AFLP analysis of progeny The AFLP fingerprints of single-spore cultures established from spores isolated from pairings of parental isolates showed evidence of biparental inheritance and exchange of genetic material. AFLP gives a genome-wide scan of polymorphic loci among individuals, thus providing a large number of reproducible loci for genotyping. All nine tested cultures showed evidence of genetic exchange using AFLP fingerprinting. None of the single-spore progeny showed exactly one of the parental genotypes. This suggests that hyphal fusions and eventual exchange of genetic material occur relatively frequently in in vitro cultures.
A fundamental concern with the application of arbitrary genetic markers, such as AFLP, to genotyping single-spore progeny is that processes other than genetic exchange may influence the genotype of the progeny. In particular, in the case of a heterokaryotic mycelium where genetically different nuclei coexist (Kuhn et al., 2001; Hijri & Sanders, 2005), spores inheriting nuclei through a random process (i.e. genetic drift) could be genetically different, even though they originate from the same mycelium (Sanders, 2002). However, the heterokaryosis hypothesis has been disputed in AMF (Pawlowska & Taylor, 2004) and no data on genetic differences among nuclei are available for G. intraradices. In our control experiment, we were able to distinguish between potential segregation of nuclei within isolates and genetic exchange. Relative to the genetic difference among the different parental isolates, only a small amount of genetic variation was found among progeny spores from one isolate. Our results corroborate the occurrence of genetic exchange among parental isolates because the AFLP data cannot be explained solely by segregation of genetically different nuclei that coexist within an isolate. Furthermore, this control experiment also shows that potential artefacts from AFLP genotyping, such as random shifts in frequency of markers or detection thresholds, are negligible.
Genetic entities in AMF comprise nuclei in a continuous cytoplasm, but also mitochondria and potentially endosymbiotic bacteria. Even though endosymbiotic bacteria were not observed in in vitro cultures of G. intraradices (M. Hijri & I. R. Sanders, unpublished), AFLP does not provide information about the location in the genome where particular loci occur. Thus evidence for genetic exchange based on AFLP may be partly the result of exchange of mitochondria and/or endosymbiotic bacteria. However, the nuclear DNA content is approx. 15 Mb (Hijri & Sanders, 2004). The G. intraradices genome must therefore be significantly larger than the mitochondrial genome. Thus, the large proportion of parental specific loci that were inherited by offspring spores suggests that the genetic exchange is likely to be mostly based on nuclear AFLP markers.
Not all specific AFLP loci of each of the two parents were found in the progeny, suggesting either shifts in frequency or complete disappearance of these particular genetic markers (see Tables S2–S4 for full AFLP data). If a given locus in a DNA sample becomes rare, AFLP fingerprints may indicate that locus as missing, although it may be present but below the detectable limit of AFLP. Similarly, AFLP fingerprints may indicate an appearance of a putatively new locus in the case where a locus at low frequency simply becomes more frequent. These two scenarios might account for disappearance or appearance of loci in fingerprints of progeny. In this way, the AFLP data could reflect changes in frequencies in loci rather than novel loci, or complete disappearance (see columns ‘Loci common to parental isolates missing in progeny’ and ‘Loci found in progeny not observed in parental isolates’ in Tables S2 and S3).
Several single-spore progeny showing evidence of biparental inheritance were very similar based on their AFLP genotype (S1, S3, S5 and Sa–Sd), as they share a large proportion of loci (Table S4). It is notable that S1, S3 and S5, as well as Sa–Sd were isolated from independent replicate plates of pairings of parental isolates. Single-spore progeny showing evidence of genetic exchange and having very similar AFLP genotypes suggest that nonrandom processes could play a role during genetic exchange between two parental isolates. These processes could include compatibility mechanisms selecting for specific combinations of parental genomes. In other fungi, genomic conflict was suggested to occur after fusion of genetically different individuals (Roca et al., 2003, 2004). Whether mixing of parental genomes in progeny provides an opportunity for conflict in AMF remains to be investigated.
In order to overcome the limiting amounts of DNA from single spores and to ensure high reproducibility, cultures originating from germinated spores were clonally subcultured. Even though we were able to control for potential segregation among single-spore lines during the subculturing, the ideal experimental design would be based on genotyping hyphae and spores directly produced by a fused mycelium of different isolates.
Detecting genetic exchange through sequence-based markers The sequence-based markers used in this study were previously developed to show genetic differences in a much larger sample of the same G. intraradices population (Croll et al., 2008b). The large number of SNPs and indels makes these sequence-based markers suitable for a reliable identification of isolates (Croll et al., 2008a). The lengths of short repeat motifs or indels at each locus were identified by capillary electrophoresis (see Supporting Information in Croll et al., 2008b) and the electropherograms exhibit typical stutter peaks of repeat locus amplifications (Griffiths, 1996; Fig. 2). The sequencing of the alleles in the progeny further confirmed the results of our analysis. The results obtained with the sequence-based markers showed biparental inheritance in one single-spore line (S2) from the pairing of isolates C2 and C3 but not in others. This shows that, in at least one single-spore line, nuclei of genetically different parents mixed and formed viable offspring. This is not contradictory to the results from AFLP showing biparental inheritance in all nine lines, as it could mean either that not all nuclei from both parental isolates were inherited simultaneously or that the alleles from one parental isolate were present at very low frequency, below detection by amplification. Relative heights of inherited alleles in offspring S2 suggest that parental nuclei were not inherited in equal proportions. Alleles specific to parental isolate C3 appear to be more abundant than alleles specific to parental isolate C2. This suggests that parental nuclei potentially mix in different proportions to form single-spore progeny.
There was a general congruence between AFLP fingerprints and sequence-based marker genotypes across all single-spore progeny, to the extent that all progeny of the parental isolates C3 and D1 showed very similar AFLP fingerprints and, in all cases, the specific sequences of parental isolate C3. Among single-spore progeny of C2 and C3, S4 showed an AFLP fingerprint closest to parental isolate C2 and inherited the specific allele from the same parent. Furthermore, single-spore progeny S1, S3 and S5 all showed AFLP fingerprints most similar to parental isolate C3, in accordance with their sequence-based marker genotype. The general correlation between AFLP fingerprints and sequence-based marker genotypes was also shown to occur within a population of G. intraradices isolates (Croll et al., 2008b).
Using ribosomal gene copy number, single-spore progeny S2 showed an intermediate copy number, suggesting that a mixing of genomes of the parental isolates can produce intermediate copy numbers for ribosomal genes. The intermediate copy number shown by progeny S2 supports the genetic analyses based on the other markers in our study.