Identification of Hippophae species (Shaji) through DNA barcodes

Abstract

Background: The morphological identification of different Hippophae species (Shaji) was difficult. This study aims to discriminate between medicinal and non-medicinal Hippophae species by DNA barcodes, the ITS2, psbA-trnH, and a combination of ITS2 and psbA-trnH (ITS2 + psbA-trnH).

Methods: DNA was extracted from the dried fruit samples. Primer pairs ITS2F/3R for ITS2 and psbAF/trnHR for psbA-trnH were used for PCR amplification. The purified PCR products were bidirectionally sequenced. Genetic distances were calculated according to the Kimura 2 parameter model and phylogenetic tree was constructed based on neighbor-joining (NJ) method, barcoding gap was also analyzed to assess identification efficiency.

Results: Amplification and sequencing efficiencies for both ITS2 and psbA-trnH were 100 %. Sequence data revealed that ITS2 + psbA-trnH was the most suitable candidate barcode at the species and subspecies level. The closely related Hippophae species were effectively differentiated in the NJ tree.

Conclusion: The combination of the two loci, ITS2 + psbA-trnH is applicable to the identification of medicinal and non-medicinal Hippophae species.

Fig. 1

Variable sites and deletions for Hippophae species based on ITS2 sequences. The specific variable sites and deletions are highlighted

Fig. 2

Variable sites and insertions for Hippophae species based on psbA-trnH sequences. The specific variable sites and deletions are highlighted

Fig. 3

Barcoding gap between Hippophae species based on intra- and inter-specific distances. Minimum inter-specific K2P distance vs. maximum intra-specific K2P distance for ITS2, psbA-trnH, and ITS2 + psbA-trnH. Each data point represents a species, and each species located above the 1:1 line has a barcoding gap

Fig. 4

NJ tree of Hippophae constructed using ITS2. An E. angustifolia sequence downloaded from GenBank was included as an outgroup. The bootstrap scores (1000 replicates) are shown (≥50 %) for each branch. Each color represents one species

Fig. 5

NJ tree of Hippophae constructed using psbA-trnH. An E. pungens sequence downloaded from GenBank was included as an outgroup. The bootstrap scores (1000 replicates) are shown (≥50 %) for each branch. Each color represents one species

Fig. 6

NJ tree of Hippophae constructed using ITS2 + psbA-trnH. The bootstrap scores (1000 replicates) are shown (≥50 %) for each branch. Each color represents one species