Species discrimination and hybrid detection in terrestrial orchids using Bar-HRM: A case of the Calanthe group

Abstract

The Calanthe group of terrestrial orchid species comprises three genera as Calanthe R.Br., Phaius Lour. and Cephalantheropsis Guillaumin that have similar morphological characteristics. Orchid species taxonomy is generally investigated based on the features of the flowers. This is a difficult task and requires an expert taxonomist, while it is often impossible to identify species if the specimens have none or incomplete flowers. Nowadays, standardized short DNA, so-called DNA barcoding, is widely used for species identification. Here, the DNA barcode, internal transcribed spacer-1 (ITS1) in the nuclear DNA or trnL in the chloroplast DNA, combined with high resolution melting (HRM) analysis was used to identify 13 orchid species in the Calanthe alliance and also to determine hybrid species. Results showed that the melting temperature (Tm) and melting curve obtained by HRM using mini-ITS1 significantly differed among Calanthe alliance species, with an accuracy of 92% compared to HRM using mini-trnL. HRM using mini-ITS1 provided a good marker for hybrid determination with two peaks from parental species. To determine the hybrids, HRM using mini-trnL was necessary for assigning the maternal parent; however, in some cases the melting shapes obtained from HRM using mini-trnL of parental species were different. Results suggested that HRM using mini-ITS1 gave good species differentiation and hybrid detection for species of the Calanthe alliance, while HRM using mini-trnL was exploited for assessing the maternal parent of hybrid species.