SCAR markers to detect mycorrhizas of an American Laccaria bicolor strain inoculated in European Douglas-fir plantations

Abstract

The American strain S238N of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton has been used to inoculate Douglas-fir [Pseudotsuga menziesii (Mir.) Franco] plantations in France over the last two decades. Laccaria fruit bodies are scarce in mature plantations, which precludes further assessment of its persistence by fruit body surveys. Our objective was to develop new markers to identify this strain and its eventual non-fruiting progeny on root tips. We converted nine random amplified polymorphic DNA markers into sequence characterized amplified region (SCAR) markers. Two of these SCAR markers enabled us to detect S238N on roots of seedlings and mature trees. No amplification of non-fungal (host plant, bacterial, etc.) DNA was observed. Moreover, both SCARs were amplified from Laccaria-like mycorrhizas in a Douglas-fir plantation inoculated 14 years ago, demonstrating the long-term persistence of the inoculant strain. We also obtained a SCAR marker to detect one strain of European origin (L. bicolor 81306), indicating that SCARs are potential markers to type the naturally occurring genets. Thus, SCAR markers are of great value in studying the persistence of inoculant strains and the effects on local populations of introducing foreign strains.

Molecular markers detecting an ectomycorrhizal Suillus collinitus strain on Pinus halepensis roots suggest successful inoculation and persistence in Mediterranean nursery and plantation

“…Indeed, a study published when this paper was under review implicitly suggests that ISSR pattern is amplifiable from ECMs without interference of host DNA for Suillus pictus (Hirose et al , 2004). Compared to SCARs, ISSR fingerprinting (1) produces more markers in a single step, (2) requires limited preliminary work before typing, and (3) avoids loss of specificity during SCAR choice, due to the design of internal primers (Weber et al , 2002). Direct ISSR thus has interesting features that counterbalance risks of amplifying plant endophytes or contaminants.…”

“…However, selfing could preserve an ISc‐like pattern, with a probability ranging from 1 (assuming a very unlikely situation where all markers are homozygous in Sc‐32) to 0.5 6 =3.1 × 10 −2 (assuming that each ISSR marker is heterozygous). Selfing can arise by mating after basidiospore germination in soil (Weber et al , 2002) or by direct formation of dikaryotic basidiospores on Sc‐32 basidia (as in some suilloids Jacobson & Miller, 1994; Bonello et al , 1998). This seems highly unlikely, as no S. collinitus fruitbodies were seen in our regular investigations, either in the nursery or plantation.…”

“…For instance, specific primers designed from sequences of the internal transcribed spacer (ITS) of nuclear ribosomal RNA genes or from the random amplified polymorphic DNA (RAPD), led to sequence‐characterized amplified region (SCAR) markers, which successfully identified Tuber species at the vegetative and reproductive phases (Amicucci et al , 1998; Bertini et al , 1998; Mello et al , 1999). Persistence of inoculated strains was recently investigated on ECM for two species: in Paxillus involutus , RFLP patterns of an intergenic spacer of the ribosomal DNA (IGS1) allowed detection of a subgroup of strains, including the inoculant one, among nursery‐collected ECMs (Hönig et al , 2000); for Laccaria bicolor , SCAR markers generated from RAPD (Selosse et al , 1998a, b) allowed the typing of an inoculant strain on ECMs both in the nursery and plantation (Weber et al , 2002). Besides these studies, few genetic data are available on in situ survival of introduced ECM strains.…”

Development and Study of SCAR Markers in Pea (Pisum sativum L.)

“…Upon use of SCAR primers in PCR, only one fragment corresponding to a single specific locus is amplified more often [4,8]. Indeed, for each of the 13 RAPD markers, one SCAR or STS marker was found.…”

“…Usually, this occurs in the cases, when the original RAPD polymorphism had been caused by a point mutation at the site of primer annealing. This fact may be explained on an assumption that a single nucleotide mismatch at the priming site hampers the association of a short RAPD primer with this site but was tolerated by the longer SCAR primer tightly bound to this region [3,7,8]. Moreover, in the case of using SCAR primers, the mismatch position that underlies original RAPD polymorphism is shifted to the middle of SCAR primer and generally does not change the effectivity and specificity of amplification [7].…”