1. LINKAGE AND GENETIC MAPPING
Lecture 7
Molecular Genetics

2. INTRODUCTION Eukaryotic genomes contain hundreds to thousands of genes
Yet most species have fewer than 50 chromosomes in a haploid set
Humans have only 23 chromosomes per haploid set but have ~25,000 genes
Therefore, each chromosome carries hundreds or even thousands of different genes
The transmission of such genes may go against Mendel’s law of independent assortment

3. LINKAGE & CROSSING OVER
Genetic linkage is the tendency of genes that are located close to each other on a chromosome to be inherited together during meiosis.
Genes whose loci are nearer to each other are less likely to be separated onto different chromatids during chromosomal crossover, and are therefore said to be genetically linked.

4. Chromosomes are called linkage groups
They contain a group of genes that are linked together on the same DNA molecule
The number of linkage groups is equal to the number of types of chromosomes of the species
For example, in humans
22 autosomal linkage groups
An X chromosome linkage group
A Y chromosome linkage group
If no crossing over, the alleles of all genes located on the same chromosome would be inherited together

5. Crossing Over May Produce Recombinant Phenotypes
In diploid eukaryotic species, linkage can be “undone” during meiosis as a result of crossing over
Crossing over
Occurs during prophase I of meiosis
Non-sister chromatids of homologous chromosomes exchange DNA segments

8. The arrangement of linked alleles has not been altered
IF NO CROSSING OVER IN REGION BETWEEN THE TWO GENES
= 100% Non-Recombinants

9. Non-recombinant gametes
IF CROSSING OVER IN REGION BETWEEN THE TWO GENES
= 50% Non-Recombinants and 50% Recombinants

10. Recombination Frequency
Recombination fraction or frequency is a measure of the distance between two loci.
Two loci that show 1% recombination are defined as being 1 centimorgan (cM) apart on a genetic map.
1 map unit = 1 cM (centimorgan)
Two genes that carry out independent assortment have recombination frequency of 50 percent and are located on nonhomologous chromosomes or far apart on the same chromosome = un-linked
Genes with recombination frequencies less than 50 percent are on the same chromosome = linked

11. Calculation of Recombination Frequency
The percentage of recombinant progeny (off-springs) produced in a cross is called the recombination frequency, which is calculated as follows:
Recombinant frequency =
Number of recombinant offspring
Total number of offspring
X 100

12. Recombination Frequency

13. Genetic map
Genetic maps allow us to estimate the relative distances between linked genes, based on the probability that a crossover will occur between them

14. The units of distance are called map units (mu)
Experimentally, the percentage of recombinant offspring is correlated with the distance between the two genes
If the genes are far apart  many recombinant offspring
If the genes are close  very few recombinant offspring
Recombinant frequency =
Number of recombinant offspring
Total number of offspring
X 100
The units of distance are called map units (mu)
They are also referred to as centiMorgans (cM)
One map unit is equivalent to 1% recombination frequency

15. Genetic mapping experiments are typically accomplished by carrying out a testcross
A mating between an individual that is heterozygous for two or more genes and one that is homozygous recessive for the same genes
This cross concerns two linked genes affecting bristle length and body color in fruit flies
s = short bristles
s+ = normal bristles
e = ebony body color
e+ = gray body color
One parent displays both recessive traits
It is homozygous recessive for the two genes (ss ee)
The other parent is heterozygous for the two genes
The s and e alleles are linked on one chromosome
The s+ and e+ alleles are linked on the homologous chromosome

16. Recombinant offspring are fewer in number than nonrecombinant offspring
Chromosomes are the product of a crossover during meiosis in the heterozygous parent

17. Estimate the distance (recombinant frequency) between the two genes
Number of recombinant offspring
Total number of offspring
X 100 =
X 100
= 12.3 map units
Therefore, the s and e genes are 12.3 map units apart from each other along the same chromosome

18. Trihybrid Crosses
Data from trihybrid crosses can also yield information about map distance and gene order. The following experiment outlines a common strategy for using trihybrid crosses to map genes
In this example, we will consider fruit flies that differ in body color, eye color and wing shape
b+ = gray body color > b = black body color
pr+ = red eye color > pr = purple eye color
vg+ = normal wings > vg = vestigial wings

19. Step 1: Cross two true-breeding strains that differ with regard to three alleles.
Female is mutant for all three traits
Male is homozygous wildtype for all three traits
The goal in this step is to obtain a F1 individuals that are heterozygous for all three genes

20. Step 2: Perform a testcross by mating F1 female heterozygotes to male flies that are homozygous recessive for all three alleles
During gametogenesis in the heterozygous female F1 flies, crossovers may produce new combinations of the 3 alleles

21. In the offspring of crosses involving linked genes,
Analysis of the F2 generation flies will allow us to map the three genes
The three genes exist as two alleles each
Therefore, there are 23 = 8 possible combinations of offspring
If the genes assorted independently, all eight combinations would occur in equal proportions
It is obvious that they are far from equal
In the offspring of crosses involving linked genes,
Non-recombinant (phenotypes of grandparents) occur most frequently
Double crossover phenotypes occur least frequently
Single crossover phenotypes occur with “intermediate” frequency

22. Number of Observed Offspring (males and females)
Step 3: Collect data for the F2 generation
Phenotype
Number of Observed Offspring (males and females)
Gray body, red eyes, normal wings
411
Gray body, red eyes, vestigial wings 61
Gray body, purple eyes, normal wings 2
Gray body, purple eyes, vestigial wings 30
Black body, red eyes, normal wings 28
Black body, red eyes, vestigial wings 1
Black body, purple eyes, normal wings 60
Black body, purple eyes, vestigial wings
412

23. Step 4: Calculate the map distance between adjacent pairs of genes
The combination of traits in the double crossover tells us which gene is in the middle. A double crossover separates the gene in the middle from the other two genes at either end
Thus, the gene for eye color lies between the genes for body color and wing shape
We know that pr is in the middle, so map b-pr and then pr-vg
b pr vg

24. Body color / eye color
Non-recombinant offspring
Total
recombinant Offspring
Gray body, red eyes
( )
472
Gray body, purple eyes
(30 + 2) 32
Black body, purple eyes
( )
Black body, red eyes
(28 + 1) 29
944 61
The map distance between body color and eye color is
Recombinant frequency = 61
X 100
= 6.1 map units

25. Eye color / wing shape
Parental offspring
Total
Nonparental Offspring
Red eyes, normal wings
( )
439
Red eyes, vestigial wings
(61 + 1) 62
Purple eyes, vestigial wings
( )
442
Purple eyes, normal wings
(60 + 2)
881
124
The map distance between eye color and wing shape is
Recombinant frequency =
124
X 100
= 12.3 map units

26. Step 5: Construct the map
Based on the map unit calculation the body color and wing shape genes are farthest apart
The eye color gene is in the middle
The data is also consistent with the map being drawn as vg – pr – b (from left to right)
Total distance b – vg = 18.4 mu

27. Mapping Function
When mapping two genes that are relatively far map distance underestimated because most double-crossovers cannot be detected
Four types of double crossovers possible
Two strand
Three strand
Four strand
Two strand counted as no crossovers
Three strand counted as single crossover
Four strand is the only one counted as a double crossover
Overall, double crossovers result in 50% recombinants and 50% non-recombinants, as if they had not occurred at all!

29. 1. Two Stranded crossing over
Types of Crossing over according to the number of strands involved in crossing over
1. Two Stranded crossing over
Result in new recombination

30. 2. Four stranded crossing over
Result in 50% new recombination and 50% parental or non recombinant

31. A chiasma
A chiasma (plural: chiasmata), in genetics, is thought to be the point where two homologous non-sister chromatids exchange genetic material during chromosomal crossover during meiosis (sister chromatids also form chiasmata between each other, but because their genetic material is identical, it does not cause any change in the resulting daughter cells)
. The chiasmata become visible during the diplotene stage of prophase I of meiosis, but the actual "crossing-over" of genetic material is thought to occur during the previous pachytene stage.
When each tetrad, which is composed of two pairs of sister chromatids, begins to split, the only points of contact are at the chiasmata.

32. chiasma frequency
chiasma frequency = 2 x recombination frequency where recombination frequency is:
recombination frequency = (no. of recombinants x 100) / (total no. of progeny)
The phenomenon of genetic chiasmata (chiasmatypie) was discovered and described in 1909 by Frans Alfons Janssens, a Jesuit professor at the University of Leuven in Belgium. [1][2] A bivalent refers to the two homologous chromosomes (4 chromatids).
The chiasmata refers to the actual break of the phosphodiester bond during crossing over. The larger the number of map units between the genes, the more crossing over occurs.

33. Kinds of Crossing-over:
Depending upon the number of chiasmata appeared, kinds of crossing-over of can be
(i) Single cross-over:
In this case, only one chiasma is formed which leads to formation of single cross-over gametes. It is most common type of cross-over.
(ii) Double cross-over:
In double cross-over, two chiasmata develop. These chiasmata may appear between the same chromatids or between different chromatids. This type of crossing over forms double crossing-over gametes.
(iii) Multiple cross-over:
Here, ‘more than two chiasmata are constituted. It may be further classified into triple (3 chiasmata), quadruple (4 chiasmata) and so on. Multiple crossing-over is of rare occurrence.

35. Factors influencing crossing-over:
Distance between the genes. More the distance between two genes on same chromosome, higher the frequency of crossing over. Significance of crossing-over:
(i) This process provides a vast store of genetic variability in sexually reproducing organisms.
(ii) Useful recombinations are used by plant and animal breeders. Breeders try to break up the linkages by crossing-over to get combinations of useful traits in the progeny.
(iii) This process produces new combination of genes (recombination). Green revolution is mainly due to selective picking up of useful genetic recombination developed by the process of crossing-over.

36. Asignment
The following experiment outlines a data from trihybrid crosses of fruit flies that differ in body color, eye color and wing shape define
1- single crossing over
2- double crossing over
3-calculate a map distance and draw gene order
S+ = normal bristles > s = short bristles
W + = red eye color > w = white eye color
Cu + = normal wings > cu = curly wings

37. Back cross result Frequency Genotypes Phenotypes 435 Cu + S+ W + 1
normal wings normal bristles red eyes 1
Cu S W
normal wings normal bristles white eyes 16
Cu S W +
normal wings short bristles red eyes 45
Cu S W
normal wings short bristles white eyes 38
Cu S W +
curly wings normal bristles red eyes 17
Cu S W
curly wings normal bristles white eyes
Cu S W +
curly wings short bristles red eyes
425
Cu S W
curly wings short bristles white eyes