Development of a DNA test for four Paphiopedilum species - A preliminary study

One of the difficulties in plant identification is the lack of floral part of the questioned specimen. Although DNA analysis is an alternative species identification method, in many cases the presence of polymorphisms within the selected DNA marker region may not be sufficient. Here, we report the development of a PCR-based DNA test to distinguish four Paphiopedilum species, i.e., P. bellatulum, P. concolor, P. godefroyae, and P. niveum, using sequence and insertion-deletions (indels) polymorphisms within the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) gene. PCR primers were designed by using anchored primer strategy, in which 4 PCR primers were designed to assemble 3 primer pairs that gave 4 different sizes of the PCR product. PCR conditions were optimized, and primer specificity was tested. Experiments suggested that the test should be performed by firstly distinguish P. bellatulum and P. concolor from P. godefroyae and P. niveum using the first primer pair. Then, each species of the 2 groups would then be subsequently distinguished by using the other 2 primer pairs.