Abstract
In this study, ITS, ITS2, matK, rbcL and psbA-trnH in Rehmannia were successfully amplified and sequenced, but some ITS sequences need to be proofread according to ITS2 sequences. Compared with rbcL, matK and psbA-trnH, ITS and ITS2 had higher mutation rate and more information sites, and ITS2 had higher interspecific diversity and lower intraspecific variation in Rehmannia, but the interspecific genetic variation of rbcL and matK was lower. Furthermore, the obvious barcoding gap was found in psbA-trnH or ITS2 + psbA-trnH, and the overlap between interspecific and intraspecific variation of ITS, ITS2 or matK was less. In addition, the phylogenetic tree based on ITS or ITS2 indicated that R. glutinosa, R. chingii or R. henryi with obvious monophyly could be successfully identified, but R. piasezkii and R. elata were clustered into one branch, R. solanifolia could not be distinguished from R. glutinosa, and R. chingii was closer to R. henryi. In phylogenetic tree based on psbA-trnH or ITS2 + psbA-trnH, cultivars and wild varieties of R. glutinosa could be distinguished, were clearly separated from other Rehmannia species, and cultivars or wild varieties of R. glutinosa could be also distinguished by matK. Taken together, ITS2 has great potential in systematic study and species identification of Rehmannia, the combination of ITS2 and psbA-trnH might be the most suitable DNA barcode for Rehmannia species.
Highlights
Rehmannia is composed of six species such as R. solanifolia, R. chingii, R. henryi, R. piasezkii, R. elata and R. glutinosa, except R. glutinosa distributes in East Asia and Japan, other Rehmannia species are only distributed in China[1]
Some markers in chloroplast genome or plastid DNA regions have been explored as DNA barcodes, such as matK, trnH-psbA, rbcL, atpF-atphH, rpoB, psbK-psbIr and rpoC16, and some nuclear ribosome DNA sequences including internal transcribed spacer (ITS), internal transcribed spacer[1] (ITS1), internal transcribed spacer 2 (ITS2) and so on have been evaluated[3], but each of these sequences does not conform to the principle of DNA barcoding because of some drawbacks, for example, low rate of variation and amplification, poor universality of primer, gene deletion and so on
PCR amplification conditions of target sequences were optimized and established, the procedure of PCR amplification was 30 cycles followed by final extension for 10 min at 72 °C, each cycle was composed of pre-degeneration for 3 min at 94 °C, degeneration for 30 s at 94 °C, annealing for 30 s at suitable temperature, extension for 1 min at 72 °C, the annealing temperature for amplification of ITS, ITS2, rbcL, matK and psbA-trnH was 46 °C, 48 °C, 50 °C, 48.6 °C or 55 °C, respectively, and amplification band of target sequences in Rehmannia was single, bright and specific
Summary
Rehmannia is composed of six species such as R. solanifolia, R. chingii, R. henryi, R. piasezkii, R. elata and R. glutinosa, except R. glutinosa distributes in East Asia and Japan, other Rehmannia species are only distributed in China[1]. R. glutinosa, R. solanifolia, R. piasezkii, R. henryi and R. elata were clustered together in subcluster I, subcluster II was composed of R. piasezkii and R. chingii, but R. chingii was found in Cluster II (Fig. 4), suggesting Rehmannia species could not be distinguished with rbcL, similar result was found in phylogenetic tree based on matK (Fig. S6).
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