Triple Test Cross

Triple test cross:

crossing of randomly selecte F2 plants with both the parents(p1 ,p2)involves in the
cross ,and with their F1 hybrid.
The most efficient analysis of randomly mating populations is possible when two
contrasting inbred lines are available using extension of design North Carolina Design III.
This was first devise by kearsy and jinks (1968). This allows for the test of one of the
assumption s that there is no non allelic interaction. Thus the epistatic component can be
worked out, in this design selected individuals is crossed not only to two inbred parents but
also to their F1 .in other words ,randomly selected F2 plants are back crossed to both the
inbred parents and their F1 .
MAIN FEATURES:
Triple test cross has been described by Mather and jinks, 1971 and Singh and chauhary, 1985. The
important features of this genetic design are briefly presented below,
1. This design involves P1 ,P2,F1 and F2 generations of a single cross in developing experimental
material.
2. This technique requires 4 crop seasons to complete study. Three crop season are require for
the development of breeding material and the t he fourth one for evaluation of material
3. The analysis is based on second order statistics; therefore, calculation is more difficult than
generation mean analysis.
4. The design provides test for non allelic interaction, which is one of the assumptions
5. This evaluates the above material in terms of genetic components of variation.
Main steps
1. Analysis of triple test cross involves five important steps, viz...
2. Making single cross
3. Raising F 1 progeny
4. Making back crosses evaluation of material
5. Biometrical analysis
Making single crosses: in the first season crop, two inbred lines having contrasting characters are
selected from the germplasm. In other words two inbred lines with high and low manifestation of
character are chosen, these lines are mated to obtain single cross. The cross seed is harvested at
maturity.

Males P1 P2 F1

M1 * * *

M2 * * *

M3 * * *

M4 * * *

M5 * * *

M6 * * *

M7 * * *

M8 * * *

M9 * * *
M10 * * *

RAISING F2 PROGENY; in the second crop season, the F1 progeny of above cross is raised. All the f1
plants are self pollinated to obtain F2 seeds.
Making back crosses:
In the third crop season, F2 progeny of above single cross is raised form the seed obtained form the F1
plants in the previous year. In F2 generation, some plants are selected randomly and designed as
males, these randomly selected plants are back crossed with inbred parents and their F1.which are
used as females. The number of back crosses to be made is equal to 3n, where n is the number of F2
plants are each mated to P1,P2 and F1, 30 crosses are obtained for evaluation

Evaluation of material: in the fourth crop season, the back cross progenies along with inbred parents
(P1, P2) an F1 and F2 are evaluated in the replicated field trail, an observations are recorded on various
quantitative traits.

Biometrical analysis:

The biometrical analysis of triple test cross is one according to Singh and Chaudhary(1985).

Triple test cross analysis is extension of North Carolina Design 3 of Comstock and Robinson (1952).
However, it Differs from the later in many aspect. For example triple test cross involves four
generations in developing experimental material, where as the later involves three generations: and it
provide test for epistasis,which is not possible in case of north Carolina design 3.

Difference between triple test cross and north Carolina design 3:

No Triple test cross north Carolina design 3

1 It involves 4 generation of a single cross It involves 3 generations of a single cross in

in analysis crossing

2 F2 plants are mate to inbred parents from F2 plants were mate to inbred parents from which
which F2 was derive an also to their F1 F2 was derived

3 Provides test for epistasis It does not provides test for epistasis

4 It allows different generations into It does not allow different generations into analysis
analysis

Test for epistasis

This design provides information about the presence or absence of non allelic interactions. In
the absence of linkage, the genetic gamatic production of F1 is equal to mixture of its two
inbred parents as per the relationship given below:

L1+L2-2L3 =0 where L1, L2, and L3 are the mean values of the progenies derived from mating

of F2, with P1, P2, F1 respectively. The relationship provides test for the presence or absence
of epitasis. If the value is equal to zero, it shows absence of non allelic interactions. In the

presence of epistasis this presence will not be zero. There are two ways to test the

significance of epistasis.either it can be tested with help of t testing a with the help of t test in

a manner comparable to scaling test or it can be tested with help of f test. In the absence of

epistasis, The F2*F1 crosses can be provide additional information about the additive

components of variation. in the absence of epistasis ,calculation of D and H component can

be carried out without transformation of data. But when epistasis is present, proper

transformation ata is necessary before calculation of D and H components.

Modifications of this technique has been proposed by some researchers .according to

the modified approach, the testers L1,L2 and their hybrid (L3) are crossed with a number of

unrelated strains, instead of crossing with randomly selected F2 individuals of the cross

L1*L2.this modified scheme is similar to triple test cross, has similar statistical treatment and

yield comparable information.

Merits and demerits:

Merits:

Triple test cross provides reliable information about the presence or absence of epistasis, In

addition to the estimates of additive genetic variance and dominant variance; it provides

independent estimation of additive genetic variance and dominance variance.

Demerits:

In this technique the main problem is the choice of contrasting pair of inbred lines to get

reliable estimates of additive genetic variance. This technique takes more time for evaluation

of material and statistical analysis.

Triple test cross analysis has been used in some crops. However, this like biometrical

techniques is not common in use in plant breeding unlike diallel,partial diallelan line *test

cross analysis.

Comparison of various biometrical techniques:

There are several biometrical techniques which are use by the plant breeers for evaluatin of

genotypes or strains in terms of their genetic value,the important biometrical techniques

which are used for the study of genetic components of the variation the plant population

include diallele cross, partial diallel analysis ,line *test cross analysis triallele ,quadriallel

generation mean analysis ,biparental an triple test cross.the comparative study provides the

clear picture of the important features of each technique and secondly it helps in better

understanding of the subject.

Criteria for comparison

Several criteria used for the comparison of various biometrical techniques. The important

criteria which are used for this purpose include

1. Genetic material used for evaluation

2. Information obtained components of genetic variance

3. Degree of statistics involved in the estimation

4. Duration of the experiment

5. Cost of experimentations

6. Genetical assumptions

7. Efficiency

8. Practical utility

9. Specific features

Comparison of diallel, triallel, quadriallel crosses:

s.no Diallel cross Triallel cross Quadrilles crosses

1 Includes all possible Includes all possible three Includes all possible three

single crosses among the way crosses among the way crosses among the

parents i.e.., n(n-1)/2 parents i.e.., n(n-1)(n-2)/2 parents i.e.., n(n-1)(n-2)/2

2
3
4
5
6
7
8
9
10
11
12

Genetic mapping
In order to do the triple test cross it is essential that we can able to:
1. Detect linkage by contingency Chi-square

2. Detect % recombination by observing the number of recombinant phenotypes in a

backcross as compared to the total.
3. It is possible to construct maps showing the order and distance between genes on a
chromosome.
4. Such mapping usually accomplished with three genes at a time. We call this the triple
test-cross.
Since this is a linkage test cross and we are going to consider three genes at a time.
First mate a triple heterozygote to a completely recessive individual. If these three genes
were independently assorting, then the triple heterozygote would make 8 gametes and have
progeny showing 8 different phenotypes in equal frequencies.
Example:
In rum bunnies:
Spock ears (D) are dominant to earless (d)
Red eyes (A) are dominant to blue eyes (a)
Spinner eyes (E) are dominant to nonspinner (e)

P1 ADE/ADE X ade/ade

(if we know the cross, the parental

gametes are those seen in the
F1 ADE/ade
parental generation,
i.e., ADE and ade)

Test cross ADE/ade x ade/ade

Observed Phenotypes Genotype Number

Red Spock Spinner ADE/ade 8576
Blue Earless Nonspinner ade/ade 8808
Red Spock Nonspinner ADe/ade 681
Blue Earless Spinner adE/ade 716
Blue Spock Spinner aDE/ade 1002
Red Earless Nonspinner Ade/ade 997
Red Earless Spinner ADe/ade 4
Blue Spock Nonspinner adE/ade 1
Total 20875

We can use the above information to construct a statistical map giving the relative order of
the three genes and the relative distances between those genes. This statistical map can
eventually be related to the physical map of the chromosomes.

Important Information for mapping

1. From previous work it is known that the three genes are all on the same chromosome.
2. The genes are arranged in one of three orders*
ADE
AED
DAE
* Since we are constructing a relative map, the order ADE and EDA are considered
to be the same.
3. The statistical distance between the genes is the frequency at which they recombine.
(Recombination % = map units or centimorgans).
4. A single crossover event occurs more frequently than a double crossover event (that
is, two single crossovers occur simultaneously). Thus the least frequent pair of
phenotypes represents gametes that arose from a double crossover event.
5. Single crossover phenotypes (representing gametes) define the ends of the map. The
double crossover gametes (when seen) define the middle of the map).
6. The gametes resulting from each type of crossover events is compared to the parental
gametes.
In the example given above, parental gametes (the most frequent ones)
are ADE and ade. The least frequent pair of gametes is AdE & aDe. These are the double
crossover gametes.
When we compare the double crossover (DC) gametes to the parental gametes we see that
the Spock/earless allele has "switched" chromosomes.

Parental DC Gametes
Gametes
ADE AdE
ade aDe
This means that the Spock-earless gene (D) is in the middle and the map order is:
A----------------D-----------E

Now I calculate the distances from the % of single and double recombinant gametes
(phenotypes). For convenience I will call the gametes from one of the single crossover
events Pair X and the gametes of the other single crossover event Pair Y. When I compare
Pair X to the parental types (Pair W) I see that spinner-nonspinner gene (E) has "switched"
chromosomes. This represents a crossover between the gene in the middle (D) and the gene
on one end (E). The distance between D and E is the % recombination shown by the Pair
X plus the frequencies of Double crossovers (since the DC's also represent a recombination
event between D and E)

DC Pair X
Parental Gametes
Gametes Gametes
ADE AdE ADe
ade aDe adE

Distance D-E = [(681 + 716)/20875] + [(1 + 5)/20875]

Distance D-E = 6.72% + .02% = 6.74% = 6.74 map units

Similarly, I compare SC-Pair Y to the parental gametes In this case the alleles for Red
eyes/blue eyes (A) have "switched" chromosomes. This represents a crossover between
gene A on the end and gene D in the middle. The distance is the single crossover frequency
plus the DC frequency.

DC Gametes Pair Y Pair X

Parental Gametes
Gametes Gametes
ADE AdE aDE ADe
ade aDe Ade adE

Distance A - D = 9.62% + .02% = 9.64 map units

 The Genetic Map is:
A-------------- D--- -----------E
9.64 6.74

Example where double crossover types are not observed.

In rumbunnies:
Bulb nose (B) is dominant to pug nose (b)
Mossy teeth (M) is dominant to slick teeth (m)
Bad breath (S) is dominant to minty (s)

P1 Bms/Bms x bMS/bMS
F1 Bms/bMS (parental gametes are Bms and bMS)
B1 Bms/bMS x bms/bms

Expected Phenotypes - with three loci we expect 2 x 2 x 2 = 8 phenotypes in a 1:1:1:1:1:1:1:1

ratio. The eight phenotypes will be (B:b) x (M:m) x (S:s)
BMS bulb mossy bad breath
BMs bulb mossy minty
BmS bulb slick bad breath
Bms bulb slick minty
bMS pug mossy bad breath
bMs pug mossy minty
bmS pug slick bad breath
bms pug slick minty

The observed phenotypes are:

Gamete Phenotype Number
BMS mossy bad breath 443
BMs bulb mossy minty 293
Bms bulb slick minty 3458
bMS pug mossy bad breath 656
bmS pug slick bad breath 281
bms bms pug slick minty 445

That is, we are missing:

BmS bulb slick bad breath 0
bMs pug mossy minty 0
Since these phenotypes occur in the lowest frequency (that is, 0%) these must be the double
crossover types.
Now, for ease of working I am going to "pair up" the single crossover and parental types.
That is, if one genotype had the B allele, the paired partner must have the b allele; if one has
the M allele the other has the m allele, and if one has the S allele the other has the s allele,

Programmatic Way to Do a Triple Test Cross Problem

.
The triple test cross has proven to be quite difficult for many students. Every one eventually
finds her/his own way to solving these types of problems. My personal approach is outlined
below. This approach works for "paper and pencil" questions as well as for laboratory
exercises. The problem solving approach will be illustrated with the following example.
Example:
Dr. Alicia Tofuson is constructing linkage maps of Emotion bees. In these bees happiness
(h), fear (f) and anger (a) are all recessive and located on the same chromosome. (The Wild
type phenotypes are not-happy (H), not-afraid (F) and not-angry (A). Dr. Tofuson crosses a
homozygous afraid, happy, not-angry emotion bee to a homozygous and not-afraid, not-
happy, angry emotion bee to attain a triple heterozygote (Fha/fHA). She crosses the triple
heterozygote to a complete recessive afraid, happy, angry emotion bee. The results of a triple
test cross involving 50,000 emotion bees are given below.
Gamete
Phenotype Number Genotype
Type
not-afraid, not happy, not angry 87 FHA/fha
afraid, not happy, not angry 3058 fHA/fha
not-afraid, happy, not angry 4241 FhA/fha
not-afraid, not happy, angry 18229 FHa/fha Parental
afraid, happy, not angry 16933 fhA/fha Parental
afraid, not happy, angry 4597 fHa/fha
not-afraid, happy, angry 2780 Fha/fha
afraid, happy, angry 75 fha/fha
a. Draw the genetic map (order and distance).

Step 1. Identify the Parental Gametes: Probably the easiest thing to do. If you set up the
initial F1 cross, the parental gametes are the same as the phenotypes of the P1 and P2 parents.
In this example, that would be FHa (not-afraid, not happy, angry) and fhA (afraid, happy, not
angry).
If you did not know the original cross, you could use the definition of statistical linkage to
determine the parental gametes. "Statistical linkage is a deviation from independent
assortment in the direction of an excess of parental gametes". Since the phenotypes
corresponding to FHa and fhA are most frequent, they must be the parental gametes.
Step 2: Determine the double crossover gametes. Probably the second easiest thing to do.
Double crossovers are the least frequent gametes. They are so infrequent that often they do
not even show up!!! That is, they have a frequency of zero. You must always remember that
a triple heterozygote is expected to make 8 different gametes. If you only see 6 phenotypes
among the test cross progeny, the missing phenotypes (in most cases) correspond to the
double crossovers. In the above example all 8 phenotypes are present and the double
recombinant gametes are fha and FHA.
Step 3: Determine the relative order of the genes on the map. The parental pair of gametes is
FHa/fhA and the double cross over gamete is FHA/fha. The difference between the two pairs
is that the A gene (in red) has switched chromosomes. This mean that the map order is:

The Genetic Map is:

F--------------- A--- -----------H

Step 4: Now pair up the other gametes: Wherever you have a capital letter in one gamete
(e.g., F) you must have a lowercase letter in the complementary gamete (e.g., f)
DC Gametes Pair X Pair Y
Parental Gametes
Gametes Gametes
FHa FHA Fha fHa
fhA fha fHA FhA
Step 5: Add up the number of each pair and calculate the frequency of each pair of gametes.
(Divide the number in each pair by the total of 50,000)
DC Gametes Pair X Pair Y
Parental Gametes
Gametes Gametes
FHa FHA Fha fHa
fhA fha fHA FhA
35162 162 5838 8838
.003 .117 .177

Step 6: Now select one of of the pairs of gametes that represent a single crossover - for
example Pair X (Fha/fHA) and compare this pair to the parental gametes (FHa/fhA). You are
looking for the alleles that appear to have switched chromosomes. In this case, the allele pair
is H/h. The pair that appeared to have switched is on one end and has crossed over with the
gene in the middle of the map (Gene A). The distance between H and A is is the frequency
of Pair X gametes and the Double crossovers (as percentages or map units).
Distance H ------ A = 11.7% + .03% = 12% = 12 map units

The Genetic Map is now

F--------------- A--- -----------H
12 map units

Step 7: Now select the other pair of gametes that represent a single crossover --- that is, Pair
Y (fHa/FhA) and compare this pair to the parental gametes (FHa/fhA). You are looking for
the alleles that appear to have switched chromosomes. In this case, the allele pair is F/f. The
pair that appeared to have switched is on one end and has crossed over with the gene in the
middle of the map (Gene A). The distance between F and A is is the frequency of Pair Y
gametes and the Double crossovers (as percentages or map units).
Distance H ------ A = 17.7% + .03% = 18% = 18 map units

The Genetic Map is finally:

F------------- A--- -----------H
18 map units 12 map units

Parentals Bms bulb slick minty 3458

(Pair W) bMS pug mossy bad breath 3656

Single Crossover BMs bulb mossy minty 293 293

(Pair X) bmS pug slick bad breath 281 281

Single Crossover bms pug slick minty 445

(Pair Y) BMS bulb mossy bad breath 443

Double Crossover BmS bulb slick bad breath 0

(Pair Z) bMs pug mossy minty 0
Total = 8676

DC Gametes SC SC
Parental Gametes
(Pair Z ) Gametes Gametes
(Pair W)
(Pair X) (Pair Y)
BMs BmS BMs bms
bmS bMs bmS BMS

Comparing the DC gametes to the parental gametes we see that the S locus has switched
positions. It must be in the middle and the B and M loci are on the ends. The order of the map
is.
B--------------S---------M

Now look at one of the single crossovers (Pair X) and compare those gametes to the parental
gametes.
The difference is that the M locus has crossed over with the middle (S). The distance is the %
of single crossover phenotypes (for Pair X) plus any Double crossover types (zero in this
case)
Distance M --- S = (293 + 281)/8676 = 6.6% + 0 map units.
Now look at the the single crossover gametes (Pair Y) and compare those gametes to the
parental gametes.
The difference is that the B locus has crossed over with the middle S locus.
Distance B ----- S = (445 + 443)/8676 = 10.2% = 10.2 map units. The final map is then:
10.2 6.6
B--------------S---------M
Analysis of a Triple Testcross Design With Recombinant Inbred Lines Reveals a Significant
Role of Epistasis in Heterosis for Biomass-Related Traits in Arabidopsis
Barbara Kusterer*, Jasmina Muminovic*, H. Friedrich Utz*, Hans-Peter Piepho , Susanne
Barth , Martin Heckenberger*, Rhonda C. Meyer , Thomas Altmann and Albrecht E.
Melchinger*,1

Primary causes of heterosis are still unknown. Our goal was to investigate the extent and
underlying genetic causes of heterosis for five biomass-related traits in Arabidopsis thaliana.
We(i) investigated the relative contribution of dominance and epistatic effects to heterosis in
the hybrid C24 x Col-0 by generation means analysis and estimates of variance
components based on a triple testcross (TTC) design with recombinant inbred lines (RILs),
(ii) estimated the average degree of dominance, and (iii) examined the importance of
reciprocal and maternal effects in this cross. In total, 234 RILs were crossed to parental lines
and their F1's. Midparent heterosis (MPH) was high for rosette diameter at 22 days after
sowing (DAS) and 29 DAS, growth rate (GR), and biomass yield (BY). Using the F2-metric,
directional dominance prevailed for the majority of traits studied but reciprocal and maternal
effects were not significant. Additive and dominance variances were significant for all traits.
Additive x additive and dominance x dominance variances were significant for all traits but
GR. We conclude that dominance as well as digenic and possibly higher-order epistatic
effects play an important role in heterosis for biomass-related traits. Our results encourage the
use of Arabidopsis hybrid C24 x Col-0 for identification and description of quantitative trait
loci (QTL) for heterosis

Triple test cross and six-population techniques for partitioning the components of genetic
variance in faba bean (Vicia faba)

B. R. BAKHEIT a1 c1, M. Z. EL-HIFNY a1, M. M. EISSA a2 and S. B. RAGHEB a3

a1
Department of Agronomy, Faculty of Agriculture, Assiut University, Assiut, Egypt
a2
Department of Agronomy, Faculty of Agriculture, Zagazig University, Egypt
a3
Food Legume Section, Field Crops Research Institute, Agriculture Research Center,
Giza, Egypt

Abstract
The efficiency of the triple test cross (TTC) and the six-population biometrical analyses was
compared in terms of assessing and quantifying the components of genetic variance for two
faba bean crosses: Triple White×Giza 843 and NA112×Giza 429. Several traits were studied
including days to first flower, plant height, branches/plant, pods/plant, seeds/pod, 100-seed
weight and seed yield/plant. The results supported the triple test cross biometrical approach
as it uses first degree statistics and can be applied to any population irrespective of its genetic
architecture. Absence of a scalar relationship between triple test cross families
(orthogonality) ensures independence between means and variance with no restrictive
assumptions. Both methods provided evidence for epistasis, and both additive and dominance
genetic components in the genetic control of the studied traits.
(ReceivedRésumé / Abstract
Maize (Zea mays L.) breeders have successfully exploited heterosis by crossing
inbred lines to develop hybrids. Epistatic effects can contribute to the expression
of heterosis for specific hybrids. The hybrid B73 x Mo17 was a widely grown
hybrid with exceptional performance in the central U.S. Corn Belt during the late
1970s and early 1980s. The objective of this study was to determine if epistatic
effects contribute significantly to the performance of B73 x Mo17. With use of
the triple testcross design, 100 random F2 plants from B73 x Mo17 were
testcrossed to B73, Mo17, and their F1. The 300 testcrosses were evaluated at
three locations in 1992 and two locations in 1993. Triple testcross analysis
suggested that epistatic effects were important for ear length, number of kernel
rows, ear height, and flowering traits. In the analysis of variance, additive by
additive effects were not significant for grain yield, whereas additive by
dominance and dominance by dominance effects were significant. The additive
by additive by environment interaction was more important than additive by
additive effects per se for grain yield. Epistatic deviations from the comparison of
testcross means suggest that B73 had favorable additive by additive effects for
grain yield, barren plants, kernel-row number, ear height, and silk delay. Inbred
Mo17 had favorable additive by additive effects for ear length. The presence of
significant positive epistatic effects may have contributed to the expression of
heterosis and could explain why B73 x Mo17 was an exceptional and widely
grown hybrid. April 18 2002)