Linkage and Recombination

Question 1

Mendel’s 1st law?

Answer 1

2 homologous alleles that separate during gamete formation in equal proportion

Question 2

Mendel’s 2nd law?

Answer 2

Alleles at different loci assort independently

Question 3

When is the 2nd law not true?

Answer 3

When genes are located nearby on the chromosome they are linked.

Question 4

Who discovered linkage?

Answer 4

Thomas Hunt Morgan
Was working on Drosophila, and saw linkage between sex and eye colour.

Crosses with mutant white eye males and red eye females produced all red-eye females and males in a 1:1 red and white ratio. It is thought that the gene for red eye colour was on the X chromosome.

Question 5

Two types of linkage?

Answer 5

Sex or autosomal.

Autosomal - genes on the same chromosome show linkage but genes in different linkage groups/chromosomes assort independently.

Question 6

What does linkage mean?

Answer 6

Parental combinations of genes are co-inherited more frequently than predicted by Mendelian laws.

Question 7

What is recombination?

Answer 7

Produces non parental phenotypes, although not in Mendelian ratios. It occurs due to exchange of genetic material between chromatids during crossing over.

Question 8

When does crossing over occur?

Answer 8

Meiosis Prophase I when the homologous chromosomes form chismata.

Question 9

What evidence is there for physical exchange?

Answer 9

abnormally structured plant chromosomes with structures that identify both poles, and linked genetic markers in the middle. A heterozygote with repulsion configuration was created, and recombination produced a coupling configuration (and structural abnormaities transferred to other chromosome). Test cross performed and recombinants observed to reveal if non parental marker combinations were present.

Question 10

What is the suppression of recombination?

Answer 10

Keeps allele combinations together, important for alleles involved in sex determination. Suppression results from chromosomal inversion, where looping disrupts crossing over to produce chromosomes with 2 or no centromeres.

This causes segregation to fail at meiosis, halting it or producing non-viable gametes.

Question 11

Process of genetic analysis?

Answer 11

 Formulate the biological question (what gene or mechanism is behind this trait?)

 Identify genetic variation that alters the phenotype using a genetic screen (finds a mutation that affects the trait of interest). Natural variation can be the source or induced mutations using chemical mutagens (point mutations), radiation (deletions) or transposable elements (insertions).

 Identify causative mutation using molecular complementation, insertion mutagenesis, linkage mapping or whole genome sequencing.

Question 12

What is molecular complementation?

Answer 12

Break whole yeast genome into fragments and insert them into plasmids (vector) to create a genomic library. Insert the vector into a yeast mutant, which is transformed. If the mutant is for example unable to synthesise adenine, replica plate the colonies onto a selective medium without adenine. The complemented colonies can then be selected.

Question 13

What is linkage mapping?

Answer 13

Based on the idea that different pairs of genes show different recombination frequencies, related to the distance between the genes.

Question 14

How are recombination frequencies calculated?

Answer 14

number of recombinants/total progeny x 100

OR more accurately

number of recombinations/total progeny x 100

The latter is more accurate as sometimes 2 recombinations can occur on the same chromosome, and so the number of recombinants should be muliplied by two.

Question 15

What is the max recombination freq for unlinked genes?

Answer 15

50% as recombinants would make up 50% of the offspring in a 1:1:1:1 genotypic ratio.

Question 16

Why do recombination frequencies vary along the chromosome?

Answer 16

There are hotspots and also low recombination at the centromere.

Physical maps don’t always match up with genetic maps due to multiple recombination events occurring between genes.

The physical maps are usually in the same order, the distances differ.

Question 17

What is a molecular marker?

Answer 17

Indels (insertions and deletions of short sequences) - variable numbers of repeats that can be detected by PCR.

SNP (single nucleotide polymorphism). Changing a base could introduce a restriction site that cuts a fragment creating shorter fragments that can be detected by PCR.

Question 18

How do you generate a mapping population?

Answer 18

 In F1 cross parents which are homozygous at both loci for either mutant or wildtype gene and its associated marker. Then cross or self the F1 heterozygous progeny. In the gametes of the F1 progeny, recombination may have occurred. The F2 progeny that result from these gametes are recombinants.

 The mapping population should be selected based on which individuals have a distinguishable phenotype that corresponds to a homozygous recessive phenotype (know they’re double mutant or WT, because it produces a recessive trait).

 Mapping population will constitute individuals homozygous for either the mutant or wildtype version of the allele, and will show no recombination, one recombination or two recombinations.

E.g. recessive mutant mapping population, individuals will have two mutant markers, one mutant and one wild type marker (recombination) or two WT markers (double recombination).

 Use the recombination events in mapping pop to calculate recombination frequency

 This produces distances between genes that can be used to construct a map, the problem is that they are based on probability, and can be made more accurate by using larger populations (events are random). The solution is to map mutations relative to individual recombination events…

Question 19

How to locate mutations using mapping recombination events?

Answer 19

 Use flanking markers and see which markers the mutation co-segregates with and which markers show recombination. The region were the recombination events don’t occur must be where the mutation lies, i.e. it must lie below x in the region without recombination and above y in the region which hasn’t recombined. It sets up a mapping interval.

 Using additional markers can pin-point recombination events and narrow down the locus of the mutation.

 The results can be confirmed by inserting the fragment of DNA within which the mutation is thought to lie, but from a WT organism, into a mutant. If it complements then it was correct (molecular complementation).

 Bulk Segregant Analysis is the process used to identify linkage groups associated with given mutations). It can be used to map chromosomes in addition to whole genome sequencing. The mapping population should be generated, and the pool mutants (bulk) and extract the DNA. The DNA should be sequenced and the region near the mutation must be homozygous whereas the unlinked regions (away from the mutation) will be 50:50 between the two parents (one copy of the region from each parent).