Gene Mapping Flashcards
Why is hypocotyl elongation repressed in light
Because a long hypocotyl above ground is not beneficial; light signals the plant to stop elongating
What is the phenotype of hy mutants when grown in light
Long hypocotyls due to impaired light perception or signaling
What is the phenotype of the hy3 mutant
Long hypocotyls in red light; hy3 is red-light insensitive
What does linkage mapping calculate
The recombination frequency between a mutant gene and genetic markers.
What are genetic markers
DNA sequence variations with known genetic and physical positions in the genome
What is a molecular marker
A DNA polymorphism detectable with molecular techniques
What type of inheritance do molecular markers usually show
Codominant – both alleles can be detected in heterozygotes
How does PCR detect molecular markers
Differences in length due to insertions or deletions (SSLPs or indels)
How are different PCR products detected
Gel electrophoresis separates DNA fragments by size
What is a CAPS marker
A SNP that alters a restriction enzyme site; different alleles yield different digestion patterns
What is the first step in map-based cloning
Bulk Segregant Analysis (BSA) – identifying the linkage group of the mutation
What is the second step in map-based cloning
Linkage mapping – calculating recombination frequency with nearby markers
What is fine mapping
Identifying recombination events to narrow the gene’s location to <50 kb
What follows fine mapping
Sequencing candidate genes to find the mutation
What is the final step in map based cloning
Complementation assay – introducing WT gene into mutant to see if phenotype is rescued
How many chromosomes does Arabidopsis thaliana have
Five chromosomes
What does BSA help determine
The linkage group (chromosome region) associated with the mutation
What did BSA reveal about the hy3 mutation
Co-segregates with INDEL9 on the lower arm of Chromosome 2
How is recombination frequency detected
By genotyping plants at marker loci and looking for Col/Ler allele combinations
What is the genotype of hy3 mutant plants
Homozygous Col at the Hy3 locus
What does a Col/Ler or Ler/Ler genotype at a marker suggest
Recombination between the marker and the Hy3 locus
What molecular method follows DNA extract from F2 plants with desired characteristics
PCR amplification of INDEL9 and ER markers
What is done to the ER PCR product
Digested with DdeI restriction enzyme
How are the DNA products analysed
Agarose gel electrophoresis
What is the purpose of PCR
to amplify specific DNA sequences
What are the key components of a PCR reaction
DNA template, Taq polymerase, primers, dNTPs, Taq buffer, MgCl₂
What are the three main temperature steps of PCR
Denaturation, Annealing, Extension
How does agarose concentration affect separation
Higher agarose = better resolution of smaller fragments
What is the graphical relationship between DNA mobility and mass
logarithmic over a limited range
What are the two main types of DNA binding dyes
Intercalating dyes and minor groove binders
Give examples of intercalating dyes
Ethidium bromide, SYBRSafe, propidium iodide
How does ethidium bromide work
Intercalates between DNA bases and fluoresces under UV light
