Genetic Linkage and Gene Mapping Notes

Genetic Linkage & Gene Mapping

Learning Objectives

Sex-linkage: Genes located on sex chromosomes (e.g., X or Y).
Linkage: Genes located close to each other on the same chromosome.
Three-point crosses: Used for chromosome mapping.
Chromosome mapping:
- The distance between two genes on a chromosome is relative to the number of crossover events separating them.
- The distance between two linked genes is estimated from the proportion of recombinants observed and is measured in centimorgans (cM).

The Sex Chromosomes

In many animals (and some plants), one sex has a pair of unlike chromosomes, usually denoted XY.
In humans (& flies):
- Female: XX
- Male: XY

The Sex Chromosomes

Autosomal genes
X chromosome genes:
- Sex-linkage discovered by Morgan (1911)
- Notation:
  - $a$ = mutant
  - $a^+$ = wildtype
  - $\frac{a \ b}{a^+ \ b}$
- Example:
  - $w^+$ gives wild-type (red) eyes
  - $w$ gives mutant (white) eyes
  - $\frac{w}{w^+}$
  - $\frac{w}{Y}$

X-LINKAGE

Cross 1: Homozygous red-eyed female crossed with white-eyed male
- F1 generation: Males and females are red-eyed (normal wildtype red eye color is dominant).
Cross 2: White-eyed female crossed with red-eyed male
- F1 generation: All males are white-eyed and all females are red-eyed.

X-LINKAGE Examples

Illustrations of Cross 1 (Homozygous red-eyed female crossed with white-eyed male) and Cross 2 (White-eyed female crossed with red-eyed male) are shown, depicting the inheritance patterns of X-linked traits.

Sex-Linked Inheritance

Y-linked Inheritance
X-linked Recessive Inheritance

Linkage & Crossing Over

Genes on the same chromosome are 'linked,' meaning they segregate together unless crossovers and genetic recombination occur.
Chiasmata (identified by Janssens in 1909) demonstrated crossover leads to recombination.
Crossing over occurs in Pachytene (Prophase I) of meiosis I and can occur between strands 1&3, 2&3, 1&4, or 2&4

A Normal Dihybrid Self-Cross

When the two genes (i.e., loci R & loci Y) are NOT linked:
- They assort independently.
- Therefore, we expect an F2 phenotypic ratio of 9:3:3:1.
- Or a 1:1:1:1 ratio from a testcross.

Linkage & Recombination Frequency

Parental cross: $AABB \times aabb$
F1: $AaBb$ dihybrid
Diagram shows parents prior to replication, gamete formation, and F1 generation.

Testcross & Linkage

F1 dihybrid testcross:
- $AaBb \times aabb$
- Complete linkage is shown by a 1:1 F2 ratio with only parental gametes produced (i.e., no $Ab$ & $aB$ recombinants; therefore, RF = 0%).

Testcross & Linkage

Incomplete linkage:
- Non-parental phenotypes are observed (i.e., $Ab$ & $aB$ ) in F2.
- Recombination frequency (RF) = 0-50%
- Low frequency of non-parental phenotypes.

Genes & Chromosomes

Genes are arranged linearly on chromosomes.
Each combination of X-linked genes gave different RF, chromosomes must contain units (i.e., genes) whose recombination depends on the linear distance between them.

Linkage & Recombination

The probability of recombination increases with larger distances between loci (i.e., greater inter-locus distance).
Crossing over and Recombination occur in pachytene of meiosis I.
This happens during gamete formation in the F1 heterozygote.

Linkage & Recombination

Linkage can be detected through a two-point test cross.
Parental cross: $AABB \times aabb$
F1 Dihybrid: $AaBb$
F1 Gametes: $AB$ and $ab$
Testcross: $AaBb \times aabb$

Linkage & Recombination

Linkage can be detected through a two-point test cross.
F1 cross: $AaBb \times aabb$
F2 Genotypes: $AaBb$ , $Aabb$ , $aaBb$ , $aabb$
F2 Phenotypes: $AB$ , $Ab$ , $aB$ , $ab$
Expected Ratio without linkage: 25% for each phenotype
Example with observed numbers: 80, 10, 20, 90 (Total = 200)

Linkage & Recombination

Linkage can be detected through a two-point test cross.
Determining if genes are linked based on observed vs. expected numbers.
Example: Are the genes linked if the observed numbers are 80, 10, 20, 90?

Gene Configuration

The Recombination frequency (RF) indicates location:
- 0-50% RF: Syntenic loci (linked).
- 50% RF: Syntenic loci (not-linked) - same chromosome but far apart.
- 50% RF: Non-syntenic loci - different chromosomes, affected by independent assortment.
- NOTE: %RF is the same as cM!

Gene Configuration

Example: Testing for synteny and linkage using recombination frequencies.
Initial experimental two-point testcross gives $y \times B = 50\%RF$ (Bar eyes, B, and yellow body, y).
Further two-point testcrosses:
- $sn \times B = 36\%RF$ (singed bristles, sn)
- $y \times sn = 21\%RF$
Therefore: $y \rightarrow B = 57cM$ , implying synteny but not linkage (>'57%RF' cannot be realized in a $y \times B$ cross!)

Gene Configuration

Two-point testcrosses are laborious; three crosses are needed in total to map the relative positions of three genes.
Cross 1: $A \times C = \%RF$
Cross 2: $A \times B = \%RF$
Cross 3: $B \times C = \%RF$

Three-Point Crosses

Three monogenic, bi-allelic traits determined by 3 different genes (loci), each with two alleles:
- $DD$ : Tall stem
- $EE$ : Purple flower
- $FF$ : Blue spots
- $dd$ : Dwarf stem
- $ee$ : White flower
- $ff$ : No spots

Three-Point Cross

Parental cross: $DDEEFF \times ddeeff$
F1 trihybrid: $DdEeFf$
Testcross: $DdEeFf \times ddeeff$
Score the number of F2 offspring for each possible genotype (8) from F1 trihybrid testcross.
Set up numerous F1 testcrosses to produce 100-1000s F2 progeny.

Test Crossing

F2 phenotypes and observed numbers are recorded. Parental classes will be the highest frequency because there is no recombination. Recombinant classes result from single (SCO) or double crossovers (DCO).

F2 phenotype	No. Obs
DEF	380
def	390
DEf	3
deF	2
Def	90
dEF	85
DeF	20
dEf	30
Total	1000

Par = parental gametic genotypes
Rec = recombinant gametic genotypes

Calculating % RF

Calculating % Recombination Frequency (RF) for different loci combinations.

Loci	Par	Rec	%RF
D/E	DE, de	De, dE	22.5
D/F	DF, df	Df, dF	18.0
E/F	EF, ef	Ef, eF	5.5

Example calculations shown for each loci pair (D/E, D/F, E/F).

Gene Configuration

All loci have % RF <50, so all are linked and thus syntenic.
- $D/E = 22.5cM$
- $D/F = 18.0cM$
- $E/F = 5.5cM$
- Different possible gene configurations with distances shown (e.g., D-F-E, E-F-D).

Crossing Over

Occurs during Prophase I of meiosis.
All four chromatids of tetrad are shown. The F1 is heterozygous for three genes to be mapped.
Vertical lines represent crossover intervals:
- Single Cross Over (SCO) between D and F occurs at i.
- SCO between F and E occurs at ii.
- Double Cross Over (DCO) can occur (at both i & ii).

Identifying DCO

Identifying Double Crossover (DCO) events where the F1 trihybrid undergoes recombination during Pachytene I, resulting in the rarest categories (DfE and dFe) at Telophase II/Metaphase II.

Crossing Over

F2 phenotypes, observed numbers, and crossover types are listed. Double crossovers (DCO) are the least frequent.

F2 phenotype	No. Obs	Crossover
DEF	380	None
def	390	None
DEf	3	i & ii
deF	2	i & ii
Def	90	i
dEF	85	i
DeF	20	ii
dEf	30	ii
Total	1000

Crossing Over

Recombination frequency calculations including single and double crossovers.
Showing how including the double cross over events affects the final Recombination Frequency (RF).

Loci	Rec	%RF (SCO only)	%RF (SCO + DCO)
D/E	De, dE	22.5	23.5

Three-Point Testcross Summary

Work out RF for each pair of loci.
See which pairs are syntenic (i.e., with <50%RF).
If all are syntenic, work out order from RF.
Check which classes are double crossovers (DCO), i.e., least frequent phenotype observed in F2.

Diagram illustrating the crosses from parental purebreds to F1 trihybrid, and then to F2 genotypes.

Locating Genes

Loci on the same chromosome are syntenic (e.g., yellow body & singed bristles on X).
Loci on different chromosomes are non-syntenic (e.g., yellow body & dumpy wings, 2).
Syntenic genes may or may not be linked.

Gene Linkage Summary

Genes on Sex Chromosomes exhibit non-Mendelian inheritance.
Non-syntenic genes randomly segregate through independent assortment.
Crossovers of homologous chromosomes occur between syntenic genes during the pachytene stage of Prophase I (via chiasma) resulting in recombination.
Linked genes are syntenic & are inherited together (diverging from Mendel’s law of independent assortment) unless crossovers occur.
Genes which are further apart have higher rates of crossing over (%RF).
Three-point test crosses are used to map configuration & distance of 3 linked genes.
The maximum %RF is 50% with values above implying genes are not linked.