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.
RghBNG, a gene of unknown function, was cloned from Rehmannia glutinosa by reverse transcription PCR and rapid amplification of cDNA ends. The full-length cDNA of RghBNG was 548 bp with a282-bp open reading frame. It encoded a polypeptide of 93 amino acids with a predicted molecular weight of 10.5 kDa and a theoretical isoelectric point of 9.25. Bioinformatics analysis indicated that RghBNG had no homology to any known plant genes, whereas the RghBNG polypeptide was highly similar to other plant proteins and possessed one conserved B12D protein family functional domain. Phylogenetic analysis revealed that RghBNG encoded for a dicot protein. RghBNG spatial and temporal expression patterns and responses to abiotic stresses and plant growth regulators were investigated by qRT-PCR. RghBNG transcripts were detected in roots, stems, leaves, petals, receptacles, stamens and pistils with the highest and lowest levels respectively observed in petals and leaves of mature plants. Additionally, RghBNG transcripts were detected at three developmental stages of roots, stems and leaves; the highest levels were observed in roots at seedling stage; Transcript levels changed to varying degrees in different tissues and stages; We also studied the effects of abiotic stress and plant growth regulators in roots and leaves. RghBNG expression was significantly increased (p < 0.01) by chromium, gibberellic acid and NaCl, with the highest levels induced by chromium stress; In contrast, 6-benzyladenine reduced expression. These results strongly suggest that RghBNG is involved in R. glutinosa growth, development and response to plant growth regulators and abiotic stresses.
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