Gene discovery and genetic mapping in eukaryotes

Question 1

Describe forward genetic approaches

Answer 1

• 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

Question 2

Describe reverse genetic approaches

Answer 2

aim: to identify phenotypic changes caused by a particular sequence variation
- sequence variation -> phenotype
- tests a hypothesis about the gene function

Question 3

Describe the process of a forwards genetics approach

Answer 3

• isolation of individual(s) with inheritable change of the phenotype of interest via mutagenesis/natural variation
• identification of causative DNA variation(s)

Question 4

Give some examples of natural variations

Answer 4

disease resistance, fur colour, herbicide tolerance

Question 5

Describe induced variations

Answer 5

• 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)

Question 6

Give a chemical mutagen

Answer 6

Ethyl methane sulfonate (EMS)

Question 7

List some methods to identify mutant genes in eukaryotes

Answer 7

• insertion mutagenesis (Drosophila and plants)
• linkage mapping + map-based cloning
• whole genome sequencing

Question 8

Describe insertion mutagenesis

Answer 8

• 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

Question 9

Explain why onsertion mutagenesis is of limited utility

Answer 9

• applicable only to a few well studied organisms
• mutation efficiency is low
• many induced or natural mutations are single- base substitutions

Question 10

Describe mutation rate in Drosophila

Answer 10

• new mutations at random sites about once every 150–300 kb

Question 11

Describe linkage and recombination

Answer 11

• 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

Question 12

Recombination frequency

Answer 12

• the frequency of crossing-overs between two loci
• (total number of recombination events / total number of gametes tested) x 100

Question 13

Recombination events can be detected only in

Answer 13

gametes derived from a heterozygous parent

Question 14

Describe linkage mapping

Answer 14

• 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

Question 15

Additive recombination frequencies

Answer 15

can exceed 50%

Question 16

Describe genetic maps

Answer 16

• 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

Question 17

Genes near opposite ends of a chromosome are

Answer 17

• effectively unlinked
• exhibit ~50% recombination

Question 18

Linkage groups are established by combining

Answer 18

short-range linkages

Question 19

Describe the basics of the genetic map

Answer 19

based on recombination frequency (cMorgan)

Question 20

Describe the basics of the physical map

Answer 20

based on DNA sequence (base pairs)

Question 21

Although genetic maps and physical maps are

Answer 21

• colinear (same gene order)
• genetic map distances are often not the same as physical map distances
• poor quantitative correlation

Question 22

Describe recombination rates across the chromosome

Answer 22

• 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

Question 23

Describe cross-over interference

Answer 23

one cross-over interferes with the coincident occurrence of another cross-over in the same pair of chromosomes

Question 24

How to identify the position of mutation with respect to classical and modern genetics?

Answer 24

Classical genetics: co-segregation of mutant phenotypes Modern genetics: co-segregation of the mutant phenotype with naturally occurring DNA polymorphisms (molecular markers)

Question 25

Describe molecular markers

Answer 25

• 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)

Question 26

How to generate a mapping population to measure genetic distance and position in the genome

Answer 26

• Cross F1 heterozygous progenies
  or self-fertilise
• F2: Select individuals with distinguishable homozygote phenotype
• identify recombination events between gene of interest and molecular markers

Question 27

What do you need to generate a mapping population?

Answer 27

• homozygous mutant
• non-related individual with wild-type phenotype, which carries large number of sequence polymorphisms

Question 28

How do you know if a recombination event has occurred?

Answer 28

if the other parental allele of a linked marker appears in an homozygote mutant individual

Question 29

What is the problem with mapping by recombination frequency?

Answer 29

based on estimates of frequency: probabilistic

Question 30

Describe map-based cloning

Answer 30

maps the mutation relative to individual recombination events, rather than estimating recombination frequencies

Question 31

Describe mapping by sequencing

Answer 31

• 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

Question 32

How to generate a mapping population in mapping by sequencing

Answer 32

• 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’

Question 33

Causality is tested with

Answer 33

complementation

Question 34

Testing causality of a recessive mutation

Answer 34

• 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

Question 35

Genetic distances are additive

Answer 35

• over short distances only (owing to multiple cross-overs)
• but allow linkage maps to be constructed.