MEIOSIS AND ANALYSIS OFCROSSING OVER

Objective: to study crossing over in the fungus Sordaria fimicola

To achieve the objective:a) understand the meiosisb) study the life cycle of S. fimicolac) set up a cross between the wild-type and the

mutant tan of S. fimicola

What is meiosis?Meiosis, or reduction division, is a nuclear division process thatreduces the chromosome number of a diploid (2n) cell by half, i.e.to the haploid (n) number. Meiosis takes place in male and femalereproductive organs.

Stages of meiosisMeiosis is composed of two successive cell divisions:

a) meiosis I (reduction division)b) meiosis II (equational division)

What is crossing over?Crossing over is a term to describe the process of reciprocalchromosomal interchange by which recombinants arise.

When does this occur?It starts at pachytene and continues into diplotene of meiosis 1.

How?

Meiosis IProphase I

Leptotene……………

Zygotene………………

Pachytene…………….

Diplotene….............

Metaphase IAnaphase I Telophase I

Meiosis II Diakinesis……………Prophase IIMetaphase IIAnaphase IITelophase IIDaughter cells

Tetrad

Bivalent

Crossing over in human chromosomes

Linkage vs Crossing Over

Alleles of genes that liein the same chromosomeremain together duringmeiosis. This is calledlinkage.

A

B

A

B

a

b

a

b

A

B

A

B

aProphase I

A

B

A

B

a

b

a

b

A

b

A

B

a

B

a

b

Anaphase I

A

B

A

B

a

b

a

b

A

B

A

b

a

B

a

bTelophase II

Parental type Recombinants=50%

The 50% can be used as a quantitative measure of the genetic distance between the two gene pairs.

b

a

b

Analysis of crossing over in Sordaria fimicola

The life cycleSordaria is an ascomycete fungus, closely related to Neurospora(the bread mold).

Sordaria is haploid with the chromosome number n=7.

Sordaria does not have distinct sexes, but has mating types.

During asexual (non-sexual) phase, haploid spores germinate andform haploid hyphae that grow and branch into expanding patchwork called mycelia.

The life cycle

During sexual reproduction, haploid hyphae of different mating types come into a physical contact resulting in fertilization.

Fertilization Zygote (2n)

Meiosis I

Meiosis II

Mitosis

Ascus

Ascospores (n)

Hyphae

Physical contact

Mating type I Mating type II

The life cycle

Formation of a mature ascus in detail

n 2nn

n

n1st

meioticdivision

2nd

meiotic division

Mitosis

or

A

Meiosis I

Meiosis II Mitosis

B 1-3 B 1-4

or

A

Meiosis I

Meiosis II Mitosis

B 2-3 B 2-4

How to calculate map distance

Disregard parental and aberrant types

Consider followings:No. of non-crossovers (NCO)….ascospore arrangement 4+4 No. of crossovers (CO)………...ascospore arrangement 2+4+2

2+2+2+2

% crossing over = {CO / (CO+NCO)} x 100Map distance = % crossing over / 2

Procedure

1. Identify the strains:-the wild-type strain has black perithecia and black ascospores-the tan mutant strain has tan (light brown) perithecia and tanascospores

2. Prepare the cross as shown:you must work close to a lit burnerto prevent plate contamination.

3. Let grow for two weeks (seestep 5).

4. From the original plate, prepare squashes from wild-type and mutant strains to view the asci and the ascospores.

Note: This is a difficult technique; practice it several timesusing the perithecia from the original culture; study the asci andthe ascospors. They should look like this

5. At the time specified by the laboratory instructor, prepare squashes from perithecia, identify crossovers and non-crossovers, and then calculate the map distance between the mutant gene of interest and the centromere of the chromosome.

Cover slip

Slide dH2O

Perithecia

In today’s experiment, your results should look like theimage shown below.