Gene discovery and genetic mapping in eukaryotes Flashcards
Describe forward genetic approaches
- aim: to identify the sequence variation(s) responsible for a particular phenotype
- phenotype -> sequence variation
- requires no assumption
about the function and the nature of the gene product
Describe reverse genetic approaches
aim: to identify phenotypic changes caused by a particular sequence variation
- sequence variation -> phenotype
- tests a hypothesis about the gene function
Describe the process of a forwards genetics approach
- isolation of individual(s) with inheritable change of the phenotype of interest via mutagenesis/natural variation
- identification of causative DNA variation(s)
Give some examples of natural variations
disease resistance, fur colour, herbicide tolerance
Describe induced variations
- generated random mutations
- chemical mutagens (point mutations, C to T or A to G)
- UV- light ( point mutations)
- X-rays or gamma rays (deletions)
- transposable elements (insertions)
Give a chemical mutagen
Ethyl methane sulfonate (EMS)
List some methods to identify mutant genes in eukaryotes
- insertion mutagenesis (Drosophila and plants)
- linkage mapping + map-based cloning
- whole genome sequencing
Describe insertion mutagenesis
- transposons or Transposable Elements (TE) create mutations when they insert into genes
- if the DNA sequence of the TE is know it can be used to identify and clone the mutant gene
- molecular cloning methods can identify genomic DNA fragments containing TE
- flanking DNA sequence encodes the gene of interest
Explain why onsertion mutagenesis is of limited utility
- applicable only to a few well studied organisms
- mutation efficiency is low
- many induced or natural mutations are single- base substitutions
Describe mutation rate in Drosophila
- new mutations at random sites about once every 150–300 kb
Describe linkage and recombination
- linkage of genes in a linkage group was rarely absolute
and produced recombinant progeny - different pairs of genes in a linkage group showed different but characteristic rates of recombinants
- recombination frequency for a gene pair is related to the distance between these genes on the chromosome
Recombination frequency
- the frequency of crossing-overs between two loci
- (total number of recombination events / total number of gametes tested) x 100
Recombination events can be detected only in
gametes derived from a heterozygous parent
Describe linkage mapping
- 1% recombination is sometimes called 1 Map Unit (MU) or 1 ‘centi Morgan’ (cM)
- relative position of genes in a linkage group
- generated from combining the recombination frequencies for multiple pairs of genes
- a series of mapping steps can establish the map of a
whole linkage group
Additive recombination frequencies
can exceed 50%
Describe genetic maps
- often have multiple recombination events 1 per chromosome
- as the physical distance increases, genetic distances (recombination frequencies) are under-estimated
- good linearity of measured and additive recombination frequency between genes up to 25-30cM apart
- tends towards the maximum 50% recombination frequency
Genes near opposite ends of a chromosome are
- effectively unlinked
- exhibit ~50% recombination
Linkage groups are established by combining
short-range linkages
Describe the basics of the genetic map
based on recombination frequency (cMorgan)
Describe the basics of the physical map
based on DNA sequence (base pairs)
Although genetic maps and physical maps are
- colinear (same gene order)
- genetic map distances are often not the same as physical map distances
- poor quantitative correlation
Describe recombination rates across the chromosome
- vary slightly
- low near centromeres
- ‘hot-spots’ and ‘cold-spots’ occur all along the chromosome
- can be seen in whole-genome sequencing of 486
recombinant lines of Arabidopsis thaliana
Describe cross-over interference
one cross-over interferes with the coincident occurrence of another cross-over in the same pair of chromosomes
How to identify the position of mutation with respect to classical and modern genetics?
Classical genetics: co-segregation of mutant phenotypes Modern genetics: co-segregation of the mutant phenotype with naturally occurring DNA polymorphisms (molecular markers)
Describe molecular markers
- a site of DNA polymorphism not associated with any observable phenotype, but can be detected with molecular techniques
- reference points in the genome (can be used for mapping if different alleles are present in homozygote form in parents)
- each sequence variation is an allele
- naturally occurring variations
- very numerous ( 10,000s per genome)
How to generate a mapping population to measure genetic distance and position in the genome
- Cross F1 heterozygous progenies
or self-fertilise - F2: Select individuals with distinguishable homozygote phenotype
- identify recombination events between gene of interest and molecular markers
What do you need to generate a mapping population?
- homozygous mutant
- non-related individual with wild-type phenotype, which carries large number of sequence polymorphisms
How do you know if a recombination event has occurred?
if the other parental allele of a linked marker appears in an homozygote mutant individual
What is the problem with mapping by recombination frequency?
based on estimates of frequency: probabilistic
Describe map-based cloning
maps the mutation relative to individual recombination events, rather than estimating recombination frequencies
Describe mapping by sequencing
- generate a mapping population
- sequence DNA from the two parents and the bulked mapping population
- genome sequence data identifies all mutated genes in this region
How to generate a mapping population in mapping by sequencing
- cross the homozygous mutant with an unrelated individual with high sequence polymorphism-> heterozygous F1 population
- cross two F1s to generate F2 population
- recombination between parental alleles occurs
- select mapping population from F2 progeny and combine all individual into one sample, a ‘bulk’
Causality is tested with
complementation
Testing causality of a recessive mutation
- introduce the wild-type (dominant) sequence of gene into the recessive mutant
- if: WT; complementation -> mutation causes phenotype
- if mutant-type; no complementation -> mutation does NOT cause phenotype
Genetic distances are additive
- over short distances only (owing to multiple cross-overs)
- but allow linkage maps to be constructed.
