Plant-specific primers for the amplification of the nrDNA ITS region in fungus-associated Pulsatilla species

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA is the most frequently used marker for distinguishing plant species. Previously published polymerase chain reaction (PCR) primers available for amplifying the ITS region from environmental plant samples provide a high degrees of success while maintaining a broad range of compatibility difficult to discriminate against fungal DNA. Thus, the plant-specific ITS primer design is required to achieve the preferential amplification of plant DNA with discrimination against fungal DNA from environmental plant samples. In this study, two primers, ITS1-F2 and ITS4-R, were newly designed and the specificity of these primers were tested against 14 accessions of Pulsatilla tongkangensis and 2 accessions of Pulsatilla cernua. Our results showed that ITS4-R, when paired with either a universal primer ITS5 or the newly designed primer ITS1-F2, efficiently amplified DNA from environmental P. cernua samples and discriminated against parasitic fungal DNAs, while another newly designed primer ITS1-F2, when paired with either a universal primer ITS4 or ITS4-R, could not preferentially amplify plant DNA or discriminate against fungal DNA. For P. tongkangensis, the dismatch of ITS5 primer resulted in noneffective or wrong amplification when paired with either ITS4-R or ITS4, while ITS1-F2, when paired with ITS4-R and ITS4, efficiently amplified plant DNA and both plant and fungal DNAs, respectively. This result suggested that the combination with ITS1-F2 and ITS4-R were intended to be specific to Pulsatilla species. This study will be particularly useful for detection and analysis of plant sequences from environmental or fungus-associated materials.   Key words: Pulsatilla, nrDNA ITS, plant-specific primer design, fungus-associated plant discrimination, molecular identification.