Development of Hybrids and Use of

Testers

Abebe Menkir
Maize Breeders Training Workshop, August
18 to September 6, 2013, Nairobi, Kenya
What is a hybrid?
 A cross between tow or more unrelated parents
 A product of North American agriculture with a commercial seed
production in 1926
 Increased dramatically in the USA and covered 100% maize
acreage in 1955 due to its better performance under drought
years
 Spread to Europe and increased hybrid production occurred
starting from 1984
 Expansion in hybrid maize production in Eastern and Southern
Africa occurred beginning from the 1960s and 1970s
 Hybrid maize
 The finest and most productive innovation in plant breeding

 It started a revolution in
 Agricultural productivity

 Uniformity of products

 Fixing specific traits that serve as trade marks

 Permitted the production and marketing of seed

 Breaking the organic link between the production of
seed and grain

 Catalyzed the establishment of a specialized business (private

sector)
Why are hybrids important?
 Hybrids allow optimal exploitation of heterosis that can enable breeders to
create superior cultivars with high yield potential
 By mixing the genes of two or more parents
 To enhance productivity to meet the increasing demand for food

 Heterosis occurs when a hybrid exhibit phenotypic characteristics that are

superior to
The average of the two parents (mid-parent heterosis)
Or the better of the two parents (high parent heterosis)

 Heterosis (hybrid vigor) is manifested in increased size, yield, and

performance resulting from heterozygosity created in hybrids
 Hybrid performance and heterosis
Measurement of heterosis
Mid-parent heterosis (%) = [(F1-MP)/MP] x 100

High-parent heterosis (%) = [(F1-HP)/HP] x 100

 F1 = performance of the hybrid
 MP = Average performance of the parents of the hybrid
 HP = Performance of the best parent of the hybrid

 Heterosis is expressed when two genetically unrelated parents are

crossed to create a hybrid

 Heterotic response (Σdy2) is realized when we have differences in gene

frequencies between the parents (y2) for alleles having dominance
effects (d)
 Underlying genetic basis of heterosis
The genetic bases of heterosis has been a focal point of continuous
debate for nearly 100 years

The earlier theories proposed as mechanism of heterosis were based on

quantitative genetic models
 Dominance hypothesis, overdominance hypothesis and epistasis

The advances made in molecular genetics has renewed interest that

sparked extensive studies of the genetic basis of heterosis

The concepts of dominance and overdominance were based on different

genetic phenomenon but led to similar expectations
 Inbreeding leads to a reduction in vigor while crossing leads to
increased vigor
 The decline in vigor is proportional to the decrease in heterozygosity
 Underlying genetic basis of heterosis
1.Dominance hypothesis
Heterosis results from masking the expression of deleterious recessive alleles
in presence of beneficial dominant alleles in the hybrid from the two parents
 The hybrid formed from such parents exhibits superior characters due to
the contribution of dominant alleles from one parent

Line 1 Line 2
Parental genotype aaBBccDD x AAbbCCdd

Phenotypic value-Line 0+5+0+5=10 5+0+5+0=10

Hybrid AaBbCcDd

Phenotypic value-Hybrid 5+5+5+5=20

Note that AABBCCDD=20
Assumption: AA= Aa and AA>aa, homozygous dominant contributes 5 unit to
the phenotype while homozygous recessive contributes 0 to the phenotype
 Underlying genetic basis of heterosis
2. Overdominance hypothesis
Heterozygosity results in greater catalysis of metabolic pathways that lead
to increased growth and performance of the hybrid over either of its parents

Line 1 Line 2
Parental genotype AAbbCCdd x aaBBccDD

Phenotypic value-Line 5+0+ 5+0=10 0+5+0+5=10

Hybrid AaBbCcDd

Phenotypic value-Hybrid 8+8+8+8=32

Assumption: Aa > aa or AA, dominant genotype contributes 5 units to the

phenotypes, recessive genotype contributes 0 unit to the phenotype, and
heterozygote genotype contributes 8 units to the phenotype
 Underlying genetic basis of heterosis

3. Epistasis
 Interaction between alleles at different loci may also
contribute to heterosis
Number of Internode
Genotype nodes length Height

Line 1 6 2 12

Line 2 2 6 12

Hybrid (add x add) 4 4 16

Hybrid (dom x dom) 6 6 36

 Underlying genetic basis of heterosis
Hallauer (2007)
 Since a hybrid is heterozygous across all genetic loci that differ
between the two parents, the level of heterosis depends on:
The interaction of alleles at the same loci (partial dominance,
dominance, overdominance)
Interaction between alleles at different loci (epistasis)

 Each hybrid is a unique cross between two or more parents

(Inbred, partially inbred, or non-inbred)
Having a unique combination of alleles from its parents

The relative importance of additive, dominant, and epistatic

effects will thus be different for each hybrid
 How do we form hybrids?
1. Crossing two or more unrelated inbred lines

2. Crossing an open-pollinated variety, or a synthetic

to an unrelated inbred line

3. Crossing two unrelated open-pollinated varieties,

or synthetics, or populations
 What are the properties of parents of maize hybrids?
1.Inbred lines
 A homozygous and homogenous parent of hybrid maize
 Homozygous – A genotype with Identical alleles present at a
gene locus on each of the two homologous chromosomes that
control a particular trait
A A A A A A
b b b b b b
C C C C C C

 Homogenous – An inbred line is homozygous at almost all

gene loci and has a uniform population of true breeding plants
Importance of homozygosity and homogeneity
 The inbred lines with desirable traits can be
maintained indefinitely without major genetic changes

 Seed company can duplicate the exact characteristics

of a particular hybrid every time it produces seeds
from the parental inbred line
What are the properties of parents of maize hybrids?
2. Open pollinated varieties, 3. Synthetics and 4. Populations
 These parents consist of individual plants that are both heterozygous
and heterogeneous
 Heterozygous – Each plant contains two different alleles at each
locus
A a D d G g J j
B b E e H h K k
C c F f I i L l

 Heterogeneous – A mixture of individual plants with different

alleles at each locus
 Major steps in hybrid maize development

1. Classifying maize inbred lines into heterotic

groups

2. Testing inbred lines in hybrid combinations

3. Evaluate the potential of selected best inbred

lines for developing different types of hybrids

4. Testing and identifying superior hybrids for

commercial seed production and use by farmers
 1. Classifying inbred lines into heterotic groups
 To exploit heterosis effectively for increasing productivity of
hybrids, inbred lines should be separated into heterotic groups

 Parental crosses within a heterotic group result in low level of

heterosis

 Greater heterosis is obtained by crossing lines from

divergent heterotic groups

 Heterotic groups represent broad but divergent germplasm

 The number and choice of heterotic groups are arbitrary decisions

 Two or many
Advantages of separating inbred lines into heterotic groups
 Heterotic pattern provide defined structures to breeding
materials

 Simplify management of germplasm

 Provides suitable tester for assessing the breeding value of

exotic germplasm

 Facilitate selection of parents for developing

 Bi-parental crosses for inbred line development (same

heterotic group)

 Hybrid combinations for testing (opposite heterotic groups)

 Dividing breeding materials into heterotic groups
1. Small numbers of lines
 Evaluate diallel crosses of limited number of genotypes (GCA and SCA)
 Impractical for a large number of genotypes

2. Large numbers of lines and testers are available

 Evaluate testcrosses with testers chosen from opposite heterotic groups
 Significant and negative SCA with tester B and positive SCA with tester A
is assigned in one group (A)

 Significant and negative SCA with tester A and positive SCA with tester B
are assigned in second group (B)

 Lines that combine well with the two testers, which cannot fit into the two
heterotic groups
 Can be maintained as a separate group
 The breeder can decide where they should be placed
Dividing breeding materials into heterotic groups
3. Large number of lines and no established testers
 Group germplasm based on genetic similarity
 DNA markers
 Pedigree Information
 Make factorial or diallel crosses among lines representing different
 Clusters
 Ancestry (pedigrees)
 Use testers
 Initial testers can be inbred parents of hybrids with proven
performance in your breeding program
 Use established testers from other breeding program
 Adapted populations isolated by time and space are promising
candidate testers to define heterotic groups
 Testcross evaluation in multiple locations in a single season would provide
adequate data to classify the lines into heterotic groups
 Dividing breeding materials into heterotic groups
 All inbred lines included in a particular heterotic group
may not be related

 Sub-groups within a heterotic group should be used

for inbred recycling

 We should use inbred lines from the opposite heterotic

groups to improve defects of a line in another group

 Heterotic groups are not constant

 The genetic composition changes over time
 2. Testing inbred lines in hybrid combinations
 Goal of inbred line development is to identify lines
that produce high-yielding hybrids

 The correlation between traits of the inbred lines

and their hybrids is weak
 Empirical and simulation studies show
correlations of less than 0.40

 Inbred lines should thus be tested in crosses to

identify promising parents of productive hybrids
 2. Testing inbred lines in hybrid combinations
 Testing a large number of inbred lines in all possible
combinations is not practically feasible

 Formula for all possible single crosses – n(n-1)/2

 n = 10, 45 hybrids

 n = 20, 190 hybrids

 n = 100, 4950 hybrids

 n = 500, 124750 hybrids

 It is necessary to identify promising lines with good combining

ability before their extensive evaluation in hybrid combinations
 2. Testing inbred lines in hybrid combinations

Cross all inbred lines to form limited number of hybrids for

testing
 New inbred lines are crossed to a common parent
referred to as a tester

 This approach is called top-cross or testcross

evaluation

 With such a common tester parent, any difference in

hybrid performance can be ascribed to differences in
the combining ability of the inbred lines
 2. Testing inbred lines in hybrid combinations
What is a desirable tester?
 Allows differentiation of lines for combining ability and desirable
agronomic traits
 Permits identifying productive hybrids for direct use
 Simple to use in a breeding program

Choice of tester
1. Type of gene action
 Broad-based tester (open pollinated varieties, synthetics,
populations, composites and double-crosses)
 Presumed to provide test for additive effects (GCA)

 Narrow-based testers (Inbred line and single-cross)

 Provide test for non-additive effects (SCA)
 Studies have shown that inbred testers provide tests for
both GCA and SCA
Choice of tester
2. Type of hybrid product
 Use inbred testers to identify lines that form superior single crosses hybrids
 Use single cross tester to identify lines that form superior 3-way cross and
double-cross hybrids
 Use a non-inbred tester for identifying lines that form superior non-
conventional hybrids

Crossing to an inbred tester-Currently most commonly used line

 Cross all inbred lines to an inbred tester (testers)
 Evaluate agronomic performance of testcrosses in a few locations using two
replications

Crossing to a broad-based tester-Used in developing courtiers

 Cross all inbred lines to an open-pollinated variety
 Evaluate agronomic performance of top-crosses in a few locations using two
replications
3. Evaluating the best lines to form different types of hybrids
Once few promising lines are identified based on the results of
testcross performance, they can be used in the production of single-
cross, three-way and double-cross hybrid
 Number of single crosses = n(n-1)/2
 Number of three-way crosses = (n(n-1) x (n-2))/2
 Number of double crosses = (n(n-1) x (n-2) x (n-3))/8

Example for 25 promising inbred lines

 Single crosses = 300
 Three-way crosses =6900
 Double crosses = 37950
The number of three-way and double crosses are too large for
evaluation
Predictive formula developed by Jenkins (1934)
 Three-way crosses = 0.5 {(P1 x P3) + (P1 + P4)}
 Double crosses =0.25{(P1 x P3) + (P1 + P4) + (P2 +
P3) + (P2 + P4)}

Evaluate agronomic performance of the selected best

hybrids based on predicted performance in few locations
with two replications
3. Evaluate the best inbred lines to produce different
types of hybrids
Double-cross hybrid
AxB CxD

Heterotic group A Heterotic group B

(A x B) x (C x D)
Three-way cross hybrid
AxB C

Heterotic group A Heterotic group B

(A x B) x C
 3. Types of Maize Hybrids
3.1. Conventional Maize Hybrids (Involve inbred parents)
 Single Cross (A x B)
 Three-Way Cross ((A x B) x C)
 Double-Cross ((A x B) x (C x D))

3.2. Non-Conventional-Low Cost Hybrids (Involve one or two

inbred parents)
 Top-Cross (Variety x Inbred)
 Double Top-Cross (Single Cross x Variety)
 3. Types of Maize Hybrids
3.3. Non-Conventional-Low Cost Hybrids (Do not involve
inbred parents)

 Variety cross (Variety A x Variety B)

 Synthetic cross (Synthetic A x Synthetic B)

 Population cross (Population A x Population B)

 Family cross (Half-Sib A x Half-sib B or Full-sib A x

Full-sib B)
 3.1. Conventional Maize Hybrids
3.1.1. Single-Cross Hybrid
 A single-cross hybrid is a cross between two unrelated lines (A x B)
Advantages
 Provide better opportunity for expression of heterosis
Offers a particular advantage over other hybrids in favourable
production environments

 A field planted with single-cross seed is impressive because plant

height, ear height, tasseling, silking, pollen shedding and all other
characteristics are extremely uniform

 Because of the availability of productive female parents, single-cross

hybrids represent almost 90% of hybrid corn seed market in the USA
3.1.1. Single-Cross Hybrid
Advantages
 Only two inbred parents are involved, a higher level of resistance to
diseases, insects and unfavourable weather is evident
 Production and maintenance of two parental lines is easier

Disadvantage
 The female parental linen is usually low yielding resulting in high cost
of seed production and the price of seed
 If any component is susceptible, it will affect all of the single-cross
plants, resulting in lower performance
 Pollen shed occurs during a shorter period since all the plants are
genetically alike, with the potential for lower yields, especially under
stress conditions.
3.1.2. Modified Single-Cross Hybrid
 A modified single-cross hybrid is formed by crossing an Fl hybrid
between two related inbred lines, A x A’, as a female to an unrelated
inbred line, C, as a male parent (A x A’) x C)

Advantages
 Use of an F1 hybrid between related lines as a female reduces the
cost of seed production and the price of seed to growers

Disadvantage
 Requires the production and maintenance of three parental inbred
lines
3.1.3. Three-Way Cross Hybrid
 This is a cross between a single-cross (A x B) hybrid as a seed parent
and an unrelated inbred line (C) as a pollen parent (A x B) x C)

Advantages
 Lower cost of production and price to growers due to the high yield of
the single-cross hybrid used as a seed parent

Disadvantage
 The hybrid is less uniform in height and other traits than the single-
cross hybrid
 As the pollinator is an inbred parent, this may add some cost to seed
production
 Requires the production and maintenance of three parental inbred lines
3.1.4. Modified Three-Way Cross Hybrid
 This is a cross between a single-cross (A x B) hybrid as a seed parent
and a single-cross (C x C’) between related lines as a pollen parent (A x
B) x (C x C’)
Advantages
 Use of an F1 hybrid between related lines as a male produces large
quantity of pollen, which ensure good seed setting
 Use of an F1 hybrid between unrelated lines as a female can significantly
reduce cost of seed production and price of seeds
Disadvantage
 The hybrid is less uniform in height and other traits than the three-way
cross hybrid
 Requires coordinated production of two hybrids for making the modified
three-way cross hybrid
 Requires the production and maintenance of four parental inbred lines
3.1.5. Double-Cross Hybrid
 Produced by crossing two different single crosses [(A x B) x (C x D)],
permitting breeders to bring more different desirable traits together into
one hybrid than is possible in a single cross

Advantages
 The plants may be “buffered” more against unfavorable situations,
which frequently occur at one or more times during the growing season
 Use of different F1 hybrids as both female and male parents produces
abundant pollen and plenty of seeds and thus reduces the cost of
production and price of seeds
 Double-cross plants also have a longer pollination period, which tends
to provide more complete filling of the ear with seed, often resulting in
higher yields.
3.1.5. Double-Cross Hybrid

Disadvantage

 The hybrid is less uniform in height and other traits than

the three-way cross hybrids

 Requires coordinated production of two hybrids for

generating the double-cross hybrid

 Requires the production and maintenance of four

parental inbred lines
 3.2. Non-Conventional-Low Cost Hybrids
3.2.1. Top-Cross Hybrid
 It is a crossing between an open-pollinated variety (OPV) or synthetic
(SYN) and an inbred line (OPV x A or SYN x A).

Advantages
 Use of an open-pollinated variety as a female produces plenty of seed
and thus reduces the cost of production and price of seeds to growers
 The inbred parent harvested from detasseled production fields can be
used as a male parent, allowing the seed company to multiply only the
female parent in a separate field.

Disadvantage
 The hybrid is not very uniform for the different traits
3.3.1. Variety-Cross, Synthetic-cross and Population-cross Hybrids
 It is produced by crossing tow unrelated open-pollinated varieties,
synthetics or populations (VAR A x VAR B or SYN A x SYN B or POPA x
POPB).

Advantages
 Use of an open-pollinated variety (synthetic) as a male produces abundant
pollen that enhances seed setting
 Better synchrony between tasseling and silking
 Use of an open-pollinated variety (synthetic) as a female produces plenty
of seeds and thus reduces the cost of production and price of seeds

Disadvantage
 The hybrid is the least uniform and productive among the group
4. Performance evaluation of hybrids formed from inbred lines
Stage 1: Performance evaluation of a large number of hybrids formed
from crosses of S4 lines with one tester
 In two replications at selected few locations
 Select the best lines for further inbreeding and evaluation with
testers

Stage 2: Performance evaluation of hybrids formed by crossing

inbred lines selected in Stage 1 to three testers
 In two to three replications at 4 to 6 locations
 Select the best lines for further evaluation with few testers

Stage 3: Performance evaluation of hybrids formed by crossing

inbred lines selected in Stage 2 to three single-cross testers
 In three replications in 6 to 8 locations
 Select promising hybrids for testing in NPT and on-farm trials
4. Performance evaluation of hybrids formed from inbred lines
NPT and On-farm testing
 Promising hybrids identified in Stage 3 are further tested in NPT
and on-farm trials

 Identify broadly adapted and superior hybrids for registration

and release

 Generate the requisite data for variety release

 Start multiplying seeds of parental materials for the production

and marketing of superior hybrids
 5. Important considerations in selecting suitable hybrids
for production and marketing
5.1. Target Production and Marketing Environment and Farmers Needs
 Adaptation to prevalent climatic conditions

 Resistance to major diseases and insect pests

 Resistance to parasitic plants

 Tolerance to drought and suboptimal soil nitrogen

 Resistance to stalk and root lodging (good standability)

 Cost of seed production and purchasing power of the growers

5.2. Stage of Development of the Private Sector and Other Seed
Producers to Multiply, Harvest, Process, Store and Market

 Emerging indigenous companies – Low cost hybrids

 Established seed companies –Single-cross and three-way

cross hybrids

 Multi-national and other companies with established breeding

programs – Genetically modified and conventional single-cross
and other hybrids based on niche markets
5.3. Maturity Groups Appropriate to Target Market Niche
Short-Season Hybrids (Early)
 Areas with short growing seasons

 In all production zones where planting is delayed

due to shift in the start of the rain

Full-Season Hybrids (Intermediate-Late)

 Areas with long growing seasons and adequate

rainfall
5.4. Productive Capacity of the Seed and Male Parent
Grain yield of the female seed parent determine the
cost of production of hybrid seed
 Use female parents that are high yielding

Choose male parents with good pollen production

capacity and high yield potential
 Summary
Inbred-hybrid breeding method
 Facilitates precise identification of productive hybrids for on-farm
testing and commercialization

 Allows the development of hybrids that meet the demands of the

consumers fairly quickly

 Permits staking of multiple traits into a single cultivar

 Increases gain in yield potential through improvements in

defensive traits
 Summary
Provide Flexibility to Seed Producers in Choosing Hybrids
 Critically look at the hybrid types most appropriate to
prevalent diverse circumstances to the producer and
grower
 Concentrate on hybrids involving parents that
 Can be managed easily in terms of maintenance of
parents and seed production
 Allow you to harvest large quantity of seeds from the
female parent at reasonable cost to reduce the price
of seeds to farmers
Thank You