Plant breeding and genetics

PRINCIPLES OF GENETICS

Genetics and Cytogenetics: Definitions and Historical Developments

● Genetics: The science of heredity and variation, explaining similarities between parents and progeny and differences among individuals of a single species.

● Heredity: The process causing biological similarity between parents and offspring

● Inheritance: The transmission of genetic information from parents and ancestors to offspring

● Variation: Differences among individuals of a single species for a particular character

● Genes: Functional units governing the development of characters; units of inheritance

● Cytology: The study of cell structure and functions of cell organelles

●     Cytogenetics: The study of chromosomes and their effects on organism development. It combines cytology and genetics

Key Discoveries and Scientists:

● 1665: Robert Hooke discovered the cell and coined the term "cell".

● 1831: Robert Brown discovered the presence of the nucleus in cells.

● 1838: Schleiden and Schwann formulated the Cell Theory.

● 1870: Fredrick Meischer isolated Nucleoprotein.

● 1879: Flemming described chromatin in the nucleus.

● 1882: Flemming described cell division (Mitosis).

● 1888: Waldeyer described chromosomes.

● 1903: Sutton proposed the Chromosome Theory.

● 1905: Farmer and Moore coined the term Meiosis.

● 1910: T.H. Morgan established the chromosome theory of heredity and showed genes are linked on chromosomes. He received the Nobel Prize in 1933.

● 1950: Barbara McClintock discovered transposons (jumping genes) in Maize, receiving the Nobel Prize in 1983.

● 1953: Watson and Crick, based on Wilkinson's X-ray diffraction studies, proposed the double helix model of DNA. They received the Nobel Prize in 1962.

● 1958: F.H.C. Crick proposed the Central Dogma of Molecular Biology.

● 1962: Jacob and Monad explained gene regulation through the Operon concept, receiving the Nobel Prize in 1965.

● 1973: Stanley Cohen and Herberd Boyer were involved in Genetic Engineering.

●     1977: Maxam and Gilbert, and Sanger and Coulson developed methods for DNA sequencing.

Cell-Structure and Function

Cell Theory: States that the cell is the smallest building element of multicellular organisms, each cell has a specific work, and cells are produced from pre-existing cells by division.

Prokaryotic vs. Eukaryotic Cells:

Ø  Primitive organisms (bacteria, blue-green algae) have unorganized nuclei called prokaryotic

Ø  Evolved organisms have organized nuclei called eukaryotic

Gross Morphology of a Cell:

·         Cell Wall:

Ø  A non-living, rigid outer coat in plant cells (absent in animal cells)

Ø  Provide shape, mechanical support, and strength

Ø  Has three parts:

§  Middle Lamella

§  Primary Cell Wall

§  Secondary Cell Wall

·         Plasma Lemma (Plasma Membrane):

Ø  Encloses the cytoplasm, composed of lipids and proteins

Ø  Chief function is to regulate the movement of molecules into and out of the cytoplasm via passive and active transport

·         Cytoplasm:

Ø  The substance within the plasma lemma, excluding the nucleus

Ø  Contains various membranous and other structures like Endoplasmic Reticulum, Ribosomes, Golgi bodies, Lysosomes, Mitochondria, and Plastids

Ø  Mitochondria and plastids contain DNA, making them semi-autonomous

·         Endoplasmic Reticulum (E.R.): An extensive network of membrane-enclosed spaces in the cytoplasm.

Ø  Smooth E.R.: Smooth surfaces, found in cells with little or no protein synthesis.

Ø  Rough E.R.: Ribosomes attached to outer surfaces, mainly composed of cisterns, found in cells active in protein synthesis.

Ø  Functions:

§  Provides structural base for protein, lipid, and phospholipid synthesis

§  Transports materials

§  Facilitates mRNA export from nucleus

§  Embeds enzymes.

·         Ribosomes:

Ø  Dense granular nucleoprotein structures in cytoplasm, mitochondria, and chloroplasts

Ø  Primary sites of protein synthesis

·         Golgi Complex (Dictyosomes in plants):

Ø  Discovered by Camilio Golgi in 1890.

Ø  Functions:

§  Absorption of compounds

§  Enzyme and hormonal production

§  Protein storage

§  Plant cell wall formation (pectin, hemicellulose, cellulose microfibrils)

§  Cell plate formation during mitosis

·         Plastids:

Ø  Living cytoplasmic inclusions in most plants, categorized into

§  Chromoplasts (pigmented)

§  Leucoplasts (colourless, for storage)

§  Chloroplasts (green, primary sites for photosynthesis).

·         Nucleus:

Ø  The most important organelle, regulating all cell activities

Ø  Discovered by Robert Brown (1831)

Ø  Parts:

§  Nuclear membrane

§  Karyolymph (Nuclear sap)

§  Chromonemata

§  Nucleolus

Ø  Nuclear Membrane: Double membrane with nucleopores, facilitates communication between nucleus and cytoplasm.

Ø  Nucleolus:

§  A spheroidal body regulating the synthetic activity of the nucleus, rich in RNA.

§  Active site of RNA synthesis and produces ribosomal RNA and ribosome precursors.

Cell Division: Mitosis and Meiosis

● Cell Division: The process by which chromosomes and cytoplasm divide into daughter cells.

● Functions of Cell Division:

Ø  Growth and development

Ø  Regeneration of damaged tissues

Ø  Production of new tissues

Ø  Reproduction

Ø  Maintaining cell size within a range

Mitosis:

●First described by Fleming in 1882.

● Occurs in meristematic tissues in plants.

Stages:

Ø  Interphase:

§  The longest stage where chromosomes are extended and DNA replication occurs

§  Divided into G1, S (DNA synthesis), and G2 phases

Ø  Prophase:

§  Chromosomes condense, becoming shorter and thicker

§  Nucleolus and nuclear membrane remain present

§  Relational coiling of sister chromatids occurs

Ø  Metaphase:

§  Nucleolus and nuclear membrane disappear

§  Chromosomes, at their shortest and thickest, align on the equatorial plate

§  Spindle apparatus is present

Ø  Anaphase:

§  Sister chromatids separate and migrate to opposite poles

§  Centromeres lead the movement

Ø  Telophase:

§  Chromosomes uncoil

§  Nucleolus reappears

§  Nuclear membrane reorganizes around each group of chromosomes

Cytokinesis:

• Division of the cytoplasm, usually completed by the end of Telophase

• Resulting in two daughter cells with the same chromosome number as the parent cell

Significance of Mitosis:

Ø  Responsible for zygote development into an adult

Ø  Normal growth

Ø  Tissue repair

Ø  Production of new plant parts (roots, leaves, stems)

Ø  Production of identical progenies in vegetatively propagated crops

Ø  Replacement of old tissues in animals (e.g., blood cells)

Meiosis:

● Occurs during gamete formation in reproductive cells

● Reduces the somatic chromosome number (2n) by half (n) in gametes

● Restores the diploid chromosome number (2n) upon fertilization, maintaining species chromosome number across generations

● Involves two successive divisions:

o   First Meiotic Division

o   Second Meiotic Division

● Pre-Meiotic Interphase: Chromosome replication occurs, but 0.3% of DNA replicates during zygotene of Prophase I.

● First Meiotic Division (Reduction Division):

             Characterized by pairing of homologous chromosomes, crossing over, and separation of homologous chromosomes.

Ø  Prophase I: Divided into five substages:

§  Leptotene:

o   Increased nuclear volume

o   Chromosome condensation (visible as fine threads)

o   Each chromosome with two chromatids

§  Zygotene:

o   Initiation of pairing (synapsis) between homologous chromosomes

o   Completion of remaining DNA replication (Z-DNA synthesis)

o   Synthesis of specific nuclear protein, and development of synaptonemal complex

§  Pachytene:

o   Further chromosome condensation

o   Homologous chromosomes easily recognizable with four chromatids (bivalents)

o   Distinct nucleolus

o   Crossing over occurs

§  Diplotene:

o   Homologous chromosomes begin to separate but remain attached at chiasmata (points of crossing over)

o   Chiasmata slowly move towards chromosome ends (chiasma terminalization)

§  Diakinesis:

o   Bivalents move to cell periphery

o   Nucleolus and nuclear envelope disappear

o   Spindle apparatus organizes

o   Bivalents migrate to the equatorial plate

Ø  Metaphase I: Bivalents arrange at the metaphase plate, with centromeres of homologous chromosomes on either side

Ø  Anaphase I: Homologous chromosomes separate and migrate to opposite poles, resulting in each pole receiving half the chromosome number at each pole

Chromosomal Aberrations

·         Chromosomal aberrations are spontaneous variations in chromosome number or structure.

·         Grouped into two classes:

Ø  Structural

Ø  Numerical

Structural Chromosomal Aberrations

  These aberrations alter chromosome structure, affecting the number, sequence, or kind of genes.

·         Four types exist:

Ø  Deletion (deficiency)

Ø  Duplication (repeat)

Ø  Inversion

Ø  Translocation

Deletion (Deficiency): Loss of a chromosome segment.

§  Terminal Deletion: A segment between the centromere and telomere is lost.

§  Interstitial Deletion: A segment between two breaks is lost.

  Duplication (Repeat): A chromosome segment is present in more than two copies in the same                                 nucleus.

§  Tandem Duplication: Additional segment is immediately after the normal segment with the same gene sequence.

§  Reverse Tandem Duplication: Same as above, but the gene sequence of the additional segment is inverted.

§  Displaced Duplication: Additional segment is in the same chromosome but away from the normal segment.

§  Translocation Duplication: Additional segment is in a nonhomologous chromosome.

Inversion: A chromosome segment contains genes in a sequence reverse to the normal.

§  Paracentric Inversion:

o   Does not contain the centromere

o   Produces dicentric bridge and acentric fragment during anaphase I in heterozygotes

§  Pericentric Inversion:

o   Contains the centromere

o   May change centromere location

Translocation: Chromosome segment(s) integrated into nonhomologous chromosome(s). Main mechanism for changes in chromosome number and morphology in nature

§  Simple Translocation (Shift): A segment of a chromosome integrated into a nonhomologous chromosome.

§  Reciprocal Translocation (Exchange):

o   A segment from one chromosome is integrated into a nonhomologous chromosome, from which a segment is integrated into the first one

o   Produces characteristic ring or chain of four or more chromosomes at Metaphase I in heterozygotes

Numerical Chromosomal Aberrations (Heteroploidy)

Ø  Any deviation from this diploid state is a numerical chromosomal aberration, also known as heteroploidy

Ø  Heteroploid states are categorized into:

§  Aneuploidy

§  Euploidy

Aneuploidy

·         Aneuploidy is the loss or gain of one or a few chromosomes compared to the somatic chromosome number of a species (2n).

·         It does not involve the entire genome, only one or a few chromosomes.

Terminology of Aneuploidy

Ø  Monosomic (2n-1): Deficit of one chromosome from the somatic number.

Ø  Nullisomic (2n-2): Absence of a chromosome pair.

Ø  Double Monosomic (2n-1-1): Two nonhomologous chromosomes missing.

Ø  Trisomic (2n+1): Individuals with one extra chromosome.

Ø  Tetrasomic (2n+2): Individuals with an additional chromosome pair.

Ø  Double Trisomic (2n+1+1): Two additional nonhomologous chromosomes.

History of Aneuploidy

·         First discovered by Bridges in 1916 (XO male and XXY female Drosophila).

·         Belling demonstrated trisomy in Datura stramonium (globe mutant) in 1920.

·         Monosomics in tobacco were extracted by Clausen and associates in the 1930s.

·         Complete sets of monosomics and nullisomics have been developed in crops like wheat, Ishacon, cotton, and oats.

Nullisomy (2n-2)

·         One pair of chromosomes is missing from the somatic complement.

·         Morphological Effects:

o   Nullisomics of hexaploid wheat are weaker and smaller.

o   Nullisomics in tobacco usually do not survive.

·         Origin and Propagation: Nullisomics are difficult to propagate and are often obtained from monosomic plants (approx. 3% in selfed progeny).

Monosomy (2n-1)

·         One chromosome is missing from the somatic chromosome complement.

·         Morphological Effects: Creates major genetic imbalance, generally not tolerated by diploid species.

·         Monosomics of polyploid species (e.g., wheat, cotton, oats, tobacco) are fully viable.

·         Monosomics show variable reduction in vigor, size, and fertility; maize and tomato monosomics are weak, while wheat monosomics are comparable to normal plants.

Trisomy (2n+1)

·         Somatic cells contain three copies of one chromosome.

·         Grouped into three categories:

o   Primary

o   Secondary

o   Tertiary

·         Primary Trisomics:

Ø  The additional chromosome is an unaltered member of the haploid complement.

Ø  There can be 'n' different primary trisomics (e.g., 7 in barley, 10 in maize and tomato).

·         Secondary Trisomics:

Ø  The extra chromosome is an isochromosome, meaning its two arms are identical in gene content.

Ø  Each chromosome can form two different isochromosomes, making 2n different secondary trisomics possible.

·         Tertiary Trisomics: Involve a translocated chromosome.

Tetrasomy (2n+2)

·         Somatic cells contain one pair of a chromosome in excess, meaning one chromosome is present in four copies.

·         Morphological Effects:

Ø  Generally more deleterious than trisomy, so fewer tetrasomics of diploid species are viable.

Ø  Viable in polyploid species like wheat.

HEREDITY

Pre-Mendelian Ideas About Heredity

Before Mendel's principles were rediscovered in 1900, various theories attempted to explain heredity:

·         Vapour and Fluid Theory (Pythagoras, ~500 B.C.): Proposed that hereditary information existed as vapours and fluids from body organs that formed the embryo in the uterus.

·         Magnetic Power Theory (Harvey, 1578-1657): Suggested the uterus had a "magnetic power" to consume an embryo.

·         Pre-formation Theory (17th Century Biologists): Believed new individuals were pre-formed in miniature within gametes (sperms or eggs).

·         Epigenetic Theory (Wolf, 1738): Proposed that each egg had a granule that gradually developed into various organs of the embryo.

·         Particulate Theory (Maupertius, 1698-1759): Suggested both parents contribute gametes, and each organ of the embryo is made of two parts, one from each parent.

·         Lamarck's Theory (1744-1829): Proposed that environmental changes cause modifications in organisms, and these "acquired characters" are transmitted to subsequent generations. (e.g., giraffe necks lengthening due to stretching and being inherited).

·         Darwin's Theory of Pangenesis (1868): Proposed that "gemmules" or "pangenes" produced by every part of the body accumulate in germ cells, transmitting parental characters and acquired modifications to offspring.

·         Weismann's Germplasm Theory (1887): Suggested that hereditary particles (genes on chromosomes) constitute the germplasm, which is distinct from the somatoplasm (body). He argued that acquired characters are not inherited, proven by experiments like cutting mouse tails for generations and ovary transplantation in guinea pigs.

·         De Vries' Mutation Theory (1901): Introduced the term "mutation" for large, discontinuous hereditary changes and proposed that these sudden changes lead to evolution. He observed these variations in the evening primrose (Oenothera lamarckiana).

Mendel's Work

·         Gregor John Mendel (1822-1884): Conducted experiments on garden peas (Pisum sativum) from 1856-1863.

·         Experimental Material: Pea plants offered advantages such as

Ø  Distinct contrasting characters

Ø  Self-pollinating flowers (avoiding contamination)

Ø  Large flowers for easy artificial hybridization

Ø  A short growing season (allowing multiple generations per year)

Ø  Easy cultivation

·         Studied Seven Contrasting Characters:

Ø  Seed shape (Round/Wrinkled)

Ø  Petal colour (Purple/White)

Ø  Cotyledon colour (Yellow/Green)

Ø  Pod colour (Green/Yellow)

Ø  Pod shape (Full/Constricted)

Ø  Position of flower (Axial/Terminal)

Ø  Length of stem (Tall/Dwarf)

·         Published Work: Presented "Experiments in Plant Hybridization" in German, but its importance was not recognized until 1900.

Gene Interactions and Inheritance Patterns

·         Epistasis: One gene masks or modifies the expression of another gene. Different types of epistasis include:

o   Dominant Epistasis (12:3:1): A dominant allele of one gene masks the expression of alleles of another gene.

o   Recessive Epistasis (9:3:4): The recessive homozygous state of one gene masks the expression of alleles of another gene.

o   Dominant and Recessive Epistasis (13:3):

§  One dominant gene produces a phenotype, and its recessive allele in homozygous state produces a contrasting phenotype.

§  A second dominant gene can stop the expression of the first gene.

o   Duplicate Recessive Epistasis (9:7): Requires the presence of dominant alleles of both genes for one of the two phenotypes; if either or both genes are homozygous recessive, the contrasting phenotype is produced.

o   Duplicate Dominant Interaction (15:1): Both dominant genes produce the same phenotype.

o   Duplicate Gene with Cumulative Effect (9:6:1): Genes have an additive effect.

·         Lethal Genes: Genes that cause the death of an organism, typically when in a homozygous state, before it reaches adulthood.

• • Recessive Lethal: Express their lethal effect only in the homozygous state.

  • Dominant Lethal: Lethal in homozygous conditions and produce defective phenotypes in heterozygous conditions.

  • Balanced Lethal System: Both homozygous dominant and homozygous recessive individuals die, leaving only heterozygotes.

  • Conditional Lethal: Genes that are lethal under specific environmental conditions.

  • Gametic Lethal: Genes that cause the inviability of gametes or make them incapable of fertilization.

·         Pleiotropy: A single gene controls more than one phenotypic character of an organism.

·         Penetrance: The ability of a gene or gene combination to be expressed phenotypically to any degree.

• • Complete Penetrance: Most dominant and recessive genes show their phenotypic expression in homozygous and many completely dominant genes in heterozygous conditions.

  • Incomplete Penetrance: Heterozygous conditions may not fully express the normal phenotype.

·         Expressivity:

Ø  The degree of phenotypic expression of a gene in different individuals.

Ø  Environmental factors influence expressivity

§  Temperature,

§  Nutrition,

§  Hormone deficiency

·         Phenocopy: A phenotype altered by the environment to resemble another phenotype produced by known genes, but it is not inherited.

·         Multiple Alleles: More than two alleles at the same locus, adding variability for a character.

• • Control the same character, but expression differs based on the allele present.

  • No crossing over occurs within a multiple allelic series.

  • Wild type is always dominant in a series of multiple alleles.

  • Crosses between two mutant alleles produce an intermediate mutant phenotype.

  • Examples

    • Fur colour in rabbits

    • Wing/eye colour in Drosophila

    • Self-incompatibility alleles in plants

    • ABO blood groups in humans

·         Pseudoalleles: Non-alleles that are very closely linked and often inherited as a single gene, but can be separated by crossover studies.

·         Isoalleles: Different wild-type alleles affecting the same character, showing similar allelic dominance or differing in expression detectable only in special combinations.

·         Modifying Genes: Genes that alter the expression of a major gene but have no effect on the allele itself. They have small, cumulative effects and are often numerous.

·         Quantitative Inheritance (Polygenic Inheritance): Traits that show continuous variation and are governed by a large number of genes (polygenes) with cumulative effects.

• • Follows Mendelian principles.

  • Contrast with Qualitative Characters which show discontinuous variation and are governed by one or two major genes.

  • Nilsson-Ehle's experiment on wheat kernel colour demonstrated quantitative inheritance, showing that multiple duplicate dominant alleles cumulatively determine the intensity of red colour.

  • Transgressive Segregation: When the range of phenotypes in the F2 progeny extends beyond the original parents, resulting in individuals with higher or lower intensity of expression than both parents.

Linkage and Crossing Over

·         Linkage: The tendency of two or more genes to stay together during inheritance because they are located on the same chromosome.

• • Linked genes do not show independent segregation.

  • A linkage group is a group of genes on the same chromosome, and its number equals the haploid chromosome number.

  • Chromosome Theory of Linkage:

    • Genes are carried on chromosomes, arranged linearly, and tend to be inherited together.

    • The strength of linkage is inversely proportional to the distance between genes.

  • Coupling Phase (Cis-arrangement):

    • Dominant genes of both pairs are on one chromosome, and their recessive alleles are on the homologous chromosome

    • Example: AB/ab

  • Repulsion Phase (Trans-arrangement):

    • Dominant gene of one pair and a recessive gene of another pair are on one chromosome, with the inverse on the homologous chromosome

    • Example: Ab/aB

  • Completely Linked Genes:

    • Genes very close on a chromosome, moving together to gametes with no recombination.

    • Example: Male Drosophila

  • Incompletely Linked Genes: Genes on the same chromosome showing a moderate level of crossovers.

·         Crossing Over: The exchange of homologous segments between non-sister chromatids of homologous chromosomes during meiosis (pachytene stage of prophase I).

• • Produces recombinant chromatids and parental chromatids.

  • Occurs at the "four strand stage."

  • Leads to new combinations of linked genes (recombination).

  • Frequency of recombinants can vary from 0-50%.

  • Chiasma: The point where segments are exchanged between non-sister chromatids.

  • Chiasma Terminalization: The movement of chiasma away from the centromere towards the ends of tetrads.

  • Types include single, double, and multiple crossing over, based on the number of chiasmata.

Sex Determination

Ø  Sex differentiation leads to morphological, physiological, and behavioural differences between males and females, a phenomenon called sexual dimorphism.

Ø  A sex-determination system is a biological mechanism that dictates the development of sexual characteristics in an organism.

·         Categories of Organisms Based on Sex Organs:

o    Monoecious:

§  Organisms with both male and female sex organs in the same individual, producing both types of gametes

§  Example: maize, earthworms, hydra

o    Dioecious:

§  Organisms with separate male and female individuals, each producing only one type of gamete

§  Example: papaya, palm

o    Hermaphrodites:

§  Plants/flowers where both male and female organs occur together

§  Example: paddy, Hibiscus

·         Gynandromorphs:

§  Individuals that show male characteristics in some parts of their body and female characteristics in others, typically due to irregularities in cell division during embryonic development

§  Example: Drosophila

How Sex is Determined:

·Sex Chromosome: Heteromorphic chromosomes (like X or Y) whose distribution in a zygote determines the organism's sex.

·Barr Body: An inactive X chromosome found in female cells (e.g., human beings), rendered inactive through a process called Lyonization.

Chromosomal Mechanisms:

Ø  XX Female, XY Male:

§  Most common (humans, mammals, some insects, some plants)

§  Females are homogametic (XX)

§  Males are heterogametic (XY)

Ø  XY Female, XX Male:

§  Found in birds and some insects

§  Females are heterogametic (XY)

§  Males are homogametic (XX)

Ø  XX Female, X0 Male:

§  Found in grasshoppers, crickets, cockroaches

§  Males have one X chromosome (X0)

§  Females have two (XX); the '0' signifies the lack of a second X

Ø  X0 Female, XX Male: Observed in Fumea.

Ø  Haplodiploid:

§  Sex determined by ploidy level (e.g., honeybees, ants, wasps)

§  Females are diploid

§  Males are haploid

                        Genic Balance Theory (Bridges, 1921):

oExample: Drosophila sex is determined by the ratio of X chromosomes to sets of autosomes.

oThe X chromosome primarily carries female-determining genes, while autosomes carry male-determining genes.

oThe balance between these determines the sex.

                        Environment: In some species, environmental variables like

o Temperature (reptiles like alligators, some turtles, tuatara)

o Social variables (e.g., dominant clownfish becoming female) determine sex

o Infection by bacteria (e.g., Wolbachia) can determine sex in some arthropods

Sex-Linked Inheritance

• Sex Linkage: The location of a gene on a sex chromosome.

• Criss-Cross Inheritance:

  •  A pattern where a sex-linked gene passes from a male to his female offspring and then back to male offspring.

  • This occurs when the gene is located on the X chromosome (X-linked gene), and the Y chromosome has no gene for that character.

  • Example: Morgan's experiments on eye colour in Drosophila demonstrated that the gene for eye colour is X-linked.

• Y-linked (Holandric) Genes:

  •  Genes located on the Y chromosome, with alleles absent on the X chromosome

  •  Example: Gene for hypertrichosis causing hairy ear lobes in humans).

• XY-linked Genes:

  • Genes present on both X and Y chromosomes in homologous regions

  • Example: Genes for colour blindness, xeroderma pigmentosum, retinitis pigmentosa, nephritis in humans).

• Sex-Influenced Characters:

  • Characters expressed differently in the two sexes even with identical genotypes, typically due to hormonal influence.

  • The genes are autosomal.

  • Dominance can be reversed between sexes

  • Example: Horns in sheep where horned is recessive in females but dominant in males; baldness in humans, recessive in females, dominant in males

• Sex-Limited Characters:

  • An extreme form of sex influence where a particular phenotype can only be expressed in one sex, despite identical genotypes in both.

  • These genes are autosomal, but physiological differences prevent expression in one sex

  • Example: Cock-feathering in domestic poultry, limited to males due to female sex hormones inhibiting its production in hens

Cytoplasmic Inheritance and Maternal Effects

• Cytoplasmic Inheritance (Extrachromosomal Inheritance): The transmission of hereditary characters through the cytoplasm, typically involving genes located in cytoplasmic organelles like plastids and mitochondria.

  • It is not controlled by Mendel's laws and is usually inherited uniparentally, often from the female parent, as the cytoplasm is primarily contributed by the egg.

  • Plasmon: The totality of heredity transmitted through the cytoplasm.

  • Plasmagenes: Cytoplasmic particles that exhibit genic properties like self-duplication, specificity, and mutability.

• Examples of Cytoplasmic Inheritance:

   Mitochondrial Inheritance in Yeast (Petite Mutants):

• • • Small colonies called petite mutants have slow growth due to inefficient aerobic metabolism, often caused by large deletions in mitochondrial DNA (mtDNA).

    • Neutral petites, when crossed with wild type, produce only wild type colonies, indicating uniparental inheritance linked to mtDNA.

Inheritance of Kappa Particles in Paramecium:

• • • Bacterial inclusions called kappa particles in the cytoplasm produce a "killer" phenotype, maintained by a dominant nuclear gene (K).

    • This trait is cytoplasmically transmitted, and its expression depends on the interaction between the nuclear gene and cytoplasmic kappa particles.

  • Inheritance of Plastids in Mirabilis jalapa (Four O'Clock Plant): Leaf variegation (green, pale green, white patches) is determined by agencies transmitted through the female parent, specifically chloroplasts, which are self-duplicating and transmitted via egg cytoplasm.

  • Maternal Inheritance by 'iojap' gene in Maize:

    • The iojap gene (Ij), located on chromosome VII, controls plastid inheritance.

    • Even with the same nuclear genotype (Ij ij), plants can have different phenotypes (normal green, striped, white) due to differences in plastids inherited maternally.

  • Cytoplasmic Male Sterility in Maize: This character is transmitted only through the female and is controlled by genes in the mitochondria (mitochondrial DNA alterations).

• Maternal Effect (e.g., Shell Coiling in Snail):

  • The phenotype of the offspring is determined by the mother's genotype, not directly by the offspring's own genotype or extranuclear factors.

  • The orientation of shell coiling (dextral or sinistral) in Limnaea peregra is predetermined in the egg cytoplasm by the mother's genotype.

Protein Synthesis and Genetic Code

• One Gene-One Enzyme Hypothesis (Beadle and Tatum, 1941):

  • Genes control the production of specific enzymes, and defects in genes can lead to loss or defect of specific enzymes.

  • This was later refined to "one gene-one polypeptide concept" by Ingram (1957).

• Protein Structure:

  • Proteins are made of polypeptide chains, which are composed of amino acids.

  • They have primary, secondary, tertiary, and quaternary structures.

• Colinearity: Crick (1958) proposed that the sequence of nucleotides in DNA is directly proportional to the sequence of amino acids in the corresponding protein.

• Process: DNA transcribes messenger RNA (mRNA), which then translates into protein on ribosomes, ultimately leading to a phenotypic trait.

CONVENTIONAL BREEDING METHODS

Selection (Pure line selection, Mass selection)

Selection in Self-Pollinated Crops

• Prerequisites for Successful Selection: Variation must be present and heritable in the population.

Effect of Self-Pollination on Genotype

• Self-pollination consistently increases homozygosity while decreasing heterozygosity

• For a single heterozygous gene (Aa), each generation of selfing reduces heterozygosity by 50% from the previous generation, with a corresponding increase in homozygotes (AA and aa)

• After 10 generations of selfing, most plants in the population become homozygous.

• The rate of homozygosity can be calculated by the formula [ (2^m - 1) / 2^m]^n

  • m is the number of generations of self-pollination

  • n is the number of segregating genes.

• The number of segregating genes and linkage between them do not affect the percentage of homozygosity in the population.

Genetic Advance Under Selection

• The improvement in the mean genotypic value of selected families over the base population.

• Factors: Depends on

  • Genetic variability

  • Heritability of the character

  • Selection intensity

• Calculation: GS = K * σP * H

  • GS is Genetic Advance

  • K is Selection differential

  • σP is Phenotypic standard deviation

  • H is Heritability.

Pure Line Theory

A pure line is the progeny of a single self-fertilized homozygous plant.

Johannsen's Experiment:

• Proposed by Johannsen based on his studies with beans (Phaseolus vulgaris) variety 'Princess'.

• He observed variation in seed size in a commercial lot, which had a genetic basis.

• He concluded that the market lot was a mixture of pure lines and that any variation within a pure line was solely due to environment.

Origin of Variation in Purelines

Variation within purelines can arise from:

1. Mechanical mixtures

2. Natural hybridization

3. Chromosomal aberrations

4. Natural mutation

5. Environmental factors

Pure Line Selection

·        A large number of plants are selected from a self-pollinated crop, harvested individually, and their progenies are evaluated.

·        The best progeny is yield tested and released as a variety.

Characteristics of Purelines:

• All plants within a pure line have the same genotype.

• Variation within a pure line is environmental and non-heritable.

• Over time, purelines can become genetically variable due to

• 1. Natural hybridization

  2. Mutation

  3. Mechanical mixtures.

General Steps:

• Year 1: Obtain variable population, select superior plants (e.g., 1,000).

• Year 2: Plant progeny rows of superior plants (e.g., 200), compare and reject unwanted progenies.

• Year 3-5: Select plants from superior rows to advance (e.g., 25-50).

• Year 6: Preliminary yield trials (e.g., 15 lines).

• Year 7-10: Advanced yield trials with checks (e.g., 10 lines), followed by multilocation trials and adaptive research trials in farmer's fields for release.

Advantages:

• Achieves maximum possible improvement over the original variety.

• Extremely uniform in appearance, making identification and seed certification easy.

• Rapid and inexpensive.

• Applicable to improving traits with low heritability as selection is based on progeny performance.

• Only the best pure line is selected for maximum genetic advance.

Disadvantages:

• Narrow adaptability and lower stability due to improvement in only the local variety.

• Takes more time than mass selection.

• Improvement is limited by the genetic variability in the base population.

• More demanding on the breeder due to careful progeny tests and yield trials.

• Narrow genetic base, making it susceptible to biotic and abiotic stresses.

• Does not create new genotypes; merely isolates the best from a mixture.

• Promotes genetic erosion by excluding other genetic variants.

• Progeny rows require more resources.

Mass Selection

·        A large number of plants with similar phenotypes are selected, and their seeds are mixed to form a new variety.

·        The resulting population is more uniform phenotypically than the original, but still genetically diverse.

Steps:

• First Season: Select 200-2000 phenotypically similar plants from the base population and bulk their seeds.

• Second Season: Plant the bulked seed in preliminary yield trials with controls, evaluate phenotype, and select higher-yielding plots.

• Third to Sixth Season: Conduct coordinated yield trials, and if outstanding, release as a new variety. Seed multiplication for distribution follows.

Merits:

• Varieties have wider adaptability and buffering action against abnormal environments due to genetic diversity.

• Less time is required for variety release.

• Genetic variability of the original population is maintained.

• Less demanding on the breeder as expensive yield trials are not always necessary for large selections.

Demerits:

• Less uniform than pure line varieties, making certification difficult.

• Superiority of selected plants cannot be definitively attributed to genotype without progeny testing.

• The variety is inferior to the best pure line in the population because it's a mixture, and most included purelines will be inferior to the best one.

Modified Mass Selection:

• Detasseling: Practiced in maize to eliminate inferior pollen by removing tassels from undesirable plants.

• Panmixis: Pollen from selected plants is collected, mixed, and used to pollinate selected plants, ensuring full control over the pollen source.

• Stratified or Grid or Unit Selection:

  • The field is divided into smaller units (e.g., 40-50 plants/plot).

  • Equal numbers of plants are selected from each plot, and their seeds are bulked.

  • This minimizes environmental variation.

  • Followed in maize improvement.

Comparison between Pure Line and Mass Selection

Feature                          Pureline Selection                                                                Mass Selection

Variety Type                  A new variety is a pureline.                                          A new variety is a mixture of purelines.

Uniformity                    Highly uniform; variation purely environmental         Genetic variation of quantitative characters

Progeny Test                 Selected plants are subjected to progeny test               Progeny test is generally not carried out

Adaptability                  Narrower adaptation and lower stability                      Wider adaptation and greater stability

Selection                       Plants are selected for desirability                                Selected plants must have similar phenotypes

Demands                       More demanding due to careful progeny tests             Less demanding

 Hybridization

Objective of Hybridization:

•  The primary goal is to create variation by combining genes from two genotypically different parents in the F1 generation, leading to new gene combinations through segregation and recombination in subsequent generations.

• The extent of variation depends on the number of heterozygous genes in F1, which in turn depends on how many genes the parents differ for.

Steps in Hybridization:

1.  Objective Setting:

§  Define the breeding objective, as it guides parent selection.

§  For resistance breeding, one parent must be a donor.

2.  Selection of Parents: The female parent is a locally adapted variety, and geographically diverse parents are chosen for intervarietal hybridization to achieve superior segregants.

3.  Evaluation of Parents: Parents, especially new ones, must be evaluated for adaptability and homozygosity.

4.  Sowing Plan:

§  If flowering durations are similar, parents can be sown simultaneously; otherwise, staggered sowing is used.

§  The ovule parent is usually planted in the center, with the pollen parent on the border for each combination.

5.  Emasculation and Dusting:

§  Emasculation is the removal of immature anthers from a bisexual flower.

§  The practice varies by crop and anthesis time.

§  For example, in rice, emasculation is done early morning, followed immediately by pollen dusting.

6.  Labelling and Bagging: After hybridization, label with parent details and crossing date, then cover to prevent foreign pollen contamination.

7.  Harvesting and Storage of Seeds:

§  Seeds are typically set 15-20 days after crossing.

§  Crossed pods in pulses may appear shrunken with reduced seed set.

§  Harvest seeds on an individual plant basis, store with proper labels in appropriate containers.

Combination Breeding

• Aim:

  • To transfer one or more desirable characters (oligogenes or polygenes) from other varieties into a single variety.

  • This approach improves yield by correcting weaknesses in yield-contributing traits (e.g., tiller number, grains per panicle).

• Examples: Disease resistance achieved through backcross breeding and the pedigree method.

Transgressive Breeding

    Production of F2 generation plants superior to both parents for one or more traits.

• Mechanism: Achieved by accumulating favourable genes from both parents due to recombination.

• Requirements: Parents should combine well and preferably be genetically diverse to contribute different "plus genes" that combine through recombination.

• Methods: Pedigree method and population approach are designed to produce transgressive segregants.

Pedigree Method

Procedure:

·         Individual plants are selected from F2 and subsequent generations, and their progenies are tested.

·         Detailed records (Pedigree Record) are maintained, tracking the ancestry of selected plants using a Crossing Ledger.

oF1 Generation:

§  Space planted for full expression, parents can be raised as border rows.

§  F1s are harvested as single plants.

oF2 Generation:

§  2,000-10,000 plants per cross are planted.

§  50-500 desirable plants are selected and harvested individually.

§   Selection intensity is typically 5-10% and depends on the breeder's skill.

oF3 Generation:

§  Individual plant progenies are space planted, and desirable plants are selected.

§  The term "family" is introduced for lines selected from each cross.

o F4-F5 Generations:

§  Similar selection is followed until F4 or F5, after which selected plants are bulked to form a family.

§  Many families attain homozygosity in F5 and can be harvested as row bulk.

oF6-F10 Generations:

§  Row bulks are assessed in multi-row trials. Segregating families are studied separately.

§  Preliminary and advanced yield trials are conducted over multiple locations and years, leading to cultivar release.

Merits:

·         Maximizes breeder's skill and judgment in selection.

·         Well-suited for simply inherited characters.

·         Transgressive segregants are easily identifiable through records.

·         Provides precise information about inheritance.

Demerits:

·         Time-consuming and limits handling of larger populations due to pedigree record maintenance.

·         Success highly dependent on breeder's skill; no opportunity for natural selection.

·         Ineffective selection for yield in F2 and F3.

·         Valuable materials can be lost if large populations are not maintained.

Mass Pedigree Method

Purpose: Addresses deficiencies in the pedigree method, especially for disease resistance screening where artificial epiphytotic conditions are not feasible.

Procedure:

• • Populations are carried as a mass and tested when natural disease occurrence is favourable.

  • Bulking is terminated, and single plant selection begins when conditions are suitable for disease expression.

Bulk Method

Procedure:

• •  F2 and subsequent generations are harvested as a bulk for 6-7 generations.

  • Selection can be made, but harvesting is done in bulk.

  • At the end of the bulking period, single plant selection is made and tested for yield.

  • Longer bulking periods (20-30 seasons) allow natural selection to act on homozygous lines.

  • No pedigree record is maintained, saving time and labour.

Merits:

• • Simple, convenient, inexpensive.

  • Artificial epiphytotic conditions can eliminate undesirable genotypes.

  • Natural selection operates with longer bulking periods.

  • No pedigree records.

  • Large populations increase the chance of transgressive segregants.

Demerits:

• • Takes longer to develop a new variety.

  • No natural selection in short-term bulks.

  • Requires much labour, time, and space for large progenies.

  • No information on inheritance is obtained.

Modified Bulk Method

Procedure:

• • Selection can be practiced in F2, F3, and subsequent generations without pedigree records; superior plants are bulked and carried forward.

  • In F4, superior plants are selected and harvested individually.

  • In F5, these are studied in progeny rows, and the best progenies are selected.

  • PYT (Preliminary Yield Trial) can be conducted in F6, followed by regular trials.

Merits:

• • Allows breeder's skill and judgment in selection.

  • No pedigree record maintenance.

Single Seed Descent (SSD) Method

Modification of Bulk Method:

• • A single seed from each F2 plant is collected and bulked to raise the F3 generation, and this process continues until F6 or F7.

  • After F6 or F7, single plant selection is made and studied in progeny rows.

Main Features:

• • Lack of selection until F6 or F7 (when homozygosity is attained).

  • Each F2 plant is represented until F6 or F7.

  • Population size can reduce due to pests, diseases, or poor germination.

  • Rapid generation advancement (RGA) is possible using glasshouses or off-season nurseries.

Backcross Method

• Hybrids and subsequent progenies are repeatedly backcrossed to one parent (recurrent parent), making the progeny genetically similar to the recurrent parent.

Objective: To improve one or two specific defects of a high-yielding variety.

Prerequisites:

• 1. Availability of a suitable recurrent parent with one or two defects.

  2. Availability of a suitable donor parent.

  3. Character to be transferred must have high heritability and preferably be determined by one or two genes.

  4. Sufficient backcrosses (generally six) to fully recover the recurrent parent's genotype.

Application:

oTransfer of simply inherited characters (e.g., disease resistance, seed coat colour).

oTransfer of quantitative characters (e.g., plant height, seed size).

oInterspecific transfer of simply inherited characters (e.g., disease resistance from wild species to cultivated species).

oTransfer of cytoplasm (e.g., male sterility).

oProduction of transgressive segregants (by 2-3 backcrosses to recurrent parent).

oProduction of isogenic lines.

oGermplasm conversion (e.g., producing photo-insensitive lines from photo-sensitive germplasm).

Procedure for Dominant Gene Transfer (Example: Rust Resistance):

oFirst Season: Hybridization (Donor B (resistant) x Recurrent A (susceptible)). F1 (Rr) is resistant.

oSecond Season: F1 (Rr) is backcrossed to recurrent parent A (rr) to produce BC1.

oSubsequent Seasons: Resistant plants are continually backcrossed to recurrent parent A for up to 7 seasons (BC7).

oEighth Season: BC7F1 plants resembling parent A and showing resistance are self-pollinated and harvested individually.

oNinth Season: Progeny rows are grown, and homozygous resistant plants resembling parent A are selected, harvested, and bulked.

oTenth Season: Yield trials, seed multiplication, and distribution.

Procedure for Recessive Gene Transfer (Example: Resistance):

oFirst Season: Cross Donor B (resistant, rr) x Recurrent A (susceptible, RR). F1 (Rr) is susceptible.

oSecond Season: Grow F1.

oThird Season: Grow F2 (RR:Rr:rr segregation). Select resistant plants (rr) and backcross with recurrent parent A (RR). This is BC1. Harvest BC1F1.

oSubsequent Seasons: Grow BC1F1. Grow BC1F2 (segregating). Repeat the process of selecting resistant plants and backcrossing with recurrent parent. This continues until 7th or 8th backcross.

oFinal Generations: After 8th BCF2, select plants resembling parent B and showing resistance, harvest individually. Raise progeny rows, conduct yield trials, multiply, and release as a variety. Artificial bombardment for disease is done during selection in F2 generations.

Merits of Backcross Method

• New variety's genotype is nearly identical to the recurrent parent, making outcomes predictable and reproducible.

• Extensive yield testing is often unnecessary, saving time and expense (up to 5 years).

• Environmentally independent, allowing for off-season nurseries and greenhouses to accelerate generations (2-3 per year).

• Requires smaller populations than the pedigree method.

• Removes specific defects (e.g., disease susceptibility) from well-adapted varieties without affecting performance, which farmers and industries prefer.

• Only method for interspecific gene transfers.

• Can be modified to induce transgressive segregation for quantitative characters.

Demerits of Backcross Method

• New variety generally cannot be superior to the recurrent parent, except for the transferred character.

• Undesirable linked genes may also be transferred.

• Hybridization is required for each backcross, which can be difficult, time-consuming, and costly.

• The recurrent parent might be replaced by superior varieties by the time the backcross is complete.

Multiline Varieties

Purpose: To overcome limitations of pureline varieties, such as

• • Limited adaptation,

  • Unstable performance,

  • Breakdown of disease resistance due to new pathogen races.

Composition:

• • Mixtures of several purelines that are similar in

§  Height,

§  Flowering/maturity dates,

§  Seed colour,

§  Agronomic characteristics,

§  But possess different genes for disease resistance.

• • Purelines must be compatible and not reduce each other's yield in mixture.

Development:

• • Purelines with different resistance genes are developed through backcross programs using one recurrent parent.

  • Five to ten lines are mixed based on prevalent pathogen races.

  • Susceptible lines are replaced by resistant ones.

Merits:

• • Lines are almost identical to the recurrent parent in agronomic characteristics and quality, avoiding pureline mixture disadvantages.

  • Only a few lines become susceptible in any season, minimizing cultivator loss.

  • Disease spread is slower, reducing damage, as only a small proportion of plants are susceptible.

Demerits:

• • Farmers must change seeds every few years due to changing pathogen races.

  • Possibility of a new race attacking all lines simultaneously.

Achievements:

• • Useful for controlling diseases like rusts.

  • Three multiline varieties (KSML 3, MLKS 11, KML 7406) have been released in India for wheat, using Kalyan Sona as a recurrent parent.

Dirty Multiline: A multiline containing one or two susceptible lines, intended to prevent race formation.

MUTATION BREEDING

Mutation:

Ø  A sudden heritable change in a specific character, which can be large or small.

Ø  Origin: The term "mutation" was coined by Hugo de Vries (1901) from his observations in the Evening Primrose (Oenothera lamarkiana).

Ø  Occurrence: Mutations occur frequently in nature across many organisms.

Ø  Types based on discernibility:

• • Macro mutation: Large and noticeable changes (e.g., in colour, shape).

  • Micro mutation: Small and inconspicuous changes (e.g., in yield, plant height).

Ø  Types based on cells:

• • Somatic mutations: Occur in body cells, not transmitted to the next generation (non-heritable).

  • Germinal mutations: Occur in reproductive cells, heritable.

Ø  Types based on mechanism/effect:

• • Gene mutation (Point mutation): Changes in the base sequences of genes, detectable by fine genetic analysis (e.g., in microorganisms).

  • Chromosomal mutations: Changes in chromosome structure or number (e.g., translocations, inversions, deletions, duplications). Large changes are cytologically detectable.

  • Cytoplasmic mutations: Mutant character shows cytoplasmic or extranuclear inheritance.

  • Bud mutations (Somatic mutations): Occur in buds or somatic tissues, particularly in clonally propagated crops. Depends on dominant mutations or recessive mutations in heterozygous clones.

  • Reverse mutation: Due to induced mutagenesis, an organism may revert to its original form (e.g., a dwarf plant becoming tall again).

Mutagens

Mutagen: A physical or chemical agent that can induce mutations.

Characteristics of an Ideal Mutagen:

• • Highly reactive.

  • Able to penetrate cell walls.

  • Capable of altering DNA structure.

  • Produce stable and desirable mutations.

  • Should show dose-dependent mutation frequency.

Types of Mutagens:

  Physical Mutagens:

       1)Ionizing Radiations:

o High-energy radiations that cause ionization (formation of charged particles) in the medium they pass through, leading to biological damage.

oExamples:

§  Alpha particles,

§  Beta particles,

§  Gamma rays (most penetrating),

§  X-rays,

§  Neutrons (thermal, fast).

oMechanism: Cause

§  chromosome breaks,

§  translocations,

§  inversions,

§  duplications,

§  deletions,

§  point mutations.

oCharacteristics:

v  Varies in penetration and ionization density.

v  Higher ionization density leads to more biological damage.

  Non-ionizing Radiations:

oExample: Ultraviolet (UV) light.

oMechanism: Causes dimerization of adjacent pyrimidine bases (e.g., thymine dimers), leading to errors during DNA replication.

oLimitations: Poor penetration; effective only on surface tissues (e.g., pollen, fungi, bacteria).

  Chemical Mutagens:

oExamples:

§  Mustard gas (first chemical mutagen discovered),

§  Ethylating agents (EMS, dES),

§  Base analogues (5-Bromouracil,

§  2-Aminopurine),

§  Acridine dyes (proflavine, acridine orange),

§  Alkylating agents (EMS, MMS, DES, NG).

oMechanism: Can cause

§  Base-pairing errors,

§  Transitions,

§  Transversions,

§  Deletions,

§  Frameshift mutations.

§  Acridines specifically cause frameshift mutations by inserting or deleting base pairs.

o Effects: Induce mutations with higher frequency than physical mutagens, and typically produce a higher proportion of point mutations.

Breeding Procedure in Mutation Breeding

        Objective: To improve existing varieties by altering specific undesirable traits through mutation.

        Steps:

1. Selection of Material:

§  Variety:

Ø  Should be well-adapted, high-yielding,

Ø  Possess most desirable traits, but have one or two defects (e.g., disease susceptibility, undesirable plant height).

§  Plant Part:

Ø  Seeds are most commonly treated due to ease of handling.

Ø  Other parts like pollen, spores, cuttings, dormant buds, or whole plants can also be used.

2. Choice of Mutagen:

Ø  Depends on the crop, desired mutation, and ease of handling.

Ø  Gamma rays are common, EMS for point mutations.

3.  Dose of Mutagen:

Ø  Determined by experimentation (e.g., LD50, where 50% survival occurs).

Ø  Optimal dose typically reduces germination by 50% and growth by 30%.

Ø  Expressed in Roentgen (R) for X-rays/gamma rays, or rads/kilorads.

Ø  For chemicals, concentration and treatment duration are considered.

4. Method of Treatment:

Ø  Direct (dry seeds, pollen) 

Ø  Indirect (soaking seeds in mutagen solution).

5. Handling of M1 Generation:

Ø  Seeds/treated material are sown (M1 - first mutant generation).

Ø  M1 is often chimeric. Single plants are harvested separately to ensure mutations are carried forward.

Ø  Sterility may be observed due to chromosomal aberrations.

6. Handling of M2 Generation:

Ø  M1 seeds are sown in progeny rows (M2).

Ø  This is the most critical generation for identifying desirable mutations.

Ø  Screening for specific characters (e.g., disease resistance) under controlled conditions.

Ø  Desirable mutant plants are selected and harvested individually.

7. Handling of M3 and Subsequent Generations:

Ø  Selected M2 plants are grown as progeny rows (M3).

Ø  Homozygous, true-breeding mutant lines are identified.

Ø  Yield trials are conducted in M4, M5, and M6, followed by advanced yield trials and release.

Applications of Mutation Breeding

• Creation of New Varieties: Directly by developing mutant lines into varieties (e.g., Sharbati Sonora wheat, Jagannath rice, Co2 groundnut, Co5 red gram, MCU 10 cotton, Co4 black gram).

• Improvement of Existing Varieties:

  • Yield: Enhancing yield (e.g., dwarfness, early maturity).

  • Quality: Improving grain quality (e.g., protein content, cooking quality).

  • Disease and Insect Resistance: Inducing resistance (most common application).

  • Early Maturity: Shortening the growing period.

  • Plant Architecture: Altering plant height or tillering.

• Overcoming Undesirable Linkages: Breaking unfavourable linkages between desirable and undesirable genes.

• Gene Duplication and Increased Recombination: Inducing polyploidy or increasing recombination frequency.

• Improving Adaptiveness: Enhancing environmental adaptability.

• Use in Cross-Breeding Programs: Mutants can be used as parents in hybridization.

• Overcoming Self-Incompatibility: Inducing self-compatibility in self-incompatible species.

Limitations of Mutation Breeding

• Low Frequency of Desirable Mutations: Most mutations are random, and beneficial ones are rare.

• Recessive and Deleterious Nature: Most mutations are recessive and harmful.

• Effectiveness with Polygenic Traits: Less effective for improving quantitative or polygenic characters (like yield).

• Extensive Screening Required: Requires large populations and extensive screening to identify useful mutants.

• Undesirable Side Effects: Mutagenesis can lead to undesirable pleiotropic effects, affecting quality, taste, or other agronomic traits.

• Lower Efficiency than Hybridization: Often less efficient than hybridization for significant improvements.

• Public Perception: Concerns about genetically modified organisms (GMOs) or "mutant" crops, although mutation breeding uses traditional techniques and is generally accepted.

• Not a Replacement for Hybridization: Complementary to, but not a substitute for, conventional breeding methods.

POLYPLOIDY BREEDING

Polyploidy

• Polyploidy refers to organisms that have more than two sets of chromosomes. Diploid organisms, which are more common, have two sets of chromosomes.

Characteristics of Polyploids:

• • Polyploids are generally more heterozygous than their diploid counterparts, especially allopolyploids, which contributes to heterosis or hybrid vigour.

  • This heterozygosity is stable due to preferential pairing of homologous chromosomes.

  • They exhibit genetic redundancy, allowing extra gene copies to mutate and diverge, potentially leading to new traits.

  • Polyploid populations often show extensive genomic rearrangement.

  • They are often self-fertile and apomictic, which favours their propagation.

  • Inbreeding is less deleterious for allopolyploids due to their greater heterozygosity.

  • Enzyme multiplicity provides greater biochemical flexibility and adaptability.

  • Changes in gene expression and rapid genetic/epigenetic changes contribute to increased variation and new phenotypes.

Classification of Polyploid  Heteroploidy:

o An individual with chromosome numbers other than true monoploid or diploid numbers.

o It is divided into

§  Euploidy

§  Aneuploidy.

o Euploidy:

1. An individual carries an exact multiple of the basic chromosome number.

2. It can involve a single set (monoploidy) or many multiples (polyploidy).

3. Euploidy is further divided into

§  Autopolyploid

§  Allopolyploidy.

§ Autopolyploidy: Polyploids with multiple chromosome sets derived from a single species (e.g., x, 2x, 3x, 4x, etc.).

§ Allopolyploidy: Two or more genomes derived from different, genomically unlike species are present.

§ Segmental Allopolyploids: Carry genomes with intermediate similarity and generally exhibit preferential pairing.

§ Autoallopolyploidy: Confined to hexaploidy and higher levels.

§ Amphiploidy/Amphidiploidy: Denotes polyploids derived after hybridization between two or more genomically dissimilar species, separated by chromosomal sterility.

oAneuploidy: The chromosome number is not an exact multiple of the basic set. It arises due to chromosomal non-disjunction during meiosis.

§ Hypoploidy: Lacking one or two chromosomes.

§ Hyperploidy: Having additional one or two chromosomes.

§ Monosome (Monosomic): An individual lacking one chromosome (2n-1).

§ Nullisome: Lacking a pair of chromosomes (2n-2). Nullisomics are generally not viable in diploid/tetraploid species but are tolerated in some hexaploid species like wheat.

§ Trisome: An individual carrying an extra chromosome (2n+1).

§ Limitations: Aneuploids are usually

§  Less vigorous,

§  Meiotically irregular,

§  Often sterile.

§  Have limited use as varieties in plant breeding. However, they are highly useful in genetic studies (e.g., detecting linked genes, chromosome transfer).

HYBRID SEED PRODUCTION

• Necessity of Male Sterility: Manual emasculation and pollination are labour-intensive and costly in hybrid seed production. Utilizing male sterility significantly reduces these costs and efforts.

Types of Male Sterility Systems for Hybrid Seed Production:

1. Genetic Male Sterility (GMS)

1. • Male sterility controlled by nuclear genes.

   • Gene Types: Can be conditioned by dominant genes (e.g., Ms4, Ms7, Ms10, Ms11) or recessive genes (e.g., ms1, ms2, ms3 singly; ms5, ms6, ms8, ms9 as duplicate recessives).

   • Stability: The GMS system involving ms5 and ms6 (or both) found in Greg MS is noted as a stable source used in India, Pakistan, and the USA.

   • Restorers: Genotypes carrying Ms5 or Ms6 (or both genes) act as restorers for these GMS systems.

   • Limitation for Hybrid Seed Production: Its utilization in cotton is challenging due to the 50:50 segregation ratio of sterile and fertile lines, which makes the maintenance of sterile lines laborious.

2. Cytoplasmic Genetic Male Sterility (CGMS)

2. • Male sterility resulting from the interaction between a sterile cytoplasm and specific nuclear genes (restorer genes).

   • Components: Involves

     • Two types of cytoplasm ('S' for sterile, 'N' for normal)

     • Two types of nuclear genes ('Rf' for restorer of fertility, dominant; 'rf' for non-restorer, recessive).

   • Mechanism:

     • Sterile plants possess 'S' cytoplasm and 'rfrf' nuclear genes.

     • Fertility is restored by the presence of 'Rf' genes in 'S' cytoplasm (S RfRf or S Rfrf) or any nuclear genotype in 'N' cytoplasm.

   • Advantages over GMS: CGMS systems are preferred over GMS because

     • Male sterile plants are true-breeding and do not segregate for fertility,

     • Simplifying their maintenance for hybrid seed production.

   • Line Development:

     • A-line (Sterile Line):

       1. The male sterile line used as the female parent in hybrid crosses.

       2. It has 'S' cytoplasm and 'rfrf' nuclear genes.

     • B-line (Maintainer Line):

       1. Used to maintain the A-line.

       2. It has 'N' cytoplasm and 'rfrf' nuclear genes and is morphologically similar to the A-line.

     • R-line (Restorer Line):

       1. Used as the male parent to restore fertility in the F1 hybrid.

       2. It has 'N' cytoplasm and 'RfRf' nuclear genes.

   • Sources in Cotton:

     • G. harknessii cytoplasm: The first practical CGMS system in cotton, identified by Meyer in 1975, and transferred into G. hirsutum and G. barbadense.

     • G. anomalum cytoplasm: Used to develop CGMS lines.

     • G. arboreum cytoplasm: Also used to develop CGMS lines.

   • Challenge: A significant challenge with CGMS in cotton is the often-poor restoration of fertility in commercial hybrids.

3. Transgenic Male Sterility (TMS)

3. • Male sterility induced through genetic engineering, involving the introduction of specific genes.

   • Mechanism:

     • The document mentions the barnase gene, which causes male sterility by disrupting pollen development.

     • Its effect can be reversed by the barstar gene, which inactivates barnase, allowing for fertility restoration.

   • Advantages: This system offers precise control over male sterility, making hybrid seed production more efficient and predictable.

MOLECULAR BREEDING

· Molecular breeding is a modern and advanced approach to improving desirable traits in plants and animals.

· It utilizes tools and techniques from molecular biology, genetics, and genomics to identify, select, and manipulate desirable characteristics at the DNA level, rather than solely relying on observable physical traits (phenotypes).

Key Components and Techniques:

• Molecular Markers Defined: Molecular markers are specific, detectable molecules, often DNA or protein sequences, that show easily discernible differences among species and whose inheritance can be monitored.

• Types of DNA Markers Used in Plant Breeding:

  • Restriction Fragment Length Polymorphism (RFLP)

  • Randomly Amplified Polymorphic DNA (RAPD)

  • Sequenced Tagged Sites (STS)

  • Sequence Characterised Amplified Regions (SCAR)

  • Variable Number Tandem Repeats (VNTR)

  • Minisatellites

  • Microsatellites or Simple Sequence Repeats (SSR)

  • Inter Simple Sequence Repeats (ISSR)

  • Amplified Fragment Length Polymorphism (AFLP)

  • Expressed Sequence Tags (EST)

  • Single Nucleotide Polymorphisms (SNPs)

• Constraints of Morphological Markers:

  • Less in number.

  • Confer indistinguishable phenotypes.

  • Influenced by the environment.

  • Influenced by the genetic background.

  • Influenced by ontogeny.

  • No stable inheritance.

• Properties of DNA Markers:

  • Abundant

  • Ubiquitous

  • Highly polymorphic

  • Stable inheritance

  • No environmental influence

  • No influence of ontogeny of individual

  • Codominant or dominant

• RFLP (Restriction Fragment Length Polymorphism):

  • Detects variation in DNA fragment length after endonuclease digestion.

  • Involves DNA digestion, electrophoresis, Southern blotting, hybridization with labelled probes, and detection.

  • Strengths:

    • Good repeatability,

    • Useful in comparative genome mapping.

  • Constraints:

    • Tedious,

    • Time-consuming,

    • Requires large DNA quantities,

    • Limited utility in MAS due to low assay efficiency.

• RAPD (Random Amplified Polymorphic DNA):

  • Based on differential PCR amplification using short oligonucleotide sequences.

  • Genetically dominant.

  • Strengths:

    • Requires small DNA quantities,

    • Limited investment in time and training,

    • Many commercial primers available.

  • Constraints:

    • Lack of reproducibility across labs/experiments due to sensitivity to DNA/primer concentration and thermal cycling conditions

    • inability to discern sequence homology among similarly-sized fragments.

• SCAR (Sequence Characterised Amplified Region) & STS (Sequence Tagged Site):

  • PCR-based markers derived by sequencing the termini of RFLP, RAPD, AFLP fragments, or known genes.

  • SCAR primers are 18-25 nucleotides long.

  • SCARs have greater reproducibility and utility than RAPDs.

  • SCARs can be converted to codominant markers by restriction digestion.

  • STS markers are generally mapped, codominant, show stable amplification, and good repeatability.

  • Constraint (STS): Not many polymorphic STSs are currently available in crop plants.

• SSR (Simple Sequence Repeats)/Microsatellites:

  • Tandem repeats of mono-, di-, tri-, tetra-, or penta-nucleotide units.

  • Polymorphisms are based on variation in the number of repeat units at a locus.

  • Strengths:

    • Abundant, uniformly distributed,

    • Hypervariable (many alleles),

    • Codominant with known genomic locations,

    • Highly reliable and reproducible.

  • Uses:

    • Genotype differentiation,

    • Seed purity evaluation,

    • Marker-assisted selection,

    • Population genetic studies,

    • Genetic diversity analysis.

  • Constraints:

    • Expensive and time-consuming to detect loci and design primers (though many are publicly available for some crops)

    • Not available for all plant species; primers usually species-specific.

• AFLP (Amplified Fragment Length Polymorphism):

  • Combines restriction digestion and PCR amplification.

  • Procedure involves DNA digestion with two restriction enzymes, ligation of adapters, preselective amplification, selective amplification with labelled primers, electrophoresis, and visualization.

  • Strengths:

    • Stable amplification,

    • High repeatability,

    • Hypervariability,

    • Generates many mappable loci per amplification (high assay efficiency),

    • Provides raw materials for STS derivation,

    • Can generate fingerprints of any DNA.

  • Constraints:

    • Time-consuming,

    • Requires significant technical skills and financial resources.

• EST (Expressed Sequence Tags):

  • Subsets of STSs derived from cDNA clones.

  • Derived from expressed genes (spliced mRNA, usually free of introns and repetitive DNA).

  • Strengths:

    • Represent real functional genes, more useful as genetic markers than anonymous non-functional sequences,

    • Advantageous for comparative genome analysis.

  • Constraints:

    • High development/start-up costs,

    • Currently available in very limited crop plants.

• SNP (Single Nucleotide Polymorphisms):

  • Considered 'third generation markers'.

  • Point mutations where one nucleotide is substituted for another.

  • Most common type of sequence difference, codominant, inexhaustible source of polymorphic markers for high-resolution genetic mapping.

  • Detection based on DNA amplification using primers from known gene sequences.

  • Strengths:

    • Easier to work with than SSRs or AFLPs,

    • Useful when several SNP loci are closely positioned for haplotype definition, integrate physical and genetic maps.

  • Constraints:

    • Requires sequence information for genes,

    • High development/start-up costs.

• Detection of DNA Polymorphisms:

  • Gel electrophoresis is widely used.

  • Agarose is preferred for RFLP and RAPD.

  • PAGE (Polyacrylamide Gel Electrophoresis) is used for smaller fragments from microsatellites and AFLPs.

  • Detection methods include silver staining (for SSRs) or autoradiography (for AFLPs with radiolabelled isotopes).

  • Laser technology with fluorescent dyes and computer programs are also used for microsatellites.

• Choice of Molecular Marker System:

  • Selection depends on experiment objectives, required resolution, and operational constraints.

  • Main criteria are degree of polymorphism, reproducibility, and repeatability.

  • SSRs and AFLPs are widely preferred over RAPDs and RFLPs for fingerprinting, genetic diversity analysis, and mapping.

• General Applications of DNA Markers:

  • Diversity analysis at the molecular level for germplasm characterization.

  • Marker-aided selection for pest resistance in crop improvements.

  • DNA fingerprinting of crop species from different geographical regions.

  • Establishing phylogenetic and taxonomic relationships among individuals.

  • Tagging of major and minor QTLs (Quantitative Trait Loci).

  • Physical mapping and map-based cloning of genes for producing transgenic organisms.

• Evaluation of Germplasm using DNA Markers:

  • Identification of germplasm.

  • Screening of duplicates.

  • Assessment of genetic diversity.

  • Monitoring the genetic stability of conserved germplasm.

QTL Mapping and Association Mapping:

 These techniques help pinpoint regions on chromosomes (Quantitative Trait Loci or QTLs) that contain genes influencing complex traits like yield, disease resistance, or stress tolerance.

Marker-Assisted Selection (MAS)

• MAS uses molecular markers to identify and select individuals with desirable traits at the DNA level, rather than relying on observable physical characteristics.

• Purpose: To indirectly select for desired traits by detecting specific DNA markers linked to genes of interest.

• Advantages over Traditional Selection:

  • Increased Precision: Selection is more accurate as it's based directly on the presence of genes or linked markers, reducing environmental influence.

  • Reduced Time: Enables selection at early developmental stages, shortening breeding cycles.

  • Cost-Effectiveness: Can minimize the need for extensive field trials or complex phenotyping.

  • Environmental Independence: Marker presence is unaffected by environmental conditions, ensuring consistent selection.

  • Targeted Improvement: Effective for complex traits challenging to manipulate by traditional methods.

  • Gene Pyramiding: Facilitates combining multiple desirable genes into one variety.

• Key Markers Used: SSRs (microsatellites) and AFLPs are widely preferred for fingerprinting, genetic diversity analysis, and mapping experiments, making them suitable for MAS.

• General Application: Markers aid selection for pest resistance in crop improvements.

Ø  Genomic Selection (GS): An even more advanced form of MAS that uses data from a large number of markers across the entire genome to predict an individual's breeding value for complex traits.

Ø  Genetic Engineering: In a broader sense, molecular breeding can also include direct manipulation of genes by introducing foreign genes (transgenes) into an organism to confer entirely new traits that might not exist in its natural gene pool.

Ø  Functional Genomics: This involves studying the function of genes and their interactions to understand the molecular basis of traits.

PLANT GENETIC RESOURCES

Plant genetic resources, also known as a gene pool or germplasm, are the complete collection of alleles within a species and are essential for breeding programs

Classification of Gene Pools:

Gene pools are categorized by:

● Area of collection: Indigenous or exotic.

● Domestication: Cultivated or wild.

● Conservation duration:

○ Base collection: Long-term storage at -18°C to -20°C with 5-6% moisture and 95%

viability, used for regeneration.

○ Active collection: Medium-term (10-15 years) at 0°C with 8% moisture, for

regeneration, multiplication, evaluation, and distribution.

○ Working collection: Short-term (3-5 years) at 5-10°C with 8-10% moisture.

● Crossability:

○ Primary gene pool (GP1): Easy intermating, producing fertile hybrids (same or

closely related species).

○ Secondary gene pool (GP2): Partial fertility when crossed with GP1.

○Tertiary gene pool (GP3): Sterile hybrids with GP1, requiring special techniques.

Components of Genetic Resources

● Landraces: Primitive, high genetic diversity, broad genetic base, less uniform, low yield.

● Obsolete cultivars: Older improved varieties replaced by newer ones (e.g., Wheat

varieties K68, K65, Pb591).

● Modern cultivars: Current high-yielding varieties with narrow genetic base and low

adaptability.

● Advanced breeding lines: Pre-released, developed by breeders.

● Wild forms of cultivated species: High resistance.

● Wild relatives: May cause hybrid sterility or inviability.

● Mutants: Valuable genetic resources (e.g., Dee-Geo-Woo-Gen rice, Norin 10 wheat);

over 410 varieties in seed-propagated crops released via mutation.

Crop Genetic Diversity

Ø  This refers to the variety of genes and genotypes in a crop species.

Ø  N.I. Vavilov (1926, 1951) identified eight main and three subsidiary centers of diversity, though he did not include Africa or Australia.

● Primary Centers:

§  Vast genetic diversity,

§  Dominant genes,

§  Wild characteristics,

§  Natural

§  Selection.

● Secondary Centers:

§  Lesser diversity,

§  Recessive genes

§  Desirable traits,

§  Natural and artificial selection.

● Microcenters:

§  Small areas with immense genetic diversity,

§  Useful for studying species evolution.

Germplasm Activities

1. Exploration and Collection: Gathering genetic diversity from various sources.

○ Genetic Erosion: Reduction in variability due to replacing landraces with modern

cultivars, agricultural modernization, and urban growth.

○ Extinction: Permanent loss of a crop species.

○ Sources include gene banks, sanctuaries, and farmers' fields.

○ Priorities are endangered areas and species.

○ Methods: Expeditions, personal visits, correspondence, and material exchange.

○ Sampling aims to capture high diversity (e.g., 95% of rice diversity with 50-100

individuals, 50 seeds/plant).

2. Conservation: Protecting genetic diversity.

○ In situ: In natural conditions (e.g., biosphere reserves), which can be costly.

○ Ex situ: In gene banks, which is cheaper and conserves entire diversity. This

includes long-term, medium-term, and short-term storage.

■ Orthodox seeds: Can be dried without viability loss (e.g., wheat, papaya).

■ Recalcitrant seeds: Lose viability if moisture drops below 12-13°C (e.g.,cocoa, mango).

○ Meristem conservation: For virus-free, vegetatively propagated, and perennial

plants.

3. Evaluation: Identifying gene sources and classifying germplasm.

4. Documentation:

o   Compiling, analyzing, storing, and disseminating information on genetic

resources.

o   Currently, there are 7.3 million germplasm accessions for 200 crop species.

5. Distribution: Supplying specific germplasm on demand.

6. Utilization: Using germplasm in crop improvement (as varieties, parents, or for resistance

transfer from wild forms).

II. Agencies and Germplasm Exchange

·IPGRI (International Plant Genetic Resources Institute):

• • Supervised by the Consultative Group on International Agricultural Research (CGIAR).

  • CGIAR was established in 1972 by FAO, the World Bank, and UNDP to set up international research institutes.

  • IPGRI was established by CGIAR in 1994, succeeding IBPGR (which existed until 1993, established in 1974).

  • Conducts research and promotes an international network of plant genetic resources.

·NBPGR (National Bureau of Plant Genetic Resources):

• • Established by ICAR in 1976 in New Delhi.

  • Plant introduction started at IARI, New Delhi in 1946.

  • A separate division for Plant Introduction was formed in 1961 under Dr. H.B. Singh.

·Quarantine:

• • Governed by the Destructive Insects and Pest Act of 1914.

  • Requires a Phytosanitary certificate.

  • Agencies involved include NBPGR, FRI - Dehradun, Botanical Survey of India - Calcutta, and Directorate of Plant Protection, Quarantine and Storage - Faridabad.

  • Applies to food grains and other produce imported for human consumption.