Plant Breeding Methods

1

Types of Cultivars and Modes of Plant Reproduction

Introduction

🧭 Overview

🧠 One-sentence thesis

Cultivars are classified into four types—clonal, synthetic, pure-line, and hybrid—based on their mode of reproduction, which determines whether the plants in a commercial field are genetically identical (homogeneous) or genetically different (heterogeneous).

📌 Key points (3–5)

  • Homogeneity vs. heterogeneity: cultivars are homogeneous when all plants are genetically and phenotypically identical, and heterogeneous when plants are genetically different.
  • Four cultivar types: clonal (asexual), synthetic (open pollination), pure-line (self-pollination), and hybrid (crossing different parents).
  • Genetic uniformity varies by type: clonal and pure-line cultivars produce homogeneous fields; synthetic and most hybrid cultivars produce heterogeneous fields.
  • Common confusion: heterozygosity (within one plant) vs. heterogeneity (across plants)—clonal cultivars are homogeneous (all plants identical) but each plant is heterozygous (no inbreeding).
  • Seed vs. vegetative reproduction: clonal cultivars use vegetative propagation (cuttings, tubers, grafts); the other three types use seed, but differ in pollination method.

🌱 Clonal cultivars

🌱 What clonal cultivars are

Clonal cultivars: reproduced asexually from a single plant that the breeder has selected.

  • All plants in a clonal cultivar are genetically identical (homogeneous).
  • Reproduction is vegetative, not by seed (except apomixis, which is asexual seed production).

🧬 Genetic structure

  • Each individual plant is heterozygous because no inbreeding is involved in developing the population for selection.
  • Don't confuse: the cultivar is homogeneous (all plants are clones of one another), but each plant itself is heterozygous (carries different alleles at many loci).

🔧 Propagation methods

Methods of clonal propagation include:

  • Cuttings
  • Tubers
  • Bulbs
  • Rhizomes
  • Grafts
  • Buds
  • Seed produced through apomixis (asexual seed formation)

Example: A breeder selects one superior plant and propagates it vegetatively; all commercial plants are genetically identical copies of that original plant.

🌾 Seed-based cultivar types

🌾 Synthetic cultivars

Synthetic cultivars: seed is produced sexually by open pollination.

  • Open pollination means pollen can come from any plant in the population.
  • As a result, plants in a commercial field are heterozygous and heterogeneous (genetically different from one another).

🌾 Pure-line cultivars

Pure-line cultivars: seed is produced by self-pollination.

  • Self-pollination leads to homozygous individuals (nearly all loci are fixed).
  • Plants are genetically similar to each other, so the cultivar is homogeneous.
  • Seed harvested in one season is used to plant commercial fields the next season.

Example: Rice, wheat, and tobacco are examples of crops that use pure-line cultivars.

🌾 Hybrid cultivars

Hybrid cultivars: seed used for commercial planting is produced by crossing two genetically different parents.

  • Plants are heterozygous because they result from a cross.
  • New hybrid seed must be produced each year; seed harvested from a commercial field is not genetically the same as the seed planted.

🔀 Types of hybrids

Hybrid typeNumber of inbred linesGenetic uniformity in field
Single-crossTwo inbred parentsHomogeneous (all plants genetically the same)
Three-way crossThree inbred linesHeterogeneous (plants genetically different)
Double-crossFour inbred linesHeterogeneous (plants genetically different)

Don't confuse: all hybrid plants are heterozygous (within-plant genetic variation), but single-cross hybrids produce a homogeneous field (all plants identical), while three-way and double-cross hybrids produce heterogeneous fields (plants differ from one another).

📊 Summary comparison

Cultivar typeReproduction methodHomozygosity/Heterozygosity (within plant)Homogeneity/Heterogeneity (across plants)
ClonalAsexual (vegetative)HeterozygousHomogeneous
SyntheticSexual (open pollination)HeterozygousHeterogeneous
Pure-lineSexual (self-pollination)HomozygousHomogeneous
Hybrid (single-cross)Sexual (cross two parents)HeterozygousHomogeneous
Hybrid (three-way, double-cross)Sexual (cross multiple parents)HeterozygousHeterogeneous
2

Types of Cultivars and Modes of Plant Reproduction

Types of Cultivars and Modes of Plant Reproduction

🧭 Overview

🧠 One-sentence thesis

The four main cultivar types—clonal, synthetic, pure-line, and hybrid—differ fundamentally in their reproduction mode (sexual vs. asexual) and in whether individual plants are homozygous or heterozygous and homogeneous or heterogeneous.

📌 Key points (3–5)

  • Four cultivar types: clonal, synthetic, pure-line, and hybrid cultivars are the most common types used in commercial crop production.
  • Two reproduction modes: sexual reproduction (pollen nucleus unites with egg cell to produce seed embryo) vs. asexual reproduction (propagation from somatic tissue).
  • Homozygosity vs. heterozygosity: refers to individual plant genetics—same alleles at a locus (homozygous) vs. different alleles (heterozygous).
  • Homogeneity vs. heterogeneity: refers to relationships among plants in a cultivar—genetically and phenotypically identical (homogeneous) vs. genetically different (heterogeneous).
  • Common confusion: don't confuse individual plant genetics (homozygous/heterozygous) with population uniformity (homogeneous/heterogeneous)—a cultivar can have heterozygous individuals that are all genetically identical (clonal) or heterozygous individuals that differ from each other (synthetic).

🌱 Reproduction modes and genetic concepts

🌸 Sexual reproduction

Sexual reproduction: occurs when the nucleus of a pollen grain unites with the egg cell in the ovary to produce the embryo of a seed.

  • This mode produces seeds through fertilization.
  • Used in synthetic, pure-line, and hybrid cultivars.

🌿 Asexual reproduction

Asexual reproduction: the propagation of an individual from its somatic tissue.

  • No fertilization involved; plants are cloned from vegetative parts.
  • Methods include cuttings, tubers, bulbs, rhizomes, grafts, buds, and apomixis (seed produced without fertilization).
  • Used exclusively in clonal cultivars.

🧬 Homozygosity vs. heterozygosity (individual plant level)

Homozygous: when the alleles at a locus are the same. Heterozygous: when the alleles at a locus are different.

  • How to achieve homozygosity: self-pollination of individuals, which is routine in developing pure-line cultivars or inbred lines for hybrids.
  • How heterozygosity results: crossing plants with different genotypes, either by hand or through open pollination (wind or insects).
  • Example: a plant with AA or aa at a locus is homozygous; a plant with Aa is heterozygous.

🌾 Homogeneity vs. heterogeneity (population level)

Homogeneous: when plants in a cultivar are genetically and phenotypically identical. Heterogeneous: when plants in a cultivar are genetically different.

  • This describes the relationship among plants in the same cultivar, not within a single plant.
  • Don't confuse: a cultivar can be homogeneous (all plants identical) even if each individual plant is heterozygous (clonal cultivars).

🌳 Clonal cultivars

🧪 Definition and characteristics

Clonal cultivars: reproduced asexually from a single plant that the breeder has selected.

  • Genetic uniformity: all plants are genetically identical (homogeneous).
  • Individual genetics: each plant is heterozygous because no inbreeding is involved in developing the selection population.
  • Propagation methods: cuttings, tubers, bulbs, rhizomes, grafts, buds, and apomixis.

🍇 Why clonal plants are heterozygous

  • The original selected plant was not inbred, so it carries different alleles at many loci.
  • Asexual reproduction copies the entire genotype, preserving heterozygosity across all clones.
  • Example: a breeder selects one superior potato plant and propagates it via tubers—all resulting plants are genetic copies (homogeneous) but retain the original plant's heterozygous allele combinations.

🌾 Synthetic cultivars

🌬️ Definition and characteristics

Synthetic cultivars: seed is produced sexually by open pollination.

  • Reproduction mode: sexual, via open pollination (wind or insects).
  • Individual genetics: plants are heterozygous (result of crossing different genotypes).
  • Population uniformity: plants are heterogeneous (genetically different from each other).

🌻 Why synthetic cultivars are heterogeneous

  • Open pollination means many different pollen sources fertilize the ovules.
  • Each seed results from a different genetic combination.
  • Example: in an alfalfa field, wind carries pollen from many plants, so each seed has a unique combination of parental alleles—plants in the commercial field vary genetically.

🌾 Pure-line cultivars

🌾 Definition and characteristics

Pure-line cultivars: seed is produced by self-pollination.

  • Reproduction mode: sexual, via self-pollination.
  • Individual genetics: plants are homozygous (self-pollination increases homozygosity over generations).
  • Population uniformity: plants are homogeneous (genetically similar to each other).
  • Seed use: seed harvested in one season is used to plant commercial fields the next season.

🌾 Why pure-line plants are homozygous and homogeneous

  • Self-pollination means a plant's pollen fertilizes its own ovules.
  • Repeated self-pollination over generations fixes alleles (AA or aa), eliminating heterozygosity.
  • All plants in the cultivar descend from the same self-pollinated lineage, so they are genetically similar.
  • Example: rice or wheat cultivars—each plant self-pollinates, and all plants in the field are nearly genetically identical and homozygous.

🌽 Hybrid cultivars

🌽 Definition and characteristics

Hybrid cultivars: seed used for commercial planting is produced by crossing two genetically different parents.

  • Reproduction mode: sexual, via controlled crossing of distinct parents.
  • Individual genetics: plants are heterozygous (result of crossing different genotypes).
  • Population uniformity: depends on hybrid type—single-cross hybrids are homogeneous; three-way and double-cross hybrids are heterogeneous.
  • Seed renewal: new hybrid seed must be produced each year because seed harvested from a commercial field is not genetically the same as the planted seed.

🌽 Types of hybrids and their uniformity

Hybrid typeNumber of inbred lines usedHomogeneity/heterogeneity
Single-crossTwo inbred linesHomogeneous (all plants genetically the same)
Three-way crossThree inbred linesHeterogeneous (plants genetically different)
Double-crossFour inbred linesHeterogeneous (plants genetically different)

🌽 Why hybrid seed must be renewed annually

  • The commercial field plants are F₁ hybrids (first generation cross).
  • If farmers save and replant seed from the commercial field, the next generation will segregate (genetic variation increases), losing the uniform hybrid performance.
  • Example: corn hybrid seed is produced by crossing two inbred lines; the resulting F₁ plants are uniform and high-performing, but their seed (F₂) will vary widely in traits.

📊 Summary comparison of cultivar types

Cultivar typeReproduction modeIndividual plant (homozygous/heterozygous)Population (homogeneous/heterogeneous)Seed/propagation use
ClonalAsexualHeterozygousHomogeneousVegetative tissue reused
SyntheticSexual (open pollination)HeterozygousHeterogeneousSeed replanted
Pure-lineSexual (self-pollination)HomozygousHomogeneousSeed replanted
Hybrid (single-cross)Sexual (controlled cross)HeterozygousHomogeneousNew seed each year
Hybrid (three-way/double-cross)Sexual (controlled cross)HeterozygousHeterogeneousNew seed each year

🔍 Key distinction reminder

  • Individual genetics (homozygous/heterozygous) tells you about alleles within one plant.
  • Population uniformity (homogeneous/heterogeneous) tells you whether all plants in the cultivar are genetically identical or different.
  • Don't confuse: clonal cultivars are homogeneous (all plants identical) even though each plant is heterozygous (carries different alleles at loci).
3

Sources of Parental Germplasm

Sources of Parental Germplasm

🧭 Overview

🧠 One-sentence thesis

Breeders primarily use elite cultivars and experimental breeding lines as parents to create segregating populations for new cultivar development, supplemented by plant introductions when novel traits are needed.

📌 Key points (3–5)

  • Primary source: Elite cultivars and experimental breeding lines offer the best chance of producing superior offspring suitable as new cultivars.
  • Written permission is essential: Accessing germplasm from other breeders requires documented agreements; verbal agreements are insufficient and can lead to job loss or legal consequences.
  • Plant introductions (PIs): Managed by the National Genetic Resources Program, PIs provide novel genes for traits not available in elite materials.
  • Common confusion: Not all germplasm is freely accessible—exchange restrictions vary by institution, and proper documentation is critical to verify legitimate access.
  • Why it matters: The choice of parent germplasm determines the potential for developing cultivars with desired traits.

🌱 Elite germplasm as the foundation

🌱 Cultivars and elite breeding lines

Elite cultivars: established commercial varieties that represent the best available genetics for a species.

Breeding lines: experimental lines developed by breeders that have not yet been released as cultivars but show promising traits.

  • These are the primary source of parent germplasm for most breeding programs.
  • Crosses among elite parents have the best chance of producing offspring that:
    • Are superior to the parents
    • Will be useful as new cultivars
  • Example: A breeder crosses two high-yielding elite cultivars to create a population in which to select for even higher yield combined with disease resistance.

🤝 Accessing germplasm from other breeders

  • Breeders often need to access elite cultivars and breeding lines from:
    • Private companies
    • Public institutions
    • Other breeding programs
  • This access expands the genetic diversity and trait combinations available for crossing.

📝 Written agreements and legal requirements

📝 Why written permission is mandatory

The excerpt emphasizes that written permission is extremely important and that a verbal agreement is not sufficient. Four specific reasons are given:

  1. Authority confusion: The individual may not realize they lack authority to make the verbal agreement.
  2. Memory failure: The terms of a verbal agreement may be forgotten over time.
  3. Personnel changes: The individual with whom the verbal agreement was made may leave the company or institution.
  4. Increasing restrictions: The exchange of germplasm has become more restricted for both private and public institutions.

⚖️ Legal consequences

  • Institutions may require written documentation to verify that germplasm was properly obtained.
  • Serious consequences: Cases exist where use of parent germplasm without written permission has resulted in:
    • Loss of employment
    • Legal awards of millions of dollars
  • Don't confuse: This is not about courtesy—it is a legal and professional requirement with severe penalties for violations.

📄 Material transfer agreements

  • The terms of written agreements vary among private and public institutions.
  • Example: The Iowa State University Research Foundation uses a material transfer agreement (Figure 1 in the excerpt) that specifies the terms for exchanging parent germplasm.
  • The signed document must be retained in case questions arise about how the germplasm was received.

🌍 Plant introductions as supplementary sources

🌍 What plant introductions are

Plant introductions (PIs): germplasm acquired from other countries or from breeding programs in the United States, managed by the National Genetic Resources Program (NGRP).

  • The NGRP is responsible for:
    • Acquisition
    • Characterization
    • Preservation
    • Documentation
    • Distribution of germplasm
  • Available plant germplasm and acquisition methods can be found at www.ars-grin.gov.

🧬 Value of plant introductions

  • PIs have been a valuable source of novel genes for important traits, including:
    • Insect resistance
    • Disease resistance
    • Seed quality
    • Many other traits
  • When to use PIs: It is common for breeders to evaluate plant introductions when searching for a trait that is not available in elite cultivars or breeding lines.
  • Example: A breeder needs resistance to a new insect pest not found in current elite lines, so they screen plant introductions from regions where the pest is endemic.

💰 Access terms for plant introductions

AspectDetails
CostAvailable without charge at present
ObligationRecipients are asked to provide evaluation results to NGRP for the benefit of future users
PurposeSharing results helps others interested in the trait in the future
  • Don't confuse: PIs are freely available through NGRP, but germplasm from other breeders requires written agreements—the access rules differ by source.

🔄 The breeding workflow context

🔄 First step in cultivar development

  • The excerpt states that the first step in developing any cultivar is obtaining a segregating population in which to do selection.
  • In most cases, the breeder develops this population by crossing parents that have the potential to produce progeny with desired traits.
  • The purpose of understanding parent germplasm sources is to know where to find suitable parents for these crosses.

🎯 Strategic use of different sources

Source typeWhen to useAdvantage
Elite cultivars and breeding linesPrimary choice for most crossesBest chance of producing superior, commercially viable offspring
Plant introductionsWhen novel traits are neededAccess to genes not available in elite materials
  • The choice depends on breeding goals: routine improvement uses elite materials, while introducing new traits may require PIs.
4

Population Formation by Hybridization

Population Formation by Hybridization

🧭 Overview

🧠 One-sentence thesis

Plant breeders form populations for selection by crossing parents in different ways—single crosses, three-way crosses, backcrosses, and polycrosses—depending on whether they are developing clonal, pure-line, hybrid, or synthetic cultivars, with the number of hybrid seeds and crossing method determined by the genetic makeup (homozygous vs. heterozygous, homogeneous vs. heterogeneous) of the parents.

📌 Key points (3–5)

  • Single crosses differ by parent type: crossing two clonal parents (heterozygous, homogeneous) produces a segregating population directly; crossing two pure lines or inbreds (homozygous, homogeneous) produces uniform F₁ hybrids that must be self-pollinated to create a segregating F₂ population.
  • Three-way crosses and backcrosses are used when a single cross may not provide enough desirable progeny or when combining genes from more than two parents is needed.
  • Synthetic cultivar populations are more diverse and can be formed from cultivar per se, single crosses, three-way crosses, or polycrosses of selected clones.
  • Common confusion: the number of hybrid seeds needed depends on parent genetic makeup—clonal crosses need as many seeds as individuals to evaluate, but pure-line crosses need only enough F₁ plants to produce the desired F₂ seed.
  • Polycross purpose: used to cross selected clones to each other for developing segregating populations, evaluating combining ability, testing experimental synthetics, and producing breeder seed of new synthetic cultivars.

🌱 Single-cross populations for clonal, pure-line, and hybrid cultivars

🧬 Clonal parent crosses

Clonal cultivars and lines used in crosses are highly heterozygous and homogeneous.

  • What happens in the cross: the hybrid individuals obtained from a single cross represent a segregating population used directly to begin selection for superior new clones.
  • How many hybrid seeds are needed: equal to the number of individuals the breeder wants to evaluate as clones from the cross.
    • Example: if a breeder wants to evaluate 1,000 individuals from a single-cross population, at least 1,000 hybrid seeds would be produced.
  • Why parent plant number doesn't matter: because the parents are genetically homogeneous, any plant of a parent is genetically identical to any other plant of that parent.

🌾 Pure-line and inbred parent crosses

Pure lines or inbred lines of hybrids used as parents are homozygous and homogeneous.

  • What happens in the cross: the hybrids obtained from a single cross are heterozygous and homogeneous (uniform F₁).
  • Why F₂ is needed: to develop a segregating F₂ population, the hybrid F₁ plants must be self-pollinated.
  • How many hybrid seeds are needed: determined by the number of F₁ plants needed to produce the desired number of F₂ seed.
    • Example: if the breeder wants 1,000 F₂ seeds and each F₁ plant produces an average of 100 seeds, only 10 hybrid plants would be sufficient.
    • Accounting for germination and accidental self-pollination: assuming 80% germination and 10% accidental self-pollinations, 14 hybrid seeds would be desired (10 divided by 0.80 and 0.90).
  • Don't confuse: clonal crosses produce segregating populations immediately, but pure-line crosses produce uniform F₁ that must be advanced to F₂ for segregation.

🔀 Three-way crosses and backcrosses

🔀 When and why to use them

  • Purpose: used whenever the expected frequency of progeny with the desired traits from a single-cross population may not be adequate.
  • Three-way cross advantage: makes it possible to combine genes for desirable traits from more than two parents.
  • Backcross limitation: seldom used in developing clonal cultivars because homozygosity will occur in the progeny that is negatively associated with performance of individuals.

📊 Genetic contribution comparison

Population typeParent 1 contributionParent 2 contributionParent 3 contribution
Three-way cross25%25%50%
Backcross25% (non-recurrent)75% (recurrent)
  • Genetic variability: the genetic variability expected in a three-way population is greater than that for a backcross population.

🎲 How many hybrid seeds are needed

  • Key principle: the number of hybrid seeds that need to be obtained from the three-way cross or backcross is dependent on the number of gametes that the breeder wants to sample from the single-cross F₁ plants.
  • Why sampling matters: every gamete produced by the heterozygous, single-cross F₁ will be genetically different.
  • Probability logic: the chance that a gamete with superior alleles from the two single-cross parents will unite with a gamete from the third parent or backcross parent is directly associated with the number of hybrid three-way or backcross seeds obtained.

🌿 Synthetic cultivar populations

🌿 Why synthetic populations are more diverse

  • The types of populations used for development of synthetic cultivars are more diverse than for clonal, pure-line, or hybrid cultivars.
  • Multiple formation methods are used depending on breeding goals.

🔄 Cultivar per se

A synthetic cultivar per se can be used as a breeding population because of its inherent heterozygosity and heterogeneity.

  • Example from excerpt: the cultivar FreedomMR red clover—the initial population used for selection was the cultivar Freedom; the breeder used approximately 10,000 seeds of Freedom to begin selection.
  • Why it works: the cultivar itself is already heterozygous and heterogeneous, so it can serve directly as a breeding population.

✖️ Single cross of synthetic cultivars

  • Parent characteristics: when two synthetic cultivars are crossed, the parents per se would be highly heterozygous and heterogeneous.
  • Offspring characteristics: the hybrid seeds obtained from crossing the two parents would be heterozygous and heterogeneous.
  • How many hybrid seeds: equal to the number of individuals from the population that the breeder wants to evaluate.
  • Crossing strategy: as many different plants as possible of each parent would be used for crossing to capture the genetic heterogeneity of the parent.
  • Example from excerpt: the cultivar Intercross ryegrass was selected from the single cross of 'Axcella' x 01-ARG; the F₁ seeds from the single cross were planted in the field and allowed to cross-pollinate; selection began with the cross-pollinated seed.

🔺 Three-way cross of synthetic cultivars

  • Purpose: makes it possible to combine favorable genetic traits from three synthetic cultivars and lines.
  • Example from excerpt: Nelson annual ryegrass was developed from a three-way cross; the synthetic breeding lines TXR2000-T2 and TXR2002-T17 and the synthetic cultivar Jumbo were each planted in adjacent rows; any plants that did not have good forage characteristics were removed and the remaining plants were allowed to cross-pollinate; the seed harvested from individual plants of the three parents was bulked to form the initial population for selection.

🔁 Polycross

In a polycross, selected plants (clones) are grown in an isolated nursery where cross-pollination occurs by wind or insect pollination.

  • Example from excerpt: for the development of Warrior, a synthetic cultivar of indiangrass, the synthetic cultivar Oto was used as the base population (cycle 0); every plant in Oto was genetically different; 29 selected clones from Oto were grown in two replications of an isolated polycross nursery and allowed to cross-pollinate by wind; seed was harvested from individual clones and an equal amount from each was bulked to form the population for the next breeding cycle (cycle 2).
  • Iterative cycles: in cycle 2, 742 clones were evaluated of which 39 were selected and mated in a polycross to produce seed for cycle 3; in cycle 3, 875 clones were evaluated, of which 38 were selected and mated in a polycross; the seed harvested from the polycross of the 38 clones became the cultivar Warrior.

🎯 Polycross purposes and methods

🎯 Four main uses of polycross

A polycross is commonly used by breeders of synthetic cultivars to cross selected individuals (clones) to each other. The number of clones involved in a polycross can vary from a few to more than 100.

  1. Develop a segregating population from which new superior clones can be selected.
  2. Obtain half-sib seed from individual clones that can be used for evaluating their general combining ability.
  3. Obtain seed of an experimental synthetic for testing.
  4. Obtain Syn.1 breeder seed of a new synthetic cultivar that will be released for commercial use.

🐝 Pollination methods

  • Natural open-pollination: the majority of polycrosses are done through natural open-pollination by wind or insects.
  • Manual polycross: for a few crops, a polycross can be done manually by transferring pollen among selected clones.
    • Example: for alfalfa, pollen is manually transferred among the clones by tripping their flowers with a folded piece of paper; pollen from different clones is mixed together as the flowers are tripped; the mixed pollen on the paper sticks to the stigma of a flower and fertilizes the eggs.
  • Insect-pollinated species in cages: selected clones are transplanted to cages where they will be intermated by bees; a bee trips the flowers in the process of collecting nectar; the pollen from the anthers sticks on the body of the bee; as the bee travels from one flower to the next, a mixture of pollen from different plants is formed; each time a flower is tripped, the pollen on its body is transferred to the stigma of the tripped flower and fertilizes its eggs; the resulting seed is a heterogeneous mixture of genotypes.
  • Wind-pollinated species in isolated field plantings: selected clones are transplanted vegetatively to an isolated field; at the time of flowering, the pollen from the clones will be dispersed among the clones by wind, resulting in a heterogeneous mixture of seed on each plant.
    • Example: a rye border can be used to isolate the polycross of tall fescue clones.

🧩 More complex populations

  • Goal: to combine together favorable alleles for multiple traits from a range of synthetic cultivars and lines.
  • Example from excerpt: Au Red Ace red clover was selected from a complex population; it was developed from five populations that together included the parentage of 18 commercial cultivars and five plant introductions.
5

Techniques for Artificial Hybridization

Techniques for Artificial Hybridization

🧭 Overview

🧠 One-sentence thesis

Artificial hybridization techniques vary by flower type (perfect, monoecious, or dioecious) and require understanding of reproductive structures, timing, and whether manual labor is needed to control parentage and obtain hybrid seed for breeding programs.

📌 Key points (3–5)

  • Three basic flower types: perfect (both male and female organs in one flower), monoecious (male and female organs on different parts of the same plant), and dioecious (male and female organs on different plants).
  • Manual vs. non-manual crossing: manual labor is used when floral parts are large enough, control of parentage is desired, and hybrid plants can be distinguished from self-pollinated plants; non-manual methods rely on natural cross-pollination, male sterility, or self-incompatibility.
  • Purpose distinction: hybridization for developing segregating populations (breeding programs) differs from commercial hybrid seed production.
  • Common confusion: emasculation (removing male organs) is not always necessary—in soybean, if the stigma is pollinated early, it becomes unreceptive by the time the flower's own anthers mature.
  • Success rates vary: even with careful technique, only about 50% of pollinated flowers may produce pods, and seed number per pollination varies by species.

🌸 Flower types and their implications

🌸 Perfect flowers

Perfect flowers contain both the male and female organs.

  • Both reproductive structures are present in a single flower.
  • Species with perfect flowers include barley, common bean, field bean, soybean, oat, triticale, canola, and wheat.
  • Crossing strategy depends on flower size and whether self-pollination can be controlled or distinguished.

🌽 Monoecious flowers

Species with monoecious flowers have the female and male organs on different parts of the plant.

  • Example: maize has staminate (male) and pistillate (female) flowers on the same plant.
  • Manual crossing involves protecting female flowers from unwanted pollen and applying pollen from selected male flowers.
  • Example: watermelon requires protection of buds (using paper clips, foam cups, or screen cages) and hand-brushing male flowers against female flowers.

🌳 Dioecious flowers

Species with dioecious flowers have the female and male organs on different plants.

  • Example: buffalograss and pistachio.
  • Crossing requires access to both male and female plants.
  • Timing challenge: male and female flowers may not mature simultaneously, requiring pollen collection and storage.

🔬 Manual crossing techniques for perfect flowers

🫘 Soybean as a model system

  • Soybean has small perfect flowers; magnifying lenses may be helpful.
  • The flower is used for crossing when the female is receptive but anthers are not yet mature enough to shed pollen.
  • All other flowers and young buds at the node are removed to reduce nutrient competition.

🧹 Preparation of the female flower

Step-by-step process:

  • Remove sepals surrounding the petals with tweezers.
  • Remove petals to expose reproductive organs.
  • The scar from sepal removal marks pods from artificial hybridization, distinguishing them from naturally self-pollinated pods.
  • The female organ (stigma at the end of the style) looks like a drop of water and is surrounded by 10 anthers.

🚫 Why emasculation is not always needed

  • In soybean, emasculation (removing anthers) is unnecessary.
  • If the exposed stigma is not pollinated early, it dries up and becomes unreceptive by the time the flower's own anthers mature.
  • As anthers mature, filaments lengthen so anthers surround the stigma when they shed pollen.
  • Don't confuse: this timing-based approach only works when the stigma can be pollinated before the flower's own pollen is ready.

🎨 Using flower color to detect contamination

  • Soybean petals are primarily white (recessive) or purple (dominant).
  • Strategy: use the white-flowered plant as the female parent.
  • Benefit: accidental self-pollinations can be readily detected when hybrid plants are grown (purple flowers indicate true hybrids).

🤝 Pollination process

  • When petals of a male parent flower are fully exposed, anthers are ready to shed pollen.
  • Use tweezers to remove male and female reproductive organs from the male parent flower.
  • Gently touch anthers on the stigma to release pollen.
  • One male flower can pollinate up to three female flowers if pollen production is good.

📊 Success rates and seed yield

  • About seven days after pollination, a pod will be visible if crossing was successful.
  • Only about 50% of female flowers successfully produce a pod.
  • Soybean pods contain only about two seeds.
  • Average yield: only one hybrid seed is obtained from the female flowers used for crossing.
  • The dried stigma remains at the pod tip when mature, and the seed scar from sepal removal is clearly visible.

🌾 Non-manual crossing methods

🌾 When manual labor is not used

Manual labor is avoided when:

  • Floral parts are extremely small.
  • Self-pollination is minimized or eliminated through self-incompatibility or male sterility.
  • Cross-pollination is common by wind or insects.
  • Individuals from self-pollination can be readily distinguished from hybrid individuals.

🎋 Sugarcane example

  • Sugarcane has perfect flowers but manual labor is generally not used.
  • The inflorescence (flowering arrow or tassel) consists of thousands of both perfect and imperfect flowers.
  • Imperfect flowers result from abortion of pistils or (more commonly) stamens.
  • Parental genotypes show broad variation in fertility and seed production due to male sterility and self-incompatibility.

Crossing procedure:

  • Parental clones are selected before flowering, potted, and maintained in an isolated facility.
  • Manual emasculations generally are not done (too many small flowers per inflorescence; whole arrow takes time to complete flowering).
  • Breeders select female parents that are largely male sterile.
  • Pollination methods:
    • Place male parent arrow above female parent arrow so pollen falls on female flowers, OR
    • Collect pollen from male flowers and dust over female clone arrow.

Seed purity:

  • Male-sterile female parent produces only hybrid seed.
  • Male-fertile female produces both selfed and hybrid seed.
  • Selfed individuals are eliminated during selection (poor performance due to inbreeding depression) or distinguished using molecular markers.

🌿 Polycross method

  • Used for species that mate multiple parents to form a population for selection.
  • Examples: alfalfa, bromegrass, orchardgrass, red clover, and switchgrass.
  • Relies on natural cross-pollination rather than controlled manual crosses.

🍉 Crossing monoecious and dioecious species

🍉 Watermelon (monoecious)

  • Manual labor is used for artificial hybridization.
  • Naturally pollinated by insects in commercial production.

Protection from contamination:

  • Female flowers protected using paper clips (to restrict buds from opening), foam cups, or small screen cages and nets.
  • Male flowers also protected to prevent pollen contamination from other plants.
  • Protection may be unnecessary in greenhouses with insect-excluding structures.

Pollination process:

  • Harvest male flowers.
  • Open petals.
  • Brush male flower against female flower.
  • Keep female flowers protected for one day after pollination to prevent accidental pollination.

🌰 Pistachio (dioecious)

  • Manual labor is used for artificial hybridization.
  • Naturally wind-pollinated in commercial production.

Timing challenge:

  • Male trees normally bloom earlier than when female flowers are fully mature and ready for pollination.
  • Solution: collect pollen from male flowers and store at 4°C until female flowers are ready.

Crossing procedure:

  • Protect female flowers using paper bags before they are fully developed.
  • Apply stored pollen to female flowers using a brush.
  • Cover pollinated flowers to prevent accidental pollination.

📋 Key considerations across all methods

📋 Synchronizing flowering

  • Artificial hybridization requires knowledge of the species' mode of reproduction.
  • Must understand conditions necessary to obtain flowers of both parents at the same time.
  • Environmental conditions control flowering; strategies are needed to synchronize parent flowering.

📋 Distinguishing hybrid from selfed seed

MethodWhen usedExample
Morphological markersDominant/recessive traits (e.g., flower color)Soybean: white female × purple male
Male sterilityFemale parent cannot self-pollinateSugarcane with male-sterile female
Performance selectionSelfed plants show inbreeding depressionSugarcane: eliminate poor performers
Molecular markersDNA-based identificationSugarcane: distinguish selfed from hybrid
Physical markersScars or structures from crossing processSoybean: sepal removal scar on pod

📋 Purpose of hybridization in breeding

  • The production of hybrid seed for developing segregating populations is not done in the same manner as production of seed of hybrid cultivars for sale to end users.
  • Focus: obtaining segregating populations from which superior individuals can be selected.
  • Commercial hybrid seed production will be covered separately in other materials.
6

Mutation Breeding

Mutation Breeding

🧭 Overview

🧠 One-sentence thesis

Mutation breeding enables plant breeders to create novel genetic variation through induced mutagenesis when naturally occurring germplasm lacks the desired traits, making it possible to develop improved cultivars with characteristics not found in existing populations.

📌 Key points (3–5)

  • When mutation breeding is needed: when no available germplasm of a plant species has a trait needed by a breeder, despite evaluating cultivars and plant introductions.
  • Two main approaches: treating elite cultivars directly (which may yield a new cultivar immediately) or creating mutant alleles to incorporate into elite germplasm through hybridization.
  • Key decision in population management: breeders must choose between testing more progeny from fewer M₁ plants versus fewer progeny from more M₁ plants.
  • Common confusion—generation terminology: M₁ is the first generation grown from treated seed; M₂ is where segregation occurs and mutants are first identified; M₂:₃ refers to progeny testing to confirm stable inheritance.
  • Why it matters: mutation breeding has successfully created valuable traits like short stature in rice, herbicide tolerance in wheat and barley, improved oil composition in soybean, and disease resistance in multiple crops.

🌱 When and why mutation breeding is used

🎯 The fundamental problem

  • Breeders sometimes need traits that do not exist anywhere in the available germplasm of a species.
  • Example from the excerpt: soybean breeders wanted to modify oil composition to improve shelf-life, but none of the cultivars or thousands of plant introductions had the necessary trait.
  • Through mutagenesis, genes were developed that made it possible to create cultivars with improved oil characteristics.

🔬 What mutagenesis provides

  • Creates novel genes that would not be available through traditional selection or hybridization.
  • Expands the genetic variation beyond what naturally occurs in the species.
  • Can target specific traits while working within an otherwise elite genetic background.

🧪 Designing a mutagenesis program

🌾 Selecting the starting material

The advantage of using an elite cultivar for mutagenesis is that it may be possible to identify a mutant that is suitable for release as a new cultivar without additional breeding.

  • Elite cultivar approach: Example 1 (rice Calrose 76) used the cultivar Calrose, which allowed the mutant to be released directly as a new cultivar.
  • Alternative outcome: A plant with the desired mutation may not be suitable for release because it may contain undesirable mutations for other traits; in those cases, the mutant allele is incorporated into elite germplasm by hybridization and selection.
  • Example 2 (St. Augustinegrass) selected cultivar Raleigh because it had superior cold tolerance and was widely used.

☢️ Choosing mutagen type and dosage

Physical mutagens:

  • Cobalt-60 gamma radiation used in Examples 1 and 2.
  • Example 1 tested five dosages (0, 15, 20, and 25 kR) on rice seeds.
  • Example 2 tested seven dosages (0, 50, 60, 70, 80, 90, 100 Gy) on grass cuttings; 1 Gy = 0.1 kR.

Chemical mutagens:

  • Example 3 used sodium azide (1mM for 2 hours) to treat barley seed.
  • About 500 grams of seed treated in that example.

Why multiple dosages:

  • Using multiple dosages increases the chance of finding one that results in adequate mutation frequency.
  • A control treatment (0 dosage) makes it possible to assess how each dosage influences germination and mutation frequency.

💧 Pre-treatment steps

  • Example 1: rice seeds brought to 14% moisture before cobalt-60 treatment.
  • Example 3: barley seed pre-soaked at 0°C for 16 hours, then at 20°C for 8 hours before sodium azide treatment.
  • Rationale: it is assumed the breeder had prior knowledge that these steps would be useful with the chosen mutagen.

🌿 Treating vegetative tissue

  • Example 2 treated single-node cuttings from stolons (not seed).
  • About 140 cuttings of uniform size were used initially.
  • Why uniform size matters: to ensure radiation penetration was uniform for all materials treated.
  • Also tested callus tissue at different doses (0, 25, 50, 100, 200 Gy) to determine if this tissue type is amenable to gamma irradiation in grass species.

🌾 Managing mutagenized populations

🌱 M₁ generation (first generation from treated seed)

  • Planting and isolation: M₁ seeds are planted in the field; even for self-pollinated species like rice, M₁ plants should be isolated from untreated genotypes to prevent outcrossing.
  • Example 1: rice M₁ plants isolated by 11 meters from untreated rice.
  • Why isolation matters: If outcrossing occurred and gametes with the wild-type allele from untreated genotypes fertilized the M₁ plant, the frequency of homozygous mutant M₂ progeny would be reduced below the expected ¼, making it harder to find desired mutations.

🌾 Harvest strategy for M₁ plants

Two approaches described in Example 1:

ApproachNumber of plantsAdvantageDisadvantage
Individual harvest200 plantsCan grow more progeny from each M₁ plant (e.g., 11 M₂ individuals per line)Fewer total M₁ families tested
Bulk harvest~1,400 plantsMore M₁ plants representedFewer M₂ progeny per M₁ plant (e.g., only 2 on average)
  • The key tradeoff: testing more progeny of fewer M₁ plants versus fewer progeny from more M₁ plants.
  • To be 95% sure of recovering at least one homozygous M₂ plant from a heterozygous M₁ plant, 11 individuals must be evaluated in each M₁:₂ line.

🔍 M₂ generation (where mutants are identified)

  • When recessive mutations segregate: If an M₁ plant had a recessive mutation, its M₂ progeny would segregate in a 1:2:1 ratio.
  • Screening methods vary by trait:
    • Example 1 (rice): at maturity, short-statured M₂ plants were harvested individually; undesirable plants (haploids, triploids, many semi-steriles) were discarded.
    • Example 3 (barley): about 2 million M₂ plants screened in greenhouse for herbicide resistance by soaking seeds in herbicide solution, planting in flats, and visually inspecting seedlings after four weeks.
    • Example 2 (grass): plants screened first for freezing tolerance, then survivors screened for morphological changes.

Common abnormalities:

  • It is common to find abnormalities in plants from mutagenized seed, particularly when physical mutagens such as gamma radiation are used.

🧬 M₂:₃ and later generations (confirmation and selection)

  • Progeny testing is essential: to confirm that an individual plant has a mutant allele and whether the allele is stable from one generation to the next.
  • Example 1: progeny of M₂ plants grown as M₂:₃ lines to identify those homozygous for short stature; eleven lines selected for further evaluation.
  • Example 3: M₂ plants showing herbicide tolerance were self-pollinated and harvested individually; 5–10 M₃ progeny grown from each M₂ plant for seed increase; M₂:₄ lines evaluated for tolerance; out of 2 million M₂ seedlings, only one line was homogeneous for herbicide tolerance.

🔬 Genetic characterization

  • Important step: conducting a genetic study to determine inheritance of the mutant trait.
  • Example 1: short stature in Calrose 76 found to be controlled by a single recessive allele designated sd1.
  • This information is important for using the allele effectively in a breeding program.

🌟 Outcomes and further breeding

✅ Direct release as cultivar

  • Example 1: experimental line D7 became the cultivar Calrose 76 after yield evaluation in subsequent generations.
  • This is possible when the mutant line has no other undesirable traits.

🔄 Backcrossing to eliminate negative traits

It is common that mutant lines have undesirable traits that preclude their use directly as a cultivar. The breeder must attempt to eliminate the negative traits by conventional hybridization and selection.

  • Example 3: herbicide tolerance in barley was associated with a slight delay in seed germination, so the new mutant line was backcrossed to the original cultivar Bob.
  • The mutant allele is incorporated into elite germplasm by hybridization and selection.

📊 Examples of successful mutation breeding

CropMutant GermplasmMutation MethodNew Trait
RiceCalrose 76Gamma raysShort stature
WheatFS4Sodium azideHerbicide tolerance
SoybeanA29Ethyl methanesulfonateReduced linolenate
SoybeanC1726Ethyl methanesulfonateReduced palmitate
SoybeanA22N-nitroso-N-methylureaReduced palmitate
OatsAlamo-XX-raysDisease resistance
BermudagrassTifEagleGamma raysBetter turf quality when mowed
BermudagrassTifWay IIGamma raysNematode and cold tolerance, faster growth
St. Augustine grassTXSA 8202Gamma raysDisease resistance
St. Augustine grassTXSA 8218Gamma raysDisease resistance
  • Calrose 76 was the first semi-dwarf rice cultivar in the USA produced by gamma irradiation; it is 75% shorter than cultivars without the mutant sd1 allele.
  • Gamma field irradiation has also been used to create flower color mutants in Chrysanthemum.
7

Novel Traits from Molecular Genetics and Biotechnology

Novel Traits from Molecular Genetics and Biotechnology

🧭 Overview

🧠 One-sentence thesis

Molecular markers and genetic engineering provide plant breeders with powerful tools to select genes of interest more efficiently than phenotypic selection and to introduce novel traits from other organisms or modified genes into elite cultivars.

📌 Key points (3–5)

  • Molecular markers (SSR and SNP) allow breeders to identify desired alleles without phenotypic testing, saving time and cost in many cases.
  • Common confusion: Not all traits benefit from molecular markers—herbicide resistance is cheaper to test by spraying plants than by using markers.
  • Genetic engineering enables insertion of transgenes (e.g., Bt insect resistance, glyphosate tolerance) from bacteria or other species into plants via transformation.
  • Inheritance of transgenes follows Mendelian patterns; hemizygous (single-copy) transgenic plants segregate like heterozygotes.
  • Backcrossing is the standard method to move novel alleles (natural, induced, or transgenic) into elite germplasm for cultivar development.

🧬 Molecular markers: what they are and why they matter

🧬 Definition and purpose

Molecular marker: part of the DNA of a plant associated with the DNA of a gene of interest; may be linked to the gene (linked marker) or part of the gene itself (perfect, direct, or functional marker).

  • Molecular markers are an alternative to phenotypic selection for identifying plants with a desired allele.
  • They are especially useful when phenotypic evaluation is unreliable, expensive, or slow (e.g., traits expressed only at maturity, long life cycles in trees).

🔍 When to use markers vs. phenotypic selection

Advantages of markers:

  • More reliable and less expensive than phenotypic tests in some cases.
  • Example: The dominant Rag1 allele for aphid resistance in soybean can be identified by a marker, avoiding the need to infect plants with aphids and risk false positives from accidental escape.
  • Useful for traits expressed late (e.g., sex in asparagus at 2+ years, gingko at sexual maturity).

When phenotypic selection is better:

  • Breeders must decide case-by-case if markers are more efficient and cost-effective.
  • Example: Herbicide resistance transgenes in soybean and maize are tested by spraying herbicide and killing susceptible plants—cheaper than molecular markers.

Don't confuse: Molecular markers are not always the best choice; cost and ease of phenotypic testing matter.

🧪 Common types of molecular markers

🧪 Simple Sequence Repeat (SSR)

SSR: pieces of DNA containing repeated nucleotide sequences (e.g., two bases like AT, AG, TC; or three bases like ATT).

Two criteria for effective use:

  1. Tight linkage to the gene of interest so crossovers during meiosis do not separate them; ideally, SSRs flank both sides of the gene.
  2. Polymorphism in the parents: individuals with the desired allele must have one SSR sequence, and those with the other allele must have a different SSR sequence.

How SSRs are detected:

  • PCR amplifies the SSR region thousands of times.
  • Gel electrophoresis separates fragments by size: longer fragments (more repeats) move slower; shorter fragments move faster.
  • Example: Rag1 parent had 20 ATT repeats (faster band), susceptible IA3027 had 26 ATT repeats (slower band); heterozygotes showed both bands.

🧬 Single Nucleotide Polymorphism (SNP)

SNP: single nucleotide differences in the DNA of two individuals (e.g., one has adenine A, another has guanine G at the same location).

Two uses:

  1. Linked marker: must be tightly linked and polymorphic in parents.
  2. Direct marker: a SNP within the gene itself is ideal because no crossover can separate it from the allele of interest.

Detection method (RFLP):

  • SNPs cannot be distinguished by size on a gel (same length).
  • Restriction Fragment Length Polymorphism (RFLP): a bacterial restriction enzyme cuts DNA at specific sequences.
  • Example: The fan3(A29) mutant allele (low linolenic acid in soybean oil) has an A instead of G. The HpaI enzyme cuts the wild-type Fan3 (G) but not the mutant fan3 (A). Homozygous wild-type shows two bands (cut), homozygous mutant shows one band (uncut), heterozygote shows three bands.

Don't confuse: SNPs and SSRs differ in detection—SSRs are separated by size; SNPs often require restriction enzymes or other assays (SimpleProbe, Taqman).

🔬 PCR and detection technology

🔬 What PCR does

  • PCR (Polymerase Chain Reaction) amplifies minute quantities of DNA into thousands of copies for detection.
  • Invented by Kary Mullis in the mid-1980s.

🧰 Materials for PCR

  1. DNA from the individual (seed or young leaf tissue).
  2. Taq polymerase enzyme (from bacterium Thermus aquaticus; stable at 95°C, optimum at 72°C).
  3. DNA primers that match sequences flanking the region to be amplified (made by DNA synthesizers).
  4. Deoxynucleoside triphosphates (dNTPs) containing the four nucleotides (A, G, C, T) for new DNA strands.
  5. Thermocycler to control temperature.

⚙️ Three PCR steps (repeated in cycles)

  1. Denaturation (~95°C): heat separates the two DNA strands.
  2. Annealing: primers bind to complementary regions on the separated strands.
  3. Extension: Taq polymerase uses primers and dNTPs to synthesize new DNA strands.

📊 Gel electrophoresis

  • DNA is negatively charged and migrates toward the positive pole.
  • Longer fragments move slower; shorter fragments move faster.
  • Ethidium bromide (EtBr) stain binds DNA; under UV light, DNA fluoresces orange, making bands visible.

🧫 Genetic engineering and transgenes

🧫 What is a transgene?

  • Transgene: a novel gene (from another organism or modified from the same species) inserted into a plant by transformation.
  • The DNA used is called a construct with four essential components linked together.

🧩 Four components of a transgene construct

  1. Gene of interest (e.g., insect resistance, herbicide tolerance).
  2. Promoter to turn the gene on/off in the cell (e.g., CaMV 35S from cauliflower mosaic virus; NOS-Pro nopaline synthase promoter).
  3. Selectable marker to identify cells that successfully received the construct (e.g., NPT II for kanamycin resistance; herbicide resistance genes).
  4. Terminator sequence (e.g., nopaline synthase NOS gene) to mark the end of the transgene for proper expression.

🎯 Events and selection

  • Each random insertion of the construct into the genome is an event.
  • Useful events are rare; selectable markers (e.g., antibiotic or herbicide resistance) allow identification of successful transformants.
  • Scientists screen many potential transformants to find useful events.

Example: Buffalograss transformed with glyphosate tolerance gene—control calli (lacking the gene) are killed by herbicide in the media; transformed calli survive and grow.

🌱 Transformation methods

🌱 Two common methods

  1. Biolistics (particle bombardment): high pressure propels tungsten or gold beads coated with DNA into plant cells.
  2. Agrobacterium tumefaciens-mediated transformation: uses a soil bacterium that naturally inserts DNA into plant cells (causes crown gall disease); scientists modified it to remove disease-causing ability but retain DNA transfer capability.

🦠 Agrobacterium transformation steps

  1. Infect select tissue with Agrobacterium carrying the transgene construct (with antibiotic resistance as selectable marker).
  2. Grow tissue in medium containing the antibiotic; untransformed cells die.
  3. Surviving transformed tissue is removed from antibiotic and regenerated into whole plants.
  4. Monitor transgenic plants in controlled environment (growth chamber, greenhouse) before field trials.

🐛 Examples of novel transgenes

🐛 Insect resistance: Bt toxin

  • Bacillus thuringiensis (Bt) is a bacterium producing crystal proteins toxic to insect larvae when ingested.
  • The Bt toxin gene was isolated, modified for better expression in plants, and introduced via transformation.
  • Example: Bt cotton and corn expressing bacterial toxin for insect control.

🌿 Herbicide tolerance: glyphosate

  • Glyphosate (active ingredient in Roundup) destroys the plant EPSP enzyme needed for amino acid production, killing the plant.
  • A bacterial EPSP enzyme is not destroyed by glyphosate.
  • Transgenic plants with the bacterial EPSP gene are tolerant to glyphosate (Roundup Ready crops).

🌾 Herbicide tolerance: glufosinate

  • Glufosinate (active compound in Liberty herbicide).
  • Transformed crops contain bar or pat transgenes (from bacteria) that destroy glufosinate, decreasing herbicide toxicity.

Don't confuse: Different herbicide tolerance traits use different mechanisms—glyphosate tolerance uses a resistant enzyme; glufosinate tolerance uses a detoxifying enzyme.

🧬 Inheritance and breeding with transgenes

🧬 Hemizygous and segregation

Hemizygous: cells with only one copy of the transgene in their genome (hemi = half, zygous = zygote).

  • Transformation inserts the transgene into one chromosome.
  • Segregation in progeny of a hemizygous plant is the same as for a heterozygous plant.
  • Self-pollination of a hemizygous diploid plant yields a 3:1 ratio (same as heterozygous × heterozygous): 1 homozygous dominant : 2 heterozygous : 1 homozygous recessive.

🔄 Backcrossing to incorporate transgenes

  • Tissue culture regeneration may introduce genetic variation unrelated to the transgene.
  • The parent line used for transformation is often selected for transformation efficiency, not agronomic performance.
  • Backcrossing is used to incorporate transgenes into elite commercial cultivars.

🌽 Stacking multiple traits

Pure-line cultivars:

  • Multiple novel alleles must be stacked together in the same line.
  • Example: Herbicide resistance (H) and insect resistance (I) → cultivar genotype HHII.

Hybrid cultivars:

  • One dominant allele (e.g., H) can be in one inbred line; another (e.g., I) in a different inbred line.
  • The F₁ single-cross hybrid (HhIi) has both traits.

Don't confuse: Pure-line cultivars require all traits in one line; hybrid cultivars can split traits across parental inbreds and combine them in the F₁.

8

Backcrossing

Backcrossing

🧭 Overview

🧠 One-sentence thesis

Backcrossing is a breeding method that efficiently transfers novel alleles into elite germplasm while recovering nearly all the desirable traits of the original elite parent, with strategies varying by cultivar type (pure-line, hybrid, clonal, or synthetic).

📌 Key points (3–5)

  • Purpose: Backcrossing incorporates novel alleles (from natural mutations, mutagenesis, or genetic engineering) into elite germplasm for cultivar development.
  • Goal for pure-line/hybrid cultivars: Recover a line containing the novel allele that is as good as the recurrent parent for all other important traits.
  • Strategy varies by cultivar type: Pure-line and hybrid programs use a single recurrent parent; clonal and synthetic programs change the elite parent each generation (modified backcrossing) to avoid inbreeding depression.
  • Common confusion: Stacking alleles in pure-line vs. hybrid cultivars—pure-line cultivars require all novel alleles stacked together in one line (e.g., HHII), whereas hybrids can place different dominant alleles in separate inbred parents (e.g., Hh in one, Ii in another) that combine in the F₁.
  • Efficiency factors: Success depends on choosing similar donor/recurrent parents, minimizing seed numbers through probability calculations, and using multiple seasons per year to reduce total time.

🌱 Backcrossing strategies by cultivar type

🌾 Pure-line and hybrid cultivars

Goal: Recover a pure line or inbred that contains the novel allele and is as good as the recurrent parent for all other important traits.

  • Single recurrent parent: The same elite parent is used repeatedly across all backcross generations.
  • Stacking alleles in pure-line cultivars: All novel alleles must be combined in the same line.
    • Example: To stack herbicide resistance (H) and insect resistance (I), the final cultivar must be HHII.
  • Stacking alleles in hybrid cultivars: Different dominant alleles can be placed in separate inbred lines.
    • Example: Put H in one inbred and I in another; the commercial F₁ hybrid (HhIi) expresses both traits.
    • This approach is possible because the farmer grows the heterozygous F₁, not the inbreds themselves.
  • Don't confuse: Pure-line stacking (all alleles in one line) vs. hybrid stacking (alleles split across parents).

🌿 Clonal cultivars

Modified backcrossing: The elite parent is changed every backcross generation to avoid homozygosity and inbreeding depression.

  • Why heterozygosity matters: Self-pollination or mating of related individuals causes homozygosity and inbreeding depression, leading to unacceptable performance in clonal cultivars.
  • Modified backcrossing procedure: Because a single recurrent parent would cause homozygosity, breeders change the elite parent each generation.
  • Outcome: A population from which individuals with the novel allele and other desirable traits can be identified for clonal evaluation as potential cultivars.

🌾 Synthetic cultivars

Heterozygosity and heterogeneity are maintained by changing the elite parent each generation and sampling as many different plants as possible.

  • Avoiding inbreeding depression: Like clonal cultivars, synthetic cultivars (forage and turf species) require heterozygosity, so the elite parent changes each backcross generation.
  • Sampling genetic heterogeneity: Use as many different plants of the elite parent as possible each generation.
  • Autopolyploid species (e.g., alfalfa): It is not practical to incorporate the novel allele into every plant.
    • Goal: Have an adequate percentage of plants in the cultivar with the novel allele.
    • Example: Glyphosate resistance (Genuity® Roundup Ready® Gene) is incorporated into synthetic alfalfa cultivars; the majority of plants contain the transgene, and farmers increase seeding rate to account for plants that die when sprayed.

🔧 Managing an efficient backcrossing program

🎯 Step 1: Selecting donor and recurrent parents

  • Donor parent: Should have as many traits similar to the recurrent parent as possible to minimize the number of backcross generations needed.
    • Example (Table 1): LD05-15601 was chosen as the donor because it was the highest-yielding line with the Rag1 allele; other traits (seed size, protein content) were also considered for similarity to IA3027.
  • Recurrent parent: Should remain competitive when the backcross is completed (often several years later).
    • Example: IA3027 was chosen in 2006 because the breeder believed the backcross-derived version would still be competitive in 2010.
    • Alternative: Use elite experimental lines likely to become future cultivars, reducing the time gap; risk is that the program may be discarded if the experimental line fails commercial acceptance.

🧮 Step 2: Minimizing seed and plant numbers

  • Why it matters: Artificial hybridization can be a limiting factor for self-pollinated species.
  • Key calculation: Determine the minimum number of plants or seeds needed to obtain the required number with the allele(s) of interest at 95% or 99% probability.
    • q: Expected genotypic frequency of individuals with the desired allele(s).
    • r: Number of individuals with the desired allele(s) needed.
    • p: Probability of successfully obtaining r individuals.
    • Also account for seed germination percentage.
  • Multiple alleles: Decide whether to backcross each allele individually and combine them later, or backcross them together.
    • Individual programs require less seed per generation but may need an extra season to combine alleles.
  • Optional selection: Obtain more than the minimum number of plants, genotype with molecular markers, and choose those with the greatest frequency of recurrent-parent markers to reduce the number of backcross generations needed.

⏱️ Step 3: Minimizing the number of years

  • Use multiple seasons per year: Enhances the likelihood that the backcross-derived version remains competitive.
    • May require making backcrosses before knowing if plants have the desired allele(s); discard seed from plants lacking the allele.
    • Most breeders prefer extra work to reduce total years.
  • Challenges: Environmental conditions in some seasons may not favor artificial hybridization.
    • Example (Table 1): Problems obtaining hybrid seed in Puerto Rico and adverse germination conditions at Ames; breeders often revise plans to deal with unforeseen circumstances.
  • Minimize backcross generations: Influenced by donor/recurrent parent similarity, required performance similarity, and testing strategy.

🧪 Testing backcross progeny: two alternatives

AlternativeDescriptionAdvantageDisadvantage
Alternative 1Grow multiple backcross-derived lines; select those similar for high-heritability phenotypic traits; bulk seed of selected lines for release.Requires less time and resources.New cultivar may not perform as well as recurrent parent for important quantitative traits. Example (Table 1): Bulk did not perform as well as recurrent parent.
Alternative 2Conduct replicated tests of individual backcross lines; bulk seed only from lines with acceptable performance to form breeder seed.Ensures performance similarity to recurrent parent for quantitative traits.Requires more time and resources.
  • When to choose Alternative 2:
    • First time working with the allele from the donor parent.
    • Donor and recurrent parent differ for multiple quantitative traits.
    • Example (Table 1): Donor LD05-15621 was significantly lower than IA3027 in seed size and protein content (both quantitative traits); testing 30 lines allowed discarding 12 with lower seed size and protein.

📋 Case study: IA3027RA1 soybean variety

🌱 Objective and timeline

  • Goal: Backcross the Rag1 allele for aphid resistance from LD05-15621 into the cultivar IA3027.
  • Timeline: Program began in 2006; commercial seed available in 2010 (4 years).
  • Recurrent parent (IA3027): Highest yield among cultivars with large seed and high protein for soyfood industry.
  • Donor parent (LD05-15621): Developed by University of Illinois; highest-yielding line with Rag1 allele in the breeder's program.

🔄 Generation advancement (2006–2008)

  • 2006 (March, Puerto Rico): Made cross IA3027 × LD05-15621 to obtain F₁ seeds.
  • 2006 (May, Ames, IA): Planted F₁ seeds; crossed F₁ plants to IA3027 to obtain BC₁F₁ seeds.
  • 2006 (October, Puerto Rico): Planted BC₁F₁ seeds for next backcross; no BC₂F₁ seeds obtained due to unfavorable conditions. Harvested leaves for molecular analysis (SSR linked to Rag1); harvested heterozygous BC₁F₁ plants individually.
  • 2007 (January, Puerto Rico): Planted BC₁F₂ seeds; identified homozygous resistant plants with SSR; backcrossed 5 plants to IA3027 to obtain BC₂F₁ seeds.
  • 2007 (May, Ames): Planted BC₂F₁ seeds and 5 BC₁F₂:₃ lines; BC₂F₁ plants died due to adverse weather. Screened BC₁F₂:₃ lines for aphid resistance in greenhouse; crossed 2 lines with scores similar to donor to IA3027 to obtain BC₂F₁ seed.
  • 2007 (October, Puerto Rico): Planted BC₂F₁ seeds; confirmed heterozygosity with SSR; crossed to IA3027 to obtain BC₃F₁ seeds.
  • 2008 (January, Puerto Rico): Planted BC₃F₁ seeds; identified heterozygous plants with SSR; harvested individually.
  • 2008 (May, Ames): Planted BC₃F₂ seeds; identified homozygous resistant individuals with SSR; scored for aphid resistance in field during natural infestation; harvested plants with resistance and similar maturity to IA3027 individually.
  • 2008 (Puerto Rico): Planted BC₃F₂:₃ lines individually for seed increase.

🧪 Testing and release (2009–2010)

  • 2009 (March, greenhouse): Tested BC₃F₂:₄ seed of each line for aphid resistance; bulked sample from 30 lines for yield testing.
  • 2009 (field): Evaluated bulk in Iowa Specialty Test (3 replications, 4-row plots, 5 locations); tested 30 BC₃F₂:₄ lines individually (2 replications, 4 locations). Grew BC₃F₂:₄ lines at Ames for seed increase; bulked seeds of 18 lines with similar agronomic and seed characteristics as IA3027 to form breeder seed of IA3027RA1.
  • 2009/10: Licensed variety to companies via Iowa State University Research Foundation; companies produced foundation seed in Argentina for planting in Midwest in 2010.

🔍 Key lessons from the case

  • Setbacks are common: Unfavorable conditions in Puerto Rico (2006) and adverse weather in Ames (2007) required plan revisions.
  • Molecular markers accelerate selection: SSR linked to Rag1 allowed identification of heterozygous and homozygous plants before phenotypic evaluation.
  • Testing revealed performance issues: Initial bulk (Alternative 1) did not perform as well as recurrent parent; individual line testing (Alternative 2) allowed selection of 18 lines with acceptable performance.
  • Multiple seasons per year: Use of Puerto Rico and Ames locations reduced total years despite setbacks.
9

Inbreeding

Inbreeding

🧭 Overview

🧠 One-sentence thesis

Inbreeding through self-pollination is the fastest method to develop homozygous lines for pure-line and hybrid cultivars, with the choice of inbreeding method depending on available environments, cost, and the desired generation for line derivation.

📌 Key points (3–5)

  • When inbreeding is used: only for developing pure-line and hybrid cultivars; not for clonal or synthetic cultivars due to severe inbreeding depression.
  • Self-pollination is fastest: reaches homozygosity much faster than half-sib or full-sib mating, so the latter are rarely used.
  • Method choice factors: available environments (local vs off-season nurseries), cost (labor vs genetic variability), and the generation at which lines are derived.
  • Common confusion: complete homozygosity is rarely achieved in practice; breeders derive lines when homogeneity is "adequate" for commercial acceptance, not when 100% homozygous.
  • Pure-line vs hybrid inbreds: pure-line cultivars are often derived earlier (e.g., F₃ or F₄) than inbreds for hybrids (e.g., F₇ or later), because hybrids require more uniformity to prevent off-types and undesirable segregation.

🌱 Why and when inbreeding matters

🌱 Cultivar types that require inbreeding

Inbreeding for the development of homozygous and homogeneous lines is only important for the development of pure-line and hybrid cultivars.

  • Pure-line cultivars: a single homozygous line released as a variety.
  • Hybrid cultivars: two or more homozygous inbred lines crossed to produce hybrid seed for commercial planting.
  • Not used for clonal or synthetic cultivars: self-pollination causes severe inbreeding depression in these types, so inbreeding is avoided.

⏱️ Speed of reaching homozygosity

  • Self-pollination is the fastest route to homozygosity.
  • Half-sib or full-sib mating can also increase homozygosity, but require many more generations (see Figure 1 in the excerpt).
  • Result: half-sib and full-sib matings are rarely used for cultivar development because they are too slow.

🧮 Mathematics of homozygosity

🧮 Formula for proportion of homozygotes

The proportion of completely homozygous plants in a selfing generation is:

[(2^m – 1) / 2^m]^n

where:

  • m = number of selfing generations (F₂ = 1, F₃ = 2, etc.)
  • n = number of segregating loci in the F₁

📉 Practical implications from Table 1

Number of segregating lociF₂ (%)F₄ (%)F₆ (%)F₈ (%)
150.0087.596.8799.22
46.2558.6288.0796.91
100.0126.3172.7992.45
100~00.00064.1845.64
1000~0~0~00.04
  • With only 1 segregating locus, 50% of F₂ plants are homozygous.
  • With 4 loci, only 6.25% of F₂ plants are homozygous.
  • With 100 or more loci, the proportion of completely homozygous plants is extremely small even by F₆ or F₈.
  • Don't confuse: "homozygous at all loci" vs "homozygous at major loci of interest." Breeders rarely wait for complete homozygosity; they derive lines when uniformity is acceptable.

🎯 When lines are derived in practice

  • Parents are chosen for genetic differences, so the F₁ has many heterozygous loci.
  • Complete homozygosity is rarely achieved except with doubled haploids.
  • Instead: breeders derive lines at a generation when progeny are "sufficiently uniform in appearance and homogeneous for major genes of interest."
  • Example: wheat cultivar Camelot was F₃-derived; maize inbred GT603 was derived after seven generations of selfing.

🛠️ Factors influencing method choice

🌍 Available environments

One of the key factors that influences the method of advancing a population from the F₁ to later generations is the environments available to the breeder.

  • Local environment: the target environment for which the cultivar is being developed.
  • Off-season nurseries: greenhouses, growth chambers, or locations with different climates (e.g., Yuma, Arizona for South Dakota wheat; Isabela, Puerto Rico for Michigan beans).

🔍 Effectiveness of selection in off-season nurseries

  • Trait-dependent: seed yield often cannot be evaluated in off-season nurseries, but other traits may be selectable.
  • Example: for cranberry bean cultivar Bellagio, breeders could select for upright vine, lodging resistance, pod load, seed size and color, and disease freedom in Puerto Rico.
  • Bulk method risk: if seed production in the off-season nursery is not representative of the local environment, desirable genotypes may be lost. A plant producing 40 seeds is less likely to be represented in the next generation than one producing 200 seeds.
  • Pedigree method risk: if a trait is not adequately expressed in the off-season nursery, selection is ineffective.
  • Common strategy: combine methods—e.g., select among F₂ plants in the local environment, advance F₃ in an off-season nursery without selection, then resume selection among F₃:₄ lines in the local environment.

💰 Cost

  • Genetic variation peaks early: half of the genetic variation among individuals is expressed in the F₂ generation.
  • Goal: maximize the number of different F₂ plants represented in the population when lines are selected for evaluation.
  • Methods that trace every line to a different F₂ plant:
    • Pedigree method
    • Single-seed procedure of single-seed descent
    • Single-hill procedure of single-seed descent
    • Doubled haploid (traces to individual gametes from F₁, each genetically different)
  • Trade-off: these methods require more labor than the bulk method or multiple-seed procedure of single-seed descent.
  • Breeders must balance cost vs genetic variability.

📅 Generation in which lines are derived

  • Pure-line cultivars: commonly derived in earlier generations (e.g., F₃ or F₄).
  • Inbreds for hybrids: commonly derived in later generations (e.g., F₇ or later).

🔍 Why hybrids need more homozygosity

  1. Recognize and remove off-types: in hybrid seed production fields, off-type plants must be identified and removed before crossing occurs.
  2. Prevent undesirable segregation: if two inbreds are not adequately homozygous and homogeneous, undesirable segregation may occur in the commercial hybrid field.

📚 Inbreeding methods and examples

🌾 Bulk method

In the bulk method, the seed planted in one generation is obtained by sampling from a bulk of seed harvested the previous generation.

  • How it works: harvest all plants together; sample from the bulk to plant the next generation.
  • Selection pressure: plants producing more seeds are more likely to be represented in the next generation.
  • Risk: if seed production in the nursery is not representative of the local environment, desirable genotypes may be eliminated.

🌾 Example: Gadsby barley (bulk + mass selection)

  • Mass selection: increase the frequency of individuals with a desired trait by selecting heavier seeds (assumed to be from scald-resistant plants).
  • Steps:
    1. F₂ plants inoculated with scald; F₃ seed harvested in bulk.
    2. F₃ seed screened on gravity table; heavier seed saved.
    3. Repeated for F₄ and F₅ bulks.
    4. F₆ bulk grown; individual plants harvested and threshed separately to form F₆-derived lines.
  • Don't confuse: mass selection during inbreeding (increases homozygosity each generation) vs mass selection in recurrent selection (maintains heterozygosity by intercrossing selected plants).

🌱 Single-seed descent (single-seed procedure)

  • How it works: harvest one seed from each plant; bulk the seeds to plant the next generation.
  • Advantage: every line traces to a different F₂ plant, maximizing genetic variability.
  • Labor: more than bulk method (must handle individual plants).

🌱 Example: Duclair wheat

  • F₂ planted in greenhouse; one head removed from each plant; one healthy seed from each head bulked for planting.
  • F₃ seeds planted; each plant harvested individually to form F₃:₄ lines.
  • F₃:₄ lines planted in field; individual lines selected and single plants harvested.
  • F₄:₅ lines evaluated; one F₄-derived line became cultivar Duclair.

🌿 Single-seed descent (multiple-seed procedure)

  • How it works: harvest two to three pods (each containing two to three seeds) from each plant; thresh in bulk.
  • Advantage: faster than single-seed procedure; still maintains good genetic variability.

🌿 Example: UA 4910 soybean

  • F₂: ~600 plants; 2–3 pods per plant threshed in bulk.
  • F₃: 2–3 pods per plant threshed in bulk.
  • F₄: 60 plants harvested and threshed individually to form F₄-derived lines.

🌾 Pedigree method

  • How it works: select among plants in one generation; grow progeny rows (lines) from selected plants; select among lines and then among plants within selected lines.
  • Key feature: maintain records of the pedigree (family relationships).
  • Selection hierarchy:
    1. Select best families (lines tracing to the same earlier plant).
    2. Select best lines within selected families.
    3. Select best plants within selected lines.

🌾 Example: Georgia-09B peanut

  • F₂ plants grown and harvested individually; one F₂:₃ line chosen for backcrossing.
  • BC₁F₁ seed planted; BC₁F₂ seed harvested.
  • F₂ plants selected; grown as F₂:₃ lines; lines and plants within lines selected.
  • F₃ plants grown as F₃:₄ lines; families, lines, and plants selected.
  • F₄ plants grown as F₄:₅ lines; best lines selected and threshed in bulk.
  • F₄-derived lines evaluated; Georgia-09B released.

🧪 Early-generation testing

  • What it is: evaluate lines in replicated tests for quantitative traits (e.g., yield) before they are adequately homozygous and homogeneous.
  • Purpose: select lines whose progeny have the potential to become cultivars, saving time and resources.
  • Don't confuse: early-generation testing does not mean the lines are ready for release; it means testing them to decide which to advance.

🧪 Example: Barlow wheat (modified bulk + pedigree + early-generation testing)

  • F₂: 200 plants selected; 20 spikes threshed individually.
  • F₃ headrows (F₂:₃ lines) grown in New Zealand; selected lines threshed in bulk (modified bulk).
  • F₂:₄ lines planted in yield test (early-generation test); lines with desirable yield selected.
  • 10 spikes from selected F₂:₄ lines planted as F₄:₅ lines in New Zealand; one F₄:₅ line selected and threshed in bulk.
  • F₄-derived line became cultivar Barlow.

🔬 Doubled haploids

  • What they are: lines derived from individual gametes of an F₁ plant, each genetically different.
  • Advantage: completely homozygous from the start (no need for multiple selfing generations).
  • Mentioned but not detailed: the excerpt notes that doubled haploids are an exception to the rule that complete homozygosity is rarely achieved, but does not describe the method in detail.

🔄 Combining methods

🔄 Flexibility in practice

  • Breeders commonly use combinations of methods during generation advance.
  • Example: select among F₂ plants in the local environment → advance F₃ in an off-season nursery without selection → resume selection among F₃:₄ lines in the local environment.
  • The choice depends on:
    • What traits can be evaluated in each environment.
    • Cost and time constraints.
    • Desired level of genetic variability.

🔄 Modified methods

  • When breeders use the term "modified," it means they changed the standard procedure.
  • Example: "modified bulk" in the Barlow wheat example meant harvesting each F₂:₃ line in bulk instead of harvesting individual plants (as in the standard pedigree method).
  • To understand what was modified: carefully compare what the breeders did to the standard procedure.
10

Recurrent Selection

Recurrent Selection

🧭 Overview

🧠 One-sentence thesis

Recurrent selection is a cyclical breeding method that progressively increases the frequency of favorable alleles in a population through repeated cycles of selection and intermating, with different approaches suited to synthetic, clonal, pure-line, and hybrid cultivar development.

📌 Key points (3–5)

  • What recurrent selection does: cycles of selecting superior individuals, intermating them, and using the resulting population as the starting point for the next cycle, progressively enriching favorable alleles.
  • Synthetic cultivars: the improved population itself becomes the cultivar; most extensive use of recurrent selection, especially in forage and turf species.
  • Selection timing matters: eliminating undesirable plants before flowering (so they don't contribute pollen) produces greater frequency shifts than eliminating them after flowering.
  • Common confusion: clonal vs. pure-line vs. hybrid cultivars require different recurrent selection strategies because they differ in how easily self-pollinated or hybrid seed can be obtained, the impact of inbreeding depression, and the role of combining ability.
  • Phenotypic vs. genotypic selection: phenotypic selection evaluates individual plants directly; genotypic selection evaluates families (half-sib, full-sib, or selfed progeny) to assess combining ability or breeding value.

🔄 Core mechanism: cycles and population improvement

🔄 What a cycle is

The initial population used for recurrent selection is referred to as cycle 0. Each subsequent cycle of selection is identified with a consecutive number.

  • Each cycle involves: evaluate individuals → select the best → intermate them → harvest seed in bulk → that seed becomes the next cycle population.
  • Example: Cycle 0 → select resistant plants → intermate → bulk seed = Cycle 1 → repeat for Cycle 2, 3, etc.
  • The goal is to increase the frequency of favorable alleles step by step.

🧬 Why intermating selected individuals matters

  • Key principle: By allowing only selected (e.g., resistant) plants to intermate, the frequency of favorable alleles in the next cycle will be greater than if selected and unselected plants had intermated and seed was harvested only from the selected ones.
  • This principle is detailed on pages 174–178 of chapter 15.
  • Example: If you let resistant and susceptible plants intermate, susceptible pollen fertilizes resistant plants, diluting the favorable allele frequency; if you remove susceptible plants before flowering, only resistant pollen is available.

🌱 Clones and genotypes

  • In recurrent selection, each plant is a different genotype.
  • Individual plants (genotypes) are commonly referred to as clones.
  • Breeders often evaluate large numbers of individuals each cycle (e.g., 10,000 plants in the FreedomMR example).

🌾 Synthetic cultivars: the population is the product

🌾 What synthetic cultivars are

  • The improved population itself is used as the cultivar, not individual selected plants.
  • Most extensive use of recurrent selection is for synthetic cultivars, particularly for forage and turf species.
  • Phenotypic selection alone or in combination with genotypic selection is common practice.

🦠 Example: FreedomMR (phenotypic selection only)

Cycle 0: The synthetic cultivar Freedom was the starting population.

Cycle 1:

  1. ~10,000 plants germinated in greenhouse, exposed to natural powdery mildew infections.
  2. ~40% susceptible plants discarded.
  3. Remaining 60% transplanted to field; ~8.7% more discarded as susceptible.
  4. Remaining resistant plants allowed to intermate by insect pollination.
  5. Seed harvested in bulk with a combine = Cycle 1 population.

Cycles 2–5: Same procedure repeated; selection intensity (percentage of resistant plants selected) varied among cycles.

Final cultivar: Cycle 5 resistant plants further selected for absence of pubescence; resistant, non-pubescent plants intermated in a polycross to produce breeder (Syn 1) seed of FreedomMR.

🌾 Example: Warrior indiangrass (phenotypic + genotypic selection)

Cycle 0: The cultivar Oto (from native prairies in southern Nebraska and eastern Kansas).

Cycle 1 (genotypic selection via half-sib families):

  1. 146 individual clones from Cycle 0 planted in a polycross.
  2. Clones intercrossed naturally by wind pollination.
  3. Seed harvested from each of the 146 clones (each clone served as the tester; the 146 clones' pollen fertilized each other).
  4. Progeny from each clone planted in two field replications; data collected for quality and yield.
  5. 29 of 146 clones selected based on performance.
  6. Two cuttings from each of the 29 clones planted in a polycross with two replications.
  7. 29 clones intercrossed by wind; seed from each clone harvested separately; equal quantity from each bulked = Cycle 1 population.

Cycle 2 (restricted recurrent phenotypic selection, RRPS):

  • Gridding: 53 rows, each with 14 plants; ~3 best plants per row harvested; plants in one row not compared to plants in other rows.
  • 39 plants selected and grown in a polycross; seed from polycross = Cycle 2 population.

Cycle 3 (RRPS again):

  • 38 selected plants grown in a polycross; seed from polycross = cultivar Warrior.

🔬 Polycross details

A polycross is a nursery in which selected clones are intermated.

  • Breeder options:
    • Grow a single plot of each plant OR clonally propagate each plant into multiple replications.
    • Harvest seed from all clones in bulk OR harvest clones individually and bulk equal quantities of seed from each.
  • Details on alternative procedures: pages 181–188 of chapter 15.

🧪 Half-sib families and general combining ability

  • For species with open pollination (wind or insects), half-sib families are formed in a polycross.
  • Seed harvested from each clone in the polycross has a common parent (the tester).
  • The clones themselves serve as the tester because their pollen fertilizes each other.
  • Evaluation of half-sib seed determines the general combining ability of the clones for quality and yield.
  • Note: Starting with half-sib family evaluation (as in Warrior Cycle 1) is less common than conducting recurrent phenotypic selection first for highly heritable traits, then evaluating half-sib families in later cycles.

🌿 Maintenance nursery

  • During field testing of progeny, each clone is grown in a maintenance nursery.
  • If a clone is selected for future use, vegetative tissue for propagation is taken from the plant in the maintenance nursery.

🧬 Clonal propagation principle

  • By clonally propagating selected individuals (e.g., the 29 clones in Warrior Cycle 1), all female and male gametes involved in producing the next cycle seed come from selected individuals.
  • This principle is described on pages 106–110 of chapter 8.

🌿 Clonal, pure-line, and hybrid cultivars: improving source populations

🌿 Key difference from synthetics

  • For clonal, pure-line, and hybrid cultivars, recurrent selection improves populations from which superior individuals are selected.
  • Unlike synthetic cultivars, the improved population is not used as a cultivar per se.

🌿 Clonal cultivars

  • Most common method: recurrent phenotypic selection.
  • Why: Combining ability is not a factor in identifying individuals that will perform well as a clonal cultivar, which minimizes the value of testing half-sib or full-sib families.
  • Inbreeding depression limits the value of testing selfed progeny.

🌾 Pure-line cultivars

  • Most common method: using self-pollinated individuals and their progeny, because self-pollinated seed is readily obtained.
  • Recurrent phenotypic selection: can be used for quantitative traits with high heritability, if enough hybrid seed can be obtained for the next cycle when intermating selected individuals.
  • Half-sib selection: possible if enough seed can be obtained from an individual when crossed to a tester.
  • Genetic male sterility: has been used to facilitate production of hybrid seed by open pollination for recurrent phenotypic and half-sib selection (chapter 16).
  • Full-sib selection: limited by difficulty of producing hybrid seed when crossing two individuals.

🌽 Hybrid cultivars

  • All methods of recurrent selection are technically possible for improving populations from which inbred lines are obtained for use in hybrids.
  • Comparison of methods for genetic gain will be discussed in Principles of Cultivar Development under maximizing genetic gain.

🔍 Factors influencing method choice

The type of recurrent selection is influenced by:

  • Feasibility of obtaining self-pollinated seed.
  • Impact of inbreeding depression.
  • Feasibility of obtaining hybrid seed.
  • Role of combining ability in assessing genetic potential of an individual.

🧮 Selection timing: before vs. after flowering

🧮 Why timing matters

  • Key principle: Eliminating undesirable plants before flowering produces a greater shift in allele frequency than eliminating them after flowering.
  • Reason: If undesirable plants flower, their pollen can fertilize desirable plants, diluting the favorable allele frequency in the next generation.

🧮 Worked example scenario (from review questions)

Setup: Random-mated maize population; 64% susceptible, 36% resistant; resistance controlled by dominant allele P.

Scenario 1: Eliminate susceptible plants after flowering

  • Susceptible plants have already contributed pollen.
  • Seed is harvested only from resistant plants, but some of that seed was fertilized by susceptible pollen.
  • Result: Cycle 1 population will have a certain frequency of P and p alleles.

Scenario 2: Eliminate susceptible plants before flowering

  • Susceptible plants do not contribute pollen.
  • Only resistant plants contribute both female and male gametes.
  • Result: Cycle 1 population will have a higher frequency of the P allele than in Scenario 1.

🧮 Genotypic and phenotypic frequencies

  • After selection and intermating, the next cycle population will have different genotypic frequencies (PP, Pp, pp) and phenotypic frequencies (resistant vs. susceptible).
  • If resistant S₀ plants are self-pollinated, some S₀:₁ lines will be heterogeneous (segregating) for resistance if the S₀ parent was Pp.
  • Within a heterogeneous line, genotypic and phenotypic frequencies follow Mendelian ratios.

🔬 Phenotypic vs. genotypic selection

🔬 Phenotypic selection

  • Evaluates individual plants directly based on their observable traits.
  • Example: FreedomMR used only phenotypic selection—plants were visually screened for disease resistance and pubescence.
  • Common for highly heritable traits.

🔬 Genotypic selection

  • Evaluates families (progeny) to assess the breeding value or combining ability of parents.
  • Types of families:
    • Half-sib families: progeny from one known parent and multiple unknown pollen donors (common in polycross).
    • Full-sib families: progeny from two known parents.
    • Selfed progeny: progeny from self-pollination.
  • Example: Warrior Cycle 1 evaluated 146 half-sib families to determine general combining ability.

🔬 Combining phenotypic and genotypic selection

  • Some breeding programs use phenotypic selection in early cycles (to quickly improve highly heritable traits) and genotypic selection in later cycles (to assess combining ability or breeding value for complex traits).
  • Example: Warrior used genotypic selection (half-sib families) in Cycle 1, then phenotypic selection (RRPS) in Cycles 2 and 3.

🧩 Restricted recurrent phenotypic selection (RRPS)

🧩 What RRPS is

  • A variant of recurrent phenotypic selection described on page 181 of chapter 15.
  • Features include gridding and intercrossing only selected individual clones.

🧩 Gridding

  • The field is divided into a grid (e.g., 53 rows, each with 14 plants).
  • A fixed number of best plants are selected from each row (e.g., ~3 plants per row).
  • Key principle: Plants in one row are not compared to plants in other rows.
  • This ensures selection is distributed across the entire population, not just concentrated in a few high-performing areas.

🧩 Intercrossing selected clones

  • After evaluation, selected plants (e.g., 39 plants in Warrior Cycle 2) are grown in a polycross.
  • Seed harvested from the polycross constitutes the next cycle population.

📚 Key definitions and terms

Recurrent selection: A cyclical breeding method involving repeated cycles of selection and intermating to progressively increase the frequency of favorable alleles in a population.

Cycle 0: The initial population used for recurrent selection; each subsequent cycle is identified with a consecutive number.

Clone: An individual plant (genotype); in recurrent selection, each plant is a different genotype.

Polycross: A nursery in which selected clones are intermated by open pollination (wind or insects).

Half-sib family: Progeny from one known parent (the tester) and multiple unknown pollen donors; used to assess general combining ability.

General combining ability: The average performance of a clone's progeny when crossed with multiple other clones.

Maintenance nursery: A field nursery where clones are grown during progeny testing; vegetative tissue for propagation is taken from plants in this nursery if the clone is selected.

Restricted recurrent phenotypic selection (RRPS): A method using gridding and intercrossing only selected clones.

Gridding: Dividing the field into a grid and selecting a fixed number of best plants from each grid unit, without comparing across units.

Synthetic cultivar: A cultivar in which the improved population itself is the product, not individual selected plants.

🔄 Don't confuse

ConceptWhat it isWhat it is NOT
Synthetic cultivarThe improved population itself is the cultivarIndividual selected plants are the cultivar
Phenotypic selectionEvaluates individual plants directlyEvaluates families (progeny)
Genotypic selectionEvaluates families to assess breeding valueEvaluates individual plants directly
Half-sib familyOne known parent, multiple unknown pollen donorsTwo known parents (that's full-sib)
Eliminating plants before floweringOnly selected plants contribute pollenSelected and unselected plants both contribute pollen (after flowering)
Clonal cultivar breedingCombining ability not a factor; phenotypic selection most commonCombining ability is key (that's hybrid breeding)
Gridding in RRPSPlants in one row not compared to other rowsAll plants compared across the entire field