How to identify genes involved in a process Flashcards
EMS mutagenesis in Arabidopsis
Mutagenisis vs. Gene
Ethyl methane sulfonate
- widely used produces point mutations
- high mutagenicity and low mortality
- alkylates guanine bases, which results in base mispairing -> alkylated guanine will pair with a thymine base, resulting primarily in G/C to A/T transitions, which ultimately results in an **amino acid change or deletion. **
- then check for interesting phenotypes
- Advantages: 1) generates a high density of nonbias irreversible mutations in the genome, which permits saturation mutagenesis without having to screen a large number of individual mutants
2) generates novel mutatnt phenotypes, not only loss-of-function but gain of function etc.
3) cheap - Disadvantage: Some AA not reachable because of the dgenearated nature of the geentic code (Lysin bleibt Lysin auch mit A statt G) and non codning RNA genes difficult to access
If we find more than one mutant with the same phenotype, how do we find out whether the two mutations affect the same gene or different ones?
complementation test with two mutants:
* if the phenotype is recued after crossing, the two mutations compliment each other and are located on different genes complementation
* if the pehnotype is not rescued, mutaion in same gene: Non-cpmlementaioion
Suppressor/modifier mutations
to look for supressor or modifier muattion: mutate already mutated plant and look for phenotypes that are enhanced/weakend or wildtype
* suggest an interaction between the two encoded gene products (either
biochemically physically or functionally)
* easy to find viadescibed mutatant screening
Redundancy
- Picture: Genes G1 und G2 are redundant regarding their function in the head
- Mutant phenotype in head only seen in double mutant
- Redundancy due to genes duplicated in tandem e.g .>15% of all A. thaliana genes occur in tandem duplications!
- microRNAs, non coding functional RNAs as small and/or mutationally insensitive targets also pose problems for EMS mutagenesis.
Example: cup shaped cotyledons
* CUC1 and CUC2 encode
two very similar transcription factors
* F2 of 9:3:3:1 tells you double mutatnt
Fast neutron mutagenesis advantages and disadvantages
mutageneisis by exposing seeds or tissues to high-energy neutron radiation
Advantages:
- causes large deletions and chromosomal rearrangements
- particularly useful for generating knockout mutations in genes and whole gene clusters -> studying functional genomics in plants like Arabidopsis thaliana
- No sequence bias, allowing for random mutagenesis across the genome.
- Effective for studying essential genes, as partial deletions may reveal functions without complete lethality.
Disadvantage:
- Mutations are often large and complex, making it difficult to pinpoint the exact affected gene.
- Higher chance of multiple gene disruptions, which can complicate phenotype-genotype associations.
- Requires specialized equipment and radiation safety measures, making it less accessible than EMS mutagenesis.
Prerequisites for a mutagenesis screen
- suitable genetic background e.g. if u want to study fertalisation use a line which needs fertalisation
- tight, robust, easy, fast screening procedure
- Use of reporter genes to aid in mutagenesis screens for “difficult” phenotypes e.g. Hsa32 expression as a read out for the heat stress memory
SHORE mapping
- extract genomic DNA from a pool of >100 F2 individuals with the mutant phenotype
- Illumina sequence the pooled DNA
- map reads back to Arabidopsis reference genome sequence
- score frequency of paternal alleles in the obtained reads within a 200 kb window, using sliding window
analysis - search for region with a clear deviation of the allele ratio from 50:50
- search for ‘new’ mutations in genes within this region
QTL
mapping-
mapping populations
Backcross
1) Standard F 2 Design:
* high statistical power
* sometimes not feasible (F1 sterile)
2) Backcross to parental lines:
* additive effects only half maximal and backcrosses to both parents needed
* -> reduced statistical power
* simpler genetic structure is easier to analyze
Key Limitations:
* Only one individual per genotype → no repeated measurements → imprecise genotype value estimates
* Genotype × environment interactions cannot be studied
* Requires immortalized F₂ populations for long-term genetic analysis
How
to get from a QTL to the gene ?
Mendelization refers to the process of isolating a quantitative trait locus (QTL) to behave like a single Mendelian gene, allowing for precise genetic analysis.
Two possibilities for Mendelization : 1) Near Isogenic Lines (NILs) – Nearly identical to parental lines except for a small genomic region.
2) Heterogeneous (or Heterozygous) Inbred Families (HIFs) – Maintain genetic variation within defined regions.
Key Considerations:
* Assess phenotype: Does it resemble a homozygous reference (e.g., red)?
* If uncertain, conduct progeny testing:
1) QTL in a homozygous region → Progeny show little variation.
2) QTL in a heterozygous region → Progeny show greater variation.
* Find enough recombinants to narrow down the region of interest as precisely as possible.
The principle of genome
wide association mapping (GWAS)
- exploits high levels of historical recombination between causative polymorphisms and linked polymorphisms -> potential for very high mapping resolution
- requires high density genotyping (the higher the shorter the range of linkage
disequilibrium is; typical values are: 1 kb [ Drosophila , maize], 10 kb Arabidopsis
thaliana ], 5 100 kb [humans]) - BUT: only works if causal/interesting alleles are present in a reasonable proportion of the population (>5%)
- BUT: confounding due to population structure is a big risk
Comparison QTL mapping vs. GWAS
QTL mapping
Advantages
* higher power to detect causal
variation
* population structure not an issue
Disadvantages
* resolution is limited
* limited to variation between the two parents for the cross
GWAS
Advantages
* very high resolution
* no fine mapping required
* uses a larger set of natural variation than QTL mapping
Disadvantages
* only see common variants
(>5% of the population)
* confounded by population
structure
Best of both worlds: Multiparent Advanced Generation Intercross (MAGIC) lines.
* QTL but start with many parents
* cross one with all e other each
* Intermating
* get homozygous lines through selfing
Comparison mutagenesis vs. natural variation
Natural variation
Advantage: see natural adaption nd trace evelotion of popuations
DIsadvantage: less stronger effects so harder to find
Mutagenesis
Advantages: trong mutant genes, discover genes that are essential whith less or no variation in nature
Disadvantages: artificial, does not tell much about nature
Example of Mendelization: Identifying a Drought-Resistant Gene in Plants
Problem:
A scientist wants to find a specific gene responsible for drought resistance in a crop like rice or wheat. This trait is complex because multiple genes (QTLs) contribute to it. To study it like a single Mendelian gene, Mendelization is used.
1)Create a Mapping Population
* Cross a drought-tolerant variety (Parent A) with a drought-sensitive variety (Parent B).
* Grow the offspring (F₂ generation), which will have varied resistance to drought.
2) Develop Near Isogenic Lines (NILs)
* Select plants that show some drought resistance.
* Backcross these with Parent B (sensitive parent) several times, but keep the region containing the QTL from Parent A.
* This results in a NIL that is almost identical to Parent B, except for the small region with the QTL from Parent A.
3) Test the NILs for Drought Resistance
- Grow the NILs in dry conditions.
- If the NILs are resistant like Parent A, the QTL is likely responsible for the trait.
- If the NILs are sensitive like Parent B, the QTL does not control the trait.
4) Progeny Testing for Confirmation
- Self-pollinate the NILs and observe the next generation.
- If little variation in drought resistance is seen, the QTL is likely in a homozygous state.
- If the plants show different levels of resistance, the QTL is likely in a heterozygous state, and further fine mapping is needed.
5) Fine Mapping and Gene Identification
* Find recombinants with breakpoints within the QTL region.
* Narrow down the region to one or a few genes.
* Test candidate genes using gene expression analysis or gene editing (e.g., CRISPR).
Final Outcome:
Pinpointing the gene responsible for drought resistance. Can now be use for breeding drought-resistant crops efficiently.
