Development of linkage map using Mapmaker/Exp3.0

Transcription

1 Development of linkage map using Mapmaker/Exp3.0 Balram Marathi 1, A. K. Singh 2, Rajender Parsad 3 and V.K. Gupta 3 1 Institute of Biotechnology, Acharya N. G. Ranga Agricultural University, Rajendranagar, Hyderabad, Andhra Pradesh 2 Division of Genetics, Indian Agricultural Research Institute, New Delhi 3 Indian Agricultural Statistics Research Institute, New Delhi MAPMAKER is a linkage analysis software program that can construct linkage maps of markers segregating in experimental crosses. MAPMAKER Version 3.0 can analyze data derived from progeny of several types of crosses, including: F 2 intercross, F 2 backcross (e.g. BC 1 ), F 3 intercross (by self-mating), Recombinant Inbred Lines (by self or sib-mating). It performs full multipoint linkage analyses i.e., estimation of all recombination fractions from the marker data for dominant, recessive and co-dominant markers. MAPMAKER Version 3.0 was developd by Stephen E. Lincoln, Mark J. Daly, and Eric S. Lander of the Whitehead Institute for Biomedical Research and the M.I.T Center for Genome Research, Massachusetts Institute of Technology, Department of Biology. It can be loaded on computers that are compatible for running Microsoft DOS, Sun SPARCStation Running SunOS, Apple Macintosh A/UX. There are two basic stages to construct a linkage map with Mapmaker/EXP: 1. To get the data into the format that Mapmaker. 2. To construct a genetic map for the marker data 1. Get the data into the format that Mapmaker needs For linkage mapping analysis of marker data in Mapmaker, prepare a data file with *txt extension containing information on mapping population type, genotype data of number of markers, number of phenotypic data of quantitative traits, coding scheme of your data set. The first line in data file is the cross type. data type where is one of the data types that mapmaker can analyze, either: f2 intercross f2 backcross f3 self ri self ri sib

2 The second line of the raw file should contain a list of three numbers, separated by spaces, such as: The first is the number of individuals scored for phenotype (310), second is number of markers scored (126) and third is the number of quantitative traits scored (12). This may be zero, if there is no quantitative trait data is present. Additional information like the coding scheme you use for genotypes may be optionally supplied at the end of this line. By default, the codes for F 2 intercross data are: 'A' Homozygote for the allele from parental strain a of this locus. 'B' Homozygote for the allele from parental strain b of this locus. 'H' Heterozygote carrying both alleles a and b. 'C' Not a homozygote for allele a (either bb or ab genotype.) 'D' Not a homozygote for allele b (either aa or ab genotype.) '-' Missing data for the individual at this locus For RI data, the default codes are: 'A' Homozygote for parental genotype a. 'B' Homozygote for parental genotype b. '-' Missing data for the individual (or line) at this locus. For F 2 backcross (a.k.a. BC1) data are: 'A' Homozygote for the recurrent parent genotype. 'H' Heterozygote. '-' Missing data for the individual at this locus. If you are not following default code schemes for marker data, specify the coding scheme at the end of second line like symbols 1=A 2=B 0=- After the first two lines, the file should contain the genetic locus data, in the following format: For each locus, list (1) the name of the locus, preceded by an asterisk ("*") followed by one or more spaces and the genotypic data for all individuals in order.

3 data type RI self *RM3732 B A B B A *RM324 B A B - B After preparation of data file in.txt format start mapmaker/exp. When started, MAPMAKER/Exp responds with its start-up banner and a prompt for the first command: ****************************************************************** * Output from: Thu Jul 23 01:20: * * * * MAPMAKER/EXP * * (version 3.0b) * * * ****************************************************************** 1> Whenever a prompt like this >1 is displayed, any valid MAPMAKER/Exp command may be entered. All commands consist of one or more words separated by spaces, and may be followed by one or more "arguments" to the command. Arguments might include locus numbers, computer file names, or numeric values. The following output from mapmaker shows how to get data in to map maker, how develop linkage map information by different commands. MAPMAKER works on DOS systems which produces output one screenful at a time and do not remember text that has scrolled off the screen. Hence it is better start saving Mapmaker output file with command Photo followed by file name (xxx), which copies present information on screen to the named file. 1>photo MAPMAKER\MAP\CHRO9.OUT' (To save output of current file) 'photo' is on: file is 'C:\MAPMAKER\MAP\CHRO9.OUT' (Output by Mapmaker 3.0) 2> prepare data c:\mapmaker\ch126.txt (To load a raw data set from a file you already created and saved i.e., c:\mapmaker\ch126.txt into Mapmaker for analysis) preparing data from file 'C:\MAPMAKER\CH126.TXT'... ok RI (selfing) data (310 individuals, 126 loci)... ok

4 unable to run file 'C:\MAPMAKER\CH126.PRE'... skipping initialization saving genotype data in file 'C:\MAPMAKER\CH126.DAT'... ok saving map data in file 'C:\MAPMAKER\CH126.MAP'... ok 3> units (To know default map function) the 'units' are currently (Haldane) centimorgans 4> cent func k (To change map function to Kosambi) centimorgan function: Kosambi 5> print names on (To see name of markers) 'print names' is on. 6> triple error detection on (To know error probabilities and associated LODerror values) 'triple error detection' is on. 7> default linkage 3 50 (To know min. LOD an d max.cm distance to declare linkage between markers) default LOD score threshold is 3.00 default centimorgan distance threshold is > sequence rm285-rm201 (To load marker data for linkage analysis) sequence #1= rm285-rm201 9> group (Separate Markers in Sequence into Linkage Groups) Linkage Groups at min LOD 3.00, max Distance 50.0 group1= RM285 RM5688 RM219 RM6920 GNMS3037 RM24233 RM6570 RM242 RM278 RM160 RM unlinked= RM257 10> default linkage 2 50 (To change min. LOD an d max.cm distance to declare linkage between markers) default LOD score threshold is 2.00 default centimorgan distance threshold is 50.00

5 11> group (Separate Markers in current Sequence into Linkage Groups based on two-point analysis) Linkage Groups at min LOD 2.00, max Distance 50.0 group1= RM285 RM5688 RM219 RM6920 GNMS3037 RM24233 RM6570 RM242 RM278 RM160 RM unlinked= RM257 12> order (To Automatically Build Map Orders from Scratch) Linkage Groups at min LOD 2.00, max Distance 50.0 Starting Orders: Size 5, Log-Likelihood 3.00, Searching up to 50 subsets Informativeness: min #Individuals 1, min Distance 0.9 Placement Threshold , Threshold , Npt-Window 7 =========================================================== Linkage group 1, 11 Markers: 94 RM RM RM RM GNMS RM RM RM RM RM RM201 All markers are informative... Searching for a starting order containing 5 of all 11 loci... Failed to find a starting order at log-likelihood threshold =========================================================== 13> three point (To compute three point for each subset of three markers and corresponding log-likelihoods of the three orders and to know best order of markers for each subset of three markers) Linkage Groups at min LOD 2.00, max Distance 50.0 Triplet criteria: LOD 3.00, Max-Dist 37.2, #Linkages 2 'triple error detection' is on. counting...38 linked triplets in 1 linkage group log-likelihood differences count markers a-b-c b-a-c a-c-b 1: RM285 RM5688 RM : RM285 RM5688 RM : RM285 RM5688 GNMS : RM285 RM5688 RM : RM285 RM219 RM

6 6: RM285 RM219 GNMS : RM285 RM219 RM : RM285 RM6920 GNMS : RM285 RM6920 RM : RM285 GNMS3037 RM : RM5688 RM219 RM : RM5688 RM219 GNMS : RM5688 RM219 RM : RM5688 RM6920 GNMS : RM5688 RM6920 RM : RM5688 GNMS3037 RM : RM5688 RM24233 RM : RM219 RM6920 GNMS : RM219 RM6920 RM : RM219 GNMS3037 RM : RM219 RM24233 RM : RM6920 GNMS3037 RM : RM6920 RM24233 RM : GNMS3037 RM24233 RM : RM24233 RM6570 RM : RM24233 RM242 RM : RM24233 RM278 RM : RM24233 RM278 RM : RM6570 RM242 RM : RM6570 RM242 RM : RM6570 RM242 RM : RM6570 RM278 RM : RM6570 RM278 RM : RM6570 RM160 RM : RM242 RM278 RM : RM242 RM278 RM : RM242 RM160 RM : RM278 RM160 RM > lod (To print all two-point data for the current sequence) Bottom number is LOD score, top number is centimorgan distance:

7 RM RM RM285 RM219 GNMS3037 RM257 RM242 RM5688 RM6920 RM24233 RM6570 RM278 RM GNMS RM RM RM RM RM RM RM RM > sequence rm285-rm24233 rm6570-rm201 (To select the loci and order(s) of markers to analyze) sequence #2= rm285-rm24233 rm6570-rm201

8 16> map (To calculate and display the maximum likelihood map for the order of markers specified by the current sequence) ============================================================== Map: Markers Distance 94 RM cm 95 RM cm 96 RM cm 97 RM cm 98 GNMS cm 99 RM cm 101 RM cm 102 RM cm 103 RM cm 104 RM cm 105 RM cm 11 markers log-likelihood= ============================================================== 17> sequence rm285 (To load current marker) sequence #3= rm285 18> make chromosome chro9 (To declare one or more named chromosomes to exist) chromosomes defined: chro9 19> anchor chro9 (To specify the anchor loci i.e., current loci for a chromosome) RM285 - anchor locus on chro9 chromosome chro9 anchor(s): RM285 20> sequence rm285-rm24233 rm6570-rm201(to load current marker data) sequence #4= rm285-rm24233 rm6570-rm201 21> asign (Typing error of command assign) unrecognized command: 'asign'

9 22> assign (To assign markers to a chromosome by using simple pairwise linkage data) RM285 - anchor locus on chro9...cannot re-assign RM assigned to chro9 at LOD 36.8 RM219 - assigned to chro9 at LOD 41.3 RM assigned to chro9 at LOD 67.7 GNMS assigned to chro9 at LOD 52.3 RM assigned to chro9 at LOD 5.9 RM assigned to chro9 at LOD 2.5 RM242 - assigned to chro9 at LOD 26.1 RM278 - assigned to chro9 at LOD 56.3 RM160 - assigned to chro9 at LOD 20.4 RM201 - assigned to chro9 at LOD > frame chro9 (To declare the framework order of the specified chromosome) setting framework for chromosome chro9... ============================================================== chro9 framework: Markers Distance 94 RM cm 95 RM cm 96 RM cm 97 RM cm 98 GNMS cm 99 RM cm 101 RM cm 102 RM cm 103 RM cm 104 RM cm 105 RM cm 11 markers log-likelihood= =============================================================== 24> q (To quit mapmaker program) save data before quitting? [yes] y saving map data in file 'C:\MAPMAKER\CH2.MAP'... ok saving two-point data in file 'C:\MAPMAKER\CH2.2PT'... ok saving three-point data in file 'C:\MAPMAKER\CH2.3PT'... ok...goodbye...

10 To make perfect linkage map more Mapmaker commands like compare, try, ripple, order, build can be utilized. The "compare" command calculates the maximum likelihood map for all orders of markers specified by the current sequence. The "try" command is used to place an unmapped marker or markers relative to a known order of loci to which it is known to be linked. The ripple command tests a map order by comparing the likelihood of the original map order to those found when the order of neighboring loci are permuted. The build command sequentially adds markers to a known map order, which should be set using the sequence command prior to typing the build command. Once information on number of linkage groups, number and name of markers of a linkage group and map distances of all chromosomes is generated from mapmaker it can be used for QTL mapping.