and the graph window is modified. How many QTL are detected by SIM and on what chromosomes are they located? What is the major difference from SIM? Question 4. How many Steptoe alleles at the detected QTL decrease the character, and if there are any, on what chromosomes are they located? Task 4. Multiple Interval Mapping of Physiological Leaf Spotting 1. Ensure that the node with your MCD file is highlighted and then click the MIM icon (10th from Left) 2. This opens a box to select the triat for analysis (MIM only woks on one trait at a time). It should have selected Pspot_Mn and, if so, press ‘MIM’. If not, choose Pspot_Mn from the drop-‐down menu and press ‘MIM’ 3. This opens a box where ‘No MIM Model Exist’ is shown in a drop down menu. Press the ‘New Model’ button immediately to its right. 4. This opens up the ‘Create New MIM Model’ dialog box with a range of options to choose from. For simplicity, turn the ‘Scan through QTL mapping result file’ radio button on. 5. This activates the ‘Criterion’ button where you change the LOD threshold value to 3 and the Min cM between QTL to 15. Press ‘OK’ 6. The ‘Open QTL Result File’ is now active. Press that and locate a file that has the same name as the second file under ‘Results’ in the tree on the LHS (it should end in ‘…-‐C.qrt’. Select it and press Open. 7. You should now have a list of 5 QTL in the window. Press OK. Task 5. Refining your QTL model via Multiple Interval Mapping. There are many options here and we will choose the following path for illustration and comparison. 1. The dialog box at the top of the screen will now be populated with the list of your 5 QTL. Note the Likelihood value in the L(5) box and QTL Effects to get a list of the estimated effect at each QTL. Then press Refine Model 2. This opens a dialog box with three main functions. Firstly, switch the ‘Optimising QTL Positions’ button on if it is not already on, increase the Window Size to 15cM and press Start. 3. This runs quite quickly and note that the Likelihood has increased by just over 2. 4. Press the ‘Summary’ button to obtain a summary of the results of this MIM step. Press OK to any dialog boxes that open up. 5. When it has run, a graph window will open up showing the results of the LOD profile and estimated effects for chromosome 1. 6. Select ‘All Chromosomes’ from the ‘Chrom’ menu in the chromosome window to display the results from all chromosomes 7. Press Refine Model again and this time switch on the ‘Search for New QTL’ button with the ‘Main QTLs’ button on. Set the window to 15cM again and press Start. 8. This should have added in 3 QTL to make a total of 8 and the Likelihood will have further increased by about 14. Task 6. Checking for Epistasis between your identified QTL. 1. Press Refine Model again and this time switch on the ‘Search for New QTL’ button with the ‘QTL Interactions’ button on. Set the window to 15cM again and press Start. 2. This should have added in 5 QTLxQTL interactions and the Likelihood will have further increased by about 17. 3. Press Summary to get a summary of the effects and a graphical output. 4. When it has run, a graph window will open up showing the results of the LOD profile and estimated effects for chromosome 1. 5. Select ‘All Chromosomes’ from the ‘Chrom’ menu in the chromosome window to display the results from all chromosomes. Task 7. Testing your QTL 1. Press Refine Model again and this time switch on the ‘Testing for existing QTLs’ button with the ‘Main QTLs’ button on. Press Start and then OK if all are accepted. 2. Press Refine Model again and this time switch on the ‘Testing for existing QTLs’ button with the ‘QTL Interactions’ button on. Press Start and there should be no change. 3. Click Close on the MIM window and then Save the file according to default instructions. QUESTION 5. Click on the Tools Menu of the chromosome menu and choose the ‘Show QTL Information’ Option and then Automatic locating QTLs with a minimum of 15cM between QTL and LOD 3 between valleys. Then press OK and the graph window is modified. How may QTL are detected by MIM and on what chromosomes are they located? What are the differences from CIM Question 6. How many Steptoe alleles at the detected QTL decrease the character, and if there are any, on what chromosomes are they located?

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