Mapping Course 2007

Jorge Dubcovsky Project funded by USDA-CSREES

Before Mapping
Nulli-tetrasomic lines: missing 1 pair of chromosomes that is compensated with a double dose of
one homoeologous chromosomes. The chromosome location is identified by
lack of amplification. Example: microsatellite located on chromosome 1B

1A 1B 1D 2B 2D 3A 3B 3D 4A 4D 5A 5B 5D 6A 6B 7A 7B

Ditelosomic lines: missing 1 arm. The name indicates the arm PRESENT. Example: microsatellite
located on chromosome arm 1BL
1BS 1BL

Deletion stocks: missing 1 segment. The name indicates the segment ABSENT.
Example: microsatellite located on bin 1BL1. C-1BL6-0.32
1BL6-0.32-0.47
1BL3 1BL2 1BL1 1BL6 C-1BL3
1BL1-0.47-0.69

1BL2-0.69-0.85
1BL3-0.85-100
Previous mapping: check chromosome location in GrainGenes
http://wheat.pw.usda.gov/GG2/index.shtml Deletion bin map of chromosome 1B
Mapping populations

Temporary populations Immortal populations

Crossovers
Meiotic chromosomes Meiotic products

Meiosis with A1 A2 A1 A2 •Linked loci can be separated

no crossover Parental A
A1 A2 A1 A2
Parental A
by a physical exchange of
between loci
“1” and “2”. B1 B2 B1 B2
chromosome parts during
Parental B meiosis.
B1 B2 B1 B2
Parental B
•This process, called crossover
A1 A2 A1 A2 generates recombinant
Meiosis with Parental A
1 crossover A1 A2 A1 B2 haploid genotypes that differ
Recombinant
between loci
B1 B2
from the haploid parental
“1” and “2”. B1 A2
B1 B2 B1 B2
Recombinant genotypes.
Parental B

• There is an average of 1
crossover per short arm and
2 crossovers per long arm.
• Therefore the average
chromosome length is 150
cM
Recombination frequency as estimate of distance
L1 L2 L3 L4

•There is a rough proportionality

between the distance between
linked loci and the chance of a
crossover between non-sister
chromatids between loci.
•By determining the frequency
of recombinants, it is possible to
obtain a measure of the map
distance between the genes.
•A recombinant frequency of
0.01 is the distance between loci
for which one product of
meiosis out of 100 is
Parental A1 A2= 7 Parental A3 A4= 10 recombinant
Parental B1 B2= 7 Parental B3 B4= 10
Recombinant A1 B2= 5 Recombinant A3 B4= 2
Recombinant B1 A2= 5 Recombinant B3 A4= 2

Rf= 10/24= 42% Rf= 4/24= 16%

Chiasma interference

Recombination frequency per length unit in

wheat (Lukaszewski and Curtis 1993)

• In wheat there is strong chiasma interference

(one chiasma reduces the pb. of 2nd chiasma in
its proximity).
• As a result of chiasma interference and the
initiation of chromosome pairing from • Small genetic distances in the centromeric
telomeres, chiasma in wheat are favored at the region correspond to large genetic distances
end of the chromosomes. in the wheat physical maps.
Recombination fraction and CentiMorgans

Meiotic chromosome Meiotic product

A1 A2 A1 A2
Parental A
A1 A2 A1 A2
Parental A
B1 B2 B1 B2
Parental B
B1 B2 B1 B2
Parental B

• When two crossovers are produced between two markers, no recombination is detected unless
an additional markers is included in the middle.
•Mapping functions (Haldane and Kosambi) correct the recombination fraction “r” for this
possibility.
• Haldane: does not consider interference and is not appropriate for wheat
• Kosambi: considers chiasma interference: cM=100*[¼ log [(1+2r)/(1-2r)]

Recombination fraction Kosambi cM

Rf= 0.01 1.0 cM
Rf= 0.02 2.0 cM
Rf= 0.10 10.1 cM
Rf= 0.20 21.2 cM
Rf= 0.49 114.9 cM
Three point analysis
If the distance between three linked markers is known

A--------5cM---------B
A-----3cM-----C
C------------8cM---------------B

We can determine the likelihood of each order:

C-----3cM-----A--------5cM---------B Likely
A-----3cM-----C---2cM---B Unlikely
A--------5cM---------B----------8cM-------------C Very unlikely

The Mapmaker command THREE POINT calculates the likelihood of the

three alternative orders for each three points and discards those that are very
unlikely
Expected Genotypic Classes
A1 A2
A1 A2

B1 B2
B1 B2
Expected Genotypic Classes
Population Dominant markers Codominant
markers
BC1F2 1:1:1:1 1:1:1:1

F2 9:3:3:1 1:2:1:2:4:2:1:2:1

RIL or RSL 1:1:1:1 1:1:1:1

DH 1:1:1:1 1:1:1:1
Significance of linkage
Example of observed and expected segregation values.

Example of RSL, two locus L1 L2 χ2

Genotype: AA : AB : BA : BB 0.44
Observed: 6.00 : 5.00 : 7.00 : 7.00
Expected : 6.25 : 6.25 : 6.25 : 6.25

Example of F2, two locus L1 L2 38.2

Genotype: AA : AH : AB : HA : HH : HB : BA : BH : BB
Observed: 5.0 : 2.0 : 0:0 : 0:0 :11.0 : 0:0 : 0:0 : 0:0 : 6.0
Expected: 1.5 : 3.0 : 1.5 : 3.0 : 6.0 : 3.0 : 1:5 : 3.0 : 1.5

The df of the χ2 is the number of classes minus 1.

Look for the critical value in a χ2 table: critical value α=0.05, χ23df= 7.81
Calculate χ2=Σ[(O-E)2/E]=(0.252+1.252+0.752+0.752)/6.25= 0.44
Since 0.44<7.81, there are no significant differences with the expected frequencies, and
therefore there is no significant linkage.
Calculating recombination frequencies in DH populations

In a DH, backcross, or RSL population the calculation of the

recombination fractions is very simple

Count the number of recombinant chromosomes and divide by the total

The total is the number of chromosomes for which we have information

for the two loci

RSLs
L1 A A B B B A B B - A A A A A A B A B B B A B A B B B B
L2 A A B B B B B A B B - B B B A A A B A A A B A A A A B
#CO 0 0 0 0 0 1 0 1 – 1 – 1 1 1 0 1 0 0 1 1 0 0 0 1 1 1 0

There are 12 recombiantion events in 25 scored pairs

RF= 12/25= 0.48 Mapmaker calculates it as 0.48

Calculating recombination frequency in F2 populations
In an F2, it is not possible to determine whether the H1H2 are parental or
recombinant. The following examples show two different pairs of chromosomes in an
F2 individual, one parental and one recombinant. The crossovers are indicated by “x”.
Example 1: H1H2 genotype from parental chromosomes
A A1 A2 A x B B

A x B1 B2 B B B
Score: A H1 H2 H B B

Example 2: H1H2 genotype derived from recombinant chromosomes

A A1 x B2 B B B

B B1 x A2 A A A
Score: H H1 H2 H H H

Therefore, in an F2 counting the recombinants is just an approximation.

Mapmaker uses Maximum Likelihood to estimate the distances more precisely:
Example 3 estimating RF in an F2:

L3 A H A H B H H B A H B H A A H H B B A A H H H B
L4
#CO H
1 H
0 A
0 H B 0
0 0 H 0
H 0
B 0
A 0
H 0
B 0
H 0
A 0
A 0
H 0
H 0
B 0
B 0
A 2
B 0
H 0
H 0
H 0
B

RF≅ 3/48 ≅ 0.0625 Mapmaker calculates it more precisely as 0.065

LOD scores
The maximum likelihood method of estimation is a general statistical method to estimate
the value of a parameter: the value chosen for the parameter is that which is most probable
(or most likely) given the occurrence of a certain set of observations.

LOD score (Log10 of the odds ratio) is calculated as:

Pb. of obtaining the observed data under linkage

LOD= log -------------------------------------------------------------------------
Pb. of obtaining the observed data under random assortment

The ratio between the two probabilities is transformed to log10.

The higher the value the more likely is the linkage.
For example, a LOD=3 indicates that is 1000 times (103) more likely to obtain the observed
data from linked markers than from unlinked ones.
LOD scores are used to determine the most likely order of markers in a map.
Mapmaker assigns 0 to the better order and negative LODs to alternative orders.
For example, if order a–b–c has LOD=0 and order La – Lc – Lb has a LOD score of –2, that
indicates that it is 100 (102) less likely to obtain the observed data from the 2nd order than
from the 1st order.
Recombinant inbred lines

Since RILs have more generations for recombination, recombination

frequencies need to be adjusted.
Recombination levels observed in RILs can be related to recombination in a
population derived from a single meiosis using Haldane and Waddington (1931)
formulas.

2r R
R = ------- or r = -------
1+2r 2-2R

Some examples
R r R/r
0.01 0.005 1.98
0.1 0.056 1.80 When linkages are tight, the
0.2 0.125 1.60 recombination frequency
0.3 0.214 1.40 among RILs is twice the
conventional F2 rate.
0.4 0.333 1.20
0.5 0.500 1.00
Mapping advice
• Take extreme care entering genotypic data. Merge linked markers into single group
• Review all double crossovers (if possible re-extract DNA and re run marker)
• Too many double crossovers for a marker indicate problems. Repeat PCR!
• Too many double crossovers along an individual suggest DNA problems. Recheck
conflicting markers with the same tube of DNA.
• Try to have 1 DNA for the complete project, if not possible keep track of DNA replacements
using a version number/date. Record mapping date of each markers
• If in doubt regarding the genotype of an individual, it is better to enter it as a missing value
than to guess. Each error expands your map 2 crossovers!
Map comparisons

Compare order with previous maps.

http://maswheat.ucdavis.edu/

Use our site in collaborator’s

area: user: cap05, password:
Genomic$

Map visualization tool

user: wheatcap, password:
jagger

We can enter your map there!

Japanese site for the

Wheat Gene Catalogue:
http://map.lab.nig.ac.jp:8080/cmap/
Answers to exercises
Exercise 1: You have assigned four loci to chromosome 1A of wheat using nullitetrasomics. Using fifty double
haploids from a hybrid between two varieties A and B you recover the following number of recombinant and parental
chromosomes:
Locus 1- Locus 2: AA:25 AB:0 BA:1 BB:24 -> 1/50=0.02 = 2%
Locus 1- Locus 3: AA:23 AB:3 BA:4 BB:20 -> 7/50=0.14 = 14%
Locus 1- Locus 4: AA:25 AB:1 BA:1 BB:23 -> 2/50=0.04 = 4%
Locus 2- Locus 3: AA:21 AB:3 BA:5 BB:21 -> 8/50=0.16 = 16%
Locus 2- Locus 4: AA:24 AB:2 BA:1 BB:23 -> 3/50=0.06 = 6%
Locus 3- Locus 4: AA:23 AB:2 BA:3 BB:22 -> 5/50=0.10 = 10%

Construct a map of these four loci.

Answer L2- (2%) - L1- (4%) - L4 – (10%) - L3

Exercise 2: Chi Square for Double haploids, RSLs or Backcrosses

In similar population of 100 double haploids you mapped two loci: 1 and 5. You checked parental and recombinant
chromosomes
L1 – L5 Observed: AA:28 AB:22 BA:24 BB:26
L1 – L5 Expected: AA:25 AB:25 BA:25 BB:25
|O-E| 3 3 1 1

χ2=Σ[(O-E)2/E]=(32+32+12+12)/25= 20/25=0.8 Critical χ2 = 7.81 No significant linkage

Answers to exercises

Exercise 3: χ2 for the F2 population

5+2+11+6=24 individuals and 16 classes
Expected per class: 24 individuals / 16 classes = 1.5 per class
1 : 2 : 1 : 2 : 4 : 2 : 1 : 2 :1
1.5 : 3.0 : 1.5 : 3.0 : 6.0 : 3.0 : 1.5 : 3.0 : 1.5

Genotype: A1A2 : A1H2 : A1B2 : H1A2 : H1H2 : H1B2 : B1A2 : B1H2 : B1B2
Observed: 5.0 : 2.0 : 0.0 : 0.0 : 11.0 : 0.0 : 0.0 : 0.0 : 6.0
Expected: 1.5 : 3.0 : 1.5 : 3.0 : 6.0 : 3.0 : 1:5 : 3.0 : 1.5 .
|Difference| 3.5 1.0 0.5 3.0 5.0 3.0 1.5 3.0 4.5
χ =Σ[(O-E) /E]=38.2 Critical value Df=8 χ =15.51
2 2 2

Exercise 4: Recombination fractions in RI or SSD

The following is the data for MapMaker for a Recombinant Inbred Population of 50 lines and 4 loci (RI or SSD). Calculate the
corrected RF ‘r’ between loci 1-2, 2-3 and 3-4. Remember that in a SSD r= R/(2-2R) where R is the proportion of recombinants in
the RI.
data type ri self
50 4 0
*locus1 aaaaa aaaaa bbbbb bbbbb aaaaa bbbbb aaaaa bbbbb aaaaa bbbbb
*locus2 aaaaa aaaaa bbbbb bbbbb baaaa bbbbb aaaaa bbbbb aaaaa bbbbb
*locus3 aaaaa aaaaa bbbbb bbbbb bbbbb bbbbb aaaaa bbbbb aaaaa bbbbb
*locus4 aaaaa aaaaa bbbbb bbbbb bbbbb aaaaa bbbbb bbbbb aaaaa bbbbb

L1-L2= 1/50 R= 0.02 r= 0.010

L2-L3= 4/50 R= 0.08 r= 0.043
L3-L4= 10/50 R= 0.20 r= 0.125
Answers to exercises
Answers to exercises
Answers to puzzle
Xabg601 AAAHAHAAAAAHAAAAAAHHHA AAHAAHHAAHHHAAHAAA AAHAAAAHAHHHAAAA
X X
Xabg602 AAAHAHAAAAAHAAHAAAHHHA AAHAAHHAAHHHAAHAAA AAHAAAAHHHHHAAAA
X X
XBamy AAAHAHAAAAAHAAHAAAHHHA AAHAAHHHAHHHAAHAHA AAHAAAAHHHHHAAAA
X
XksuH11 AAAHAHAAAAAHAAHAAAHHHA AAHAAHHHAHHHAAHAHA AAHAHAAHHHHHHAAA
X X X X
Xabc305 AAAHAHAAAAHHAAHAAAHHHH AAHAAHHHHHHHAAHAHA AAHHHAAHHHHHHHAA
X
Xmwg2112 AAAHAHAAAAHHAAHAAAHHHH AHHAAHHHHHHHAAHAHA AAHHHAAHHHHHHHAA
X X X X X X
Xbcd1302 AHAHAHAAAHHHAAHAAHHHHH AHHAAHHHHHHHHAHAHA AAHHHAHHHAHHHHAH
X X X X X
Xpsr164 AHAHAHAAHHHHAAHHAHHHHH AHHAHHHHHHHHHAHAHA AHHHHAHHHAHAHHAH
X X
XksuC2 AHAHAHAAHHHHHAHHAHHHHH AHHAHHHHHHHHHAHAHA AHHHHAHAHAHAHHAH
X
Xpsr1051 AHAHAHAAHHHHHAHHAHHHHH AHHAHHHHHHHHHAHAHH AHHHHAHAHAHAHHAH
X X
Xbcd1262 AHAHAHHAHHHHHAHHHHHHHH AHHAHHHHHHHHHAHAHH AHHHHAHAHAHAHHAH
X X
XDhn6 AHAHAHHAHHHHHAHHHHHHAH AHHAHHHHHHHHHAHHHH AHHHHAHAHAHAHHAH
X X
Xpsr922 AHAAAHHAHHHHAAHHHHHHAH AHHAHHHHHHHHHAHHHH AHHHHAHAHAHAHHAH
XX
Xcdo669 AHAAAHHAHHHHAHAHHHHHAH AHHAHHHHHHHHHAHHHH AHHHHAHAHAHAHHAH
X X XX X X
Xwg622 AHAAA-HAHAHHAHAHHHHAAH AH-AHHHHAAHHHAHHHH AHHHHAHAHAHHHAAH
X X
Xpsr921 AHAAAAHAHAHHAHAHHHAAAH AHHAHHHHAAHHHAHHHH AHHHHAHAHAHHHAAH