LINKAGE AND GENETIC MAPPING Lecture 7 Molecular Genetics

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

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.

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

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

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

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

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

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

Recombination Frequency

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

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

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

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

Estimate the distance (recombinant frequency) between the two genes Number of recombinant offspring Total number of offspring X 100 76 + 75 = X 100 542 + 537 + 76 + 75 = 12.3 map units Therefore, the s and e genes are 12.3 map units apart from each other along the same chromosome

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

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

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

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

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

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

Body color / eye color Non-recombinant offspring Total recombinant Offspring Gray body, red eyes (411 + 61) 472 Gray body, purple eyes (30 + 2) 32 Black body, purple eyes (412 + 60) 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 944 + 61

Eye color / wing shape Parental offspring Total Nonparental Offspring Red eyes, normal wings (411 + 28) 439 Red eyes, vestigial wings (61 + 1) 62 Purple eyes, vestigial wings (412 + 30) 442 Purple eyes, normal wings (60 + 2) 881 124 The map distance between eye color and wing shape is Recombinant frequency = 124 881 + 124 X 100 = 12.3 map units

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

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!

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

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

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.

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.

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.

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.

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

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