Selection of Molecular Markers

Two of the most important factors that need to be considered in the selection of molecular markers in any study are the taxonomic rank under consideration, and the life cycle of the fungus. As detailed above, a marker that is particularly useful at a certain taxonomic rank for one species may not be useful at the same rank for another species. One explanation for this is that different systematists or plant pathologists have had different species concepts, and so the terms species and subspecies may not be directly comparable between different fungal genera. In some genera, such as Fusarium, there has been a tradition of placing different pathogenic forms in the special form category, whereas in others, such as Colleto-trichum or Phoma, there has been a general history of describing new species. Such differences in species concepts may reflect evolutionary ages, or may reflect levels of variation in other characters. Whatever the reason, there can be significant differences in the degree of variation seen in the molecular markers chosen.

Fungi occur in asexually (imperfect, anamorphic) and sexually (perfect, teleomorphic) reproducing forms, and in some cases both forms are present at the same time (holomorphic). In the imperfect state cell division is solely by mitosis, whereas in the perfect state recombination and meiosis will occur. Recombination and meiosis can have a significant effect on results obtained from some molecular markers (see Table 1). Isoenzyme markers could be expected to be subject to allelic variation under such circumstances, as would many DNA fingerprinting markers. The degree to which a marker will be affected will vary considerably, and one example is the comparison of sibling haploid lines derived from a single dikaryotic fruit body. In these circumstances, the haploid progeny have arisen by meiosis and may show different isoenzyme or DNA fingerprints from the parental material. This has been investigated in the oil palm pathogen G. boninense where both RAPD (Pilotti et al. 2000) and simplified AFLP (Figure 2) fingerprints differed both between siblings and between siblings and parent. This variation can then be further compounded through subsequent mating and recombination.

Some molecular markers can be expected to be consistent despite meiosis and recombination. DNA sequences of major structural and functional proteins will be resistant to recombination events, and the rRNA gene cluster is one region generally considered to be maintained under such conditions (Hillis and Dixon 1991). There are however some indications that this is not always the case, and there is at least one report that in some fungi, not only can the rRNA region be affected by crossover, but also that this may occur at a higher frequency than predicted (Selosse et al. 1996).

Nonnuclear markers may be recombination insensitive, and mtDNA has been used to demonstrate a single hereditary line, where the mtDNA was inherited unilinearly (Whittaker et al. 1994). It should be remembered however that this will

Table 2 Features of Ascochyta complex species on legumes

Species

mt SSU rRNA size

b-tubulin gene RFLPa

ITS sequenceb

mtDNA RFLPs

P. exigua

749 bp

D

1

Multiple, distinct

749 bp

A

1

A. rabiei

660 bp

A

2

Multiple, distinct

A. fabae

660 bp

C

3

Multiple, distinct

A. fabae f. sp. lentis

660 bp

C

4

Single, distinct

A. pisi

660 bp

C

5

Single, distinct

P. medicaginis var. pinodella

645 bp

B

6

Multiple, distinct

A. pinodes

645 bp

B

6

Multiple, distinct

P. subboltshauseri

645 bp

E

7

Single, distinct

645 bp

F

7

645 bp

G

7

a Letters A—G designate 7 different RFLP patterns obtained by digestion of a PCR amplified fragment of the b-tubulin gene. b Numbers 1-7 designate 7 different RFLP patterns obtained by digestion of the complete ITS1/5.8 s/ITS2 region.

a Letters A—G designate 7 different RFLP patterns obtained by digestion of a PCR amplified fragment of the b-tubulin gene. b Numbers 1-7 designate 7 different RFLP patterns obtained by digestion of the complete ITS1/5.8 s/ITS2 region.

Figure 2 Simple sequence repetitively primed molecular fingerprints for 4 lines of Ganoderma derived from a single bracket. Lane 1, molcular size markers; lanes 2 and 3 monokaryotic culture derived from basidiospore a; lanes 4 and 5, monokaryotic culture derived from basidiospore b; lanes 6 and 7, monokaryotic culture derived from basidiospore c; lanes 8 and 9, dikaryotic culture obtained from b and c.

not always be the case, as not all fungi have unilinear mitochondrial inheritance, and in some cases mitochondrial recombination will occur during biparental inheritance (Borst and Grivell 1978). The range and type of variation associated with molecular markers can provide many different tools that can be used for determining the epidemiology of plant pathogenic fungi. At one level, recombination insensitive markers may be available for the detection of a particular taxon in the environment, such as species and pathogen specific probes and primers. At a lower level, recombination sensitive markers may be used to follow individuals or lines, or to determine if a disease is spread by spores or through vegetative growth.

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