University of Kentucky

Phylogenetic Analysis of the lolC Gene, Required for Production of Insecticidal Loline Alkaloids, in Neotyphodium/Epichloë Grass Endophytes

Institution

University of Kentucky

Abstract

The objectives of this project were (i) to isolate by polymerase chain reaction (PCR) a selected region of the lolC gene (a gene required for production of loline alkaloids by fungal grass endophytes of the genus Epichloë/Neotyphodium) from different fungal species/isolates, and (ii) to establish the phylogenetic relationships between lolC gene alleles in these species/isolates. With PCR on fungal genomic DNA and lolC allelespecific primers, DNA fragments were amplified from ten isolates of eight Epichloë/Neotyphodium species. The DNA fragments obtained were purified and sequenced with high-throughput sequencing technology at UK’s Advanced Genetics Technology Center (AGTC). Eleven DNA fragments containing lolC sequence from eight endophyte species were obtained by PCR, and all eleven were nearly completely sequenced (the average length of the DNA fragments was 1.8 kb). All intron sequences (lolC has five introns) were used in phylogenetic analysis. The intron sequences were aligned with each other in the MacClade Program, and the alignment exported into the PAUP 4.0 Program for construction of phylogenetic trees. Maximum parsimony and bootstrap analysis of phylogenetic trees indicated relationships between the lolC alleles in the different species/isolates very similar to relationships previously found for three housekeeping genes, suggesting that lolC was solely vertically transmitted (i.e., by meiotic events or hybridization between different endophyte species) during its evolution in the Epichloë/Neotyphodium endophytes.

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Phylogenetic Analysis of the lolC Gene, Required for Production of Insecticidal Loline Alkaloids, in Neotyphodium/Epichloë Grass Endophytes

The objectives of this project were (i) to isolate by polymerase chain reaction (PCR) a selected region of the lolC gene (a gene required for production of loline alkaloids by fungal grass endophytes of the genus Epichloë/Neotyphodium) from different fungal species/isolates, and (ii) to establish the phylogenetic relationships between lolC gene alleles in these species/isolates. With PCR on fungal genomic DNA and lolC allelespecific primers, DNA fragments were amplified from ten isolates of eight Epichloë/Neotyphodium species. The DNA fragments obtained were purified and sequenced with high-throughput sequencing technology at UK’s Advanced Genetics Technology Center (AGTC). Eleven DNA fragments containing lolC sequence from eight endophyte species were obtained by PCR, and all eleven were nearly completely sequenced (the average length of the DNA fragments was 1.8 kb). All intron sequences (lolC has five introns) were used in phylogenetic analysis. The intron sequences were aligned with each other in the MacClade Program, and the alignment exported into the PAUP 4.0 Program for construction of phylogenetic trees. Maximum parsimony and bootstrap analysis of phylogenetic trees indicated relationships between the lolC alleles in the different species/isolates very similar to relationships previously found for three housekeeping genes, suggesting that lolC was solely vertically transmitted (i.e., by meiotic events or hybridization between different endophyte species) during its evolution in the Epichloë/Neotyphodium endophytes.