Molecular identification of original plants of Spica prunellae based on ITS sequence

The ITS sequences of the original plants of Spica prunellae and its confusable species including 16 individuals belonging to 6 species from 12 localities, were analyzed. The results showed that the length of ITS sequences was 584 bp after alignment and the overhanging terminals were cut. When alignment gaps were treated as missing data, there were 150 variable sites. The percent divergence between species was 10.9%, with a transitions/transversions ratio of 1.53. There were 11 variable and 9 informative sites among the ITS sequences of 4 species in Prunella Linn. There were specific sites at 5, 474 and 525 nt of the ITS sequence of P. grandiflora. There were 122 specific sites between genus Ajuga and genus Prunella. Both phylogenetic trees constructed by Maximum parsimony method and Neighbor-Joining method indicated that Prunella vulgaris Linn., P. asiatica Nakai. and P. hispida Benth. had a very close relationship. The character of ITS sequences can be used for providing the molecular markers for identifying Chinese Prunella (S. prunellae).


INTRODUCTION
According to Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission, 2005), the dried fruit-spike of Prunella vulgaris L. (Fam.Labiatae) is used as the crude drug Spica prunellae (Common Selfheal Fruit-Spike ) in practice.P. vulgaris is generally regarded as one of the original species of S. prunellae according to Modern Chinese Materia Medica (Xiao, 2002), which mainly distributes in Jiangsu, Anhui, Hubei and Henan provinces.Especially the species from Jiangsu with long spike and short spike-stalk has the best curative effect and is called 'Jing Common Selfheal'.Besides, fruitspikes of Prunella asiatica Nakai and Prunella hispida Benth are regarded as other origins of S. prunellae.Wang et al. (1996) found that the three species could be accurately distinguished according to their characters *Corresponding author.E-mail: hangyueyu@yahoo.com.cn.
Delectis Florae Reipublicae Popularis Sinicae Agendae of bract, calyx and so on.But in practice it is still not easy to distinguish them.Ajuga decumbens Thunb.and Ajuga multiflora Bunge from the same family are also used as S. prunellae in some areas of China (Xiao, 2002;Academiae Sinicae Edita, 1977a).
Besides the introduced Prunella grandiflora (Linn.)Jacq., there are three other Prunella species including P. vulgaris, P. asiatica, and P. hispida in China according to Flora Reipublicae Popularis Sinicae (Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae Edita, 1977b) and Flora of China (Wu and Raven, 1994), Jiangsu Flora (Jiangsu Institute of Botany, 1982) and Anhui Flora (Cooperation Group of the Anhui Flora, 1992) record that P. vulgaris, but not P. asiatica distributes in Jiangsu and Anhui, whereas Flora Reipublicae Popularis Sinicae records that P. asiatica instead of P. vulgaris distributes in Jiangsu and Anhui.This contradiction indicated that the confusion of distribution and taxonomy of P. vulgaris existed, then botanical origins of S. prunellae might be mixed and the original species of the genuine medicinal material Jing Common Selfheal were uncertain.
The internal transcriped spacer (ITS) of ribosomal DNA is becoming a useful molecular marker in identification of the origin of medicinal plants in addition to systematic development and classification researches due to its especial characteristics such as high repeatability, rapid mutation, stability under processing conditions (Lau et al., 2001;Zhao et al., 2001;Xu et al., 2006;Shen et al., 2001;Shen et al., 2005;Yu et al., 2003).In this paper, we reported to identify the original plants of S. prunellae and their confusable species based on ITS sequence.

MATERIALS AND METHODS
Samples of the 4 Prunella species including P. vulgaris, P. asiatica, P. hispida and P. grandiflora were collected from different habitats (Table 1); the species were wild except P. grandiflora.Voucher specimens were deposited in the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences and were identified based the morphological features according to Flora of China.

DNA isolation
Total DNA was extracted by the CTAB method described by Paterson et al. (1993).The extracted DNA was purified using E.Z.N.A. TM Cycle Pure Kit (Beijing Bio-lab Materials Institute) and dissolved in double distilled water and stored at -20°C until use.Purified DNA was subjected to electrophoresis through a 0.8% agarose gel and the concentration was estimated by using an UV lamp.

Sequence analysis
Sequences were aligned with the program Clustal X (Thompson et al., 1997).Genetic distances were computed with MEGA3.1 (Kumar et al., 2001) using Kimura-two parameter model, and phylogenetic tree of NJ, MP was reconstructed by PAUP 4.0 (Swofford, 1999) with MP (Maximum parsimony) and NJ (Neighbor-Joining) methods respectively.The search algorithm was heuristic, and the reliability of each branch was estimated from bootstrap test (1000 replications).Alignment gaps were treated as missing data.

Sequence features of ITS regions
Because of the inconsistency about the boundary of ITS1,5.8S and ITS2 regions with that of P. vulgaris or A. decumbens by Clustal X, and the overhanging terminals were deleted.Aligned sequence of 584 bp (including partial ITS1 and complete 5.8S and ITS2) was obtained.When alignment gaps were treated as missing data, 584 bp contained 150 variant sites and 149 informative sites.The percentage of sequence divergence between species (Table 2) was 10.9%, and the transitions/transversion ratio was 1.53.The mean nucleotide composition of all the sequences was 18.2% T, 32.8% C, 18.3% A, and 30.7%G; the percentage of G-C content was 63.5%.

Identification of species by ITS sequence analysis
The sequences were calculated with MEGA3.1 after pairwise arrangement, as shown in Table 3 and 4. Prunella species had 11 variant sites and 9 informative sites, and there were no variation at the coding region.P. vulgaris, P. asiatica, and P. hispida had 6 variant sites.
When being compared with the three bases of 5(C), 474(C) and 525(T) in the other three species, P. grandiflora had three different bases: 5(G), 474(A) and 525(C).Therefore, it could be identified from the other 3 species.Further, it was easy to discriminate the counterfeits of S. prunellae -A.decumbens and A. multiflora based on the 122 informative sites present at genus Ajuga and genus Prunella.
There existed a certain degree of variation in ITS sequence among four species in Prunella.P. vulgaris from Jiangxi had the same ITS sequence as well as P. asiatica from Jiangsu, Anhui and Hubei had.The ITS sequences of P. vulgaris from Hunan and Guizhou were identical.The sequence of P. vulgaris from Sichuan was only different at 376 and 518 nt with populations from Hunan and Guizhou.The four populations of P. asiatica had same ITS sequences except 143 nt.
The sequences of two P. hispida individuals were the same, which had identical bases at 103 nt, 143 nt, 376 nt and 518 nt with P. vulgaris from Hunan and Guizhou, and the same bases at 181nt and 182nt with P. asiatica.P.hispida 1 P.hispida 2 P. vulgaris (Yingxiu, Sichuan) P. vulgaris (Guiyang, Guizhou) P. vulgaris (Hunan) P. asiatica (Jurong, Jiangsu) P. vulgaris (Xinyu, Jiangxi) P. asiatica (Xishui, Hubei) P. asiatica (Vuhu, Anhui) P. asiatica (Panan, Zhejiang) Though P. hispida could not be distinguished at a single site, a combination of six bases (103,143,181,182,376 and 518 nt) could be used as a standard to identify P.

hispida.
In brief, ITS sequence could be used to identify P. grandiflora, A. decumbens, A. multiflora and P. hispida.P. vulgaris and P. asiatica could not be distinguished only based on ITS sequences.

Phylogenetic analysis of Spica prunellae
Phylogenetic trees were constructed by PAUP 4.0 with MP (Maximum parsimony) method and NJ (Neighbor-Joining) method respectively.The topology constructed by two methods was rather similar, only different in bootstrap values (Figure 1).It showed that Prunella species located in the same class, A. multiflora and A.
decumbens assembled in another one.P. grandiflora had a rather distant phylogenetic relationship with the other 3 Prunella species.P. hispida was close to P. vulgaris.P. vulgaris and P. asiatica had an obscure relationship, as reflected by the relatively low bootstrap values of branches.In phylogenetic tree, P. vulgaris from Xinyu did not belong to the cluster of other P. vulgaris populations, but it had a closer relationship with P. asiatica, which indicated that it might be transition between the two species.

Molecular Identification of Spica prunellae
There were related reports about genuineness of Traditional Chinese Medicine, for example, P. vulgari is prescribed by Chinese Pharmacopoeia as the botanical origin of Spica prunellae from its closely related plant P.
grandiflora and confusable species in genus Ajuga.Genuineness of traditional Chinese drugs refers to genuineness of species, genuineness of producing area or genuineness of effective components.In this study, we focused on the genuineness of species.A lot of researches on genuineness of species have been done (Ge et al., 2007;Wen et al. 2007;Huang et al., 2007).The specific sites in ITS sequences could be used to identify P. vulgaris.Jiangsu Flora (Jiangsu Institute of Botany, 1982) and Anhui Flora (Cooperation Group of the Anhui Flora, 1992) record that P. vulgaris grow in Jiangsu and Anhui, and that P. asiatica don't.Whereas Flora Reipublicae Popularis Sinicae records that P. asiatica instead of P. vulgaris grow in Jiangsu and Anhui.This contradiction indicated that there existed the confusion of distribution and taxonomy of P. vulgaris, so the botanical origins of Spica prunellae could be mixed and the original species of the genuine medicinal material Jing Common Selfheal is uncertain.As mentioned above, there are a few contradictions in the description of the morphological characters and distribution of P. vulgaris and P. asiatica in different literatures.Wang et al. (1994;1996;1999) found that P. vulgaris, P. asiatica and P. hispida had similar features of morphology, chemical composition and pharmacological effects, and that the cross distribution made it rather difficult to distinguish P. vulgaris from P. asiatica.One P. vulgaris population as well as P. asiatica had the same ITS sequence, and 4 specific sites of P. hispida were the same as those of P. vulgaris, with 2 specific sites of P. hispida identical with those of P. asiatica, which suggested that P. asiatica, P. vulgaris and P. hispida should be treated as one specie and P. asiatica should be regarded as the geographic subspecies of P. vulgaris.
Our results showed that P. vulgaris, the botanical origin of S. prunellae and local origins P. asiatica and P. hispida had a very close phylogenetic relationship, while taxonomy of three species should need more detailed evidences in future.

The origin of S. prunellae and genuineness of the species
S. prunellae is one of the genuine medicinal materials in Jiangsu.P. vulgaris is prescribed as the botanical origin of S. prunellae in Chinese Pharmacopoeia (2005).However, according to Flora Reipublicae Popularis Sinicae, not P. vulgaris but P. asiatica distributes in Jiangsu.Based on this study, it is suggested that the original plants of S. prunellae should be P. vulgaris and P. asiatica, and that the botanical origin of Jing Common Selfheal should be P. asiatica.

Figure 1 .
Figure 1.Phylogenetic treef Spica prunellae and their confusable species based on ITS sequences from original plants.Numbers above branch indicated bootstrap values (%) of Maximum parsimony method; numbers below branch indicated bootstrap values (%) of Nei ghbor-Joining method, respectively.

Table 1 .
List of samples.

Table 2 .
Percent divergence of ITS sequences from original plants of S. prunellae and their confusable species.
Note:The numbers indicate Std.errors and percent divergences, respectively.

Table 3 .
Variable sites in the ITS sequence of four species in Prunella.