D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp

Abstract

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.

Fig. 1.

Nucleated rhizoids of Orpinomyces intercalaris by phase-contrast microscopy (A) and fluorescence microscopy (B). Magnification, ×400.

Fig. 2.

Different patterns (as indicated by arrows) of sporangial development. (A) Terminal end in O. joyonii. Magnification, ×400. (B) Intercalary position in O. intercalaris. Magnification, ×200.

Fig. 3.

PCR-amplified product (≈780 bp) of the LSU region of O. joyonii (lanes 1 to 5) and O. intercalaris (lanes 6 to 10). M, 100-bp DNA marker.

Fig. 4.

Patterns of variation obtained after multiple sequence alignment of isolates with an extra restriction site (highlighted area) for AluI in O. intercalaris.

Fig. 5.

Neighbor-joining phylogenetic tree of Orpinomyces spp. obtained from the sequences of the LSU. The evolutionary history was inferred using the neighbor-joining method (19). The optimal tree with the sum of branch length of 0.03107997 is shown. The tree is drawn to scale, with branch lengths (below the branches) in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method (21) and are in the units of the number of base substitutions per site. The analysis involved 11 nucleotide sequences. The codon positions included were the 1st, 2nd, 3rd, and noncoding positions. All positions containing gaps and missing data were eliminated. There were a total of 743 positions in the final data set. Evolutionary analyses were conducted in MEGA5 (22).

Fig. 6.

AluI-digested PCR product of the LSU regions of O. joyonii (lanes 1 to 5) and O. intercalaris (lanes 6 to 10). M, 50-bp DNA marker.