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User Manual for QTLMapper Version 1.0

1. A gt B gt Select Esc gt Next Figure 15 Dialogue box for setting mapping ranges Trait range The default setting for trait range is AI namely all the traits will be analyzed To change trait range press letter A in Setting Mapping Ranges dialog box Then an input box Figure 16 will appear ast 7s Manual for QTLMapper Version 1 0 Trait Range Setting Al Enter gt Ok Esc gt Cancel Figure 16 Input box for setting trait range In the input box type the order numbers based on the order of trait names in the data file of the traits you want to analyze and press Enter key At least one white space is required between the order numbers A dash symbol can be used for simplifying the input for several consecutive order numbers For example 1 3 5 7 is equivalent to 13 5 67 But no white spaces are allowed between number and and between and number Note that trait names are not acceptable Genomic range The default setting for genomic range is Aut short for automatically which means that the marker intervals to be tested will be decided automatically based on selected important main effect markers see the following sections for details This is a quick way for identifying important putative QTLs To change this setting press letter B in Setting Mapping Ranges dialogue box then another dialogue box Figure 17 will appear for you to ch
2. Data file name DAQTLMAPER simu txt Marker map file name DXQTLMAPER simu map Environments yes Replications no Contents selected main effect markers Analysis method Stepwise Regression Inclusion probability 0 005000 Exclusion probability 0 005000 Date 1999 03 12 Time 12 13 07 Trait1 simTt TILEI 1 Environment code Total selected markers 3 General R 2 0 4039 Marker Name F value Prob R 2 42 MK42 44 6769 0 0000 0 1359 32 MK32 56 7282 0 0000 0 1725 25 MK25 29 5473 0 0000 0 0899 TI LE2 2 Environment code Total selected markers 4 General R 2 0 5242 Marker Name F value Prob R 2 33 MK33 78 7107 0 0000 0 1921 42 MK42 98 2873 0 0000 0 2398 j Manual for QTLMapper Version 1 0 27 MK27 20 9713 2 MK2 8 4631 0 0000 0 0040 For each trait there is a subset of results for each environment A string such as TI LEI File with two extension List 3 is an example of two file created for a data set from a single environment List 3 An example of two file for a single environment dh two TI LE2 indicates the trait order number and environment order number For instance TI LEI stands for the first trait and the first environment level The integer number that follows the string is the code of the environment level as a subscript for the environmental level in the data file Result file created by QTLMapper V 1 0 Data file name DAQTLMAPERMh txt
3. Marker map file name DAQTLMAPER dh map Environments no Replications no Contents selected interaction markers Analysis method Stepwise Regression Inclusion probability 0 010000 Exclusion probability 0 010000 Date 1999 02 01 Time 15 47 05 Trait ju28ph Total selected marker pairs 4 General R 2 MarkerA Name MarkerB 51 MkS1 74 10 Mk10 14 96 Mk96 152 32 Mk32 79 The description of conditions is quite similar to that for one file So we are not going to talk about it here In the result body the trait name total number of selected pairs of interaction markers and 0 2187 Name Mk74 Mk14 Mk152 Mk79 edo F value 20 1118 35 9031 17 4605 10 0271 general R 2 if using stepwise regression are given for each trait Note that general R 2 is the multiple determination coefficient of all the selected pairs of interaction markers with control over all the selected main effect markers in the one file obtained for the same data set Then there is also Manual for QTLMapper Version 1 0 a two way table showing the results for the selected pairs of interaction markers The MarkerA and MarkerB columns are the order numbers of each pair of interaction markers The order numbers are also decided according the order of the markers arranged in the data file The two Name columns give the marker names corresponding to the MarkerA order number and MarkerB order number The F value and Prob co
4. Aut to decide your genomic range to be mapped then a decision box Figure 22 will appear if the one file already exists You can decide to redo the main effect marker selection here only by stepwise regression or keep the original If the one file does not exist in the work directory the main effect marker selection will start automatically Decision Main effect marker selection already done Do it anyway Manual for QTLMapper Version 1 0 Figure 22 Decision box for main effect marker selection If you set BGV control as controlling Main amp Interaction Markers a similar dialog box not shown here will appear subsequently if the two file already exists You can decide to redo the interaction marker selection only by stepwise regression or keep the original If there is no such two file in the work directory the subroutine for selecting interaction markers will be started automatically and a new two file will be created After all the above has been done QTLMapper 1 0 will begin the process of mapping additive effect QTLs and display the progress of the analysis and some other information such as result file name and path trait being analyzed BGV control method and hypothesis for LR test etc Figure 23 Trait 1 simTt UUUD DO CODOD0 000000000000 000000 10 Figure 23 Progress of mapping additive effect QTLs for first trait named simTt Step 7 Deciding whether to filtrate putative main effe
5. Permission to use copy and distribute this software and its documentation for any purpose with or without fee is hereby granted provided that the above copyright notice appear in all copies and that both that copyright notice and this permission notice appear in supporting documentation Permission to modify the software is granted but not the right to distribute the modified code Modifications are to be distributed as patches to released version This software is provided as is without express or implied warranty AUTHORS Original Software Thomas Williams Colin Kelley Gnuplot 2 0 additions Russell Lang Dave Kotz John Campbell Gnuplot 3 0 additions Gershon Elber and many others We have packed this software with QTLMapper 1 0 in the compressed file qtlmapr exe After you unzip the files in qtlmapr exe there will be a directory named Wegnuplot as a sub directory of QTLMaper All the necessary files for Wgnuplot are saved in this directory Viewing LR graphs In Windows Explorer open the folder Wgnuplo in folder QTLMaper Double click on wgnuplot exe to open a Wgnuplot window Figure 39 Press Open button or select Open item in File menu to bring up a Load dialog box Select the drive and the work directory in which your graph files are saved Plie Piat Expewteione Pigctions ewval duet hat jtrles 3b dele fepe Figure 39 Windows interface of Wgnuplot 55 Manual for QTLMapper Vers
6. Environments yes Replications no Contents Bayesian test results for epistatic QTLs Jackknife based on D QTLMAPER simu fle BGW control method A control marker main amp interaction effects Threshold probability 0 005000 Date 1999 03 10 Time 21 44 53 Trait 1 simTt Ch Ini Int Namei Sitei M Ch Inj Int Namej Sitej M AEil Prob AEBjl Prob AAEijl Prob 1 2 MK2 MK3 0 00 2 6 MK24 MK25 008 J 0 034 0 835 2 230 0 000 2 808 0 000 1 2 MK2 MK3 0 00 2 14 MK32 MK33 002 0 043 0 788 0 247 0 263 1 491 0 000 1 2 MK2 MK3 0 02 3 8 MK41 MK42 0 10 asa 0 000 1 000 3 121 0 000 0 916 0 001 2 6 MK24 MK25 0 06 3 8 MK41 MK42 0 10 2 161 0 000 3 191 0 000 1 112 0 000 2 7 MK25 MK26 0 00 2 14 MK32 MK33 0602 2 169 0 000 0 143 0 442 1 264 0 000 2 14 MK32 MK33 0 02 3 9 MK42 MK43 002 J 0 252 0 263 3 171 0 000 2 724 0 000 End 49 Manual for QTLMapper Version 1 0 6 Contribution files ctq and cte files These files are created for outputting the relative contributions of detected putative additive epistasis effect to the phenotypic variation as well as the relative contributions of QE interaction effects when multiple environments are available The calculation of relative contributions is different from that for R squares in multiple regression analysis A relative contribution here is calculated as the proportion of variance cause
7. Jackknife test is a re sampling technique that has been widely used for parameter estimation and significance test Here we use it for parameter estimation and significance test of QTL genetic main effects and QE interaction effects by cutting one genotype each round when an environmental factor is involved in the data set If the data set does not include an environmental factor you are not suggested to run Jackknife test because this operation will take a long time To run Jackknife test press number 2 in Output sub menu An input box Figure 35 will appear You need to input the name of the file created from filtrating additive effect QTLs or epistatic QTLs The Jackknife test will be based on the positions of the putative QTLs presented in this file Open Filtration File Filename _ Enter gt Ok Esc gt Cancel Figure 35 Input box for opening filtration file After the filtration file has been opened you will be asked to input the map file and data file if they are not opened The method of opening the map and data files is the same as that described in Working with File Sub Menu After that the process of Jackknife test will begin and the progress and some information will be displayed After the process a result file with the same stem as the original result file and an extension of jpq for additive effect QTLs or jpe for epistatic QTLs will be created 3 Bayesian Test Bayesian test is desig
8. and s are the additive effect dominance effect and the difference between a and d respectively The LOD column is the LOD scores transferred from the LR values under LOD 0 217 LR The A column shows the additive genetic effects estimated at the testing points The title A is used for DH RIL or F populations If a BC population is used it will become S S is the difference between the additive effects and the dominance effects at the testing points the additive effect and dominance effect are not separable in a BC population It is noted that the signs of the additive effects A follow that of the parent called P1 in marker coding Positive A values imply that the P1 parent takes positive value for the additive effect while the P2 parent takes the negative Negative A values mean that the P1 parent takes the negative value for the additive effect while the P2 35 Manual for QTLMapper Version 1 0 parent takes the positive The Prob column is the two tail probabilities of student values for testing the additive effects This is used for testing the significance of the estimated additive effects If you are using a F population then two more columns will appear following the above a D column and another Prob column The D column gives the dominance effects estimated at the testing points with the Prob column is for the significance of the dominance effects The layout of the mapping results in the qtl file for the data set
9. 0 005 used for the filtration But it cannot change the default threshold probability specified in Settings sub menu Filtration Probability 0 004999 _ Enter gt Ok Esc gt Cancel Figure 33 Input box for changing the filtration probability If you do not want to change the default just press Enter key Otherwise you need to input a value between 0 and 1 Another input box Figure 34 will appear after you press Enter in the previous input box You need to input the original result file qtl or eps file in this input box Open Result File Filename _ Enter gt Ok Esc gt Cancel Figure 34 Input box for inputting original result file name The original result file must already exist in the work directory that is specified in Settings sub menu The resulted file with flq or fle extension will also be saved in the work directory 2 Jackknife Test In the mixed model approach Zhu 1998 Zhu and Weir 1998 Wang Zhu Li and Paterson 1998 the environmental factor if involved is taken as a random factor Then the QE interactions should also be random though the genetic main effects QTL additive effects and epistatic effects are fixed in the mixed model In this case the significance test for the predicted QE interaction Manual for QTLMapper Version 1 0 effects by best linear unbiased prediction BLUP has very low power using the ordinary z test based on the normal distribution
10. in Main Menu by pressing number key 4 If current status is not in Main Menu you need to press Esc one or more times to return to Main Menu first Detailed descriptions for changing settings under Settings sub menu are made below 1 Work Directory A work directory is where input files and all sorts of output files are saved Once the user has specified a work directory it would not be necessary to add the path rame before a file name when opening the file the program will automatically look for the specified file in the work directory The default work directory is where Qtlmaper exe is started It is suggested that a separate work directory be created to avoid confusion To change the default setting e g CXQTLMAPER press number T under Settings sub menu and input a new directory name in the input box Figure 3 The character string C QTLMAPER in the square brackets is the current work directory Work Directory CAQTLMAPER _ Enter gt Ok Esc gt Cancel Figure 3 Input box for changing work directory If the specified directory does not exist a Decision box Figure 4 will pop up to let you decide whether to create it Decision Directory not exist create it Enter gt Ok Esc gt Cancel Manual for QTLMapper Version 1 0 Figure 4 Decision box for creating a new work directory If Enter key pressed the directory will be automatically created and the work directory is changed to
11. 0 0023 0 0000 0 1009 0 0745 General contributions Additive A H 2 A 20 0852 Epistasis H 2 AA 0 0302 QE Interactions H 2 AE 20 5758 H 2 AAE 20 2205 53 Manual for QTLMapper Version 1 0 In the result body the layout of the results for the relative contributions is similar to that for ctq files The Ch Ini and Chi Inj columns provide the order numbers for the chromosomes and intervals on which the pair of involved putative QTLs are located The Int Namei and Int Namej columns give the flanking marker names of the two involved intervals The Sitei M and Sitej M columns show the genetic distances of the two putative QTLs from the left markers on their own intervals The H X Ai and H 2 Aj columns give the relative contributions of the two putative QTLs i and j involved in the epistasis The H 2 AAij column is the contributions of the epistasis For a BC mapping population the titles will be H 2 Si H 2 Sj and H 2 SSij respectively For the case of multiple environments more columns will be available for the contributions of QE interactions In this example the H 2 AEi and H 2Z AEj columns provides the contributions of additive by environment interaction effects for the two putative QTLs while the H Z AAEij is the contributions of epistasis by environment interactions For a BC mapping population the titles will be H 2 SEi H 2 SEj and H 2 SSEij respectively It is noted that there will be no those columns if no
12. For opening the dialogue box Figure 9 to do the settings press number 6 under Settings sub menu to change the default settings further press letter A B or C and input an appropriate value between 0 and 1 in the input box that appears subsequently Probabilities A Marker Inclusion B Marker Exclusion C Threshold Prob A gt B gt Select Esc gt Back Manual for QTLMapper Version 1 0 Figure 9 Dialogue box for changing default probabilities Probability for Marker Inclusion is used for selecting markers with stepwise regression or analysis of variance ANOVA Probability for Marker Exclusion is used only in stepwise regression for ruling out selected marker s from regression model when a new marker or marker pair is selected in Probability for Marker Inclusion cannot be larger than that for Marker Exclusion Otherwise it can cause an endless loop To prevent this from happening the program will pop up an error message when you input wrong probabilities Threshold Probability is used in mapping additive effect QTLs or mapping epistatic QTLs for testing likelihood ratio LR at a test point in mapping additive effect QTLs or a pair of test points in mapping epistatic QTLs But this setting becomes effective only when you choose to save mapping results By Threshold 7 Walking Speed Walking speed means the genetic distance in Morgan between adjacent testing points in mapping additive effect Q
13. Jackknife test for all the testing points listed in the filtration file The Chromcolumn gives the order number of chromosomes on which the putative QTLs are located The Interval column is the names of flanking marker intervals in which the putative QTLs are sited Manual for QTLMapper Version 1 0 The Site M column gives the genetic distances in Morgan of the putative QTLs from the left marker on the interval The Ai column provides the Jackknife estimates for additive effects of the putative QTLs DH or RIL populations With a BC population the title of the column will be Si the difference of the additive effect and dominance effect for the putative QTL The signs of Ai values are determined in the same way as that in qtl files The Prob column after this column is the probabilities for testing the estimates of additive effects If multiple environments are involved more columns that give the results for QE interaction effects will appear after the above columns In the example the AEix columns for DH or RI populations shows the predicted values of interaction effects between putative QTL i and x th environment The order number of a specific environment is decided in the appearing order of the subscript for the environment in the data file If a BC population is used the columns will be titled SEix Immediately following each of the AEix or SEix is a Prob column that gives the probability for testing the predicted values Fi
14. RI population it is equivalent to Hj a O and o E 1 0 where a is the additive effect of the individual testing point i and o E 1 is the variance from the main effect aj x environment interaction Item B is for testing Hp a 0 only while item C is for testing H o g 0 1 only For BC population just change a and A to s and S in the expressions for the hypotheses S or s stands for the difference between the additive effect a and the dominance effect d for the testing point s 2 a d The user can decide one in this dialog box by pressing letter A B or C then press Esc for next step Step 5 Deciding whether to continue the process for QTL mapping A decision box Figure 21 will appear and give you a chance to quit the mapping process and return to Run sub menu Decision Now begin to run Enter gt Ok Esc gt Cancel Figure 21 Decision box for continuing or quitting QTL mapping Step 6 Deciding whether to redo marker selection The mixed model approach uses important markers selected by ANOVA or stepwise regression for BGV control The selected main effect markers will be saved in a file with extension of one while the selected pair wise interaction markers are saved in another file with extension of two If you set BGV control by Settings sub menu as controlling Main effect Markers or Main amp Interaction Markers or you have used
15. according to the arrangement of the trait data in the data file Followed is the trait name if more than eight characters in the data file it will be shown here as a truncated name Then there is a large two way table showing the mapping results for each testing point using the method for saving mapping results If the method is By Threshold then only the testing points shown significant LR values are saved otherwise the results for all testing points are saved The Chrom column gives the order numbers integer numbers of the chromosomes on which each testing point is set The order numbers are decided the arrangement of chromosome columns for the genetic distances in the map file The Interval column presents the names of the marker intervals each composed of two ee 99 flanking markers on which a testing point is set A hyphen is used between the two flanking markers Here the truncation method will apply if the marker names exceed eight characters The Site1 M column is the genetic distance in Morgan of the testing points from the left end of the chromosomes on which the testing points are set The Site2 M column is the genetic distance in Morgan of the testing points from the left marker on the interval on which the testing points are set The LR column gives the likelihood ratio values for the testing points The hypothesis is Ho a 0 for DH or RI populations s 0 for BC population a d 0 for F population Here a di
16. be automatically extracted into a directory named as QTLMaper on the specified hard disk e g C QTLMaper Composition of QTLMapper 1 0 QTLMapper 1 0 is composed of Qtlmaper exe Qtlmaper hlp Qtlmaper ini and some sample files Qtlmaper exe an executable file is the core of QTLMapper 1 0 It performs all the operations necessary for QTL mapping analysis Qtlmaper hlp is a text file that provides on line help Qtlmaper ini also a text file is used for initialization of settings when Qtlmaper exe is started see a later section for more details If Qtlmaper ini does not exist Qtlmaper exe will automatically create one that contains default settings Sample input files are provided for demonstrating the format required by QTLMapper 1 0 X Warning Do not edit Qtlmaper hlp and Qtlmaper ini Starting and Exiting QTLMapper 1 0 Method of Starting QTLMapper 1 0 can be started in three ways 1 Run from Windows Explorer of Windows 95 98 TM In Windows Explorer find the folder e g C QTLMaper containing the files of QTLMapper 1 0 Double click on Qtlmaper exe 2 Run from Start menu Create a shortcut in Start menu of Windows 95 98 TM for QTLMapper 1 0 If you do not know how to do so please consult a computer expert Run the shortcut from Start menu just like running any other Windows applications 3 Run under MS DOS prompt First run MS DOS prompt from Start menu then take the Manual for QTLMapper Version 1 0 followi
17. be omitted However if you include such factors as replications and or environments it becomes necessary Environments yes specifies the status of experimental design for environments The specification yes or y case insensitive indicates that one environmental factor such as year factor or location factor is included in the phenotyping experiments If your phenotyping experiment was carried out in only one environment you can use specification no or n or simply omit this item It is noted that QTLMapper 1 0 cannot analyze data sets with designs for more than one environmental factor e g both year factor and location factor Replications no specifies the status of experimental design for replications or randomized blocks The specification no or n case insensitive indicates that only one or no replication was used in the phenotyping experiment In this case this item can also be omitted When replications or randomized blocks were used however it becomes necessary and the specification must be yes or y lIraitNumber 1 specifies the total number of traits whose data are included in the data file TotalMarker 64 specifies the total number of the markers included in the data file This number must also be equal to the summation of the numbers for _MarkerNumbers in the map file MarkerCode Pl 1 P222 Fl 3 HP1 4 FIP2 5 defines a marker coding scheme There are five possible strings for the specifications Each of the
18. from multiple environments is basically the same The only difference is that one more item will be included in the description of conditions about the hypothesis used for LR test List 6 In addition the LR and LOD values are obtained based on the hypothesis when multiple environments are included List 6 An example of qtl file for multiple environments simu qtl Result file created by QTLMapper V 1 0 Data file name DAQTLMAPER simu txt Marker map file name DAQTLMAPER simu map Environments yes Replications no Contents putative additive effect QTLs BGY control method A control marker main amp interaction effects Results saved for points significant at 0 005000 LR test for hypothesis on both genetic effects and GE interactions 3t Date 1999 03 12 Time 12 13 07 Traitl simTt Chrom Interval Site1 M Site2 M LR LOD A Prob 1 MKI MK2 0 000 0 000 13 89 3 01 0 6931 0 0051 1 MKI MK2 0 020 0 020 15 44 3 35 0 7434 0 0034 1 MKI MK2 0 040 0 040 16 74 3 63 0 7798 0 0024 1 MKI MK2 0 060 0 060 17 66 3 83 0 7991 0 0019 1 MKI MK2 0 080 0 080 18 17 3 94 0 8003 0 0017 1 MK2 MK3 0 098 0 000 18 26 3 96 0 7869 0 0017 1 MK2 MK3 0 118 0 020 17 71 3 84 0 7914 0 0019 1 MK2 MK3 0 138 0 040 16 78 3 64 0 7803 0 0025 File with eps extension List 7 is an example of eps file for a data set from a single environment The part of description of conditions is quite similar to that for qtl file
19. gt Back Y Settings Work Directory File Format BGV Control Marker Coding Map Function Probabilities Walking Speed Save Settings Output Filtrate Jackknife Test Bayesian Test Contributions Make LR Graphs 2D af wn gt 00 UY Gr OO I n 1 gt 5 gt Select Esc gt Back 1 gt 8 gt Select Esc gt Back Figure 1 Menu system of QTLMapper Version 1 0 Help ereerreerererer Display Basic Statistics Map Main effect QTLs Map Epistatic QTLs Marker Detection 1 gt 4 gt Select Esc gt Back Help 1 Contents 2 About 1 gt 2 gt Select Esc gt Back Working with Settings Sub Menu Settings are a series of conditions that control the way QTLMapper 1 0 performs analysis for QTL mapping These include work directory file format BGV control marker coding scheme map Dus Manual for QTLMapper Version 1 0 function probabilities and walking speed Usually the first thing the user needs to do is to change the settings so that QTLMapper 1 0 runs under conditions suitable for his her specific input files and objective of the analysis Settings Work Directory File Format BGV Control Marker Coding Map Function Probabilities Walking Speed Save Settings Oo o Or eO D 1 gt 8 gt Select Esc gt Back Figure 2 Settings sub menu To change the settings open Settings sub menu Figure 2
20. gt C gt Select Esc gt Back Figure 6 Dialogue box for changing BGV control 4 Marker Coding Marker data in data file need to be presented in a coding manner or exactly observations of markers need to be represented by a set of letters and or numbers each standing for one marker type The user can define his her coding scheme in the data file or use the default setting shown in Marker Coding dialogue box Figure 7 QTLMapper 1 0 also allows the user to change the default setting using this dialogue box To open the dialogue box press number 4 under Settings sub menu Marker Coding A P1 Type A B P2 Type B C F1 Type H D F1P1 Type C E F1P2 Type D A gt E gt Select Esc gt Back Figure 7 Dialogue box for changing default maker coding scheme In the above dialogue box P1 Type Marker phenotype that is the same as that of the parent named as P1 If the backcross population is used the recurrent parent must be named as P1 P2 Type Marker phenotype that is the same as that of the other parent named as P2 FI Type Marker phenotype that is the same as that of the heterozygote as Manual for QTLMapper Version 1 0 FIP1 Type Marker phenotype that is not P2 type P1 dominant or not clear between P1 type and F1 type FIP2 Type Marker phenotype that is not P1 type P2 dominant or not clear between P2 type and F1 type The letters A B H C and D on the right
21. strings looks like an equation but no white space is allowed within the string On the left side of the equation symbol is the marker genotype phenotype specification i Manual for QTLMapper Version 1 0 PI Marker phenotype that is the same as that of the parent named as P1 If BC population is used the recurrent parent must be named as P1 P2 Marker phenotype that is the same as that of the other parent named as P2 Fl Marker phenotype that is the same as that of the heterozygote FIPI Marker phenotype that is not P2 type P1 dominant or not clear between P1 type and F1 type F1P2 Marker phenotype that is not P1 type P2 dominant or not clear between P2 type and F1 type On the right side of the equation symbol 1 2 3 4 or 5 is the code for the marker type The marker code should always be a single character a number or a letter This definition of marker coding scheme will disable the settings for marker coding that can be viewed and changed in Settings sub menu It is noted that the code for missing marker data should always be a dot and cannot be defined in your data file It is not necessary to specify codes for all possible marker types except for E population For example if your marker data were collected from a DH population only the specifications for P1 and P2 types are enough Marker data body This part is embraced by two key strings MarkerBegin and MarkerEnd not case sensitive In marker data b
22. this new directory if Esc key pressed the current work directory will remain unchanged 2 File Format For performing analyses with QTLMapper 1 0 two input files are required a marker linkage map file for simplification we call it map file and a data file A map file contains information about the relative order and genetic distances of all observed markers on the chromosomes or linkage groups a data file contains observations of the markers and the traits under study for all individuals File format regulates the way of preparing these input files QTLMapper 1 0 recognizes three formats of map and data files These include the format for QTLMapper 1 0 the format for the transitive files output files of Rmap exe and Rcross exe of QTL Cartographer 1 12c and the format for Mapmaker QTL 1 1 respectively The format for QTLMapper 1 0 will be described in detail in the section about Preparing Input Files Before opening the input files the user needs to make the setting of file format consistent with that of his her specific input files Wrong setting will cause failure in opening files To view or change current file format press number 2 under Settings sub menu to open a dialogue box Figure 5 Here the current setting is the format for QTLMapper 1 0 To change the setting further press letter A B or C case insensitive and same hereafter File Format A Format for QTLMapper 1 0 Yes B Format for QTL Car
23. to obtain a text version of your input files if you use an electronic table software 2 Formats of input files recognizable QTLMapper 1 0 can open three formats of input files including the format for QTLMapper 1 0 itself the format for QTL Cartographer 1 12c transitive files outputted by Rmap exe and Rcross exe and the format for Mapmaker QTL 1 1 For details about the formats for QTL Cartographer 1 12c and Mapmaker QTL 1 1 please check their related documents and sample files If your input files are already in the format for QTL Cartographer 1 12c or Mapmaker QTL 1 1 you can open the files directly in QTLMapper 1 0 under correct setting for file format 3 Format of input files for QTLMapper 1 0 We provide two sample files sample map and sample txt for briefly demonstrating the format of input files for QTLMapper 1 0 You can open these files without removing any description lines Here we describe the format for QTLMapper 1 0 in more details Format of marker linkage map file This file contains information about the marker linkage map such as the number of chromosomes number of markers on each of the chromosomes flanking marker distances etc It consists of general description map body and some comment lines Comment lines are not a necessary part of the map file but they are helpful for reading the file General Description This part is usually put in the front of map file But it can be anywhere except inside the ma
24. 2 3 643 0 000 1 149 0 447 1 2 MK2 MK3 0 02 3 8 MK41 MK42 0 10 234 64 50 92 0 828 0 001 0 052 0 987 0 374 0 694 2 6 MK24 MK25 0 06 3 8 MK41 MK42 0 10 352 99 76 60 1 282 0 558 0 055 0 987 1 600 0 166 2 7 MK25 MK26 0 00 2 14 MK32 MK33 0 02 375 86 81 56 1 057 0 631 3 752 0 000 0 810 0 538 2 14 MK32 MK33 0 02 3 0 MK42 MK43 0 02 433 92 94 16 3 628 0 000 0 017 0 996 0 670 0 806 End 43 Manual for QTLMapper Version 1 0 List 11 An example of jkq file simu jkq Result file created by QTLMapper V 1 0 Data file name D AQTLMAPER simu txt Marker map file name D QTLMAPER simu map Environments yes Replications no Contents Jackknife test results for additive effect QTLs _ Jackknife based on D QTLMAPER simu flq BGV control method A control marker main amp interaction effects Threshold probability 0 005000 Date 1999 03 10 Time 15 30 40 Trait 1 simTt Chrom Interval Site M Ai Prob AEi1 Prob AEi2 Prob 1 MK2 MK3 0 00 0 791 0 0000 0 006 0 9454 0 006 0 9449 2 MK25 MK26 0 00 0 988 0 0000 2 245 0 0000 2 231 0 0000 2 MK32 MK33 0 04 3 687 0 0000 0 393 0 2527 0 393 0 2534 ise Manual for QTLMapper Version 1 0 List 12 An example of jke file simu jke Result file created by QTLMapper V 1 0 Data file name DAQTLMAPERNimu txt Marker map file name D QTLMAPER simu map Environments yes Replications no Contents Jackknife test results for epistatic QT
25. Ls Jackknife based on D QTLMAPER simu fle BGVW control method A control marker main amp interaction effects Threshold probability 0 005000 Date 1999 03 10 Time 21 44 53 Trait 1 simTt Ch Ini Int Namei Sitei M Ch Inj Int Namej Sitej M AEil Prob AEjl Prob AAAEijl Prob 1 2 MK2 MK3 0 00 2 6 MK24 MK25 008 0 148 0 022 2 349 0 E 2 769 0 000 1 2 MK2 MK3 0 00 2 14 MK32 MK33 002 J 0 214 0 000 0 387 0 175 1 480 0 000 1 2 MK2 MK3 0 02 3 8 MK41 MK42 0 10 0 000 1000 3 184 0 000 0 950 0 000 2 6 MK24 MK25 0 06 3 8 MK41 MK42 0 10 2 105 0 000 3 297 0 000 1 172 0 000 2 7 MK25 MK26 0 00 2 14 MK32 MK33 002 J 2 320 0 000 0 326 0 287 1 332 0 010 2 14 MK32 MK33 0 02 3 9 MK42 MK43 002 J 0 426 0 129 3 198 0 000 2 764 0 000 End 47 Manual for QTLMapper Version 1 0 Similarly the AEjx columns are the predicted interaction effects between the other QTL j involved in the epistasis and x th environment The Prob columns immediately after them provide significance tests for these effects The AAEijx columns are the predicted interaction effects between the epistasis between QTLs and j and x th environment The corresponding Prob columns are used for significance tests of these effects If the population type is BC the letter A in the titles of all these columns will be changed to S as mentioned in previous sect
26. TLs or epistatic QTLs The default setting is 0 02 M One can use a smaller value slower speed to perform more detailed search or a larger value faster speed to get a rougher knowledge of QTLs on the genome To change the setting press number 7 under Settings sub menu and input a new value in the input box Figure 10 Walking Speed M 0 02 Enter gt Ok Esc Cancel Figure 10 Input box for changing walking speed 8 SaveSettings This item in Settings sub menu is designed specially for saving the settings to file Qtlmaper ini so that all the changes can be kept when you run QTLMapper 1 0 the next time To save the settings press number 8 If you have made changes to the settings but forgot to save them then QTLMapper 1 0 will also prompt you to save the settings before exiting zs Manual for QTLMapper Version 1 0 Preparing Input Files To use QTLMapper 1 0 for QTL mapping analysis you need to get your marker linkage map and data of markers and traits into two plain text files a map file and a data file in a format recognized by QTLMapper 1 0 These files are collectively called Input Files 1 What software to use Any text file editor can be used for preparing input files But using MS Excel Lotus 1 2 3 or other electronic table software can make it much easier to prepare and manage your input files Note that QTLMapper 1 0 can only open text file so you need to use Save As
27. User Manual for QTLMapper Version 1 0 A Computer Software for Mapping Quantitative Trait Loci QTLs with Main Effects Epistatic Effects and QTL x Environment Interactions Copyright 1999 by Daolong Wang Jun Zhu Zhikang Li Andrew H Paterson Biometrics Section Anhui Agricultural University Hefei Anhui 230036 China 2 Department of Agronomy Zhejiang University Hangzhou 310029 China 3 Plant Genetics Breeding and Biochemistry Department IRRI 1099 Manila Philippines 4 Department of Soil and Crop Sciences Texas A amp M University College Station TX77843 USA This software can be freely redistributed with non commercial purpose and under the condition of no changes to the software and its related documents No warranty of any sort is provided for use of the software Manual for QTLMapper Version 1 0 System Requirements Hardware PC 486 or higher 16MB RAM or more at least 2 MB free space on hard disk Operation System Microsoft Windows 95 98 TM Installing QTLMapper 1 0 All related files for QTLMapper 1 0 were compressed into QTLMapr exe a self extracted file To install the software 1 Run QTLMapr exe under Windows Explorer the file manager of Windows 95 98 then a WinZip Self Extractor dialogue box will appear 2 In the Unzip to edit box input the name of the hard disk e g C on which the software is going to be installed then click on Unzip button All the files for QTLMapper 1 0 will
28. ackknife File is the file with jkq or jke extension from Jackknife test After you have selected a file type an input box will appear for you to input the file name of that type Subsequently you will be asked to open the map file and data file through input boxes The operations are similar to those for Jackknife test After the process of calculating relative contributions a result file with the same stem as the original result file and an extension of ctq for putative QTLs or cte for putative epistatic QTLs will be created 5 Make LR Graphs 2D This item in Output sub menu is designed to create some files containing two dimensional coordinates testing points on each chromosome as x axis the likelihood ratio LR value as y axis It is based only on the original result file from mapping additive effect QTLs These graph files can be used by Wgnuplot a free Windows application Copyright C 1986 1993 Thomas Williams Colin Kelley for showing and printing LR graphs This can make the presentation of your mapping results more attractive To make LR graphs press number 5 in Output sub menu An input box Figure 37 will appear for you to input the original result file from mapping additive effect QTLs Open Result File Filename _ Enter gt Ok Esc gt Cancel 8 Manual for QTLMapper Version 1 0 Figure 37 Input box for opening original result file The graph files will be created in a moment The
29. cided press Esc for next step A decision box Figure 30 will appear and let you decide to continue or quit the process of marker detection Decision Now begin to run Enter gt Ok Esc gt Cancel Figure 30 Decision box for continuing or quit marker detection Manual for QTLMapper Version 1 0 Press Enter key to continue and a progress bar similar to Figure 23 and some information will also be displayed Press Esc key to quit the process After the marker detection one detecting main effect markers only or two files detecting both main and interaction markers will be created The user cannot change the file names They will always be the stem name of the data file plus an extension of one for main effect markers only or two for interaction markers only For example if you have a data file named rice txt then the result file for detected maineffect markers will be rice one and the result file for detected interaction markers will be rice two Working with Output Sub Menu Output sub menu is designed for processing the original result file from mapping QTLs with additive epistatic effect so that the manual work for presentation with the original result file can be largely reduced In addition Output sub menu can also be used for obtaining the results of hypothesis test using some special methods To open Output sub menu Figure 31 press number 3 in Main Menu Output Filtrate Jackknife Test Ba
30. column are the default codes for the five marker types To change the setting further press letter A B C D or E Each marker type can be coded with only one unique letter case sensitive or number These settings will be disabled if you define your own marker coding scheme in you data file see later section But it will also be effective when you use the function Save As in File sub menu to save your current input files in a different format Note that the default setting for missing marker data cannot be changed and should always be a dot 5 Map Function The user needs to indicate the map function that was used for creating the marker linkage map so that QTLMapper 1 0 can conduct correct transformation between genetic distances and recombination fractions The user can specify it in the map file or use the default setting shown in Map Function dialogue box Figure 8 To open the dialogue box press number 5 under Settings sub menu to change the default setting further press letter A or B Map Function A Haldane Function B Kosambi Function A gt B gt Select Esc gt Back Figure 8 Dialogue box for changing default map function The current setting is Haldane Function 6 Probabilities Before doing analyses using Map Additive effect QTLs Map Epistatic QTLs or Marker Detection in Run sub menu appropriate probabilities for significance tests need to be decided
31. ct epistatic QTLs QTLMapper 1 0 provides the user with a function for filtrating putative additive effect QTLs or epistatic QTLs from the original mapping results obtained through the above steps The filtration subroutine will search for the peak points of LR values along each chromosome and compare them with the threshold value determined by the threshold probability setting If the LR value at a peak is above the threshold the testing point with this LR value will be kept and saved in a file with the same stem name as the original result file and extension of flq for additive effect QTLs or fle for epistatic QTLs A decision box Figure 24 will appear after the process of mapping additive effect QTLs has been completed The user can do the filtration at this step or use Output sub menu to do it later Decision Now filtrate putative QTLs epistasis Enter gt Ok 21 Esc gt Cancel Manual for QTLMapper Version 1 0 Figure 24 Decision box for filtrating putative QTLs epistatis Step 8 Deciding whether to conduct Bayesian test for the putative main effect epistatic QTLs If the data set includes an environmental factor a dialog box Figure 25 will appear at this step otherwise this step will be automatically skipped Decision Now run Bayesian test Enter gt Ok Esc gt Cancel Figure 25 Decision box for running Bayesian test Bayesian test is intended to test both fixed genetic main effects QTL additive
32. d With BC population it will be H 2 S H X AE is the general contributions from all the additive by environment interaction effects 1f multiple environments are involved File with cte extension List 16 is an example of cte file that is created for multiple environments The items included in the part of description of conditions are the same as those in ctq files Ses Manual for QTLMapper Version 1 0 List 16 An example of cte file simu cte Result file created by QTLMapper V 1 0 Data file name DAQTLMAPER simu txt Marker map file name D QTLMAPER simu map Environments yes Replications no Contents relative contributions H 2 for putative additive effect QTLs epistatic QTLs Calculations based on D QTLMAPER simu bye BGV control method A control marker main amp interaction effects Date 1999 03 11 Time 16 54 05 Trait 1 simTt Ch Ini Int Namei Sitei M Ch Inj Int Namej Sitej M H 2X Ai H XAj H XAAij H XAEi H XAEj H XAAEij 1 2 MK2 MK3 0 00 2 6 MK24 MK25 0 08 0 0026 0 0087 0 0060 0 0000 0 0499 0 0791 1 2 MK2 MK3 0 00 2 14 MK32 MK33 0 02 0 0027 0 0666 0 0066 0 0000 0 0003 0 0223 1 2 MK2 MK3 0 02 3 8 MK41 MK42 0 10 0 0034 0 0000 0 0000 0 0000 0 0978 0 0084 2 6 MK24 MK25 0 06 3 8 MK41 MK42 0 10 0 0082 0 0000 0 0128 0 0469 0 1022 0 0124 2 7 MK25 MK26 0 00 2 14 MK32 MK33 0 02 0 0056 0 0706 0 0033 0 0472 0 0001 0 0160 2 14 MK32 MK33 0 02 3 0 MK42 MK43 0 02 0 0661 0 0000
33. d by a specific genetic source in the total phenotypic variance So actually it could be taken as heritability contributed by that genetic source That is why we use H 2 to stand for relative contributions The calculation of relative contributions is based on the results of putative QTLs epistatic QTLs given in flx jkx or byx files x is q or e File with ctq extension List 15 is an example of ctq file that is created for multiple environments List 15 An example of ctq file simu ctq Result file created by QTLMapper V 1 0 Data file name DAQTLMAPER simu txt Marker map file name DAQTLMAPER simu map Environments yes Replications no Contents relative contributions H 2 for putative additive effect QTLs epistatic QTLs Calculations based on DAQTLMAPER simu byq BGY control method A control marker main amp interaction effects Date 1999 03 11 Time 16 47 34 Trait 1 simTt Chrom Interval Site M H 2 Ai H 2 AEi 1 MK2 MK3 0 00 0 0130 0 0000 2 MK25 MK26 0 00 0 0208 0 2277 2 MK32 MK33 0 04 0 2877 0 0000 General contributions Additive A H 2 A 20 3015 QE interactions H 2 AE 0 2277 The description of conditions includes the following items Manual for QTLMapper Version 1 0 Names of the map file and data file Experimental design for environments and replications Brief content of the file Name of the file on which the calculation is based Three types of files are possib
34. e environment List 5 An example of qtl file for a single environment dh qtl Result file created by QTLMapper V 1 0 Data file name DAQTLMAPERMh txt Marker map file name DAQTLMAPER dh map Environments no Replications no Contents putative additive effect QTLs BGY control method A control marker main amp interaction effects Results saved for points significant at 0 005000 Date 1999 02 03 Time 12 28 36 Trait 1 ju28ph Chrom Interval Site1 M Site2 M LR LOD A Prob 1 Mk17 Mk18 2 167 0 000 13 06 2 83 1 9994 0 0004 1 Mk17 Mk18 2 187 0 020 14 80 3 21 2 1550 0 0001 1 Mk17 Mk18 2 207 0 040 16 16 3 51 2 2455 0 0001 1 Mk17 Mk18 2 227 0 060 16 99 3 69 2 2630 0 0000 1 Mk17 Mk18 2 247 0 080 17 27 3 75 2 2134 0 0000 2 Mk31 Mk32 1 599 0 000 10 16 2 21 1 7219 0 0016 End Manual for QTLMapper Version 1 0 The part of description of conditions is put in the front of the file It includes the names and paths of the map file and the data file experimental design for environments and replications yes included no not included brief contents of the file BGV control method decided through Settings sub menu and method for saving the results that is decided in Step 3 Deciding how to save mapping results of the mapping analysis The layout of the result body is also trait based like in the one or two files For each trait a string e g Trait 1 is given to indicate the trait order number
35. e in the map file each item in general description is a key character string followed by certain specification s Each key string must be started with an ec 99 underline and no white space is allowed within it There are eight possible items for general description They can be arranged in any order A typical general description for data file looks like Population DH Genotypes 200 Observations 400 _Environments yes _Replications no _ TraitNumber 1 TotalMarker 64 Manual for QTLMapper Version 1 0 _MarkerCode P1 1 P2 2 F1 3 F1P1 4 F1P2 5 Population DH specifies the population type used This is a necessary item Specification DH case insensitive is for doubled haploid population derived from F between two inbred lines Other three specifications are also acceptable to QTLMapper 1 0 including RI Recombinant inbred line population derived from a line cross BC Backcross population derived from crossing F with one of the inbred parents F2 F population derived from selfing or sib mating F that is made from two inbred lines Genotypes 200 specifies the total number of individuals or genotypes sampled from the mapping population This is also a necessary item Observations 400 specifies the total number of observations for each trait under study If your experimental design does not include replications and environments the value is the same as the total number of genotypes In this case this item can
36. e saved in a file with the same stem as that for the original result file and extension of ctq for additive effect QTLs or cte for epistatic QTLs in the work directory 3 Map Epistatic QTLs This item in Run sub menu is designed for mapping both additive effect and digenic epistatic QTLs The statistical methodology used is also the mixed model approach proposed Zhu 1998 Zhu and Weir 1998 Wang Zhu Li and Paterson 1998 To map epistatic QTLs press number 3 in Run sub menu The subsequent operations are quite similar to those for mapping additive effect QTLs with the the following differences Difference 1 In step 1 Deciding a name for result file the default file name here is the stem name of the data file plus an extension of eps instead of qtl Difference 2 In step 4 Deciding a hypothesis for LR test if you select hypothesis C testing GE Interactions Only another dialog box Figure 27 will appear in which you can test separately Item B A x E or S x E for BC population interactions Gig 0 or S y 0 Item C A x E or S x E for BC population interactions Ho o p o o p 05 J J Item D AA x E or SS x E for BC population interactions Ho o E7 0 or ij 2 o SET 0 You can also test General GE Interactions Ho co 26 AE DO A E D AAE 0 by selecting item A GE Interaction Test for General GE Interactions Ai x E Interactions Only No A
37. effects and or epistatic effects and random QTL x environment QE interaction effects see later section for details for the putative QTLs filtrated in the previous step You can do Bayesian test at this step If you do not take Bayesian test at this step you will not have the predicted values for the QE interaction effects since the QE interaction effects are also predicted in this Bayesian test step The results from Bayesian test will be saved in a file with the same stem as that for the original result file and extension of byq for additive effect QTLs or bye for epistatic QTLs in the work directory Step 9 Deciding whether to calculating relative contributions for putative main effect epistatic QTLs Relative contributions of QTLs and QE interaction effects to the phenotypic variation are important criteria by which the importance of a QTL or QE interaction effect can be judged In this step the user can estimate the relative contributions for all the putative QTLs and or QE interactions based on the filtration results if Bayesian test has not been done or Bayesian test results if Bayesian test has been done in the previous steps A decision box Figure 26 will appear for you to decide Decision Now calculate relative contributions Enter gt Ok 22 Esc gt Cancel Manual for QTLMapper Version 1 0 Figure 26 Decision box for calculating relative contributions The results for the relative contributions will b
38. ents of these result files In general every result file consists of two portions description of conditions on which the result is obtained and the result body In these files there is usually a word End that ends the files In this section the contents of all types of result files will be described in detail X Warning Do not edit the part of description of conditions Some information given in this part will usually be read in as conditions for further treatme nts by Run or Output sub menu If this part in a result file has been modified particularly with BGV control method Threshold probability etc changed inappropriate results could be created in the files derived from it 1 Files from marker detection one and two files As we mentioned earlier controlling background genetic variation BGV is an important feature of QTLMapper 1 0 The selected main effect and or interaction markers are used in the mixed model for BGV control Whether you use stepwise regression or ANOVA two types of 29 Manual for QTLMapper Version 1 0 result files will be created The file with one extension contains the results for selected main effect markers and the file with two extension has the results for selected interaction markers The contents of the two types of files are described in this section File with one extension List 1 gives an example of one file obtained for a data set without environmental factor in the phenotyping exp
39. environmental factor is involved At the bottom of each two way table is General contributions The value for H 2 A is the general contribution from all the additive effects of the identified putative QTLs The H 2 AA value is the general contribution from all the epistatic effects of the identified putative QTLs H X AE and H 2 AAE provide the general contributions from all the additive by environment interactions and all the epistasis by environment interactions respectively if an environmental factor is involved For a BC population likewise the letter A in the above expressions will be changed to S 7 LR Graph 2D files plt sig 001 files etc These files are created from the original qtl file They are only used for displaying or printing two dimensional graphs of LRs against testing positions in Morgan for the results from mapping additive effect QTLs The user usually does not need to check these files for details In the section of Working with Output Sub Menu we have briefly described the contents of these files so we here will talk about how to use these files to display your main effect QTL mapping results 54 Manual for QTLMapper Version 1 0 You need to install the Wgnuplot application a free software for Windows in your computer before you view the LR graphs Here we copy the statement of the copyright shown in the help of the software Copyright C 1986 1993 Thomas Williams Colin Kelley
40. eriment The description of the conditions is placed in the front of the file It includes the names and paths of data file and map file experimental design for environments and replications yes included no not included brief contents of the file method of the analysis used and threshold probabilities for the marker selection The date and time when the file was created are also provided in this part The result body includes the results of selected main effect markers for all the traits that are inputted in the data file For each trait the output of the results include trait name following a string e g Traitl Trait2 Trait3 etc indicating its order in the data file total number of selected markers and general R 2 multiple determination coefficient of all the selected markers Note that there will be no general R 2 if you used ANOVA method Then there is a two way table showing results for the selected main effect markers List 1 Anexample of one file for a single environment dh one Result file created by QTLMapper V 1 0 Data file name DAQTLMAPER dh txt Marker map file name DAQTLMAPER dh map Environments no Replications no Contents selected main effect markers Analysis method Stepwise Regression Inclusion probability 0 010000 Exclusion probability 0 010000 Date 1999 02 01 Time 15 46 56 Trait1 ju28ph Total selected markers 6 General R 2 0 4989 Markers Name F va
41. ge Press PgDn or PgUp to turn to next or previous page press Esc to quit the display The statistics for data of each trait include the frequency distribution indicated with asterisks data automatically divided into ten groups mean standard deviation SD skewness and kurtosis 2 Map Additive effect QTLs This item in Run sub menu is designed for mapping additive effect QTLs assuming no epistasis between QTLs The statistical methodology used is the mixed model approaches Zhu 1998 Zhu and Weir 1998 Wang Zhu Li and Paterson 1999 To map additive effect QTLs press number 2 in Run sub menu Then following operations need to be carried out step by step through various dialog boxes Step 1 Setting a name for result file A dialogue box will appear and allow you to change the default name of the result file we call it original result file for saving the mapping results If you do not want to change it just press Esc Otherwise press Enter and type a new file name in another dialogue box Step 2 Setting mapping ranges After result file name has been decided a dialogue box will pop up for you to set mapping ranges trait range and genomic range Figure 15 In this step you have a chance to choose a subset of the traits a subset of chromosomes and certain marker intervals for detailed analysis instead of all the traits and the entire genome Setting Mapping Ranges A Trait Range B Genomic Range
42. iltrated results because some filtrated testing points may not be putative QTLs The constitutions of filtration files are the same for data sets with or without an environmental factor File with flq extension List 9 is an example of flq file for additive effect QTLs List 9 An example of flq file simu flq Result file created by QTLMapper V1 0 Contents filtrated putative additive effect QTLs Original result file DAQTLMAPER simu qtl BGV control method A control main amp interaction markers Threshold probability 0 0050 LR Threshold value 7 8798 3t Date 1999 03 09 Time 10 39 05 Trait 1 simTt Chrom Interval Sitel M Site2 M LR LOD A Prob 1 MK2 MK3 0 098 0 000 18 26 3 96 0 7869 0 0017 2 MK25 MK26 0 594 0 000 150 78 32 72 0 9942 0 6711 2 MK32 MK33 1 262 0 040 247 09 53 62 3 6980 0 0000 End In this file the description of conditions includes brief contents of the file name of the original result file BGV control method threshold probability used in the filtration and corresponding LR critical value The result body is just a subset of that in the original qtl file In a flq file only testing points with local maximum LR values are retained For detailed description please see the previous section about the File with qtl extension Manual for QTLMapper Version 1 0 File with fle extension List 10 provides an example of fle file The items included in the part of descript
43. ion 1 0 Find the file with plt extension and double click on it Then a LR graph will appear in another window for the first trait and first chromosome To show next graph press Enter or click on the OK button in a gnuplot pause dialog box The following graph is presented as an example rT lajxj hood Rato Map fer rat a2 Eph end chromosome Figure 40 An example of LR graph Each LR graph has a title ike Likelihood Ratio Map for trait ju28ph and chromosome 3 The string ju28ph is the name of the trait while the number 3 is the order of the chromosome The x axis is the positions in Morgan of the testing points from the left end of the chromosome The y axis is LR values for each testing point The horizontal line is the threshold line whose height is decided by the threshold probability specified in Settings sub menu The curve line is the LR line showing the relation between the testing points and the LR values The peak points above the threshold line could be taken as putative additive effect QTLs Getting On line Help The sub menu Help in Main Menu is designed to provide on line help The user can get or line help by pressing number 5 in Main Menu and then 1 in Help sub menu Figure 38 The program will load a text file named Qtlmaper hlp and display it By pressing PgDn PgUp Home or End keys one can view next previous first or last pages To view a specific page press letter P and then page nu
44. ion of conditions are exactly the same as in flq file The result body is a subset of that in the original eps file containing the testing points with local maximum LR values For detailed contents of the result body please refer to the previous section on File with eps extension 4 Jackknife files jkq and jke files These files are created from Jackknife test for genetic main effects and QE interaction effects when multiple environments are involved These files provide both estimates of genetic main effects and predicted values of QE interaction effects only for the testing points presented as putative QTLs in filtration files File with jkq extension This type of file is created from Jackknife test for additive effect QTLs List 11 is an example of jkq file for multiple environments In the description of conditions the following items are included Names of the map file and data file Experimental designs for environments and replications Brief contents of the jkq file Name of the filtration file on which the jkq file is based BGV control method used for mapping analysis for creating original result file Threshold probability used for filtration of putative QTLs in the filtration file Like in the previous sections the layout of the result body is also trait based For each trait involved trait order number in such string as Trait 1 and trait name are given first followed by a two way table that shows the results from
45. ions 5 Bayesian files byq and bye files These files are created from Bayesian test for genetic main effects and QE interaction effects usually in the presence of multiple environments The compositions of these files are quite similar to those of Jackknife files Only the brief contents indicated in the description of conditions is different Here it is Contents Bayesian test results for while in Jackknife files it is Contents Jackknife test results for List 13 and List 14 are examples of byq and bye files respectively List 13 An example of byq file simu byq Result file created by QTLMapper V 1 0 Data file name D AQTLMAPER simu txt Marker map file name D QTLMAPER simu map Environments yes Replications no Contents Bayesian test results for additive effect QTLs Bayesian test based on D QTLMAPER simu qtl BGY control method A control marker main amp interaction effects Threshold probability 0 005000 Date 1999 03 08 Time 10 41 54 Trait 1 simTt Chrm Interval Site M Ai Prob AEil Prob AED 1 MK2 MK3 0 00 0 787 0 000 0 001 0 994 0 001 2 MK25 MK26 0 00 0 994 0 000 2 326 0 000 2 326 2 MK32 MK33 0 04 3 698 0 000 0 105 0 549 0 105 End be Manual for QTLMapper Version 1 0 List 14 An example of bye file simu bye Result file created by QTLMapper V 1 0 Data file name D QTLMAPER simu txt Marker map file name D QTLMAPER simu map
46. j x E Interactions Only No oom AAij x E Interactions Only A gt D gt Select Esc gt Next Figure 27 Dialog box for deciding a hypothesis on QE interactions ads Manual for QTLMapper Version 1 0 Difference 3 Mapping epistatic QTLs with F data is not yet available in QTLMapper 1 0 whether the data set is collected from one environment or from multiple environments 4 Marker Detection This item in Run sub menu Figure 13 is designed for selecting important main effect and or interaction markers that can be used in two ways controlling BGV and automatically determining genomic ranges in mapping main effect epistatic QTLs To do marker detection press number 4 in Run sub menu A dialogue box Figure 28 will appear to prompt you to select a method from the two alternatives stepwise regression and analysis of variance Method Choice A Stepwise Regression B Analysis of Variance A gt B gt Select Esc gt Next Figure 28 Dialog box for choosing a method of marker detection After the method selected press Esc for next step Then another dialog box Figure 29 will appear Detection for A Main Effect Markers B Main amp Interaction Markers A gt B gt Select Esc gt Next Figure 29 Dialog box for detecting important markers In this dialog box you can decide to detect main effect markers item A only or both main and interaction markers item B After you have de
47. le filtration file Jackknife file or Bayesian file BGV control method used for creating the original result file The result body is also trait based Trait order number and trait name are given first Then there is a two way table showing the relative contributions of the putative additive effect QTLs and the contributions of QE interactions if multiple environments are involved The Chrom column shows the order number of the chromosomes on which the putative QTLs are located The Interval column gives the names of the flanking markers constructing the intervals in which the putative QTL are detected The Site M column provides the genetic distances in Morgan of the putative QTLs from the left markers on the intervals The H 2 Ai column is the relative contributions from the additive effects of the putative QTLs If the mapping population is BC the title will be H 2 Si and the relative contributions are from the differences between the additive effects and dominance effects for the putative QTLs The H 2 AEi column is the relative contributions from the interaction effects between the additive effect at putative QTL i and the environments With a BC population it will be H 2 SEi It is noted that there will be no such column if no environments are involved in the experiments At the bottom of each two way table are the General contributions H 2 A is the general contribution from the additive effects of all the putative QTLs involve
48. le with jke extension This type of file is created from Jackknife test for epistatic QTLs List 12 is an example of jke file for multiple environments The items included in description of conditions are the same as those in jkq files In the result body of jke file the part represented by is the columns for Ai Probi Aj Probj AAij and Probij for each trait The meanings of these columns are the same as those for eps files In the presence of multiple environments more columns for the results of QE interaction effects will be outputted after the part In the example AEix columns and Prob columns immediately after them are the predicted interaction effects between QTL involved in the epistasis and x th environment The Prob columns provide significance tests for these effects Manual for QTLMapper Version 1 0 List 10 An example of fle file simu fle Result file created by QTLMapper V1 0 Contents filtrated putative epistatic QTLs Original result file DAQTLMAPER simu eps BGVW control method A control main amp interaction markers Threshold probability 0 0050 LR Threshold value 7 8798 Date 1999 03 09 Time 17 13 35 Trait 1 simTt Ch Ini Int Namei Sitei M Ch Inj Int Namej Sitej M LR LOD Ai Probi Aj Probj AAij Probij 1 2 MK2 MK3 0 00 2 6 MK24 MK25 0 08 276 66 60 04 0 718 0 002 1 314 0 560 1 092 0 699 1 2 MK2 MK3 0 00 2 14 MK32 MK33 0 02 303 28 65 81 0 740 0 00
49. lue Prob R 2 171 Mk171 16 5499 0 0002 0 0873 54 Mk54 20 9835 0 0000 0 1107 18 Mk18 12 0779 0 0013 0 0637 67 Mk67 21 4424 0 0000 0 1131 32 Mk32 11 1240 0 0019 0 0587 124 Mk124 8 6798 0 0054 0 0458 Manual for QTLMapper Version 1 0 The Marker column shows the order numbers of selected markers The order numbers are decided according to the order of the markers arranged in the data file The order number for the first marker is 1 the second is 2 The Name column shows the names of the selected markers Note that if the name of a selected marker is longer than eight characters in the data file the truncated version of the name will be given in this one file The F value column provides the F values for testing partial regression coefficients of the selected main effect markers The Prob column gives the probabilities for the F values The R 2 column shows the partial determination coefficients of the selected main effect markers each calculated with all the other selected markers in the regression model There will be no such column if you use ANOVA for the marker detection If the data set includes an environmental factor the presentation of the results for selected main effect markers is slightly different from the above in the result body List 2 is a one file obtained for a data set with two environments List 2 An example of one file for multiple environments simu one Result file created by QTLMapper V 1 0
50. lumns gives the F test results for the selected interaction markers which is similar to that in one file The R 2 column if using stepwise regression method presents the partial determination coefficients for the selected interaction markers with the control over all the selected main effect markers in the corresponding one file and all the other selected pairs of interaction markers If ANOVA method was used there will be no such column If the data set includes an environmental factor several environmental levels the presentation of the results for selected interaction markers is slightly different from the above in the result body List 4 is a two file obtained for a data set from two environments The subset of the data for each of the environments will be analyzed separately and the results for selected interaction markers are presented separately A string like TI LEI is used for indicating the trait and the environment level The integer number that follows the string is the code of the environment level as appeared for the subscript in the data file List 4 An example of two file for multiple environments simu two Result file created by QTLMapper V 1 0 Data file name DAQTLMAPER simu txt Marker map file name DAQTLMAPER simu map Environments yes Replications no Contents selected interaction markers Analysis method Stepwise Regression Inclusion probability 0 005000 Exclusion p
51. mber To quit help press Esc key Manual for QTLMapper Version 1 0 The About item in the sub menu is for showing the About information of the software Help 1 Contents 2 About 1 gt 2 gt Select Esc gt Back Figure 41 Help sub menu Error Messages and Troubleshooting If you have any questions with our software please contact Jun Zhu Department of Agronomy Huajiachi Campus Zhejiang University Hangzhou Zhejiang 310029 China Email jzhu zjau edu cn References Zhu J 1998 Mixed model approaches of mapping genes for complex quantitative traits In Wang LZ and Dai JR eds Proceedings of Genetics and Crop Breeding of China Chinese Agricultural Science and Technology Publication House Beijing pp19 20 Zhu J and B S Weir 1998 Mixed model approaches for genetic analysis of quantitative traits In Chen LS Ruan SG and Zhu J eds Advanced Topics in Biomathematics Proceedings of International Conference on Mathematical Biology World Scientific Publishing Co Singapore pp321 330 Wang D L J Zhu Z K Li and A H Paterson 1999 Mapping QTLs with epistatic effects and QTL x environment interactions by mixed linear model approaches Theor Appl Genet in printing ses
52. n input filenames for the map file and or data file through the input boxes appearing subsequently The new map and or data file s will be saved in the work directory 3 File Summary This item in File sub menu is used for displaying the basic information of the opened map file and data file For map file file name total number of chromosomes total number of markers map function used etc will be displayed for data file file name population type total number of genotypes total number of observations total number of traits experimental design etc will be shown Checking this information the user can learn whether his her input files are opened correctly To display file summary information press number 3 in File sub menu 4 Close Files Close Files item in File sub menu is designed to close current input files and free the allocated es Manual for QTLMapper Version 1 0 memory After closing current files the user can load another set of map and data files to do QTL mapping without exiting the program To close files press number 4 in File sub menu Working with Run Sub Menu Run sub menu implements all the operations related to mapping QTLs To open Run sub menu Figure 13 press number 2 in Main Menu If current status is not in Main Menu press Esc one or more times to return to Main Menu Run Display Basic Statistics Map Main effect QTLs Map Epistatic QTLs Marker Detection 1 gt 4 g
53. ned for the same purpose as that of Jackknife test But Bayesian test is much quicker than Jackknife test The results from the two methods are rather similar To run Bayesian test press number 3 in Output sub menu The method of following operations is the same as for Jackknife test After the process of Bayesian test a result file with the same stem as the original result file and an extension of byq for additive effect QTLs or bye for epistatic QTLs will be created 4 Contributions This item in Output sub menu is designed for obtaining the relative contributions of QTLs and or QE interactions Relative contributions of QTLs to the phenotypic variation are important sI Manual for QTLMapper Version 1 0 criteria of judging a specific additive effect QTL epistatic QTLs or QE interaction To obtain relative contributions press number 4 in Output sub menu Then a dialog box Figure 36 will appear and ask you to select a file type on which the calculation of relative contribution will be based Calculation Based on A Filtration File B Bayesian File C Jackknife File A gt C gt Select Esc gt Cancel Figure 36 Dialog box for selecting a file type for calculating relative contributions Type A Filtration File is the file with flq or fle from filtrating putative additive effect or putative QTLs type B Bayesian File is the file with byq or bye extension from Bayesian test type C J
54. ng steps assuming that the folder containing QTLMapper 1 0 is C QTLMaper C Windows gt CD C QTLMaper C QTLMaper gt QTLMaper Running QTLMapper 1 0 under full screen mode will provide a better user interface File conagent pif is given for this purpose But sometimes conagent pif may cause problem In this case it needs to be renamed or deleted If the software is running under full screen mode to switch to other Windows applications press Ctrl Esc Method of Exiting To exit QTLMapper 1 0 press Esc key one or more times depending on the running status of the software However it cannot be exited in this way when it is in the process of performing analyses for QTL mapping To terminate the process and exit the software in this case press Ctrl C Menu System of QTLMapper 1 0 QTLMapper 1 0 runs on a menu system instead of DOS command style Each operation with QTLMapper 1 0 is a matter of hitting a number key or a letter key corresponding to a specific menu item Many dialogue boxes are provided for guiding users through operations The menu system of QTLMapper 1 0 consists of a main menu with five sub menu items Figure 1 The detailed usage of the menu system will be described in the following sections Main Menu fa TO MM 1 File eee Run File DI ee Output Open Files i Settings Save As i File Summary Close Files 1 gt 5 gt Select Esc gt Exit 1 gt 4 gt Select Esc
55. ody the input of marker data is genotype based That is the marker data at all marker loci for an individual genotype must be inputted before inputting marker data for next genotype The order of the marker data for different marker loci must be consistent with the order of markers on each chromosome determined in the map file Below is an example for marker data body MarkerBegin Genof Mkl1l Mk18 Mk19 Mk33 Mk34 Mk48 Mk49 Mk64 1 2 e 2 1 ve 1 1 wc 2 2 las 1 2 2 ii 2 2 m 2 1 s d 2 ds 1 200 2 2 E os d i o 2 i ows X 3 MarkerEnd The second row Geno Mk1 includes strings for genotype and marker names The marker names must be arranged in the order given in the map file In this example Mk1 Mkz2 Mk18 are located on Chromosome I Mk19 Mk20 Mk33 are located on Chromosome II Mk34 Mk35 Mk48 are located on Chromosome III Mk49 MKk50 Mk64 are located on Chromosome IV At the end of the row is a semicolon with at least one white space away from the last marker name Mk64 Like in the previous sections the strings for genotype and marker names should not contain any white space inside The marker names will be read to memory and used for outputting results later It is noted that it will be better to limit the length of each marker name within eight characters as 2 Manual for QTLMapper Version 1 0 QTLMapper 1 0 will automatically truncate the name longer than eight character
56. ontrol marker main amp interaction effects Results saved for points significant at 0 005000 LR test for hypothesis on both genetic effects and GE interactions 3t Date 1999 03 12 Time 12 15 39 Trait 1 simTt Ch Ini Int Namei Sitei M Ch Inj Int Namej Sitej M LR LOD Ai Probi Aj Probj AAij Probij 1 1 MKI MK2 0 00 1 10 MK10 MK11 0 00 18 94 4 11 0 689 0 0051 0 045 0 8588 0 2333 0 705 1 1 MK1 MK2 0 00 1 10 MK10 MK11 0 02 21 10 4 58 0 690 0 0045 0 0432 0 8597 0 2697 0 6966 1 1 MK1 MK2 0 00 1 10 MK10 MK11 0 04 23 50 5 10 0 690 0 0040 0 0414 0 8435 0 3022 0 6881 1 1 MK1 MK2 0 00 1 10 MK10 MK11 0 06 24 81 5 38 0 689 0 0036 0 0396 0 8605 0 3263 0 6799 1 1 MKI MK2 0 00 1 10 MK10 MK11 0 08 25 47 5 53 0 688 0 0035 0 0383 0 8888 0 3393 0 6716 1 1 MKI MK2 0 02 1 10 MK10 MK11 0 00 20 87 4 53 0 745 0 0033 0 0268 0 9156 0 2346 0 7134 1 1 MKI MK2 0 02 1 10 MK10 MK11 0 02 23 04 5 00 0 744 0 0030 0 0248 0 9192 0 2705 0 7052 1 1 MK1 MK2 0 02 1 10 MK10 MK11 0 04 25 43 5 52 0 744 0 0027 0 0234 0 9123 0 3023 0 6970 End 39 Manual for QTLMapper Version 1 0 3 Filtration files 7g and le files These files are created for simplifying the manual work of picking out putative additive effect QTLs or epistatic QTLs from the original result files qtl and eps The results for the testing points with the local maximum LR values along chromosomes are filtrated and saved in these files It is strongly suggested that the user check the f
57. oose from the three alternatives A C Genomic Range Setting A Automatically Yes B Whole Genome No C Manually A gt C gt Select Esc gt Back Figure 17 Dialogue box for changing genomic range Yes marks the current setting Automatically If you want to search QTLs on entire genome select item B Pressing letter C will bring out an input box Figure 18 for you to setting chromosome range Chromosome Setting Al _ Enter gt Ok Esc gt Cancel 18 Manual for QTLMapper Version 1 0 Figure 18 Input box for setting chromosome range In the input box input the order number based on the order of the chromosomes in map file and press Enter key The method of inputting chromosome order number is the same as that for inputting trait order numbers in the previous passage After chromosome setting has been done another dialogue box will appear for you to set marker intervals on each of the selected chromosomes for mapping analysis You can do this in a similar way When you have finished the setting for mapping ranges you will have to return the dialogue box Figure 15 by pressing Esc Further pressing Esc the operation will go to next step Step 3 Choosing a method for saving mapping results A new dialogue box will appear Figure 19 Through this dialogue box the user can decide how to save the mapping results How to Save Results A By Threshold B F
58. or All Testing Points A gt B gt Select Esc gt Next Figure 19 Dialogue box for selecting a way of saving mapping results Item A saving results by threshold is a default setting In this way only the results for the testing points that are likely to be putative QTLs based on the threshold probability specified in Settings sub menu will be saved This can effectively reduce the size of the result file If you want to view the results for all the testing points particularly to make graphs from the mapping results you will need to select item B Yes marks the current setting After this is done pressing Esc the operation will move to next step Step 4 Deciding a hypothesis for LR test This step happens only when your trait data include an environmental factor If no environmental factor was involved in your phenotyping experiment the program will skip to the next step In addition QTLMapper 1 0 cannot analyze F data set from multiple environments In this step a dialog box will appear for you to choose a hypothesis from three alternatives Figure 20 LR Test for Genetic Main Effects amp GE Interactions Yes Genetic Main Effects Only No GE Interactions Only No 9 A gt C gt Select Esc gt Next Manual for QTLMapper Version 1 0 Figure 20 Dialog box for deciding a hypothesis for LR test The default hypothesis is for testing Genetic Main Effects amp GE Interactions For DH or
59. p body A typical general description looks like _DistanceUnit cM _MapFunction K Chromosomes 6 2s Manual for QTLMapper Version 1 0 MarkerNumbers 21 20 19 15 6 18 There are a total of four possible items for general description They can be arranged in any order Each item in general description is a key character string or key string followed by certain ec 99 specification s Each key string must be started with an underline and there should not be any white spaces within the key string The specification s must be separated from the key word by at least one white space and there should also be at least one white space between any two neighboring specifications if two or more specifications are included for the item Key strings and specifications if characters are case insensitive DistanceUnit cM specifies the unit of genetic distances used in the map file The specification string cM stands for centi Morgan Another valid character M is for Morgan If the unit is cM this item can be omitted and QTLMapper 1 0 will use its default distance unit cM in reading the map file MapFunction K indicates the map function used in creating the marker linkage map for turning recombination fractions into genetic distances QTLMapper 1 0 needs it for correctly transferring genetic distances into recombination fractions in QTL mapping analysis Specification character K is for Kosambi function Another valid character is H fo
60. r Haldane function This item can also be omitted if you use the current setting specified for map function in Settings sub menu Chromosomes 4 is for specifying the total number of chromosomes or linkage groups involved in the map file This is an essential item and cannot be omitted MarkerNumbers 18 15 15 16 is for specifying the numbers of markers on all the chromosomes The order of the numbers must be consistent with that for genetic distance columns in the map body The key string line here indicates 18 markers on chromosome I 15 markers on chromosome II 15 markers on chromosome III and 16 markers on chromosome IV Map Body This part starts from key string MapBegin and ends at key string MapEnd Wrong typing of the key strings will cause failure in opening map file It is convenient to place map body after general description but it can also be placed anywhere in map file A typical map body looks like MapBegin Marker Chl Ch2 Ch3 Ch4 1 0 00 0 00 0 00 0 00 9 84 11 26 7 45 9 85 3 10 22 8 69 9 10 10 93 15 8 25 9 87 10 66 10 70 16 7 47 11 30 17 12 30 18 11 78 MapEnd Os Manual for QTLMapper Version 1 0 The character strings Marker Chl Ch2 Ch3 Ch4 in the second row show the contents of the columns below them QTLMapper 1 0 will skip these strings so any string can be used but they cannot be omitted The Marker column first column is for the order of all markers on each chromosome the maximum o
61. rder is equal to the number of markers on the chromosome that has the most markers among all the chromosomes The Chl column second column to Ch4 column last column each represents a chromosome and contains genetic distances between adjacent markers on the chromosome The genetic distance for the first marker on each chromosome must be set to zero as the start point of the linkage map for the chromosome The distance for the second marker is between the first and the second markers the distance for the third marker is between the second and the third markers and so on The order of Chl column second column to Ch4 last column must be consistent with that for the numbers following the key string MarkerNumbers Comment Lines Comment lines can be placed anywhere except between MapBegin and MapEnd in the map file You can put them even between general description lines or at the end of general description lines see the sample map file Format of data file This file contains information on population type number of genotypes sampled from the population number of observations observations for both markers and quantitative traits etc It is composed of four parts general description marker data body trait data body and some comment lines Comment lines are not a necessary part of data file General description This part is for specifying the basic features of the data file and is usually put in the front of the data file Lik
62. rn to Main Menu File Open Files Save As File Summary Close Files 1 gt 4 gt Select Esc gt Back Figure 11 File sub menu Manual for QTLMapper Version 1 0 The usage of the items in File sub menu is described as follows in details 1 Opening Files Loading marker linkage map file and data file into memory is the first step to do analyses using QTLMapper 1 0 Before opening your input files you will have to make the setting for file format consistent with that applied to your input files see a previous section To open input files press number 1 in File sub menu Two dialogue boxes Figure 12 will appear in turn for you to input the file names of the map file and the data file The input files must be already in the work directory you specified through Settings sub menu One can choose not to open the map file or the data file by pressing Esc to skip the corresponding dialogue box Open Marker Map File Open Data File Filename _ Filename _ Enter gt Ok Esc gt Cancel Enter gt Ok Esc gt Cancel Figure 12 Input boxes for opening map file and data file 2 Save As In File sub menu Save As item is designed for changing file formats between the three formats recognized by QTLMapper 1 0 One can use this function for compare mapping results from different software To do Save As press number 2 in File sub menu and choose one format from the three possible formats the
63. robability 0 005000 3t Date 1999 03 16 Time 21 07 34 Trait1 simTt TILEI 1 Environment code Total selected marker pairs 7 General R 2 0 4763 MarkerA Name MarkerB Name F value Prob R 2 2 MK2 25 MK25 189 3376 0 0000 0 1200 32 MK32 42 MK42 153 9681 0 0000 0 0976 2 MK2 32 MK32 94 2345 0 0000 0 0597 25 MK25 42 MK42 88 5875 0 0000 0 0562 24 MK24 32 MK32 59 4700 0 0000 0 0377 3 MK3 42 MK42 29 2651 0 0000 0 0186 33 Manual for QTLMapper Version 1 0 1 MKI 11 MKII 15 0407 0 0001 0 0095 TI LE2 2 Environment code Total selected marker pairs 4 General R 2 0 2356 MarkerA Name MarkerB Name F value Prob R 2 25 MK25 33 MK33 62 3905 0 0000 0 0785 32 MK32 42 MK42 61 481 0 0000 0 0773 2 MK2 24 MK24 45 0538 0 0000 0 0567 23 MK23 A MK41 14 7214 0 0002 0 0185 2 Original result files qtl and eps files So called original result files as defined previously are created from mapping QTLs with additive effects and epistatic effects The default extensions are qtl for additive effect QTLs and eps for epistatic QTLs But the user can change them at Step 1 Deciding a name for result file in performing mapping analysis Likewise these two types of result files are also composed of two parts description of conditions used for mapping analysis and the result body The following describes the details about the contents of these files File with qtl extension List 5 is an example of qtl file for a data set from a singl
64. s The only difference is the brief contents For qtl file it is putative additive effect QTLs while for eps file it is putative epistatic QTLs Manual for QTLMapper Version 1 0 The result body is also a trait based two way table Trait names and strings for trait order numbers are also provided in the same way as in qtl files But in eps files more columns will be given to show the results for epistatic QTLs The Ch Ini and Ch Inj columns show pairs of marker intervals expressed in order numbers on which the pair wise testing points are set For example 1 9 and 1 13 expressed a pair of marker intervals The 1 9 interval is the 9 interval counted from the left end on the 1 chromosome 1 13 interval is the 13 interval on the 1 chromosome The order numbers of chromosomes are decided in the map file The Int Namei and Int Namej columns give the corresponding names of flanking markers for Ch Ini and Ch Inj The Sitei M and Sitej M columns give the genetic distances in Morgan of pair wise testing points from the left markers of their own intervals For example Sitei 2 0 00 M means that the testing point i is set exactly on the left marker of the interval The LR column shows the likelihood ratios for all the testing points The hypothesis tested is Ho a a aa 0 for DH or RI populations Ho s s ss 0 for BC population Here aj aj Si and s are the same as defined for qtl file while aaj and
65. s and keep it ec 99 within eight characters by adding a plus a number or letter in the end This is for bettering the appearance in output files For example two marker names Markerl 1 and Markerl 21 will be read as Marker 1 and Marker 2 respectively when the data file is loaded by QTLMapper 1 0 In ee 99 addition hyphen should not be used within marker names because QTLMapper 1 0 uses it to express marker intervals The row of marker names not only gives the names of the markers but also designates the order of marker data arranged in each of the following marker data rows Each of the data rows must start with an integer number as the genotype and end with a semicolon at least one white space away from the last marker value between which is the marker data for all marker loci in the order given for the marker names There is no requirement for the seriation of genotype but you must keep the genotype unique for each genotype Electronic table software usually has a limit on the number of columns in spreadsheet For example MS Excel cannot hold data with more than 256 columns If you have more than 256 marker loci you will have to input marker data in a few consecutive rows for each genotype with MS Excel QTLMapper 1 0 has no limit on the number of marker data rows for an individual genotype But do not forget to put a semicolon in the end of the last row This is also the case for the marker name row Trai
66. sis used for the LR test The LR or LOD values are obtained for this hypothesis List 7 An example of eps file for a single environment dh eps Data file name DAQTLMAPERMh txt Marker map file name DAQTLMAPER dh map Environments no Replications no Contents putative epistatic QTLs BGY control method A control marker main amp interaction effects Results saved for points significant at 0 005000 Date 1999 01 29 Time 21 20 34 Trait 1 ju28ph Ch Ini Int Namei Sitei M Ch Inj Int Namej Sitej M LR LOD Ai Probi Aj 1 9 Mk9 Mk10 0 00 1 13 Mk13 Mk14 0 00 20 78 4 51 0 218 0 802 0 225 1 9 Mk9 Mk10 0 00 1 13 Mk13 Mk14 0 02 22 03 4 78 0 254 0 768 0 082 1 9 Mk9 Mk10 0 00 1 13 Mk13 Mk14 0 04 22 63 4 91 0 289 0 736 0 055 1 9 Mk9 Mk10 0 00 1 13 Mk13 Mk14 0 06 22 39 4 86 0 316 0 713 0 158 1 9 Mk9 Mk10 0 00 1 13 Mk13 Mk14 0 08 21 34 4 63 0 329 0 705 0 218 1 9 Mk9 Mk10 0 02 1 13 Mk13 Mk14 0 00 19 81 4 30 0 053 0 955 0 050 1 9 Mk9 Mk10 0 02 1 13 Mk13 Mk14 0 02 21 12 4 58 0 127 0 892 0 150 1 9 Mk9 Mk10 0 02 1 13 Mk13 Mk14 0 04 21 82 4 74 0 207 0 825 0 329 End 38 Manual for QTLMapper Version 1 0 List 8 An example of eps for multiple environments simu eps Result file created by QTLMapper V 1 0 Data file name DAQTLMAPER simu txt Marker map file name D QTLMAPER simu map Environments yes Replications no Contents putative epistatic QTLs BGV control method A c
67. ss are digenic additive x additive epistasis and digenic interaction effects between s and s The LOD column provides the equivalent LOD score values under LOD 0 217 LR The Ai Aj and AAij for DH or RI populations columns list the estimates of additive effect for testing point i additive effect for testing point j and additive by additive effects between the two testing points respectively The signs of Ai and Aj values are determined in the same way as that for qtl files see previous section A positive AAij value implies that the two locus genotypes being the same as those in P1 parent or P2 parent take the positive effects while the two locus genotypes of recombination between the P1 parent and P2 parent take the negative effects The case of negative AAij values is just the opposite If you are using a BC population the three columns will be named as Si Sj and SSij for giving the estimates of s s and ss of the two testing points respectively The Probi Probj and Probij columns provide the t test probabilities for the corresponding estimates of the three genetic effects equ Manual for QTLMapper Version 1 0 List 8 presents an example of eps file for a data set from multiple environments The layout of the mapping results for multiple environments will be basically the same as the above The difference is that there is one more item in description of conditions in the eps file for multiple environments the hypothe
68. strings are names of the traits These names will be read to memory for outputting results later Like the case for marker names trait names should not be more than eight characters Otherwise they will be automatically truncated and a symbol and a number or a letter are added in the ends of the names Below the row of trait names are rows of trait data In these data rows the first one or three integer numbers corresponding to the source strings are the codes of the specific environment replication and or genotype You need to specify a unique integer number for each of the environments as well as for each of the replications but the genotype must be consistent with that for the marker data observed on the same genotype Following each set of the source codes are the observations for all the traits in the order for the names given in the row of trait names In the end of each data row must be a semicolon Trait data for each individual from a specific source can also be inputted in a few consecutive rows to avoid the insufficient capacity of columns provided by some electronic table software Comment lines can be placed anywhere in the data file except within marker data body and trait data body Working with File Sub Menu File sub menu performs operations related to input files To open File sub menu Figure 11 press number 1 in Main Menu If current status is not in Main Menu press Esc one or more times to retu
69. t 1 10b No C Format for Mapmaker QTL 1 1 No A gt C gt Select Esc gt Back Figure 5 Dialogue box for changing file format The current setting is format for QTLMapper 1 0 It is noted that Mapmaker QTL 1 1 does not require a separate map file for mapping QTL So if you choose file format for Mapmaker QTL 1 1 you will be asked to choose a format for your map file from the other two alternatives one for QTLMapper 1 0 the other for QTL Cartographer 1 12c 3 BGV Control Background genetic variation BGV is one of the major factors affecting the precision resolution and power in mapping QTLs A good control of BGV is the key to obtaining high A Manual for QTLMapper Version 1 0 precision resolution and power in mapping QTLs The capability of controlling BGV is an important feature of QTLMapper 1 0 It provides three choices for BGV control A Controlling main and interaction effects of important markers B Controlling main effects of important markers C No BGV control The choice A is recommended and has been taken as the default setting But the user has the freedom of changing it To view or change current setting press number 3 under Settings sub menu Figure 6 The current setting in Figure 6 is Main amp Interaction Markers To change the setting further press letter A B or C BGV Control Main amp Interaction Markers Main Effect Markers No BGV Control A
70. t Select Esc gt Back Figure 13 Run sub menu There are four items in Run sub menu Note that these items become active only after map and data files have been opened 1 Display Basic Statistics This item is for calculating and displaying the basic statistics of the marker data and or trait data The user can use this function to check the segregation of the observed marker loci and the frequency distributions of the traits under study etc For displaying basic statistics press number 1 in Run sub menu and a dialogue box will appear for you to decide what data to display Figure 14 Display A Marker Statistics B Trait Statistics A gt B gt Select Esc gt Cancel Figure 14 Dialogue box for displaying basic statistics To display basic statistics of marker data press letter A in the dialogue box Then statistics of every four markers appear in a page Press PgDn or PgUp to turn to next or previous page press Esc to quit the display The statistics include percentage of different marker phenotypes in the 16 Manual for QTLMapper Version 1 0 sample chi square values and probabilities for testing marker segregation A brief comment is given based on the probabilities Poor for P lt 0 05 Fair for 0 05 lt P lt 0 10 Good for P 0 10 To display basic statistics of trait data press letter B in the dialogue box Then the statistics of all the traits included will be displayed page by pa
71. t data body This part is between two key strings TraitBegin and TraitEnd case insensitive The input of trait data is source based The Source includes the environment if available the replication if available and the genotype from which each set of observations was obtained for all the traits under study The following is an example for trait data body TraitBegin Env Rep Geno TraitNm 1 TraitNm2 TraitNm3 1 1 1 244 7 40 10 04 1 1 2 2 40 4 32 8 55 1 1 200 3 54 8 19 10 74 1 2 1 3 17 6 91 11 86 1 2 2 1 90 4 31 11 36 1 2 200 3 22 10 54 11 48 2 1 1 5 74 12 78 11 27 2 1 2 7 65 7 02 11 96 2 1 200 6 58 13 92 9 94 2 2 1 6 01 10 22 9 95 2 2 2 6 22 11 99 7 81 2 2 200 7 98 13 21 12 03 TraitEnd 3 Manual for QTLMapper Version 1 0 The second row includes the source strings and the names of the traits At the end of the row is also a semicolon The number of source strings depends on the experimental design If both environments and replications are taken a maximum of three strings must be inputted the first string for environment Env the second string for replication Rep and the last string for genotype Geno If only environments or replications were taken two strings must be put because the string for genotype must be included in any case If there is neither environments nor replications taken in the experiment one string for genotype must be provided Following these source
72. y include A plot file with an extension of plt containing commands for plotting 2D LR graphs by Wenuplot A series of coordinate files like ticj 00k containing the coordinates of testing positions and LR values Each chromosome for each trait has one or more such files For example t1c1 001 contains the coordinates for chromosome 1 of trait 1 the numbers are determined in map and data files If a relatively large gap was found between two testing segments on a chromosome in the original result file there will be two separate files to express the coordinates on the same chromosome for the same trait if there are more such gaps then more files will be created The extension 001 indicates that this file is the first one for the trait and the chromosome A series of threshold files like chrony sig These files contain coordinates for horizontal threshold lines y threshold Each chromosome will have one such file For example chroml sig contains coordinates for drawing a threshold line on chromosome 1 The threshold is determined by the threshold probability specified in Settings sub menu For the method of plotting 2D graphs by Wgnuplot please see the next section Understanding Files Created by QTLMapper 1 0 Several kinds of result files will be created from the analysis for mapping QTLs with QTLMapper 1 0 as described above To make inferences about the putative QTLs for the traits under study the user needs to understand the cont
73. yesian Test Contributions Make LR Graphs 2D 1 gt 5 gt Select Esc gt Back Figure 31 Output sub menu There are five items in Output sub menu Detailed usage of these items in Output sub menu is given below 1 Filtrate This function is designed for filtrating putative additive effect QTLs or epistatic QTLs from the original result file The filtration subroutine will search for the peak points of LR values along each chromosome and compare them with the threshold value determined by the threshold probability setting If the LR value at a peak is above the threshold the testing point with this LR value will be kept and saved in a file with the same stem name as the original result file and extension of flq for additive effect QTLs or fle for epistatic QTLs This can largely reduce the manual work in searching putative additive effect QTLs epistatic QTLs from the original result file a Ses Manual for QTLMapper Version 1 0 To filtrate putative additive effect QTLs or epistatic QTLs press number 1 in Output sub menu A dialog box Figure 32 will appear and let you decide what to filtrate Filtrate for A Putative Additive effect QTLs Yes B Putative Epistatic QTLs No A gt B gt Select Esc gt Figure 32 Dialog box for filtrating putative QTLs Whatever you select in the dialog box an input box Figure 33 will appear subsequently This input box let you change the default threshold probability

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