Mutation breeding In

Pre and post Genomic era

UMESH BABU B S PG15AFR7071

Introduction

Classification of mutation

Causes for mutationVariation in plant breeding,

How mutation helps?Mutation breeding in Pre and

post genomic eraCASE STUDIES

Conclusions

FLOW OF SEMINAR…

Mutation is a sudden heritable change in the genetic material not caused by recombination or segregation.(De Vries , 1903)

The term mutation breeding was first coined by Freisleben and Lein (1944)

The goal in mutagenesis breeding is to cause maximal genomic variation with a minimum decrease in viability.

The term mutant variety is simply a variety , but draws attention to the fact that it carries an important trait controlled by a known mutant gene.

Introduction

1936: The first induced mutant variety is released, tobacco var. ‘Chlorina’ using X-rays in Indonesia.

1966: First chemically induced mutant variety, Luther of barley was released in the USA.

High yielding, short culm and lodge resistant barley- It was produced by seed-treatment with diethyl sulfate, a mutagenic chemical.

1972: The “Mutation Breeding Newsletter” is launched and published by the FAO.

2000: Onwards Development of TILLING populations.

Milestones

W. C. Rontgen (1845-1923)

Hugo de Vries (1848 – 1935)

Influential people in the history

of mutation breeding

H. J. Muller (1890 – 1967)

Lewis J. Stadler (1896 – 1954)

H. Nilsson-Ehle (1879-1949)

R. Sengbusch (1898-1985)

Hans Stubbe (1902-1989)

Åke Gustafson (1908-1998)

I. Forward Mutations (+ to m)

A. Effect at DNA Level

1.Transitions: A <-> G; T <-> C.

2. Transversions : Other bp substitutions.

3. Deletions: Loss of 1 or more bp.

4. Insertions: Addition of 1 or more bp.

Classification Of Mutation

1.Neutral : Altered codon: Equivalent Amino acid. Minimal effect on protein structure, No loss of protein function.

2. Silent : Altered codon: Same Amino acid. Example: AGG (Arg) to CGG (Arg)

3. Missense : Altered codon: Different Amino acid, Protein function altered.

4. Nonsense : Premature termination of translation by stop codon in the frame.

5. Frameshift : Small additions or deletions. Not multiple of 3 bp Alters reading frame during translation

B. Effect at Protein Level

A. Exact Reversions

B. Equivalent Reversions

C. Excision of Transposon

1. Perfect: Original genomic sequence restored. Mutant allele becomes wild type

2. Imperfect: Small piece of transposon remains Footprint – evidence of excision may or may not alter protein function.

II. Reverse Mutations

Other Classifications

Systems for Mutations

I. Spontaneous: Occur in naturally. Induced: Generated in lab; exposure to

mutagen.II. Germ Line: occurs in reproductive tissues.

Somatic: occurs in somatic cellIII. Dominant: Phenotype apparent in heterozygote

Recessive: Phenotype apparent in homozygoteIV. Lethal: Mutant dies.

Viable: Mutant survives. V. Intragenic: Mutation within same gene Extragenic: Mutation in different gene

Four classes of mutations are,

1. Spontaneous mutations (molecular decay)

2. Mutations due to error prone replication bypass of naturally occurring DNA damage (error prone translation synthesis)

3. Errors introduced during DNA repair

4. Induced mutations caused by mutagens

Causes for mutation

Error prone replication by-passMajority of spontaneously arising mutations are due to error prone replication (translation synthesis)

Spontaneous mutationSpontaneous mutations on the molecular level can be caused by:Tautomerism

Depurination

Deamination

Slipped strand mispairing

Non-homologous end joining (NHEJ) is a major pathway for repairing double-strand breaks.

Removal of a few nucleotides to allow inaccurate alignment of the two ends for rejoining followed by addition of nucleotides.

Errors introduced during DNA repair

Induced mutationInduced mutations on the molecular level can be caused by:- 1. ChemicalsAlkylating agents (eg, EMS,MMS)Base analogs (e.g, 5-BU,2-AP)Acridine Dyes (e.g, Acriflavin,Proflavin)Deamination agents (e.g,Nitrous acid NHO2 ).

2. RadiationUltraviolet radiation (Nonionizing radiation). x- raysβ-raysNeutrons

Importance of variability in plant breeding

Variation is the source from which plant breeders are able to produce new and important cultivars.

Alleles of varying forms at given loci in a population can be selected and fixed within a new individual or line.

These changes in our target plant can be passed on to progeny and used for human benefit through breeding.

Importance of mutation in creating variationWe depend on recombination and independent assortment of favorable alleles to produce new and unique individuals from which to select and produce the lines that will serve as our cultivars.

With tens of thousands of genes within each crop species genome, the possibilities seem limitless.

Recombination per se cannot produce novel traits. This ability is only attainable through the act of mutation.

The occurrence of mutations within the genome of plants is rare, and in natural settings can be lethal.

Ploidy level & its effect in mutation breeding

Seems to be a strong correlation between the ploidy level and the induced mutation frequency.

Polyploid conditions can further complicate the process of reaching homozygosity for the mutation, so must be selfed for additional generations to ensure presence of the mutation.

Molecular Basis of Mutation

One allele of a gene changes into a different allele called a point mutation.

It is always true that mutations that reduce or eliminate gene function are the most abundant class.

Mispairing in the course of replication is a source of spontaneous base substitution.

Most mispairing mutations are transitions.

Replication errors can also lead to frameshift mutations. These mutations often occur at repeated bases.

γ-ray bombardment is less destructive causing point mutations and small deletions.

whereas fast neutron bombardment causes translocations, chromosome losses, and large deletions.

Genetically, Az-MNU predominantly causes GC to AT shifts, or AT to GC shifts. Thus, contrary to EMS, a shift can happen in either direction.

A generalized scheme of mutation breeding

First year M1 i

ii

Mutagen-treated seeds space planted

Seeds from individual plants harvested separately

Second year M2 i

ii

Individual plant progenies grown

Fertile, vigorous, normal looking plants harvested separately

Third Year M3 i

ii

Individual plant progenies from the selected plants grown Superior plant selected from superior progenies showing segregation

Fourth year M4 i

iiiii

Individual plant progenies from the selected plants grown Superior homogeneous lines harvested in bulkSegregating lines usually rejected

Fifth year M5 i

ii

Preliminary yield trail with a suitable check

Superior lines selected

Sixth to eight year

M6

toM8

i

ii

Replicated yield trial with at several locations

Outstanding line released as a new variety

Nine year M9 i Seed increase for distribution among farmers ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─

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Pre-genomic era

From Phenotype to Genotype

Identification of a strong genetic character in a mutagenised population by a phenotypic screening procedure is followed.

Since a mutation does not necessarily need to be in an exon of the candidate gene, identifying a mutation may be difficult if a reference genome is unavailable.

During the early part of the era of induced mutagenesis, the technique was used as a tool for improvement of traditional traits like yield, resistance to disease and pest etc. in various agricultural crops.

Subsequently the emphasis shifted to more diversified uses of crop end products, enhancing quality and nutritional value, tolerance to abiotic stresses etc.

Pre-genomic mutagenesis would be to obtain as many mutants as possible and evaluate each one separately

Since the value of the other mutants cannot be ascertained immediately, it is worthwhile to select and preserve them for future studies and possible utilization.

For example in dwarf rice mutants, their worth in breeding cannot be fully appreciated unless the genes controlling them are studied and compared to already established genes like sd1, sd4, sd6 etc.

Visual selection

Mechanical or physical method

Chemical, biochemical, physiological, physio-chemical and other screening procedures may also be needed for selecting certain kind of mutants but almost all employ visual observation.

[Selection usually start from the M2 and confirm in the M3].

HOW THE MUTANTS ARE SELECTED?

Crop & species Mutant var. Release yr. Mutagen Cotton (Gossypium sp.) M.A.-9 1948 gamma raysWheat (Triticum aestivum) NP-836 1961 x-raysSugarcane (S. officinarum) Co-6608 1966 gamma raysCastor (Ricinus communis) Aruna 1969 neutronsMulberry (Morus alba) S-54 1974 EMSBougainvillea (Bougainvillea sp.) Arjuna 1976 gamma raysBlackgram (Vigna mungo) Co-4 1978 MMSHibiscus (Hibiscus sp.) Purnima 1979 gamma raysSesame (Sesamum indicum) Kalika 1980 EMSWhite jute (Corchorus capsularis) Shyamali 1980 x-raysLentil (Lens culinaris) S-256 1981 radiationCowpea (vigna unguiculata) V-16 Amba 1981 DMSMungbean (Vigna radiata) TAP-7 1981 gamma raysChickpea (Cicer arietinum) Pusa-408 1985 gamma rays

World’s first mutant cultivars released in India

Post-genomic era

From Genotype to Phenotype

Contrary to traditional screening methods done by plant breeders, TILLING focuses on first identifying mutations within genes of interest and then linking those mutations to a specific phenotype.

This approach is only possible when a gene linked to the trait of interest is known and the gene sequence available.

Site-directed mutagenesisSite-directed mutagenesis is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule.

Also called site-specific mutagenesis or oligonucleotide directed mutagenesis

It is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.

Site-directed mutagenesis was achieved in 1973, which induces transition of GA to TC.

ZFNs can be used to disable dominant mutations in heterozygous individuals by producing double strand breaks in the mutant allele.

This technology has been applied successfully in several organisms including Arabidopsis.

Induction of ZFN expression resulted in mutations at the chromosomal target locus at frequencies as high as 0.4 mutations per cell (Lloyd et al., 2005)

ZFN-Mediated Mutagenesis

TILLING - Targeting Induced Local Lesions in Genomes

In TILLING, mutagenesis is complemented by the isolation of chromosomal DNA from every mutated line and screening of the population at the DNA level using advanced molecular techniques.

The power of TILLING was first demonstrated in model systems such as Arabidopsis and Drosophila , where it was shown that single mutations in specific genes could be identified.

A major obstacle in many crop species is the lack of complete genome sequence information.

TILLING

Methods for screening TILLING PopulationsDirect Sequencing.

Li-Cor. System

High- Performance Liquid Chromatography (HPLC)

Next-generation sequencing (NGS) has significantly accelerated the prospects of identifying mutations at the whole-genome level.

The two most commonly used NGS platforms are the 454 Genome Sequencer FLX Ti (Roche Applied Science) and the Illumina (Solexa) Genome Analyzer.

These technologies are under constant development both with regard to read length, data quality and the number of sequences generated.

As the amount of data generated from NGS is immense, some knowledge of bioinformatics and access to computational resources are invaluable during analysis.

Emerging Technologies

Case studies

01

Zhang et al. (2014)Beijing, China

Morphological measurements of the wild-type(3037) and mutant (sd37) plants.

Phenotype 3037 sd37Mature plant height (cm) 99.37+

64.0769.82+ 63.28**

Flag leaf length (cm) 33.8064.41 18.0062.51**Flag leaf width (cm) 1.5560.08

NS1.5960.11

Productive panicle per plant

13.2065.77**

19.7065.65

Length of main panicle (cm)

24.9061.02 16.9061.15**

Number of grains (per main panicle)

203.80632.90

96.20627.50**

Length of seed (mm) 9.6360.33 8.9960.46*Width of seed (mm) 2.8160.13* 3.2860.16Length-width ratio of seed 3.4360.03 2.7460.06*

1000-grain weight (g) 25.3560.30 21.9860.11**Zhang et al. (2014)Beijing, China

02

Fig : TILLING strategy for rice. Bradley et al.,2007

Bradley et al.,2007

Bradley et al.,2007

Institutes handling mutation work in India

Bose Research Institute Calcutta (BRI).

IARI, New Delhi.

Bhabha Atomic Research Centre (BARC) Mumbai.

Tamil Nadu Agricultural University (TNAU) Coimbatore.

National Botanical Research Institute (NBRI) Lucknow.

CONCLUSIONS..

Thank you…