MEIOSIS AND ANALYSIS OF CROSSING OVER

Objective: to study crossing over in the fungus Sordaria fimicola

To achieve the objective: a) understand the meiosis b) study the life cycle of S. fimicola c) 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 that reduces the chromosome number of a diploid (2n) cell by half, i.e. to the haploid (n) number. Meiosis takes place in male and female reproductive organs.

Stages of meiosis Meiosis 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 reciprocal chromosomal interchange by which recombinants arise.

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

How? Meiosis I Prophase I Leptotene……………

Zygotene……………… Tetrad

Pachytene……………. Bivalent

Diplotene…......

Metaphase I Anaphase I Telophase I Meiosis II Diakinesis…………… Prophase II Metaphase II Anaphase II Telophase II Daughter cells Crossing over in human chromosomes Linkage vs Crossing Over

Alleles of genes that lie A a A a in the same chromosome A A a a Prophase I B B b b remain together during B b B b meiosis. This is called linkage.

A A a a Anaphase I A A a a

B B b b B b B b

A A a a A A a a Telophase II B B b b B b B b

Parental type Recombinants=50%

The 50% can be used as a quantitative measure of the genetic distance between the two gene pairs. Analysis of crossing over in Sordaria fimicola

The life cycle Sordaria 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 and form 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)

Physical Meiosis I contact

Meiosis II

Hyphae Ascus

Ascospores (n) Mitosis

Mating type I Mating type II The life cycle Formation of a mature ascus in detail

1st 2nd meiotic meiotic Mitosis n division division

n n n 2n Meiosis II Mitosis Meiosis I

or

A

B 1-3 B 1-4 Meiosis II Mitosis Meiosis I

or

A

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 100 Map 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 tan ascospores

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

3. Let grow for two weeks (see step 5). 4. From the original plate, prepare squashes from wild-type and mutant strains to view the asci and the ascospores.

Cover slip Perithecia

Slide dH2O

Note: This is a difficult technique; practice it several times using the perithecia from the original culture; study the asci and the 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. In today’s experiment, your results should look like the image shown below.