Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent.
Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra-organism genetic variation. However, information about intra- vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra-isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12-40 clones per isolate. Intra-isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut-off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next-generation sequencing; and its ease of amplification in single-step PCR.
Department of Botany, University of Tartu, 40 Lai St., 51005, Tartu, Estonia.
Odile
O
Thiéry
Department of Botany, University of Tartu, 40 Lai St., 51005, Tartu, Estonia.
Martti
M
Vasar
Department of Botany, University of Tartu, 40 Lai St., 51005, Tartu, Estonia.
Teele
T
Jairus
Department of Botany, University of Tartu, 40 Lai St., 51005, Tartu, Estonia.
John
J
Davison
Laboratoire de Recherche en Sciences Végétales, UPS, CNRS 24 chemin de Borde Rouge-Auzeville, BP 42617, Université de Toulouse, 31326, Castanet-Tolosan, France.
Christophe
C
Roux
Estonian Genome Center, University of Tartu, 23b Riia St., 51010, Tartu, Estonia.
Paula-Ann
PA
Kivistik
Estonian Genome Center, University of Tartu, 23b Riia St., 51010, Tartu, Estonia.
Andres
A
Metspalu
Estonian Genome Center, University of Tartu, 23b Riia St., 51010, Tartu, Estonia.
Lili
L
Milani
Department of Botany, University of Tartu, 40 Lai St., 51005, Tartu, Estonia.
Ülle
Ü
Saks
Department of Botany, University of Tartu, 40 Lai St., 51005, Tartu, Estonia.
Mari
M
Moora
Department of Botany, University of Tartu, 40 Lai St., 51005, Tartu, Estonia.
Martin
M
Zobel
Department of Botany, University of Tartu, 40 Lai St., 51005, Tartu, Estonia.
Maarja
0000-0001-8025-7460
M
Öpik
D004271:DNA, Fungal / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D014644:Genetic Variation; D055137:Glomeromycota / Q000145:classification / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D038821:Mycorrhizae / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D054658:Ribosome Subunits, Large / Q000235:genetics*; D054679:Ribosome Subunits, Small / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer
arbuscular mycorrhizal fungi; qiime; rDNA variation; sequence-based identification