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)