1. Plant Breeding Lecture 3

2. Objectives Know essential terminology
Know some examples of traits targeted by breeding for genetic improvement of crops
Understand the foundations of plant breeding
Know breeding techniques

3. Gamete
A mature reproductive cell that is specialized for sexual fusion
Haploid (n)
Cells that have only one set of chromosomes (n). Each gamete is haploid
Cross
A mating between two individuals, leading to the fusion of gametes and progeny
Diploid (2n)
Cells with two copies of each chromosome. The diploid state is attained by the fusion of two gametes
Zygote
The cell produced by the fusion of the male and female gametes

4. Gene
The inherited segment of DNA that determines a specific characteristic in an organism
Locus
The specific place on the chromosome where a gene is located
Alleles
Alternative forms of a gene

5. Genotype
The genetic constitution of an organism
Homozygous
An individual whose genetic constitution has both alleles the same for a given gene locus (i.e., AA or aa)
Heterozygous
An individual whose genetic constitution has different alleles for a given gene locus (i.e., Aa)

6. Homogeneous
A population of individuals having the same genetic constitution (e.g., a field of pure-line soybean or a field of hybrid corn)
Heterogeneous
A population of individuals having different genetic constitutions
Phenotype
The physical manifestation of a genetic trait that results from a specific genotype and its interaction with the environment

7. Trait goals Yield = seeds, biomass, or fruit size/number
Quality traits, such as oil, flavor, color
Stress tolerance, e.g., drought
Insect and disease resistance
Introgressing transgenes into plant varieties

8. Yield
Figure 3.5 Yield of hybrid corn varieties versus year of release. Data were obtained from Duvick and Cassman (1999), based on field experiments conducted at a plant density of 79,000 plants per hectare at three locations in central Iowa in 1994.

9. Quality trait: oil quality
Hydrogenation: flavor and oxidative stability
Trans fats: health issues
FDA label mandate
cis form
saturated
trans form
H H
C C H
C C
H H
C C
Hydrogenation ;
(Source: Wilson, 2004)

10. Environmental Stress Tolerance

11. Soybean sudden death syndrome
Insect and disease resistance
Soybean sudden death syndrome

12. Deployment of transgenic traits (e. g
Deployment of transgenic traits (e.g., transfer of herbicide resistant genes in commercial varieties)

13. Foundations of plant breeding
Importance of genetic variation and selection

14. What are the causes of biological variation observed in plants?
1. Genetic causes (mode of inheritance)
single genes
multiple genes
2. Environmental
3. GxE: the interaction between the genotype of the plant and the environment in which it grows

15. Phenotype vs. Genotype P = G + E + (GxE)
P is called the phenotypic value, i.e., the measurement associated with a particular individual
G is genotypic value, the effect of the genotype (averaged across all environments)
E is the effect of the environment (averaged across all genotypes)

16. Genetic variation: the basis for improvement

17. If we could measure P in all possible environments and regard E as a deviation, then the mean of E would be zero and P = G.
The genotype responds more strongly in some environments.
Sets of environments tend to shift the trait value in one direction, other environments in a different direction.

18. Figure 3.8
Figure 3.8 In Sewall Wright’s shifting balance theory, a genotype or population is defined by coordinates in N-dimensional space, and a fitness value forms a surface in the (N + 1)th-dimension. Here, genotype coordinates are defined in two dimensions on the ground beneath a mountainous fitness surface (the third dimension). The coordinates of a given population can be changed by selection, but only in small increments. Direct selection tends to move a population toward coordinates where fitness is highest, but that may be only a local peak. Applying this concept to plant breeding, we see that to find genotypes beneath a global peak, we need to create and explore a large genotype space (i.e., genetic diversity) or to know exactly where we are going (an ideotype).

19. Methods and strategies: when and why each is useful
Typically the goal is cultivar production

20. How complex is selection?
Qualitative traits, simple inheritance, controlled by major genes
Quantitative traits, complex inheritance controlled be several gene loci

21. Qualitative traits Classified into discrete classes
Individuals in each class counted
Some environmental influence on phenotype
Controlled by a few (<3) major genes

22. Mendel’s seven traits showing simple inheritance
Figure 2.3
Often single gene traits are easy to see or measure, since environment typically has limited control over their expression
Tawny (TT or Tt) versus gray (tt) single gene locus on soybean chromosome 6

23. F1 Hybrid Plants: 100% yellow
Figure 2.4.
A. Monohybrid Cross
B. F1 Self Fertilization =
Parent 1
Parent 2
Parent 1
Parent 2 X X YY yy Yy Yy
Gametes: Y Y y y
Gametes: Y y Y y
F1 Fertilization:
F2 Fertilization: YY Yy yy Y y
Parent 1
Parent 2 Yy y Y
Parent 1
Parent 2
YY & Yy Yy
F1 Hybrid Plants: 100% yellow
F2 Plants: 75% yellow
25% green yy

24. Gene and Genotype Frequencies Example: Self pollinated diploid species
Upon selfing F2 population; 25% homozygous ‘YY’ will produce only ‘YY’ genotypes, and 25% homozygous ‘cc’ will produce only ‘yy’ genotypes. So only ‘Yy’ will segregate to produce genotypes in proportion of 0.25 (YY):0.50:
(Yy):0.25(yy).
F2 population:
0.25(YY ) 0.50 (Cc) 0.25 (cc ) YY Yy Yy yy
0.25
0.50
0.25
Produce all CC plants
Segregate into 0.25(CC ) 0.50% (Cc) and 0.25 (cc)
Produce all cc plants
Resulting F3 population will have
½ (0.25) = CC plants
½ (0.50) = 0.25 Cc plants
½ (0.25) + (0.25) = cc plants

25. Heterozygosity reduced by half in each selfing generationYY Yy yy F2 F3 F4
25% F5 F6 F7
50%
43.75%
46.88%
12.5%
48.44%
6.25%
49.22%
3.135
49.61%
1.56 F8
37.5%
0.78%
When should we select?

26. Questions based on F5 single plant derived progeny rows from one population formed from crossing two pure line parents:

27. Selfing a double het (AaBb × AaBb) produces a 9:3:3:1 phenotypic ratio only if trait governed by complete dominance
Freq
Genotype
1/16
AABB
2/16
AABb
AAbb
AaBB
4/16
AaBb
Aabb
aaBB
aaBb
aabb
Phenotypic Ratio
Underlying Genotypes 9
AABB = AABb =
AaBB = AaBb 3
AAbb = Aabb
aaBB = aaBb 1
aabb
Note: only 1 out of 16 is homozygous favorable allele for both gene loci

28. Selfing a double het (AaBb × AaBb) produces 9 genotypic classes
Freq
Genotype
No. of CAP alleles
1/16
AABB 4
2/16
AABb 3
AAbb 2
AaBB
4/16
AaBb
Aabb 1
aaBB
aaBb
aabb
Figure 3.1
Freq
No. of CAP alleles 1 4 6 2 3

29. Quantitative traits Express continuous variation (normal distribution)
Individuals measured, not counted
Significant environmental influence on phenotype
Controlled by many minor (or major) genes, each with small (or large) effects

30. X
aa, BB
(6 kg)
AA, bb
(6 kg)
Aa, Bb
(6 kg)
Note: Consider upper case letter represents the favorable allele for each gene
Self-pollinate
4 kg:
aa, bb
5 kg:
Aa, bb (x2)
aa, Bb (x2)
6 kg:
Aa, Bb (x4)
AA, bb
aa, BB
7 kg:
Aa, BB (x2)
AA, Bb (x2)
8 kg:
AA, BB
Histogram depicts dominant genotype effect with yield: “capital” alleles (0, 1, 2
Figure 3.1

31. High yielding low-phytate parental lines is the goal
Frequency distribution of seed yield for 187 different recombinant inbred lines (RIL) in the soybean population 5601T x Cx (Scaboo et al., 2009) [no transgressive segregates for this trait in this population]
5601T = 3252 Cx
High yielding low-phytate parental lines is the goal

32. 15/16 7/8 3/4 1/2 (15/16)20 (7/8)20 (3/4)20 (1/2)20 = [1-(½)G]L
Adapted from Allard, 1999
Then find the better individuals among the homozygous plants (those accumulating the greatest number of superior alleles). Can be done with DNA technologies and progeny row testing.
15/16
7/8
3/4
1/2
(15/16)20
(7/8)20
(3/4)20
(1/2)20
Even if 20 genes are involved, using the power of inbreeding 5 generations, over half the proportion of individuals will be completely homozygous!

33. The effect of reproductive behavior

34. Reproductive Behavior
Self pollinated
Pure line variety
Hybrid variety
Monoecy
Cross pollinated
Dioecy
Self-incompatible
- Synthetic variety – heterogeneous population (not a pure line)
- Hybrid variety, if inbred development is possible
Perfect flower
Clonal variety
Hybrid
No flowering/limited flowering
Vegetative reproduction

35. Figure 3.9
Figure 3.9 The pedigree breeding method is used in self-pollinated species to derive pure-line varieties when it is desirable to practice selection in early generations.

36. Cultivar development for self-pollinated species: pedigree method
Cultivar, local or exotic landraces, wild relatives
Germplasm
Hybridization
Parents are usually inbred
F1 Nursery, all plants heterozygous
Homogeneous population if parents were inbred
F2 Nursery, all plants heterozygous
Every single plant is a different genotype
F3: head rows
Select the best rows, select best plant within selected rows, proceed to F4 head rows
This is typical pedigree method of selection in self-pollinated crop. Each head row is called line. Most F6 or F7 lines are uniform enough for preliminary yield testing

37. Cultivar development for self-pollinated species: bulk method
Cultivar, local or exotic landraces, wild relatives
Germplasm
Hybridization
Parents are usually inbred
F1 Nursery, all plants heterozygous
Homogeneous population if parents were inbred
F2 population, all plants heterozygous
Collect equal amount of seed from each plant
F3: bulk population
Repeat one or two more generation, then follow head rows
This is bulk method of breeding self-pollinated crop. Most F6 or F7 lines are uniform enough for preliminary yield testing. This is less resource consuming.

38. Figure 3.10
Figure 3.10 The single-seed descent (SSD) breeding method is used in self-pollinated species to derive pure-line varieties when it is desirable to select from random homozygous lines in an advanced generation.

39. Backcross breeding and recurrent selection
Figure 3.11 The backcross breeding method is used to transfer alleles at a small number of loci from a donor parent into the genetic background of a reciprocal parent. Each generation of backcrossing reduces the proportion of alleles from the donor (D) parent by half (), as shown on the right.

40. Used extensively in cross-pollinating crops
Figure 3.13 An example of a recurrent selection strategy with progeny testing. Many variations on this type of strategy have been devised.

41. Figure 3.14 Schematic simplification of the development of a synthetic plant variety in an outcrossing species. The Syn-1 generation is produced by random mating of reproducible components (inbred lines or clones). If it is found to be desirable as a new plant variety, it can be reproduced and sold by repeating the identical crossing block. This type of breeding method is most practical in a perennial forage species. If adequate seed cannot be produced in Syn-1 generation, the Syn-2 generation (harvested from Syn-1) may be used instead.

42. Figure 3.15 Schematic simplification of the development of a hybrid plant variety. In corn, the parents (i.e., A, B, and C) are inbred lines that have been derived through other breeding methods. In other crops, the parents may be clonally propagated. Parents are grown in adjacent rows for crossing, and the female parent is emasculated so that it will not self-pollinate. Seed harvested from the female parent is tested in performance trials. If a hybrid variety is successful, the cross is repeated on a large scale for commercial production.

43. BC1F1 75 % BC2F1 87.5 % BC3F1 93.5 % BC4F1 96.9 % BC5F1 98.4 %
With Traditional Backcross Breeding
Year 1 F1 50 %
BC1F1 75 %
BC2F %
BC3F %
BC4F %
BC5F %
BC6F % Year 7
With traditional backcrossing you increase the amount of your recurrent parent each generation and it takes 6 generations to recover 99% of your chosen parent.
With the use of molecular markers we have already been able to speed up this process. Notice that these calculated values show that the BC1F1 should contain on average 75% of the desired line. A BC1F1 is produced by using the Recurrent line 5601T as the female and using the F1 as the donor or pollen source. A BC2F1 is produced by using the Recurrent line 5601T as the female and using the BC1F1 as the donor or pollen source.

44. Molecular markers allow visualization of genotypesRR RR RR rr rr rr rr Rr
Gel electrophoresis of DNA markers: we can now ‘see’ genotypes to accelerate breeding cycles

45. Figure 3.17
Figure 3.17 Visualization of SNP markers on chromosome-1 for a set of soybean varieties. Each column represents a locus position on the chromosome, and each row represents a different soybean variety. Most loci have two alternate alleles, which are colored to represent the DNA base present in a homozygous state in the corresponding soybean variety. The predicted value of each allele is determined by testing a reference population where phenotypes are known, A predicted genotypic value of each soybean variety is then derived as a summation of predicted allele values, and varieties with the highest overall genotypic values are selected.

46. Other breeding methods
Vegetative reproduction and apomixis
Mutation breeding

47. Summary
Plant breeding mostly uses sexual crosses to recombine genes and alleles in crops
Goal: usually a cultivar with improved traits
Selfing crops are improved using the pedigree method or bulk methods, such as single-seed descent
Recurrent selection and backcrossing is a useful tool, especially for outcrossing crops
Synthetic varieties and hybrids are the product of breeding outcrossing species
Mutation, vegetative cloning and apomixes are sometimes used in breeding
Marker assisted selection can speed up breeding cycles

48. Figure 3.17
Figure 3.17 Visualization of SNP markers on chromosome-1 for a set of soybean varieties. Each column represents a locus position on the chromosome, and each row represents a different soybean variety. Most loci have two alternate alleles, which are colored to represent the DNA base present in a homozygous state in the corresponding soybean variety. The predicted value of each allele is determined by testing a reference population where phenotypes are known, A predicted genotypic value of each soybean variety is then derived as a summation of predicted allele values, and varieties with the highest overall genotypic values are selected.