As we mentioned earlier, exhaustive analyses of large linkage groups are not practical. Instead, to find a map order of a larger group, we need to find a subset of markers on which we can perform an exhaustive "compare" analysis. Thus, to map group2 we could pick a subset of its 8 markers at random, although we might do better if we pick markers which are likely to be ordered with high likelihood. Generally, this is true for sets of markers which have (i) as little missing data as possible, and (ii) do not have many closely spaced markers.
To quickly see how much data is available for the markers in this group, we set MAPMAKER's "sequence" appropriately and use MAPMAKER's "list loci" command.
MAPMAKER prints a list of loci, showing each marker by both its MAPMAKER-assigned number as well as it's name in the data file. For each marker, MAPMAKER prints the number of informative progeny (out of the 333 in the data set), and the type of scoring. In this case all loci have been scored using "co-dominant" markers (e.g. RFLP-like genotypes in an F2 intercross), although clearly markers 4 and 6 are the least informative.
To also look for markers which may be too close, we use MAPMAKER's "lod table" command.
MAPMAKER prints both the distance and LOD score between all pairs of markers in the current sequence. Unfortunately, the closest pair is separated by over 6.0 cM, a distance which should almost always be resolvable in a data set with so many informative meioses.
Given the results of these two analyses, a good subset to try might be:
8 9 10 11 12Note that the above two tests could have been automatically performed using MAPMAKER's "suggest subset" command, documented in the reference section.
9> sequence 4 6 8 9 10 11 12 sequence #4= 4 6 8 9 10 11 1210> list loci Linkage Num Name Genotypes Group 4 T24 273 codom group2 6 T209 275 codom group2 8 T125 306 codom group2 9 T83 327 codom group2 10 T17 297 codom group2 11 C15 324 codom group2 12 T71 319 codom group2
11> lod table Bottom number is LOD score, top number is centimorgan distance:
4 6 8 9 10 11
6 63.1 3.33 8 16.8 56.0 39.06 4.33 9 56.3 17.8 54.8 6.77 36.70 7.68 10 106.3 27.7 - 43.3 0.89 22.51 15.08 11 14.9 74.0 6.3 65.4 - 43.78 2.20 80.87 5.76 12 28.2 43.1 18.4 24.1 89.1 30.1 22.24 9.13 39.84 32.39 2.22 23.90
As before, we now change MAPMAKER's sequence to specify the subset we wish to test, and then type the "compare" command. This time, the results are even more conclusive, with one order at least 10 to the 14.6th more likely than any other.
We also again select the best order as the current sequence using MAPMAKER's "sequence" command. This time however, we so this using a special shortcut, "order1", which is a name MAPMAKER often sets to indicate the results of its analyses.
To determine the map position of the remaining two markers in group2, we will use the following procedure: Starting with the known order of 5 markers, we will place the other two (one at a time) into every interval in this order and then recalculate the maximum-likelihood map of each resulting 6 marker order. In this analysis, MAPMAKER recalculates all recombination fractions for all intervals in each map (not just the ones involving the newly placed markers).
This function is performed by MAPMAKER's "try" command. In its output, MAPMAKER again displays relative log-likelihood of each position for the inserted markers. The relative log-likelihood of 0 indicates the best position, while the negative log-likelihoods indicate the odd against placement in each other interval.
In this case, we see that marker 6 strongly prefers to be in-between markers 9 and 10. Even the next most likely position for marker 6 is more than 10 to the 21.09th power times less likely.
The "try" command not only tries to place markers in each interval in the framework, but also tries to place each marker infinitely far away (that is, forced 50% recombination between it and the framework). The relative log-likelihoods for this position are indicated following the "INF" entry in the MAPMAKER output. In the same way that a two-point LOD score indicates the odds of linkage between two loci when they are separated by their maximum likelihood distance, these relative log-likelihoods indicate the odds supporting linkage between one locus and a framework of loci when the locus is placed in its most likely position. In the above test, we see that a log-likelihood of 44.66 supports linkage between 4 and the rest of the group.
12> sequence {8 9 10 11 12}
sequence #5= {8 9 10 11 12}
13> compare
Best 20 orders:
1: 11 8 12 9 10 Like: 0.00
2: 10 11 8 12 9 Like: -14.57
3: 8 11 12 9 10 Like: -15.23
4: 10 9 11 8 12 Like: -27.20
5: 11 8 12 10 9 Like: -29.97
6: 10 8 11 12 9 Like: -30.14
7: 9 10 11 8 12 Like: -32.23
8: 8 11 10 9 12 Like: -39.80
9: 10 9 8 11 12 Like: -39.91
10: 9 11 8 12 10 Like: -40.05
11: 11 8 10 9 12 Like: -40.25
12: 11 8 9 12 10 Like: -44.73
13: 8 11 12 10 9 Like: -45.21
14: 10 11 8 9 12 Like: -46.57
15: 8 11 9 12 10 Like: -47.46
16: 9 10 8 11 12 Like: -47.94
17: 10 8 11 9 12 Like: -49.61
18: 8 11 10 12 9 Like: -52.71
19: 9 8 11 12 10 Like: -52.74
20: 11 8 10 12 9 Like: -53.07
order1 is set
14> sequence order1
sequence #6= order1
15> try 4 6
4 6
---------------
| 0.00 -42.68 |
11 | |
|-35.57 -118.6 |
8 | |
|-19.65 -70.19 |
12 | |
|-46.80 -28.09 |
9 | |
|-51.35 0.00 |
10 | |
|-43.40 -21.09 |
|---------------|
INF |-44.66 -45.03 |
---------------
BEST -619.33 -612.03
As a last step, we now type the complete sequence for this group, adding
markers 4 and 6 into their most likely positions. Then we type "map" to see
the complete map of all markers in this group.
16> sequence 4 11 8 12 9 6 10 sequence #7= 4 11 8 12 9 6 1017> map ========================================================================= Map: Markers Distance 4 T24 14.8 cM 11 C15 6.4 cM 8 T125 18.9 cM 12 T71 24.0 cM 9 T83 18.1 cM 6 T209 28.6 cM 10 T17 ---------- 110.8 cM 7 markers log-likelihood= -688.99 =========================================================================