評估matK、rbcL及trnL-L-F分子條碼對台灣蕨類鑑種之效力

Abstract

Identification of fern species, and particularly fern gametophyte is a difficult task because there are not many morphological characters available. DNA barcoding helps to identify morphologically related gametophytes and unidentified specimens with molecular characters. However, there is only a few works on developing universal DNA-barcode in ferns. In this study, I evaluated discriminatory powers of three chloroplast barcodes, matK, rbcL and trnL-L-F, by proposed criteria such as primer universality (PCR success rate), sequence quality (mononucleotide repeats disrupting individual sequencing reads) and discrimination success. The discrimination success of species is accessed by two different methods for comparison. A distance-based analysis checks if the minimum uncorrected interspecific P-distance involving a species was larger than its maximum intraspecific distance (indication of a barcoding gap); and a Bayesian phylogenetic analysis examines whether individuals of the same species can be clustered together (checking for porportions of well-supported monophyly of species). 28 fmailies 77 genera 217 species of the Taiwanese ferns were selected to assess DNA discriminatory powers of matK、rbcL and trnL-L-F. Primer universality assessment resulted in 99.33% PCR success rate for trnL-L-F, the highest among three; mean while, 88.55% for rbcL and 67.34% for matK. To improve the success rate of matK, two-stage PCR (universal primer pairs then lineage-specific primer pairs) were performed, the new matK success rate increased to 85.86%. Sequence quality checking resulted in 100% rbcL sequences no disrupting reads caused by mononucleotide repeat (tandem repeat), whereas 98.01% in matK and 86.69 % in trnL-L-F. Barcoding gaps can be found in 95.61% trnL-L-F pairwise distance, 95.42% by rbcL and 87.70% by matK. On the other hand, the discrimination success inferred by Basyien monophyly indicated 90% of within-species trnL-L-F sequences were successfully resolved when forming a monophyletic group with sufficient bootstrap support, in rbcL this was 85% and in matK this was 82%. To sum up, trnL-L-F has the highest discriminatory power to tell different fern species apart. Because the discrimination success of trnL-L-F in our well-sampled Taiwanese ferns is above 90%, higher enough to be the most universal DNA barcode for ferns.