Parasexuality - genetic analysis of nuclear genes

without meiosisBIO341

• Meiotic analysis limited to those organisms that can be crossed sexually.

• Organism of interest may not reproduce sexually.

• Organisms of interest may be too distantly related to cross.

• Your concern may be with somatic cells.

Stages of the parasexual cycle in Aspergillus

• 1. heterokaryosis• 2. nuclear fusion - diploidy (1/106 to

1/107)• 3. propagation of diploid• 4. mitotic crossing over ( 2/103)• 5. haploidization (1/103)

Note: steps 2, 4, and 5 occur at low frequency.Also, steps 4 and 5 are completely independent of one another and occur by completely different mechanisms.

Example in Aspergillus nidulans

w pro x y ad paba

Example in Aspergillusw pro x y ad paba

• Pair strains on complex medium.• Transfer from confluent zone of colonies to minimal medium.• Yellow and white clusters of conidia mixed together are

characteristic of heterkaryons.• Each cluster is either yellow or white (indicating what?).• Harvest conidia, plate on MM to find diploids. Conidia are

uninucleate. Why is this important in this scheme?• Diploids have green conidia.• During growth colored sectors may be produced. The ploidy of

these sectors can be inferred from conidial size.• Segregants in following selected by color (From Pontecorvo and

Kafer).

Wild-type is green

White and yellow sectors

Diploid formation

A B

C

a b

c

A B

C

a b

c

Diploids (post chromatid replication)

A B

C

a bc

Diploid - metaphase plate

A B Ca b c

A B Ca b c

Normal chromosome segregation to form daughter diploids

A B Ca b c

a b cA B C

Abnormal chromosome segregation to form an

aneuploidA B Ca b c

a b

cA B C

Aneuploid - metaphase plate

A B Cc

A B Cc

Another abnormal chromosome segregation

to form a haploid

A B Cc

cA B

C

Haploid, recombinant for A/C and for B/C, but not for A/B

Mitotic crossing over:

A B

a

b

50% 1,3 and 2,4: heterozygosity at A, homozygosity at B

50% 1,4 and 2,3: heterozygosity at both A and B

A B

a b

B

Baa

b

bA1

2

3

4

Mapping chromosomes by mitotic crossing over between a selectable marker and the centromere.

Plate on MM containing histidine, proline, biotin and arginine, but no adenine.

Recover ade-suppressed, assay other markers.

his

ade

ade

+ +

su

++

argprobio

+

Ade+segregants:

50 Prototrophic200 Arg-100 Arg-Bio-150 Arg-Bio-Pro-

What is map for Arg, Bio, Pro, and the centromere?

Parasexual cross: w pro x y ad pb

Below are the phenotypes and ploidy of segregants from this parasexual cross.

Diploid:32 Prototrophic, Yellow78 Paba-, Yellow56 Prototrophic, White

Haploid19 Paba-, Ad-, Yellow25 Pro-, White25 Paba-, Ad-, White