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. 2015 Oct 13;10:28. doi: 10.1186/s13020-015-0062-9

Identification of Hippophae species (Shaji) through DNA barcodes

Yue Liu 1, Wei Sun 2, Chuan Liu 1, Yaqin Zhang 3, Yilong Chen 1, Ming Song 3, Gang Fan 1, Xia Liu 3, Li Xiang 2,✉, Yi Zhang 1,✉
PMCID: PMC4604095  PMID: 26468319

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.

Background

In Hippophae (Fam. Elaeagnaceae) (Shaji), seven species and 11 subspecies have been identified worldwide [1, 2]. In China, there are seven species and seven subspecies of Hippophae, which are mainly distributed from the Hengduan Mountains to the Qinghai-Tibet Plateau [3–6].

Both the fruits and leaves of Hippophae species possess abundant nutritional properties and bioactive compounds [7–9], i.e., high level of vitamin C [10, 11]. Hippophae species have been widely used in food, pharmaceutical, and health care products [12, 13].

Medicinal Hippophae species are used in Chinese medicine (CM) and Tibetan medicine for their antioxidant and anti-tumor activities, to improve lipid metabolism and enhance immunity [14, 15]. The dried fruits are used as remedies for cardiovascular disease; liver, stomach, and spleen disorders; as well as lung and throat phlegm [14–18]. Hippophae species are sometimes misidentified because of the similarities in vegetative morphology [2, 5]. Furthermore, the fruits of different species are labeled with the same name and mainly sold or used in the dried form or as powders. Therefore, different species cannot be identified by only morphological characteristics and accurate identification methods are needed.

With the advantages of high PCR amplification efficiencies, DNA sequencing success rates, and discrimination power, DNA barcoding has become popular with taxonomists and has gained wide acceptance as a standard and effective method in biodiversity research and conservation genetics. It can be applied without the limitation of the samples development stages, parts and gathering time, compared with the conventional identification method [19, 20]. The Consortium for the Barcode of Life (CBOL) Plant Working Group initially recommended the coding plastid regions rbcL and matK as core barcodes for plant species [21]. However, two barcodes are not precise enough because of the low identification rate [22, 23]. The psbA-trnH, ITS, and ITS2 were subsequently suggested [23–25]. Additionally, the amplification efficiency of ITS is lower than that of ITS2, because of the multiple functional copies exist in many taxa [26]. Consequently, more than 6600 plant samples that belong to 4800 species from 753 distinct genera have been barcoded by ITS2, with 92.7 % success at the species level [23, 26–34]. The psbA-trnH intergenic spacer region from plastid DNA has also been recommended as a complementary barcode to ITS2 for a broad series of plant taxa [35].

This study aims to discriminate between medicinal and non-medicinal Hippophae species by DNA barcodes, using the ITS2 and psbA-trnH regions as candidate barcodes.

Methods

Materials

Seventy-five samples (Table 1) representing seven species and seven subspecies were collected from the major distribution areas, including Sichuan, Qianghai, Tibet, Yunnan, Beijing, and Xinjiang (China), between May and November 2013. The native wild samples were identified based on morphological features by Professor Zhang Yi referred to previous Hippophae research [4, 5]. Voucher specimens were deposited in the College of Ethnic Medicine, Chengdu University of Traditional Chinese Medicine. All of the ITS2 and psbA-trnH sequences were submitted to GenBank.

Table 1.

Hippophae samples for testing potential barcodes

Scientific name Haplotype Voucher no. Location GenBank no.
ITS2 psbA-trnH ITS2 psbA-trnH
H. rhamnoides subsp. sinensis A1 M1 YC0546MT01 Wanlin, Jinchuan, Sichuan, China KJ843997 KJ854997
A2 M2 YC0546MT02 Maierma, Aba, Sichuan, China KJ843998 KJ854998
A2 M1 YC0546MT03 Shili, Songpan, Sichuan,China KJ843999 KJ854999
A2 M1 YC0546MT04 Rongrida, Rangtang, Sichuan, China KJ844000 KJ855041
A1 M3 YC0546MT05 Nanmenxia, Huzhu, Qinghai, China KJ844001 KJ855000
A1 M3 YC0546MT06 Puxi, Lixian, Sichuan, China KJ844002 KJ855001
A1 M3 YC0546MT07 Puxi, Lixian, Sichuan, China KJ844003 KJ855002
A2 M4 YC0546MT08 Chaka, Wulan, Qianghai, China KJ844004 KJ855003
A2 M5 YC0546MT09 Gatuo, Mangkang, Tibet, China KJ844005 KJ855004
A2 M1 YC0546MT10 Aba, Aba, Sichuan, China KJ844006 KJ855005
A2 M1 YC0546MT11 Luoerda, Aba, Sichuan, China KJ844007 KJ855006
A2 M3 YC0546MT12 Kehe, Aba, Sichuan, China KJ844008 KJ855007
A2 M3 YC0546MT13 Nawu, Hezuo, Gansu, China KJ844009 KJ855008
A1 M6 YC0546MT14 Laya, Kangding, Sichuan, China KJ844010 KJ855009
A2 M1 YC0546MT15 Chuanzhusi, Songpan, Sichuan, China KJ844011 KJ855010
A1 M1 YC0546MT16 Rilong, Xiaojin, Sichuan, China KJ844012 KJ855011
A1 M1 YC0546MT17 Fubian, Xiaojin, Sichuan, China KJ844013 KJ855012
A1 M1 YC0546MT18 Dawei, Xiaojin, Sichuan, China KJ844014 KJ855013
A2 M7 YC0546MT19 Baihuashan, Beijing, China KM047400 KM047406
A2 M7 YC0546MT20 Baihuashan, Beijing, China KM047401 KM047407
A2 M7 YC0546MT21 Baihuashan, Beijing, China KM047402 KM047408
A2 M7 YC0333MT09 Beijing, China KM047403 KM047409
A2 M7 YC0333MT10 Beijing, China KM047404 KM047410
A2 M2 FDC112a National Institute for Food and Drug Control, China KM047405 KM047411
H. rhamnoides subsp. mongolica B1 N1 YC0547MT01 Buerjin, Altay, Xinjiang, China KJ843986 KJ855021
B1 N1 YC0547MT02 Buerjin, Altay, Xinjiang, China KJ843987 KJ855022
B1 N1 YC0547MT03 Buerjin, Altay, Xinjiang, China KJ843988 KJ855023
H. rhamnoides subsp. yunnanensis C1 O1 YC0548MT01 Gu, Bomi, Tibet, China KJ817423 KJ854989
C1 O1 YC0548MT02 Rewa, Milin, Tibet, China KJ817424 KJ854990
C1 O1 YC0548MT03 Rewa, Milin, Tibet, China KJ817425 KJ854991
C2 O1 YC0548MT04 Jiantang, Shangri-La, Yunnan, China KJ939408 KJ939410
C2 O1 YC0548MT05 Jiantang, Shangri-La, Yunnan, China KJ939409 KJ939411
H. rhamnoides subsp. turkestanica D1 P1 YC0549MT01 Aotebeixi, Wushi, Xinjiang, China KJ844038 KJ855017
D1 P1 YC0549MT02 Aotebeixi, Wushi, Xinjiang, China KJ844039 KJ855018
D1 P2 YC0549MT03 Tucheng, Zhada, Tibet, China KJ844040 KJ855019
D1 P2 YC0549MT04 Tucheng, Zhada, Tibet, China KJ844041 KJ855020
H. rhamnoides subsp. wolongensis E1 R1 YC0550MT01 Taiping, Maoxian, Sichuan, China KJ844024 KJ855038
E1 R1 YC0550MT02 Taiping, Maoxian, Sichuan, China KJ844025 KJ855039
E1 R1 YC0550MT03 Taiping, Maoxian, Sichuan, China KJ844026 KJ855040
H. rhamnoides subsp. caucasia DLA1 – – GenBank JQ663574 –
DLA1 – – GenBank JQ663578 –
DLA1 – – GenBank JQ663579 –
DLA1 – – GenBank JQ663580 –
H. rhamnoides subsp. rhamnoide DLB1 – – GenBank AF440242 –
DLB2 – – GenBank JQ663575 –
H. rhamnoides subsp. carpatica DLC1 – – GenBank AF440245 –
DLC2 – – GenBank JQ663576 –
DLC2 – – GenBank JQ663577 –
H. rhamnoides subsp. fluviatilis DLD1 – – GenBank AF440248 –
DLD2 – – GenBank JQ289287 –
H. goniocarpa F1 S1 YC0551MT01 Galitai, Songpan, Sichuan, China KJ844018 KJ855027
F1 S1 YC0551MT02 Galitai, Songpan, Sichuan, China KJ844019 KJ855028
F1 S1 YC0551MT03 Galitai, Songpan, Sichuan, China KJ844020 KJ855029
H. litangensis G1 T1 YC0552MT01 Jiawa, Litang, Sichuan, China KJ844015 KJ854986
G1 T1 YC0552MT02 Jiawa, Litang, Sichuan, China KJ844016 KJ854987
G1 T1 YC0552MT03 Jiawa, Litang, Sichuan, China KJ844017 KJ854988
H. neurocarpa subsp. neurocarpa H1 U1 YC0553MT01 Babao, Qilian, Qinghai, China KJ844042 KJ854992
H2 U2 YC0553MT02 Jiawa, Litang, Sichuan, China KJ844043 KJ854993
H2 U2 YC0553MT03 Jiawa, Litang, Sichuan, China KJ844044 KJ854994
H2 U1 YC0553MT04 Chali, Aba, Sichuan, China KJ844045 KJ854995
H1 U1 YC0553MT05 Maierma, Aba, Sichuan, China KJ844046 KJ854996
H. neurocarpa subsp. stellatopilosa I1 V1 YC0554MT01 Gaocheng, Litang, Sichuan, China KJ844027 KJ855024
I1 V1 YC0554MT02 Gaocheng, Litang, Sichuan, China KJ844028 KJ855025
I1 V1 YC0554MT03 Gaocheng, Litang, Sichuan, China KJ844029 KJ855026
H. salicifolia J1 W1 YC0653MT01 Lebu, Nacuo, Tibet, China KJ844021 KJ855014
J1 W1 YC0653MT02 Lebu, Nacuo, Tibet, China KJ844022 KJ855015
J1 W1 YC0653MT03 Lebu, Nacuo, Tibet, China KJ844023 KJ855016
H. gyantsensis K1 X1 YC0654MT01 Qiangna, Milin, Tibet, China KJ843989 KJ855030
K1 X1 YC0654MT02 Jieba, Naidong, Tibet, China KJ843990 KJ855031
K1 X1 YC0654MT03 Ridang, Longzi, Tibet, China KJ843991 KJ855032
K1 X1 YC0654MT04 Gangdui, Gongga, Tibet, China KJ843992 KJ855033
K2 X1 YC0654MT05 Pozhang, Naidong, Tibet, China KJ843993 KJ855034
K1 X2 YC0654MT06 Jiaxing, Gongbujiangda, Tibet, China KJ843994 KJ855035
K1 X1 YC0654MT07 Mozhugongka, Mozhugongka, Tibet, China KJ843995 KJ855036
K2 X1 YC0654MT08 Jiubu, Linzhi, Tibet, China KJ843996 KJ855037
H. tibetana L1 Y1 YC0655MT01 Langkazi, Langkazi, Tibet, China KJ844030 KJ854976
L2 Y1 YC0655MT02 Duoma, Ruoergai, Sichuan, China KJ844031 KJ854977
L1 Y1 YC0655MT03 Tangke, Ruoergai, Sichuan, China KJ844032 KJ854978
L2 Y1 YC0655MT04 Riduo, Mozhugongka, Tibet, China KJ844033 KJ854979
L1 Y1 YC0655MT05 Jiangrong, Hongyuan, Sichuan, China KJ844034 KJ854980
L1 Y2 YC0655MT06 Maiwa, Hongyuan, Sichuan, China KJ844035 KJ854981
L1 Y1 YC0655MT07 Nanmenxia, Huzhu, Qinghai, China KJ844036 KJ854982
L1 Y1 YC0655MT08 Tawa, Ruoergai, Sichuan, China KJ844037 KJ854983
L1 Y3 YC0655MT09 Chali, Aba, Sichuan, China KJ855042 KJ854984
L1 Y2 YC0655MT10 Maiwa, Hongyuan, Sichuan, China KJ855043 KJ854985
L1 Y1 YC0655MT11 Keledong, Dege, Sichuan, China KJ855044 KJ854975
E. angustifolia DLE1 – – GenBank AF440256 –
E. pungens – DLDF1 – GenBank – GQ435025
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–: not acquired in this study

aFDC112: a reference crude drug that was purchased from National Institute for Food and Drug Control

Additional sequences belonging to four subspecies of H. rhamnoides which are only found in Europe were obtained from GenBank. In addition, Elaeagnus angustifolia and E. pungens sequences were downloaded from GenBank for use as outgroups in this study.

DNA extraction, PCR amplification, and sequencing

Total genomic DNA was extracted from 50 mg of fruit dried in silica gel. DNA extractions were performed by a Plant Genomic DNA Kit (Tiangen Biotech Co., Beijing, China). Plant material was ground for 2 min at 50 Hz by a DNA Extraction Grinder (Xinzhi Biotech Co., Ningbo, China) as previously described [36]. Primer pairs ITS2F (5′-ATGCGATACTTGGTGTGAAT-3′)/ITS3R (5′-GACGCTTCTCCAGACTACAAT-3′) for ITS2 and psbAF (5′-GTTATGCATGAACGTAATGCTC-3′)/trnHR (5′-CGCGCATGGTGGATTCACAATCC-3′) for psbA-trnH were used for PCR amplification. PCRs were performed in a 25-μL volume, containing 2–3 μL of genomic DNA, 12.5 μL of 2 × EasyTaq PCR MasterMix (Aidlab Biotechnologies Co., Ltd., Beijing, China), 1.0 μL of each primer, and the total volume was adjusted to 25 µL with sterile deionized water. The reaction conditions used were the same as described previously [21, 37]. The PCR products were visualized on agarose gels (the electrophoresis was run in 1 × TBE for 20 min at a constant voltage 120 V). After electrophoresis, purified PCR products were bidirectionally sequenced by the same primers that were used for PCR in a 3730XL sequencer (Applied Biosystems, Foster, CA, USA).

Data analysis

Proofreading and contig assembly of sequencing peak diagrams were performed by CodonCode Aligner 3.7.1 (CodonCode Co., Centreville, MA, USA). The ITS2 region was obtained by the HMMer annotation method based on the Hidden Markov model to remove the 5.8S and 28S sections at both ends of the sequences [38–40]. The psbA-trnH intergenic spacer boundary was determined according to the annotation of similar sequences in GenBank. All sequences were aligned (MUSCLE option) by MEGA 6.0 (Center for Evolutionary Medicine and Informatics, Tempe, AZ, USA) [41], and the genetic distances were calculated according to the Kimura 2 parameter (K2P) model. The distribution of intra- vs. inter-specific variability was assessed by DNA barcoding gaps. A neighbor-joining (NJ) tree was constructed and bootstrap resampling (1000 replicates) was conducted to assess the confidence in phylogenetic analysis by MEGA 6.0. The combination of ITS2 and psbA-trnH (ITS2 + psbA-trnH) was also evaluated by these methods.

Results

Efficiency of DNA extraction and PCR amplification

DNA was successfully extracted from all 75 samples. The PCR amplification success rates for both ITS2 and psbA-trnH were 100 %. All PCR products in correspondence to the ITS2 and psbA-trnH regions were successfully sequenced, and high-quality bidirectional sequences were obtained (Table 2).

Table 2.

Characteristics of the DNA barcodes evaluated in this study

DNA region ITS2 psbA-trnH ITS2 + psbA-trnH
Number of individuals 86 75 75
Number of species 7 7 7
PCR/sequencing success (%) 100/100 100/100 100/100
Amplified sequence length (bp) 221–223 300–313 521–530
Aligned sequence length (bp) 227 320 547
Average GC content (%) 52.72 25.62 37.18
Variable sites 43 19 59
Haplotypes 23 23 28
Intra-specific distance range (mean) 0–0.0571 (0.0041) 0–0.0340 (0.0021) 0–0.0297 (0.0025)
Inter-specific distance range (mean) 0–0.1298 (0.0594) 0–0.0489 (0.0237) 0.0019–0.0708 (0.0363)
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Sequence and inter-/intra-specific variation analysis

The sequence characteristics are summarized in Tables 2 and 3. The average G-C contents of the ITS2 and psbA-trnH regions were 52.72 and 25.62 %, respectively. ITS2 sequences ranged from 221 to 223 bp with 43 variable sites; 23 haplotypes were identified, and four indels that were 1–2 bp in length within the aligned 227 bp. The psbA-trnH intergenic spacer region ranged from 300 to 313 bp and showed less variation, with only 19/320 variable sites among 23 haplotypes.

Table 3.

Sequence information and intra/inter-specific genetic distance of ITS2, psbA-trnH and ITS2 + psbA-trnH regionss of Hippophae species

Species ITS2 psbA-trnH ITS2 + psbA-trnH
Length (bp) GC content (%) Intraspecific distance (mean) Intrespecific distance (mean) Length (bp) GC content (%) Intraspecific distance (mean) Intrespecific distance (mean) Length (bp) GC content (%) Intraspecific distance (mean) Intrespecific distance (mean)
H. rhamnoides 223 52.4 0–0.0571 (0.0174) 0.0137–0.1190 (0.0644) 307 25.5 0–0.0340 (0.0142) 0–0.0447 (0.0212) 530 37 0–0.0297 (0.0127) 0.0019–0.0623 (0.0306)
H. goniocarpa 221 54.3 0 0.0137–0.0928 (0.0354) 307 25.4 0 0–0.0376 (0.0160) 528 37.5 0 0.0077–0.0478 (0.0236)
H. litangensis 221 52.5 0 0–0.1246 (0.0566) 303 25.1 0 0.0033–0.0411 (0.0173) 524 36.6 0 0.0038–0.0623 (0.0318)
H. neurocarpa 221 52.3 0–0.0091 (0.0031) 0–0.1298 (0.0587) 305 25.6 0 0–0.0341 (0.0172) 526 36.9 0–0.0038 (0.0013) 0.0038–0.0603 (0.0331)
H. salicifolia 223 52.5 0 0.0091–0.1142 (0.0474) 300 27 0 0.0135–0.0489 (0.0365) 523 37.9 0 0.0116–0.0708 (0.0398)
H. gyantsensis 221 52.4 0–0.0045 (0.0019) 0.0091–0.1198 (0.0487) 313 25.1 0–0.0032 (0.0008) 0.0135–0.0449 (0.0317) 534 36.3 0–0.0038 (0.0013) 0.0116–0.0685 (0.0377)
H. tibetana 223 59.4 0–0.0183 (0.0060) 0.0822–0.1298 (0.1050) 303 25.8 0 0.0133–0.0413 (0.0257) 526 40.1 0–0.0077 (0.0025) 0.0435–0.0708 (0.0575)
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With these ITS2 sequences, both variable sites and deletions provided insight into the identification of H. salifocilia, H. tibetana, and three H. rhamnoides subspecies (Fig. 1). By comparing the sequences, all species except H. salifocilia have deletions from the sites 201–202; in H. tibetana, there were 15 variable sites from site 2 to site 223 which could be used for identification and discrimination from other species. Other important variable sites also provided useful information for species identification and discrimination, such as H. rhamnoides subsp. yunnanensis at site 80, H. rhamnoides subsp. turkestanica at site 153 and site 155, and H. rhamnoides subsp. wolongensis at site 34, site 207, and site 219. With psbA-trnH sequences, the variable sites and insertions enable the identification and differentiation of H. goniocarpa, H. gyantsensis, H. salicifolia, H. tibetana, and two H. rhamnoides subspecies (Fig. 2). When the sequences were compared, most species had no insertions except H. goniocarpa, which had insertions between site 90 and site 91, and H. gyantsensis, which had insertions at site 37 and from site 221 to site 229. Stable sequence variations, which provided useful information for species identification, were found in three species and two subspecies: H. salicifolia at site 38, site 94, and site 211; H. gyantsensis at site 7; H. tibetana at site 65, site 77, and site 302; H. rhamnoides subsp. mongolica at site 64; and H. rhamnoides subsp. turkestanica at site 24.

Fig. 1.

Fig. 1

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

Fig. 2.

Fig. 2

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

The intra- and inter-specific K2P genetic distances for ITS2, psbA-trnH, and ITS2 + psbA-trnH are listed in Table 2. In general, the mean inter-specific distances were higher than the mean intra-specific distances for the single-locus barcodes as well as the 2-locus barcode by the K2P model. ITS2 showed the highest intra- and inter-specific distances among the two DNA regions and the combination of the two regions, whereas the psbA-trnH exhibited the lowest intra- and inter-specific distances.

Assessment of barcoding gaps

Ideal barcode sequences should have a distinct inter-specific distance and relatively little intra-specific variation, and there need to be distinct differences between the sequences to form a spacer region, known as the “barcoding gap”. Figure 3 shows the minimum inter-specific K2P distances vs. maximum intra-specific distances, and the points that represented species distributed above the 1:1 line indicated that there were barcoding gaps for these species. With psbA-trnH and ITS2 + psbA-trnH, the species located in the area with no barcoding gap was H. rhamnoides. With the ITS2 region, there were two species, H. rhamnoides and H. neurocarpa, that had no barcoding gap. There were four points located on the 1:1 line, indicating that these species also had no barcoding gap. These four points included H. litangensis with ITS2, H. goniocarpa and H. neurocarpa with psbA-trnH, and H. neurocarpa with ITS2 + psbA-trnH.

Fig. 3.

Fig. 3

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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

Neighbor-joining tree analysis

In this study, a phylogenetic tree was constructed by the NJ method, with 1000 bootstrap replicates for ITS2 (Fig. 4), psbA-trnH (Fig. 5), and ITS2 + psbA-trnH (Fig. 6) regions. Using ITS2 + psbA-trnH was the most effective for the species differentiation: all species were clearly identified, including the medicinal and non-medicinal Hippophae species. The ITS2 single-locus barcode was the second-most effective and differentiated five species: H. rhamnoides, H. goniocarpa, H. salicifolia, H.gyantsensis, and H. tibetana. The psbA-trnH region showed relatively poor performance with regard to species identification, as only four species were identified: H. litangensis, H. salicifolia, H.gyantsensis, and H. tibetana.

Fig. 4.

Fig. 4

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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.

Fig. 5

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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.

Fig. 6

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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

At the subspecies level, four subspecies were identified by psbA-trnH (H. rhamnoides ssp. mongolica, H. rhamnoides ssp. yunnanensis, H. rhamnoides ssp. turkestanica, and H. rhamnoides ssp. wolongensis), three subspecies with ITS2 (H. rhamnoides ssp. yunnanensis, H. rhamnoides ssp. turkestanica, and H. rhamnoides ssp. wolongensis), and four subspecies with ITS2 + psbA-trnH (H. rhamnoides ssp. mongolica, H. rhamnoides ssp. yunnanensis, H. rhamnoides ssp. turkestanica, and H. rhamnoides ssp. wolongensis). Consequently, the 2-locus barcode ITS2 + psbA-trnH showed the highest efficiency for identifying Hippophae at the species and subspecies level. The single-locus barcode psbA-trnH was also suitable for identifying H. rhamnoides subspecies.

Discussion

The morphological similarities of Hippophae species caused a high chance of misidentification and misuse. Raw Hippophae products are often sold in dried and powdered forms, making morphological identification infeasible.

DNA barcoding is an important supplement and validation of conventional morphological identification [23]. In the present study, medicinal and non-medicinal Hippophae species were identified by DNA barcoding after a preliminary morphological identification, and remarkable Hippophae variation at the species level was shown. The genomic DNA could be extracted from dried fruits with both ITS2 and psbA-trnH with 100 % amplification and sequencing efficiencies. Two single-locus barcodes, ITS2 and psbA-trnH, as well as their combination were evaluated and validated. All Hippophae species were successfully identified by DNA barcoding, and four H. rhamnoides subspecies were also differentiated. The information obtained from the variable sequence sites and deletions/insertions facilitated the identification of Hippophae species; H. salicifolia, H. tibetana, and three H. rhamnoides subspecies were identified by ITS2 sequences, whereas H. goniocarpa, H. salicifolia, H. gyantsensis, H. tibetana, and two H. rhamnoides subspecies were identified by psbA-trnH sequences.

A relatively high value was observed for ITS2 + psbA-trnH with regard to the barcoding gap analysis: one species was located under the 1:1 line, and one species was located on the 1:1 line. However, three species had no barcoding gap for each of the single-locus barcodes: H. rhamnoides, H. litangensis, and H. neurocarpa for ITS2 barcode; H. rhamnoides, H. goniocarpa, and H. neurocarpa for psbA-trnH barcode. The identification efficiency of single-locus and combined barcodes by the NJ tree method showed that ITS2 + psbA-trnH was the most suitable barcode, with all seven species as well as four H. rhamnoides subspecies clearly identified. None of the selected barcodes were suitable for H. neurocarpa subspecies identification. Although it was hard to identify all H. rhamnoides and H. neurocarpa subspecies by ITS2, psbA-trnH, and ITS2 + psbA-trnH, the medicinal species were successfully distinguished from non-medicinal Hippophae species. While H. rhamnoides is the original medicinal plant according to Chinese Pharmacopeia, H. neurocarpa, H. gyantsensis, and H. tibetana are used in the Tibetan medicine [14, 15, 17, 18]. Thus, all native Hippophae species were identified by DNA barcode and the accurate and standard sequence information was gained. This information would be applicable to commercial products alignment and authenticate Hippophae species origins in the future.

There have been debates over whether H. litangensis was a subspecies of H. goniocarpa and whether H. rhamnoides subsp. wolongensis was a distinct species [3, 4, 42]. In our study, we considered H. litangensis and H. goniocarpa as two separate species, and the results demonstrated that they could be identified separately at the species level; H. rhamnoides subsp. wolongensis was a subspecies of H. rhamnoides based on the K2P genetic distance, NJ tree, and identification efficiency results.

Conclusion

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

Authors’ contributions

LX and YZ designed the study. YL, CL, YQZ, YLC and MS performed the experiment. YL analyzed the data and wrote the manuscript. LX, YZ, WS, GF and XL revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by Science Technology Support Plan Project from Science & Technology Department of Sichuan Province (No. 2013SZ01114)-“Key technology, research and development of distinctive Chinese and Tibetan medicine resources utilization” and The Major Scientific and Technological Special Project for ‘‘Significant New Drugs Creation’’ (No. 2014ZX09304307).

Competing interests

The authors declare that they have no competing interests.

Abbreviations

ITS2

internal transcribed spacer 2

K2P

Kimura 2-parameter

NJ tree

neighbor-joining tree

CM

Chinese medicine

CBOL

Consortium for the Barcode of Life

Contributor Information

Yue Liu, Email: lucindalau1225@sina.com.

Wei Sun, Email: djsunwei@gmail.com.

Chuan Liu, Email: 550590623@qq.com.

Yaqin Zhang, Email: 1207360366@qq.com.

Yilong Chen, Email: 564996126@qq.com.

Ming Song, Email: songmingcn@outlook.com.

Gang Fan, Email: fangang1111@yahoo.com.cn.

Xia Liu, Email: lrx1125@126.com.

Li Xiang, Email: xl_yzhm@163.com.

Yi Zhang, Email: 1175332408@qq.com.

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