Mitotic recombination and parasexual cycle in Aspergillus

Parasexual cycle in Aspergillus, where diploid formation through heterokaryon and haploid formation without meiotic division are shown. Visible markers in the form of coloured conidia can be used to demonstrate the parasexual cycle
Fig. 11.15. Parasexual cycle in Aspergillus, where diploid formation through heterokaryon and haploid formation without meiotic division are shown. Visible markers in the form of coloured conidia can be used to demonstrate the parasexual cycle.

Production of twin spots (yellow and green) due to somatic crossing over in Aspergillus
Fig. 11.16. Production of twin spots (yellow and green) due to somatic crossing over in Aspergillus.

Production of homozygotes cc, bbcc and aabbcc due to somatic crossing over in a heterozygote a+b+c+/abc, (or determining the linear order of genes (for details, see text).
Fig. 11.17. Production of homozygotes cc, bbcc and aabbcc due to somatic crossing over in a heterozygote a+b+c+/abc, (or determining the linear order of genes (for details, see text).
In Aspergillus, like many other fungi, normal vegetative state is haploid. Since mitotic recombination can take place only in presence of homologous chromosomes, a stable diploid condition should first be produced. Diploid strains in Aspergillus are often produced due to fusion of nuclei of two strains. In this process, heterokaryons are first produced, where similar haploid nuclei remain separate. These may occasionally fuse to form diploid nuclei, so that both haploid and diploid nuclei will exist and form haploid and diploid conidia, which can sometimes be identified by colour (white, yellow, green; Fig. 11.15). If two nutritional mutant strains say a (ab+) and b (a+b) are used, each will individually need a nutrient for growth and therefore only diploids (ab+/a+b) can survive on a minimal medium, not supplemented by any nutrient. Diploid strains can thus be established and characterised by larger conidia and double the DNA content.
Parasexual cycle in Aspergillus, where diploid formation through heterokaryon and haploid formation without meiotic division are shown. Visible markers in the form of coloured conidia can be used to demonstrate the parasexual cycle
Fig. 11.15. Parasexual cycle in Aspergillus, where diploid formation through heterokaryon and haploid formation without meiotic division are shown. Visible markers in the form of coloured conidia can be used to demonstrate the parasexual cycle.

The diploids may be established in repulsion phase (ab+/a+b) as described above or in coupling phase (ab/a+b+). These diploids may remain as diploid, but sometimes may revert back to haploid condition (haploidization). In the later case, if a and b are present on two different non-homologous chromosomes, all the four combinations (a+b+, a+b, ab+, ab) will be obtained, whether diploid is in coupling phase (a+b+/ab) or repulsion phase (a+b/ab+). But if 'a' and 'b' are located on same chromosome, haploids produced by diploids in coupling phase (a+b+/ab) will be mainly a+b+ and ab, while those produced by diploids in repulsion phase (a+b/ab+) will be mainly a+b and ab+. By such tests, one can find out whether or not a and b are linked, because if they are linked, assortment will be non-random (giving two predominant types), otherwise assortment will be random (giving all four types .in equal proportions).

The process of haploidization, may sometimes be accompanied by mitotic crossing over between two linked genes. This may lead to production of recombinant haploids. For instance, if a and b are linked, diploid (ab+/a+b) may also give rise to a+b+ and ab in addition to ab+ and a+b. This process of production of recombinant haploids from diploids has been termed a parasexual cycle, by famous British scientist Pontecorvo. The term parasexual has been used since it does not involve sexuality, but produces genetic variability in the same manner as sexual reproduction does.

Since mitotic recombination only rarely occurs during haploidization, only diploid recombinant products are analysed for chromosome mapping. As earlier discussed, somatic crossing over leads to twin spots. In Aspergillus if y stands for yellow and is recessive to y+ (green), then a green diploid (y/y+)may give twin spots (yellow and green) due to somatic crossing over (Fig. 11.16).
Production of twin spots (yellow and green) due to somatic crossing over in Aspergillus
Fig. 11.16. Production of twin spots (yellow and green) due to somatic crossing over in Aspergillus.

Utilizing several markers, diploids heterozygous for different linked markers (e.g. a, b, c)may be obtained. A heterozygote a+b+c+/abc may give rise to homozygotes, aa, bb, cc, aabb, aacc, bbcc and aabbcc in different frequencies. The frequencies will depend on gene order and distances of these markers from centromere. For instance, if the gene order 4s centromere-a-b-c, then c being farthest away will have maximum likelihood of being obtained in recombinant state (c/c+c/c + c+/c+). If recombinant homozygotes for c (cc) are examined for simultaneous homozygosity for a and b also, three classes can be obtained (Fig. 11.17) (i) cc homozygotes which are neithetr homozygotes for a, nor for b; (ii) cc homozygotes which are homozygotes for b as well, but not for a i.e. bbcc and (iii) cc homozygotes which are homozygotes for b and a also i.e. aabbcc. This shows that a recombinant for a will be homozygote for b and c also and similarly a recombinant for b will be recombinant for c as well, except in case of rare double crossovers. The reverse is not true i.e. a recombinant for c (cc) may not necessarily be a recombinant for a or b. This kind of relationship among recombinant homozygotes establishes the gene order a-b-c.
Production of homozygotes cc, bbcc and aabbcc due to somatic crossing over in a heterozygote a+b+c+/abc, (or determining the linear order of genes (for details, see text).
Fig. 11.17. Production of homozygotes cc, bbcc and aabbcc due to somatic crossing over in a heterozygote a+b+c+/abc, (or determining the linear order of genes (for details, see text).

Frequencies of the above three classes can be used for estimation of map distances. In Aspergillus nidulans three genes, pro (proline requiring mutation), paba (p-aminobenzoate requiring mutation) and y (yellow mutation) were used for preparation of linkage map using mitotic crossing over. Heterozygote pro+paba+y+/pro paba y gave following results :
(1) yly (crossing over in region paba-y) = 96/371 = 26%
(2) pabd-ylpaba-y (crossing over in pro-paba region) = 245/371 = 66%
(3) pro paba y/pro paba y (crossing over incentromere-pro region) = 30/371 = 8%

It can be seen that recombination frequencies based on mitotic crossing over were 26%, 66% and 8%. These values are different from corresponding meiotic recombination values which were respectively 36%, 18% and 4.6%. This discrepancy is attributed to the fact that mitotic crossing over is a rare event (1 in 100 divisions) and unpredictable, while meiotic crossing over takes place regularly and is predictable.

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