Sequence Analysis
The ribosomal ITS sequences of G. littoralis and its five adulterants, A. lappa Decne, C. lanceolata, S. conoidea, C. tangshen, and A. stricta, were determined to be 609 bp, 647 bp, 664 bp, 632 bp, 663 bp, and 714 bp, respectively. As expected, the multiple sequence alignment result showed that a host of DNA polymorphisms were detected in ITS region. In service of our objective to develop a DNA marker for discrimination of G. littoralis from its five adulterants, we tried to exploit polymorphic sites that are unanimously specific to the five adulterants. As shown in Fig. 1, G. littoralis contains AT at nucleotide position 43–44, but the five adulterants were replaced with CA or TA in the same position. Likewise, the five adulterants contain nucleotide G in the 321st position, while G. littoralis was replaced with C at the same location. Similarly, the chloroplast trnL-F sequences also showed a variety of different sizes among G. littoralis and its five adulterants resulting from nucleotide insertions/deletions. The PCR products of G. littoralis, A. lappa Decne, C. lanceolata, S. conoidea, C. tangshen, and A. stricta, were determined to be 993 bp, 955 bp, 1021 bp, 1060 bp, 999 bp, and 990 bp, respectively. Three SNP sites that unanimously specific to the five adulterants were exploited, which were located at nucleotide positions 88 (C/T), 281 (G/A), and 506 (C/A), respectively (Fig. 2). Consequently, species-specific primers for molecular discrimination of G. littoralis and its five adulterants can be designed based on these SNP sites exploited in ITS and trnL-F regions.
Species-specific Primer Design
Based on the species-specific SNP sites delineated in 2.1, two specific primer pairs were designed for molecular discrimination of G. littoralis and its five adulterants in ITS and trnL-F regions, respectively. In ITS region, primers ASF and ASR were designed for specific identification of the five adulterants based on the SNP sites located at nucleotide positions 43, 44, and 321. Primers BSF and BSR were designed for specific authentication of G. littoralis according to its specific sequences compared with those of the five adulterants (Fig. 1). In chloroplast trnL-F region, primers NTF and NTR were designed for specific identification of the five adulterants based on their SNP sites located at nucleotide position 88. According to the SNP sites at nucleotide positions 281 and 506, primers BTF and BTR were respectively designed for specific authentication of G. littoralis (Fig. 2). The sequences of these specific primer pairs were shown in Table 2. The substitutions of A for C in primer NTF and A for T in primer BTR, were additional mismatches introduced deliberately to ensure reliable allelic specificity for target species.
Table 2
Nucleotide sequences of specific primers
Location | Primer | Nucleotide sequence* | Mismatch |
ITS | ASF | 5′-TCATTGTCGAAACCTGCACA-3′ | CA(TA)/AT |
ASR | 5′-AGAGCCGAGATATCCGTTGC-3′ | C/T |
BSF | 5′-TCTTGTCCACAAACCACTCA-3′ | Natural |
BSR | 5′-GTTCCTGGAGCTCGCTAGG-3′ | Natural |
trnL-F | NTF | 5′-TTCAAATTCAGAGAAACACT-3′ | ACT/CCC |
NTR | 5′-TCCATTGAGTCTCTGCACCT-3′ | No |
BTF | 5′- ACTTCCGAGAAGGATATGCG-3′ | CG/GA(AA) |
BTR | 5′- TCGTCCGATTAATTAGTTCAG-3′ | AG/TT |
*Bolded nucleotides indicate the species-specific SNP sites at the 3’ terminals. Underlined bases indicate the deliberate mismatches. |
Molecular Authentication of G. littoralis
Two species-specific primer pairs designed in ITS and trnL-F regions were respectively used in multiplex PCR for molecular discrimination of G. littoralis from its five adulterants. In ITS region, the combination of two primer pairs amplified different fragment patterns for G. littoralis and its five adulterants. As shown in Fig. 3, the G. littoralis samples produced unique bands with a size of 187 bp. The five adulterants, A. lappa Decne, C. lanceolata, S. conoidea, C. tangshen, and A. stricta, generated their species-specific amplicons with sizes of 295 bp, 295 bp, 280 bp, 295 bp, and 313 bp, respectively. Similarly, with the two species-specific primer pairs designed in trnL-F region, G. littoralis samples amplified unique fragments with a size of 211 bp, while A. lappa Decne, C. tangshen, and A. stricta produced species-specific bands of 125 bp, and C. lanceolata and S. conoidea generated their amplicons of 106 bp (Fig. 4). No fragment was amplified when the same primer pairs were cross-challenged against DNAs from non-target species, and the minor differences in the amplicon sizes among five adulterants will not interfere with the authentication of G. littoralis on an agarose gel. Molecular discrimination of G. littoralis were repeated many times with the samples listed in Table 1, and the results were further validated with expected reproducibility. Therefore, the developed multiplex species-specific PCR was effective for molecular discrimination of G. littoralis from its five adulterants.
Lod Test And Botanical Origin Authentication Of Commercial Products
The absence of the longer PCR product on an agarose gel could be explained by the template DNA degradation, something that will be less visible than the shorter fragment. Therefore, chloroplast trnL-F region is selected for botanical origin authentication of commercial products because the five adulterants yielded shorter amplicons than those from G. littoralis. A total amount of 10 ng binary DNA mixtures containing 0.1%, 1%, 2%, 5%, 10%, and 50% of non-target DNA were respectively checked by the developed multiplex species-specific PCR. As shown in Fig. 5, as the content of non-target DNA increased, species-specific bands of the adulterant gradually became brighter. The results showed that the developed method could detect 0.1% of adulteration in G. littoralis down to 0.01 ng level of template DNA. To confirm the practicality of the developed method in botanical origin authentication, genomic DNAs of commercial products in the forms of slice, powder, and extract were subjected to the developed PCR assay. As expected, the commercial products yielded their species-specific amplicons representing their botanical origins, which were consistent with that described in Fig. 4. For the mixed samples of G. littoralis and adulterant extracts, two fragments with dim brightness were detected for each sample (Fig. 6), indicating the existence of adulterant and the DNA degradation in their extracts. The findings highlight that this approach is effective for botanical origin authentication of commercial G. littoralis food products and can be used for authenticity and adulteration determination of G. littoralis food products.