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An accurate and rapid method for species identification in plants: Melting fingerprint-high resolution melting (MFin-HRM) analysis
Plant Gene 20 (2019) 100203
Contents lists available at ScienceDirect
Plant Gene journal homepage: www.elsevier.com/locate/plantgene
An accurate and rapid method for species identification in plants: Melting fingerprint-high resolution melting (MFin-HRM) analysis
T
⁎
Kittisak Buddhachat , Phanupong Changtor, Sunatcha Ninket Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand
A R T I C LE I N FO
A B S T R A C T
Keywords: Melting temperature Medicinal plant Phyllanthus ISSR Real-time PCR DNA fingerprint
Species identification is required in various areas such as systematics, ecology, conservation, evolution, agriculture, forensics, pharmacology, food science, and even industry. Yet the number of taxonomists who identify based on morphological observations is limited, and some materials are incomplete and/or processed, leading to difficulty in species identification by morphological features. Thus, the molecules remaining inside, especially DNA, often have been extensively used to aid species identification. Here, we established an accurate and rapid method, melting fingerprint-high resolution melting or “MFin-HRM,” to facilitate species identification/authentication based on the melting profile of the DNA fingerprint obtained from inter simple sequence repeat (ISSR) coupled with HRM, or the “melting fingerprint”. This method generates a melting fingerprint specific to a certain species. In this study, Phyllanthus amarus and its closely related species were investigated as a model to test the performance of MFin-HRM in species identification. A final concentration of DNA template of more than 100 pg/μl is suitable for obtaining a consistent melting fingerprint by MFin-HRM. In addition, MFin-HRM can be used for adulterant determination, and its sensitivity and specificity depend on the ISSR primer chosen for generating the melting fingerprint. These results demonstrate that MFin-HRM is a reliable and fast method for species identification/authentication in a closed tube.
1. Introduction
characters, contributing to misuse that jeopardises the customers' safety and the quality of the medicinal products. Herbaceous Phyllanthus spp. is a medicinal plant for hepatoprotection and antiarthitis; however, the bioactive compounds, phyllanthin and hypophyllanthin, are present in high amounts in Phyllanthus amarus but little or none are found in other, related species (Tripathi et al., 2006; Buddhachat et al., 2017). Species confirmation, therefore, is an important step for quality control of the herbal products. With advancing biotechnology, several newly emerging molecular techniques for species identification have been developed and amended to improve their performance. Initially, DNA fingerprints derived from amplified fragment length polymorphism (AFLP) (Vos et al., 1995), restriction fragment length polymorphism (RFLP) (Lander and Botstein, 1989), random amplified polymorphic DNA (RAPD) (Welsh and McClelland, 1990; Williams et al., 1990) and inter-simple sequence repeat (ISSR) techniques (Gupta et al., 1994) have been extensively used for developing DNA markers in several applications such as
Species identification is a crucial task for various disciplines such as taxonomy, systematics, evolution, genetics, ecology, conservation, agriculture, forensics, pharmacology, food science and the study of industrial medicinal plants (Hebert and Gregory, 2005; Hajibabaei et al., 2007; Dawnay et al., 2007; Rasmussen and Morrissey, 2008; Chen et al., 2010; Bosmali et al., 2012; Buddhachat et al., 2015; Osathanunkul et al., 2016). Traditionally, plant species identification based on morphological observations has been used by experts and specialised taxonomists; thus, it has been restricted for non-experts who require species diagnosis for their purposes. In addition, specimens may be incomplete due to damage or processing, creating hurdles for species delineation based on morphological characters. For example, for industrial medicinal plants, the raw materials for manufacturing need to be confirmed as to whether they are the correct species because several medicinal plant species can share a common name or morphological
Abbreviations: DNA, deoxyribonucleic acid; PCR, polymerase chain reaction; ISSR, inter simple sequence repeat; HRM, high resolution melting; MFin-HRM, melting fingerprint derived from high resolution melting; RAPD, random amplified polymorphic DNA; RFLP, restriction fragment length polymorphism; AFLP, amplified fragment length polymorphism; Bar-HRM, DNA barcode coupled with high resolution melting; Tm, melting temperature; rbcL, Ribulose bisphosphate carboxylase large chain; matK, maturase K ⁎ Corresponding author. E-mail address: [email protected] (K. Buddhachat). https://doi.org/10.1016/j.plgene.2019.100203 Received 13 June 2019; Received in revised form 19 August 2019; Accepted 6 September 2019 Available online 10 September 2019 2352-4073/ © 2019 Published by Elsevier B.V.
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genetic improvement as well as for species-specific markers. These methods enable species differentiability without extensive DNA sequence information in both plants e.g. Dendrobium (Wang et al., 2009) and Eucalyptus (Balasaravanan et al., 2006) species and animals e.g. meat quality control (Huang et al., 2003). The RAPD and ISSR methods are less complex compared to AFLP and RFLP; therefore, the two former methods have gained more attention than the latter two, which consist of various steps (Powell et al., 1996; Patzak, 2001; Dangi et al., 2004; Garcia et al., 2004). Since the early 2000s, with Hebert et al. (2004a) as the initiators of the technique, DNA barcoding, in which a short-standardised DNA sequence is used to identify a specific species with high accuracy and reliability, has been extensively and widely used in species identification. DNA barcode repositories of many kinds of organisms such as bacteria, fungi, insects, fish, birds and plants have been created from massive amounts of data within less than twenty years (Hebert and Gregory, 2005; Hebert et al., 2004a; Hebert et al., 2004b; Ratnasingham and Hebert, 2007; Ward et al., 2009; Group et al., 2009). Several studies suggest that a combination of various barcodes is required for species identification to enable higher species resolution (Group et al., 2009; Kress et al., 2005; Kress and Erickson, 2007; Fazekas et al., 2008; Hollingsworth et al., 2011). For example, the CBOL Plant Working Group (2009) recommended a combination of rbcL and matK in plant species identification. Recently, DNA barcoding was conjugated with high resolution melting analysis (HRM) in a technique called “Bar-HRM” to facilitate species identification without DNA sequencing, allowing the completion of the reaction in a closed tube, leading to a cost-effective and rapid method. Bar-HRM has been used to identify a wide range of organisms, especially modified, damaged, processed, incomplete or invisible specimens (Osathanunkul et al., 2016; Ereqat et al., 2010; Ganopoulos et al., 2013; Faria et al., 2013; Buddhachat et al., 2015; Sun et al., 2016). Unfortunately, Bar-HRM has the limitation of having been developed as a universal method for a species group with a small number of species, due to the fact that Bar-HRM produces data for a single locus, given as a narrow range of melting temperatures (Tm). As a result, there is inadequate resolution of Bar-HRM to differentiate among many species in a large species group. Although Bar-HRM allows a combination of multiloci for identification, an overlapping of Tm occurred for Thai medicinal plant species (Osathanunkul et al., 2016). To increase species resolution by HRM, a DNA fingerprint such as RAPD or ISSR would be more powerful in differentiating many species because a single primer can produce multiple DNA bands from multiple loci in the plant genome (Welsh and McClelland, 1990; Williams et al., 1990; Gupta et al., 1994). Here, we evaluate the feasibility of combining the DNA fingerprint method with HRM, in a technique we call “melting fingerprint HRM” or “MFin-HRM,” to distinguish among closely related Phyllanthus species as a model.
Table 1 ISSR primers used in this study. ISSR primer
Sequence (5′➔3′)
UBC802 UBC807 UBC810 UBC813 UBC816 UBC820 UBC825 UBC827 UBC873 UBC877
ATA TAT ATA TAT ATA TG AGA GAG AGA GAG AGA GT GAG AGA GAG AGA GAG AT CTC TCT CTC TCT CTC TT CAC ACA CAC ACA CAC AT GTG TGT GTG TGT GTG TC ACA CAC ACA CAC ACA CT ACA CAC ACA CAC ACA CG GAC AGA CAG ACA GAC A TGC ATG CAT GCA TGC A
1.5% agarose gel electrophoresis with visualisation under ultra-violet (UV) light by a UV transmitter. DNA was dissolved with tris-EDTA buffer and stored at −20 °C for further use. 2.3. DNA fingerprint (Fin)-high resolution melting (HRM) of inter simple sequence repeats (ISSR) In this study, ISSR was chosen for generating a DNA fingerprint because of (i) no requirement of DNA sequence information, (ii) the small amount of DNA required, (iii) use of a single primer and (iv) high reproducibility (Gupta et al., 1994). Preliminarily, P. amarus was used as the DNA template for generating a DNA fingerprint using 10 different ISSR primers (Table 1). A total of 10 ISSR primers were screened to choose suitable primers that showed clear and multiple bands in the DNA fingerprint. Subsequently, the positive primers were used for making DNA fingerprints for four Phyllanthus species in order to select primers producing DNA patterns in all tested species. Each reaction contained 1× reaction buffer (RBC Bioscience, Taiwan); 1.5 mM MgCl2 (RBC Bioscience, Taiwan); 0.2 mM dNTPs (Thermo Scientific, USA); 0.4 mM primer; and 1 U Taq DNA polymerase (RBC Bioscience, Taiwan), with adjustment with deionised distilled water to a final volume of 25 μl. The PCR was performed under the following conditions: 95 °C for 5 min, 40 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR products were detected by gel electrophoresis using a 2% agarose gel with GelRed (Biotium, USA) as the visualiser under UV light. The primers that gave distinct DNA patterns among species were selected for HRM analysis to acquire the melting profiles of the different Phyllanthus species including P. amarus, P. urinaria, P. airy-shawii, P. debillis and P. virgatus. DNA amplification and HRM were performed using real-time PCR on the LightCycler® 480 instrument II (Roche Life Science, Germany). The reaction mixture for real-time PCR and HRM analysis was made in a total volume of 10 μl containing 5 μl of 2× SensiFAST HRM mix (Bioline, England), 0.4 μM primer and 1 μl of 10 ng/μl DNA. The amplification protocol was conducted using an initial denaturing step at 95 °C for 5 min followed by 40 cycles of 95 °C for 1 min and 55 °C for 1 min. The final extension was done at 72 °C for 7 min. The fluorescence data were acquired at the end of each cycle during DNA amplification. Before HRM, the products were denatured at 95 °C for 15 s, and then annealed at 50 °C for 15 s to randomly form DNA duplexes. For HRM experiments, the fluorescence data were collected at every 0.1 °C/cycle for 2 s per cycle in the temperature range of 65 °C to 95 °C. EvaGreen® was the fluorescent dye used to monitor the accumulation of amplified product during the PCR and HRM processes in order to obtain a set of melting temperature (Tm) values for a species. The DNA products after performing HRM were subjected to gel electrophoresis for DNA fingerprint and they were assessed for the percentage of monomorphic and polymorphic bands of each chosen primer as follow;
2. Materials and methods 2.1. Plant materials Four closely related Phyllanthus species comprising P. amarus (n = 6), P. urinaria (n = 8), P. debilis (n = 5) and P. virgatus (n = 6) (Supplementary Fig. 1), which are important herbaceous plants for hepatoprotection, were included for testing the feasibility of MFin-HRM in species differentiation. These samples were collected at Naresuan University, Phitsanulok, Thailand and were identified through a key from Flora of Thailand Euphorbiaceae (http://www. nationaalherbarium.nl/ThaiEuph/). 2.2. DNA isolation The leaves of Phyllanthus samples were used for DNA extraction using the Nucleospin® Plant II kit (Macherey-Nagel, Germany). The quality and quantity of DNA were measured using a Nanodrop and
number of monomorphic or polymorphic bands × 100 total bands 2
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The curve for the negative derivative of fluorescence (F) over temperature (T) (dF/dt) primarily displayed the Tm with the normalised raw curve depicting the decreasing fluorescence vs. increasing temperature. The melting profile of each species obtained from this technique provided many peaks, the so-called “melting fingerprint”, resulting from the presence of multiple loci produced from the ISSR method. In addition, we generated normalised melting curves (Wittwer et al., 2003) by setting pre- and post-melt normalisation regions to define the temperature boundaries and different melting curves, with P. amarus as the reference species. In the present study, the conceptual idea of MFin-HRM is that the different species would provide distinctive sets of molecular markers (multiloci) leading to different sets of melting peaks based on a single primer, called “melting fingerprints”.
Fig. 1. ISSR fingerprint of P. amarus derived from ten ISSR primers. The numbers 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 indicate ISSR primers including UBC873, UBC877, UBC827, UBC820, UBC825, UBC816, UBC813, UBC810, UBC802 and UBC807, respectively.
2.4. Sensitivity of MFin-HRM To determine the lowest concentration producing a clear and reproducible melting fingerprint by MFin-HRM, genomic DNA of P. amarus and P. urinaria at a starting concentration of 10 ng/μl was serially diluted to 1, 0.1, 0.01 and 0.001 ng/μl; 1 μl of each dilution was then added to each reaction to obtain final concentrations of 1000, 100, 10, 1 and 0.1 pg/μl, respectively. Distilled water was used as a negative control.
followed by UBC827 (Fig. 2A) and UBC807 (Fig. 2C and Table 3) with 6 and 5 polymorphic bands, respectively. All DNA loci produced were the polymorphic band. In addition, three primers were used for generating “melting fingerprints” obtained from the ISSR fingerprints. Each of the three primers demonstrating different ISSR fingerprints by PCR provided multiple melting peaks. The melting fingerprints obtained from UBC807, UBC825 and UBC827 displayed at least 7, 7 and 8 dominant, different peaks, respectively, among the four species, as shown in Table 3.
2.5. Adulterant detection by MFin-HRM To test the ability of MFin-HRM in adulterant detection, DNA from P. amarus (PA, 10 ng/μl) and P. urinaria (PU, 10 ng/μl) were mixed together in different proportions, given as PA:PU in various concentration (ng/μl) ratios as follows: 10:0, 5:5, 2.5:7.5, 1.2:8.8, 0.6:9.4, 0.3:9.7, 0.1:9.9 and 0:10. DNA mixtures in these ratios were used as DNA templates for MFin-HRM in triplicate.
3.2. Optimal concentration of template DNA for the MFin-HRM method We varied the final concentrations of either P. amarus or P. urinaria from 0.1–1000 pg/μl in reactions for MFin-HRM using UBC807, UBC825 and UBC827. Notably, it was obvious that the concentrations of DNA template less than 100 pg/μl led to differences in the melting fingerprint of both species for each of the three primers (Fig. 3). In addition, we noted that the three primers produced no or shifted melting fingerprints from 10 pg/μl for P. amarus or less (Fig. 3A, C, E), while the melting fingerprints of P. urinaria were generated at all DNA concentrations (1–1000 pg/μl) of three primers with the exception of UBC827 showing no melting fingerprint at a concentration of 1 pg/μl (Fig. 3B, D, F).
2.6. Testing commercial products by MFin-HRM Eleven commercial products, including capsules, tablets, dried materials and tea infusion Phyllanthus products distributed at a local market in Thailand were used for species authentication by MFin-HRM to see whether they were consistent with their labelling (Table 2). All products were extracted for DNA using the Nucleospin® Plant II kit (Macherey-Nagel, Germany). DNA obtained from each product was used as templates for species authentication by the MFin-HRM method.
3.3. Adulterant detection using the MFin-HRM method As shown in Fig. 4, we tested the ability of the MFin-HRM method to detect admixtures containing DNA from P. amarus and P. urinaria at different proportions. The patterns of the melting fingerprints of P. amarus derived from the two primers UBC807 and UBC825 were gradually transformed to those of P. urinaria, corresponding to the increasing amount of P. urinaria (Fig. 3A-D), while UBC827 exhibited a rapid change in the melting profile from P. amarus to P. urinaria with less than 50% contamination (Fig. 4E-F). To determine the level of adulterant contamination by melting fingerprint, a normalisation plot was required because we can see the alteration of the normalisation graph of P. amarus similar to that of P. urinaria, especially for UBC807 in the temperature range of 81 °C to 83 °C (Fig. 4B).
3. Results 3.1. ISSR primer screening for MFin-HRM In a preliminary evaluation, we screened ten ISSR primers for the DNA fingerprint of P. amarus by conventional PCR. Five primers including UBC873, UBC827, UBC825, UBC810 and UBC807 produced positive and clear DNA patterns as shown in Fig. 1. Subsequently, these five primers were used to generate ISSR fingerprints for four Phyllanthus species: P. amarus, P. urinaria, P. debilis and P. virgatus. Three primers including UBC807, UBC825 and UBC827 enabled positive ISSR fingerprinting and showed a distinctive DNA pattern among the four species (Fig. 2A, B and C). UBC825 showed the highest number of polymorphic bands accounting for 10 bands (Fig. 1B and Table 3)
3.4. The reliability of MFin-HRM Based on the preliminary screening of the ISSR primers to generate melting fingerprints of Phyllanthus species, we found that primer UBC807 showed greater abilities than the others because (i) the optimal concentration for generating a constant melting fingerprint at lowest concentration was 10 pg/μl and (ii) it has the capacity to detect adulterants. UBC807 was tested for reliability in MFin-HRM using an increasing number of samples of each species as templates. UBC807 produced different melting fingerprints among four species (Fig. 5A-D)
Table 2 Commercial products of P. amarus distributed at a local market in Thailand. Product code
Product form
C1, C3, C4, C8, C10, C11 C5, C6, C7, C9 C2
capsule tea infusion and dried materials tablet
3
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Fig. 2. DNA fingerprints and melting fingerprints of four Phyllanthus species derived from the MFin-HRM method using the positive ISSR primers. A, B and C represent DNA fingerprints using UBC807, UBC825, UBC810, UBC873 and UBC827. D, E and F depict the melting fingerprints of MFin-HRM using UBC807, UBC25 and UBC827, respectively. PA, PU, PD and PV represent P. amarus, P. urinaria, P. debilis and P. virgatus, respectively. M indicates 100 bp DNA marker.
C3, C4, C8 and C11) had melting fingerprints matching that of P. amarus (Fig. 6A). In addition, although C10 gave a melting fingerprint, it did not match that of any of the four species; however, its DNA fingerprint on agarose gel electrophoresis agreed with P. amarus's DNA fingerprint and was also similar to C1, C2, C3, C4, C8 and C11 (Fig. 6B). Noticeably, the tea infusion/dried materials failed to generate DNA fingerprints (Fig. 6B).
that agreed with the results from the normalisation (Fig. 5E) and difference graphs (Fig. 5F), except for two P. urinaria samples (PU8 and PU9) that showed melting fingerprints that were distinct from the others. 3.5. The performance of MFin-HRM for species authentication of commercial products
4. Discussion
In the present study, we established the MFin-HRM method in aiding species authentication, and a performance test was done using commercial products of P. amarus sold in Thai local markets using UBC807 for generating melting fingerprints. Our results revealed that seven samples yielded melting fingerprints and six out of seven (C1, C2,
Currently, species identification of plants based on molecular tools is increasingly sought in various areas, especially in food and pharmaceutical science to control product quality and to assure consumer
Table 3 The DNA fingerprint analysis and the melting peaks present in the melting fingerprints of four Phyllanthus species obtained using three ISSR primers. ISSR primer
UBC807 UBC825 UBC827 a b
DNA fingerprint analysis
Melting peak (°C)a
no. of bandb
monomorphic
polymorphic
P. amarus
P. urinaria
P. debilis
P. virgatus
6 10 5
– – –
100% 100% 100%
79.83 80.13, 81.37 74.38, 78.23, 79.9
75.03, 79.77 76.69, 81.14 79.95, 81.49
74.50, 82.20 76.92, 79.71 79.41, 81.67
80.19, 82.80 74.85 79.36
Dominant peaks with a negative derivative of fluorescence (F) over temperature (T) (-dF/dt) of more than 4. Bands are clear to be noted by naked eyes. 4
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Fig. 3. Sensitivity test of MFin-HRM using three ISSR primers. The amount of either genomic DNA of P. amarus or P. urinaria at 1, 0.1, 0.01 and 0.001 ng was used for sensitivity test in 25-μl volume. UBC807 (A, B), UBC825 (C, D) and UBC827 (E, F) with P. amarus (A, C, E) and P. urinaria (B, D, F) as a model.
DNA fingerprint with high number of bands, it seemed that the number of total DNA bands do not strongly influence the number of peaks in melting fingerprints. For example, the DNA fingerprint of P. amarus using UBC827 have a high number of clear bands (6 bands) but its melting fingerprint showed only two major peaks consisting of one sharp peak and one stutter peak, possibly relating to the major bands (Fig. 2c and f). This indicates that the number of total bands derived from DNA fingerprint may not necessarily relate to the number of the melting peak obtained from MFin-HRM. However, we believe that the high number of bands with more than 10 can affect the well-defined melting peak. This should be done in further study. We postulated that it is a limitation in the resolution of the HRM method in producing a melting profile. On the other hand, we believe that although the maximum number of generated melting peaks in a run is three melting peaks, hundreds of different patterns of melting peaks can be created because of (i) the difference in the number of melting peaks (one, two and three peaks), (ii) the divergence in melting temperature of a peak and (iii) the unique characteristics of a peak. A previous study has reported the potential of melting fingerprints derived from RAPD conjugated with HRM for differentiating pathogenic Leptospira isolates (10 serovars), with a high degree of stability as indicated by the RAPD-HRM melting curve showing the homogenetic intra-serovar clustering and by the determination of genetic changes in some reference strains (Tulsiani
safety. Some plant species share common local names or similar morphological characters, leading to their interchangeable use due to misidentification. Additionally, correct species identification/authentication based on the remains of morphological features from damaged/ incomplete plant materials such as herbal products or food materials may be difficult or impossible; thus, examination of DNA remains is necessary for species investigation in those samples. As molecular techniques advance, a large number of established methods must continually be developed over the decades. Our current study improved the procedure to identify or authenticate plant species using HRM coupled with ISSR fingerprinting to generate multiple melting peaks derived from various DNA fragments (multiloci) from a single ISSR primer of a certain species, called a “melting fingerprint”. We designated this method “melting fingerprint-HRM” or “MFin-HRM”. Here, we established MFin-HRM and used Phyllanthus amarus with its allies including P. urinaria, P. debilis and P. virgatus as a case study to test the performance of MFin-HRM in aiding species taxonomy/authentication. Preliminarily, out of ten ISSR primers, three primers, UBC807, UBC825 and UBC827, gave independently different melting fingerprints of each species. The number of melting peaks appeared to relate to the major bands (high intensity in gel) of DNA fingerprint, however, it did not exceed three melting peaks for each primer despite the DNA fingerprint having more than three fragments. As we noted 5
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Fig. 4. Adulterant detection by MFin-HRM using the three ISSR primers. The admixture between P. amarus and P. urinaria in various concentration (ng/μl) ratios as follows: 10:0, 5:5, 2.5:7.5, 1.2:8.8, 0.6:9.4, 0.3:9.7, 0.1:9.9 and 0:10. The admixtures were created for melting fingerprint by HRM using UBC807 (A, B), UBC825 (C, D) and UBC827 (E, F).
as the presence of 3% P. amarus in P. urinaria rendered a dramatic change in the normalisation plot. These results indicated that the sensitivity of a suitable primer and melting peak was also specific to the plant species; thus, we suggest that the use of MFin-HRM to detect adulteration needs further study for specific cases. More importantly, known species involved in adulterant detection need to be co-tested each time testing is conducted, as a reference for pure samples. Our previous study employed Bar-HRM using the trnL region for detecting adulteration between P. amarus and P. urinaria and it seemed to be more straightforward than MFin-HRM (Buddhachat et al., 2015). This method needs to be improved for adulterant determination. Apart from the optimal final concentration of DNA template and adulterant detection ability, we exhibited the reliability of MFin-HRM using UBC807 for several individuals of each species, generating constant melting fingerprints. Yet, there were two samples of P. urinaria (PU8 and PU9) that showed divergent melting fingerprints from the others, and the ISSR fingerprints of these samples contained an additional DNA fragment at approximately 500 bp. This result indicated that the MFin-HRM method might also allow differentiation/authentication of varieties and strains of plant species. Moreover, we employed MFinHRM using UBC807 for testing various forms (capsule, tablet, tea infusion, dried materials) of herbal products labelled as P. amarus to
et al., 2012). In addition, we observed that changes in the concentration of the DNA template can shift the melting fingerprint. The appropriate final concentration of DNA recommended as the template for generating a melting fingerprint by MFin-HRM is more than 100 pg/μl. A small amount of DNA template can be used to give a constant DNA pattern but some DNA fragments in the DNA fingerprint seemed to fade out or disappear, resulting in the alteration of the melting fingerprint due to the interaction of different sequences of DNA molecules within a reaction. Moreover, we found that the kinds of primers and species can alter the sensitivity and consistency of making the melting fingerprint at different concentrations of DNA template. Adulterant detection by MFin-HRM was possible using UBC807 and UBC825 because a transformation of the melting fingerprints in melting profile plots and an obvious, gradual change of the relative signal of P. amarus to P. urinaria in normalisation plots were seen, especially with UBC807. Within a melting fingerprint, there were a few melting peaks for each species, but some peaks could be effectively used for monitoring the adulterant level, for instance, a melting peak of 82 °C derived from UBC807. Furthermore, the sensitivity in detecting the level of adulterant differed depending on the primer used. For example, UBC807 seemed to be sensitive to the amount of P. amarus in P. urinaria 6
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Fig. 5. The consistency of MFin-HRM using UBC807 for four Phyllanthus species. Genomic DNA of (A) P. amarus; n = 6, (B) P. urinaria; n = 8, (C) P. debilis; n = 5 and (D) P. virgatus; n = 6 were used as template for reproducibility of MFin-HRM to differentiate herbaceous Phyllanthus species. The normalisation (E) and difference (F) plots of the four species are illustrated.
fingerprint pattern of each single species, whereas Bar-HRM can produce an amplicon from a locus using a primer pair. The melting peaks of a species obtained from Bar-HRM can be similar to or overlapping that of another species, as reported in a study by Osathanunkul et al. (2016). Although increasing the number of barcode regions augmented the species resolution for a Thai medicinal plant, with the highest of 99%, such resolution required at least four barcodes including rpoC1 + trnL +rbcLB+rbcLC or rpoC1 + rbcLB+rbcLC+matK. Meanwhile, MFinHRM uses a single primer but can generate data for multiple loci (Osathanunkul et al., 2016). However, what we have one important concern about MFin-HRM is to likely occur the variation among population (between individuals) within same species as ISSR primers would produce the variation of DNA bands between individual. Therefore, the suitable ISSR primers or other DNA fingerprint methods
determine whether they were made of authentic P. amarus or not. For seven out of ten products, we obtained a melting fingerprint, and all but one of these (C10) agreed with that of P. amarus. Although C3 had a similar melting fingerprint to that of P. amarus, its ISSR fingerprint differed from P. amarus's ISSR fingerprint. We postulated that the C3 product may have had other constituents admixed in a small amount, leading to the presence of extra DNA bands in the agarose gel. There were three products in the form of tea infusions/dried materials that were unable to yield any melting fingerprints. We would need to improve DNA extraction from these forms. What makes MFin-HRM more compelling than Bar-HRM as a choice for species identification is that the MFin-HRM method uses only a single primer to generate a melting fingerprint resulting from multiloci as a consequence of producing a possible large number of melting 7
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Fig. 6. Species authentication of commercial products labelled as Phyllanthus amarus using MFin-HRM with UBC807. (A) Melting fingerprint and (B) DNA fingerprint. PA, PU, PD and PV represent P. amarus, P. urinaria, P. debilis and P. virgatus, respectively. M indicates 100 bp DNA marker. The numbers indicate the code of commercial products C1-C11 and N represents the negative control.
species diagnosis, a wide range of relevant plant species needs to be analysed to create a reference database. Further, this method can be utilised for the quality control of medicinal plant products in the industrial scale. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.plgene.2019.100203.
(e.g. start codon targeted (SCoT) or sequence related amplified polymorphism (SRAP)) that enable to generate the different DNA fingerprint only between species but same within species are investigated for further studies. We suggest the protocol for species identification of other medicinal plant species using MFin-HRM as the follow. (i) DNA used for species identification using MFin-HRM should be good quality and quantity (more than 1 ng/μl in concentration). (ii) DNA is used for screening the suitable primer for creating DNA fingerprint by agarose gel electrophoresis that are different among species and high percentage of polymorphic band. (iii) the proper primers would be extended to create the melting fingerprint by MFin-HRM and the melting peak observed should have a negative derivative of fluorescence (F) over temperature (T) (-dF/dt) of more than 4 in order to facilitate for observation. (iv) Reproducibility test of MFin-HRM for species identification would be assessed using several individual of each species.
Author contributions Kittisak Buddhachat (KB) conducted and designed all experiments. Phanupong Changtor (PC) and Sunatcha Ninket (SN) performed part of the experiments. KB and SN analysed the data. KB wrote the manuscript. KB, PC and SN have read the manuscript.
Declaration of Competing Interest 5. Conclusions
The authors declare that there was no conflict of interest regarding the publication of this paper.
Taken together, our findings revealed that a melting curved-based genotyping method, MFin-HRM, provides a novel and rapid technique with high reliability and reproducibility to aid species identification or authentication for four Phyllanthus species in a closed tube and can be expanded for a large number of species in further studies. Before MFinHRM can be applied for further practical use as a routine method for
Acknowledgements This research was financially supported by research from the Faculty of Science, Naresuan University, 2018 (R2561E001). 8
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References
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Balasaravanan, T., Chezhian, P., Kamalakannan, R., Yasodha, R., Varghese, M., Gurumurthi, K., Ghosh, M., 2006. Identification of species-diagnostic ISSR markers for six Eucalyptus species. Silvae Genet 55, 119–122. Bosmali, I., Ganopoulos, I., Madesis, P., Tsaftaris, A., 2012. Microsatellite and DNAbarcode regions typing combined with high resolution melting (HRM) analysis for food forensic uses: a case study on lentils (Lens culinaris). Food Res. Int. 46, 141–147. Buddhachat, K., Osathanunkul, M., Madesis, P., Chomdej, S., Ongchai, S., 2015. Authenticity analyses of Phyllanthus amarus using barcoding coupled with HRM analysis to control its quality for medicinal plant product. Gene 573, 84–90. Buddhachat, K., Chomdej, S., Pradit, W., Nganvongpanit, K., Ongchai, S., 2017. In vitro chondroprotective potential of extracts obtained from various Phyllantus species. Planta Med. 83, 87–96. Chen, S., Yao, H., Han, J., Liu, C., Song, J., Shi, L., Zhu, Y., 2010. Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species. PLoS One 5, 1–8 e8613. Dangi, R.S., Lagu, M.D., Choudhary, L.B., Ranjekar, P.K., Gupta, V.S., 2004. Assessment of genetic diversity in Trigonella foenum-graecum and Trigonella caerulea using ISSR and RAPD markers. BMC Plant Biol. 4, 13. https://doi.org/10.1186/1471-2229-4-13. Dawnay, N., Ogden, R., McEwing, R., Carvalho, G.R., Thorpe, R.S., 2007. Validation of the barcoding gene COI for use in forensic genetic species identification. Forensic Sci. Int. 173, 1–6. Ereqat, S., Bar-Gal, G.K., Nasereddin, A., Azmi, K., Qaddomi, S.E., Greenblatt, C.L., Spigelman, M., Abdeen, Z., 2010. Rapid differentiation of Mycobacterium tuberculosis and M. bovis by high-resolution melt curve analysis. J. Clin. Microbiol. 48, 4269–4272. Faria, M.A., Magalhães, A., Nunes, M.E., Oliveira, M.B.P.P., 2013. High resolution melting of trnL amplicons in fruit juices authentication. Food Control 33, 136–141. Fazekas, A.J., Burgess, K.S., Kesanakurti, P.R., Graham, S.W., Newmaster, S.G., Husband, B.C., Percy, D.M., 2008. Multiple multilocus DNA barcodes from the plastid genome discriminate plant species equally well. PLoS One 3, e2802. Ganopoulos, I., Bazakos, C., Madesis, P., 2013. Barcode DNA high-resolution melting (Bar-HRM) analysis as a novel close-tubed and accurate tool for olive oil forensic use. J. Sci. Food Agric. 93, 2281–2286. Garcia, A.A., Benchimol, L.L., Barbosa, A.M., Geraldi, I.O., Souza Jr., C.L., Souza, A.P.D., 2004. Comparison of RAPD, RFLP, AFLP and SSR markers for diversity studies in tropical maize inbred lines. Genet. Mol. Biol. 27, 579–588. Group CPW, Hollingsworth, P.M., Forrest, L.L., Spouge, J.L., Hajibabaei, M., Ratnasingham, S., van der Bank, M., Chase, M.W., 2009. A DNA barcode for land plants. Proc. Natl. Acad. Sci. U. S. A. 106, 12794–12797. Gupta, M., Chyi, Y.S., Romero-Severson, J., Owen, J.L., 1994. Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats. Theor. Appl. Genet. 89, 998–1006. Hajibabaei, M., Singer, G.A., Hebert, P.D., Hickey, D.A., 2007. DNA barcoding: how it complements taxonomy, molecular phylogenetics and population genetics. Trends Genet. 23, 167–172. Hebert, P.D., Gregory, T.R., 2005. The promise of DNA barcoding for taxonomy. Syst. Biol. 54, 852–859. Hebert, P.D., Penton, E.H., Burns, J.M., Janzen, D.H., Hallwachs, W., 2004a. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc. Natl. Acad. Sci. U. S. A. 101, 14812–14817.
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Contents lists available at ScienceDirect
Plant Gene journal homepage: www.elsevier.com/locate/plantgene
An accurate and rapid method for species identification in plants: Melting fingerprint-high resolution melting (MFin-HRM) analysis
T
⁎
Kittisak Buddhachat , Phanupong Changtor, Sunatcha Ninket Department of Biology, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand
A R T I C LE I N FO
A B S T R A C T
Keywords: Melting temperature Medicinal plant Phyllanthus ISSR Real-time PCR DNA fingerprint
Species identification is required in various areas such as systematics, ecology, conservation, evolution, agriculture, forensics, pharmacology, food science, and even industry. Yet the number of taxonomists who identify based on morphological observations is limited, and some materials are incomplete and/or processed, leading to difficulty in species identification by morphological features. Thus, the molecules remaining inside, especially DNA, often have been extensively used to aid species identification. Here, we established an accurate and rapid method, melting fingerprint-high resolution melting or “MFin-HRM,” to facilitate species identification/authentication based on the melting profile of the DNA fingerprint obtained from inter simple sequence repeat (ISSR) coupled with HRM, or the “melting fingerprint”. This method generates a melting fingerprint specific to a certain species. In this study, Phyllanthus amarus and its closely related species were investigated as a model to test the performance of MFin-HRM in species identification. A final concentration of DNA template of more than 100 pg/μl is suitable for obtaining a consistent melting fingerprint by MFin-HRM. In addition, MFin-HRM can be used for adulterant determination, and its sensitivity and specificity depend on the ISSR primer chosen for generating the melting fingerprint. These results demonstrate that MFin-HRM is a reliable and fast method for species identification/authentication in a closed tube.
1. Introduction
characters, contributing to misuse that jeopardises the customers' safety and the quality of the medicinal products. Herbaceous Phyllanthus spp. is a medicinal plant for hepatoprotection and antiarthitis; however, the bioactive compounds, phyllanthin and hypophyllanthin, are present in high amounts in Phyllanthus amarus but little or none are found in other, related species (Tripathi et al., 2006; Buddhachat et al., 2017). Species confirmation, therefore, is an important step for quality control of the herbal products. With advancing biotechnology, several newly emerging molecular techniques for species identification have been developed and amended to improve their performance. Initially, DNA fingerprints derived from amplified fragment length polymorphism (AFLP) (Vos et al., 1995), restriction fragment length polymorphism (RFLP) (Lander and Botstein, 1989), random amplified polymorphic DNA (RAPD) (Welsh and McClelland, 1990; Williams et al., 1990) and inter-simple sequence repeat (ISSR) techniques (Gupta et al., 1994) have been extensively used for developing DNA markers in several applications such as
Species identification is a crucial task for various disciplines such as taxonomy, systematics, evolution, genetics, ecology, conservation, agriculture, forensics, pharmacology, food science and the study of industrial medicinal plants (Hebert and Gregory, 2005; Hajibabaei et al., 2007; Dawnay et al., 2007; Rasmussen and Morrissey, 2008; Chen et al., 2010; Bosmali et al., 2012; Buddhachat et al., 2015; Osathanunkul et al., 2016). Traditionally, plant species identification based on morphological observations has been used by experts and specialised taxonomists; thus, it has been restricted for non-experts who require species diagnosis for their purposes. In addition, specimens may be incomplete due to damage or processing, creating hurdles for species delineation based on morphological characters. For example, for industrial medicinal plants, the raw materials for manufacturing need to be confirmed as to whether they are the correct species because several medicinal plant species can share a common name or morphological
Abbreviations: DNA, deoxyribonucleic acid; PCR, polymerase chain reaction; ISSR, inter simple sequence repeat; HRM, high resolution melting; MFin-HRM, melting fingerprint derived from high resolution melting; RAPD, random amplified polymorphic DNA; RFLP, restriction fragment length polymorphism; AFLP, amplified fragment length polymorphism; Bar-HRM, DNA barcode coupled with high resolution melting; Tm, melting temperature; rbcL, Ribulose bisphosphate carboxylase large chain; matK, maturase K ⁎ Corresponding author. E-mail address: [email protected] (K. Buddhachat). https://doi.org/10.1016/j.plgene.2019.100203 Received 13 June 2019; Received in revised form 19 August 2019; Accepted 6 September 2019 Available online 10 September 2019 2352-4073/ © 2019 Published by Elsevier B.V.
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genetic improvement as well as for species-specific markers. These methods enable species differentiability without extensive DNA sequence information in both plants e.g. Dendrobium (Wang et al., 2009) and Eucalyptus (Balasaravanan et al., 2006) species and animals e.g. meat quality control (Huang et al., 2003). The RAPD and ISSR methods are less complex compared to AFLP and RFLP; therefore, the two former methods have gained more attention than the latter two, which consist of various steps (Powell et al., 1996; Patzak, 2001; Dangi et al., 2004; Garcia et al., 2004). Since the early 2000s, with Hebert et al. (2004a) as the initiators of the technique, DNA barcoding, in which a short-standardised DNA sequence is used to identify a specific species with high accuracy and reliability, has been extensively and widely used in species identification. DNA barcode repositories of many kinds of organisms such as bacteria, fungi, insects, fish, birds and plants have been created from massive amounts of data within less than twenty years (Hebert and Gregory, 2005; Hebert et al., 2004a; Hebert et al., 2004b; Ratnasingham and Hebert, 2007; Ward et al., 2009; Group et al., 2009). Several studies suggest that a combination of various barcodes is required for species identification to enable higher species resolution (Group et al., 2009; Kress et al., 2005; Kress and Erickson, 2007; Fazekas et al., 2008; Hollingsworth et al., 2011). For example, the CBOL Plant Working Group (2009) recommended a combination of rbcL and matK in plant species identification. Recently, DNA barcoding was conjugated with high resolution melting analysis (HRM) in a technique called “Bar-HRM” to facilitate species identification without DNA sequencing, allowing the completion of the reaction in a closed tube, leading to a cost-effective and rapid method. Bar-HRM has been used to identify a wide range of organisms, especially modified, damaged, processed, incomplete or invisible specimens (Osathanunkul et al., 2016; Ereqat et al., 2010; Ganopoulos et al., 2013; Faria et al., 2013; Buddhachat et al., 2015; Sun et al., 2016). Unfortunately, Bar-HRM has the limitation of having been developed as a universal method for a species group with a small number of species, due to the fact that Bar-HRM produces data for a single locus, given as a narrow range of melting temperatures (Tm). As a result, there is inadequate resolution of Bar-HRM to differentiate among many species in a large species group. Although Bar-HRM allows a combination of multiloci for identification, an overlapping of Tm occurred for Thai medicinal plant species (Osathanunkul et al., 2016). To increase species resolution by HRM, a DNA fingerprint such as RAPD or ISSR would be more powerful in differentiating many species because a single primer can produce multiple DNA bands from multiple loci in the plant genome (Welsh and McClelland, 1990; Williams et al., 1990; Gupta et al., 1994). Here, we evaluate the feasibility of combining the DNA fingerprint method with HRM, in a technique we call “melting fingerprint HRM” or “MFin-HRM,” to distinguish among closely related Phyllanthus species as a model.
Table 1 ISSR primers used in this study. ISSR primer
Sequence (5′➔3′)
UBC802 UBC807 UBC810 UBC813 UBC816 UBC820 UBC825 UBC827 UBC873 UBC877
ATA TAT ATA TAT ATA TG AGA GAG AGA GAG AGA GT GAG AGA GAG AGA GAG AT CTC TCT CTC TCT CTC TT CAC ACA CAC ACA CAC AT GTG TGT GTG TGT GTG TC ACA CAC ACA CAC ACA CT ACA CAC ACA CAC ACA CG GAC AGA CAG ACA GAC A TGC ATG CAT GCA TGC A
1.5% agarose gel electrophoresis with visualisation under ultra-violet (UV) light by a UV transmitter. DNA was dissolved with tris-EDTA buffer and stored at −20 °C for further use. 2.3. DNA fingerprint (Fin)-high resolution melting (HRM) of inter simple sequence repeats (ISSR) In this study, ISSR was chosen for generating a DNA fingerprint because of (i) no requirement of DNA sequence information, (ii) the small amount of DNA required, (iii) use of a single primer and (iv) high reproducibility (Gupta et al., 1994). Preliminarily, P. amarus was used as the DNA template for generating a DNA fingerprint using 10 different ISSR primers (Table 1). A total of 10 ISSR primers were screened to choose suitable primers that showed clear and multiple bands in the DNA fingerprint. Subsequently, the positive primers were used for making DNA fingerprints for four Phyllanthus species in order to select primers producing DNA patterns in all tested species. Each reaction contained 1× reaction buffer (RBC Bioscience, Taiwan); 1.5 mM MgCl2 (RBC Bioscience, Taiwan); 0.2 mM dNTPs (Thermo Scientific, USA); 0.4 mM primer; and 1 U Taq DNA polymerase (RBC Bioscience, Taiwan), with adjustment with deionised distilled water to a final volume of 25 μl. The PCR was performed under the following conditions: 95 °C for 5 min, 40 cycles of 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR products were detected by gel electrophoresis using a 2% agarose gel with GelRed (Biotium, USA) as the visualiser under UV light. The primers that gave distinct DNA patterns among species were selected for HRM analysis to acquire the melting profiles of the different Phyllanthus species including P. amarus, P. urinaria, P. airy-shawii, P. debillis and P. virgatus. DNA amplification and HRM were performed using real-time PCR on the LightCycler® 480 instrument II (Roche Life Science, Germany). The reaction mixture for real-time PCR and HRM analysis was made in a total volume of 10 μl containing 5 μl of 2× SensiFAST HRM mix (Bioline, England), 0.4 μM primer and 1 μl of 10 ng/μl DNA. The amplification protocol was conducted using an initial denaturing step at 95 °C for 5 min followed by 40 cycles of 95 °C for 1 min and 55 °C for 1 min. The final extension was done at 72 °C for 7 min. The fluorescence data were acquired at the end of each cycle during DNA amplification. Before HRM, the products were denatured at 95 °C for 15 s, and then annealed at 50 °C for 15 s to randomly form DNA duplexes. For HRM experiments, the fluorescence data were collected at every 0.1 °C/cycle for 2 s per cycle in the temperature range of 65 °C to 95 °C. EvaGreen® was the fluorescent dye used to monitor the accumulation of amplified product during the PCR and HRM processes in order to obtain a set of melting temperature (Tm) values for a species. The DNA products after performing HRM were subjected to gel electrophoresis for DNA fingerprint and they were assessed for the percentage of monomorphic and polymorphic bands of each chosen primer as follow;
2. Materials and methods 2.1. Plant materials Four closely related Phyllanthus species comprising P. amarus (n = 6), P. urinaria (n = 8), P. debilis (n = 5) and P. virgatus (n = 6) (Supplementary Fig. 1), which are important herbaceous plants for hepatoprotection, were included for testing the feasibility of MFin-HRM in species differentiation. These samples were collected at Naresuan University, Phitsanulok, Thailand and were identified through a key from Flora of Thailand Euphorbiaceae (http://www. nationaalherbarium.nl/ThaiEuph/). 2.2. DNA isolation The leaves of Phyllanthus samples were used for DNA extraction using the Nucleospin® Plant II kit (Macherey-Nagel, Germany). The quality and quantity of DNA were measured using a Nanodrop and
number of monomorphic or polymorphic bands × 100 total bands 2
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The curve for the negative derivative of fluorescence (F) over temperature (T) (dF/dt) primarily displayed the Tm with the normalised raw curve depicting the decreasing fluorescence vs. increasing temperature. The melting profile of each species obtained from this technique provided many peaks, the so-called “melting fingerprint”, resulting from the presence of multiple loci produced from the ISSR method. In addition, we generated normalised melting curves (Wittwer et al., 2003) by setting pre- and post-melt normalisation regions to define the temperature boundaries and different melting curves, with P. amarus as the reference species. In the present study, the conceptual idea of MFin-HRM is that the different species would provide distinctive sets of molecular markers (multiloci) leading to different sets of melting peaks based on a single primer, called “melting fingerprints”.
Fig. 1. ISSR fingerprint of P. amarus derived from ten ISSR primers. The numbers 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 indicate ISSR primers including UBC873, UBC877, UBC827, UBC820, UBC825, UBC816, UBC813, UBC810, UBC802 and UBC807, respectively.
2.4. Sensitivity of MFin-HRM To determine the lowest concentration producing a clear and reproducible melting fingerprint by MFin-HRM, genomic DNA of P. amarus and P. urinaria at a starting concentration of 10 ng/μl was serially diluted to 1, 0.1, 0.01 and 0.001 ng/μl; 1 μl of each dilution was then added to each reaction to obtain final concentrations of 1000, 100, 10, 1 and 0.1 pg/μl, respectively. Distilled water was used as a negative control.
followed by UBC827 (Fig. 2A) and UBC807 (Fig. 2C and Table 3) with 6 and 5 polymorphic bands, respectively. All DNA loci produced were the polymorphic band. In addition, three primers were used for generating “melting fingerprints” obtained from the ISSR fingerprints. Each of the three primers demonstrating different ISSR fingerprints by PCR provided multiple melting peaks. The melting fingerprints obtained from UBC807, UBC825 and UBC827 displayed at least 7, 7 and 8 dominant, different peaks, respectively, among the four species, as shown in Table 3.
2.5. Adulterant detection by MFin-HRM To test the ability of MFin-HRM in adulterant detection, DNA from P. amarus (PA, 10 ng/μl) and P. urinaria (PU, 10 ng/μl) were mixed together in different proportions, given as PA:PU in various concentration (ng/μl) ratios as follows: 10:0, 5:5, 2.5:7.5, 1.2:8.8, 0.6:9.4, 0.3:9.7, 0.1:9.9 and 0:10. DNA mixtures in these ratios were used as DNA templates for MFin-HRM in triplicate.
3.2. Optimal concentration of template DNA for the MFin-HRM method We varied the final concentrations of either P. amarus or P. urinaria from 0.1–1000 pg/μl in reactions for MFin-HRM using UBC807, UBC825 and UBC827. Notably, it was obvious that the concentrations of DNA template less than 100 pg/μl led to differences in the melting fingerprint of both species for each of the three primers (Fig. 3). In addition, we noted that the three primers produced no or shifted melting fingerprints from 10 pg/μl for P. amarus or less (Fig. 3A, C, E), while the melting fingerprints of P. urinaria were generated at all DNA concentrations (1–1000 pg/μl) of three primers with the exception of UBC827 showing no melting fingerprint at a concentration of 1 pg/μl (Fig. 3B, D, F).
2.6. Testing commercial products by MFin-HRM Eleven commercial products, including capsules, tablets, dried materials and tea infusion Phyllanthus products distributed at a local market in Thailand were used for species authentication by MFin-HRM to see whether they were consistent with their labelling (Table 2). All products were extracted for DNA using the Nucleospin® Plant II kit (Macherey-Nagel, Germany). DNA obtained from each product was used as templates for species authentication by the MFin-HRM method.
3.3. Adulterant detection using the MFin-HRM method As shown in Fig. 4, we tested the ability of the MFin-HRM method to detect admixtures containing DNA from P. amarus and P. urinaria at different proportions. The patterns of the melting fingerprints of P. amarus derived from the two primers UBC807 and UBC825 were gradually transformed to those of P. urinaria, corresponding to the increasing amount of P. urinaria (Fig. 3A-D), while UBC827 exhibited a rapid change in the melting profile from P. amarus to P. urinaria with less than 50% contamination (Fig. 4E-F). To determine the level of adulterant contamination by melting fingerprint, a normalisation plot was required because we can see the alteration of the normalisation graph of P. amarus similar to that of P. urinaria, especially for UBC807 in the temperature range of 81 °C to 83 °C (Fig. 4B).
3. Results 3.1. ISSR primer screening for MFin-HRM In a preliminary evaluation, we screened ten ISSR primers for the DNA fingerprint of P. amarus by conventional PCR. Five primers including UBC873, UBC827, UBC825, UBC810 and UBC807 produced positive and clear DNA patterns as shown in Fig. 1. Subsequently, these five primers were used to generate ISSR fingerprints for four Phyllanthus species: P. amarus, P. urinaria, P. debilis and P. virgatus. Three primers including UBC807, UBC825 and UBC827 enabled positive ISSR fingerprinting and showed a distinctive DNA pattern among the four species (Fig. 2A, B and C). UBC825 showed the highest number of polymorphic bands accounting for 10 bands (Fig. 1B and Table 3)
3.4. The reliability of MFin-HRM Based on the preliminary screening of the ISSR primers to generate melting fingerprints of Phyllanthus species, we found that primer UBC807 showed greater abilities than the others because (i) the optimal concentration for generating a constant melting fingerprint at lowest concentration was 10 pg/μl and (ii) it has the capacity to detect adulterants. UBC807 was tested for reliability in MFin-HRM using an increasing number of samples of each species as templates. UBC807 produced different melting fingerprints among four species (Fig. 5A-D)
Table 2 Commercial products of P. amarus distributed at a local market in Thailand. Product code
Product form
C1, C3, C4, C8, C10, C11 C5, C6, C7, C9 C2
capsule tea infusion and dried materials tablet
3
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Fig. 2. DNA fingerprints and melting fingerprints of four Phyllanthus species derived from the MFin-HRM method using the positive ISSR primers. A, B and C represent DNA fingerprints using UBC807, UBC825, UBC810, UBC873 and UBC827. D, E and F depict the melting fingerprints of MFin-HRM using UBC807, UBC25 and UBC827, respectively. PA, PU, PD and PV represent P. amarus, P. urinaria, P. debilis and P. virgatus, respectively. M indicates 100 bp DNA marker.
C3, C4, C8 and C11) had melting fingerprints matching that of P. amarus (Fig. 6A). In addition, although C10 gave a melting fingerprint, it did not match that of any of the four species; however, its DNA fingerprint on agarose gel electrophoresis agreed with P. amarus's DNA fingerprint and was also similar to C1, C2, C3, C4, C8 and C11 (Fig. 6B). Noticeably, the tea infusion/dried materials failed to generate DNA fingerprints (Fig. 6B).
that agreed with the results from the normalisation (Fig. 5E) and difference graphs (Fig. 5F), except for two P. urinaria samples (PU8 and PU9) that showed melting fingerprints that were distinct from the others. 3.5. The performance of MFin-HRM for species authentication of commercial products
4. Discussion
In the present study, we established the MFin-HRM method in aiding species authentication, and a performance test was done using commercial products of P. amarus sold in Thai local markets using UBC807 for generating melting fingerprints. Our results revealed that seven samples yielded melting fingerprints and six out of seven (C1, C2,
Currently, species identification of plants based on molecular tools is increasingly sought in various areas, especially in food and pharmaceutical science to control product quality and to assure consumer
Table 3 The DNA fingerprint analysis and the melting peaks present in the melting fingerprints of four Phyllanthus species obtained using three ISSR primers. ISSR primer
UBC807 UBC825 UBC827 a b
DNA fingerprint analysis
Melting peak (°C)a
no. of bandb
monomorphic
polymorphic
P. amarus
P. urinaria
P. debilis
P. virgatus
6 10 5
– – –
100% 100% 100%
79.83 80.13, 81.37 74.38, 78.23, 79.9
75.03, 79.77 76.69, 81.14 79.95, 81.49
74.50, 82.20 76.92, 79.71 79.41, 81.67
80.19, 82.80 74.85 79.36
Dominant peaks with a negative derivative of fluorescence (F) over temperature (T) (-dF/dt) of more than 4. Bands are clear to be noted by naked eyes. 4
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Fig. 3. Sensitivity test of MFin-HRM using three ISSR primers. The amount of either genomic DNA of P. amarus or P. urinaria at 1, 0.1, 0.01 and 0.001 ng was used for sensitivity test in 25-μl volume. UBC807 (A, B), UBC825 (C, D) and UBC827 (E, F) with P. amarus (A, C, E) and P. urinaria (B, D, F) as a model.
DNA fingerprint with high number of bands, it seemed that the number of total DNA bands do not strongly influence the number of peaks in melting fingerprints. For example, the DNA fingerprint of P. amarus using UBC827 have a high number of clear bands (6 bands) but its melting fingerprint showed only two major peaks consisting of one sharp peak and one stutter peak, possibly relating to the major bands (Fig. 2c and f). This indicates that the number of total bands derived from DNA fingerprint may not necessarily relate to the number of the melting peak obtained from MFin-HRM. However, we believe that the high number of bands with more than 10 can affect the well-defined melting peak. This should be done in further study. We postulated that it is a limitation in the resolution of the HRM method in producing a melting profile. On the other hand, we believe that although the maximum number of generated melting peaks in a run is three melting peaks, hundreds of different patterns of melting peaks can be created because of (i) the difference in the number of melting peaks (one, two and three peaks), (ii) the divergence in melting temperature of a peak and (iii) the unique characteristics of a peak. A previous study has reported the potential of melting fingerprints derived from RAPD conjugated with HRM for differentiating pathogenic Leptospira isolates (10 serovars), with a high degree of stability as indicated by the RAPD-HRM melting curve showing the homogenetic intra-serovar clustering and by the determination of genetic changes in some reference strains (Tulsiani
safety. Some plant species share common local names or similar morphological characters, leading to their interchangeable use due to misidentification. Additionally, correct species identification/authentication based on the remains of morphological features from damaged/ incomplete plant materials such as herbal products or food materials may be difficult or impossible; thus, examination of DNA remains is necessary for species investigation in those samples. As molecular techniques advance, a large number of established methods must continually be developed over the decades. Our current study improved the procedure to identify or authenticate plant species using HRM coupled with ISSR fingerprinting to generate multiple melting peaks derived from various DNA fragments (multiloci) from a single ISSR primer of a certain species, called a “melting fingerprint”. We designated this method “melting fingerprint-HRM” or “MFin-HRM”. Here, we established MFin-HRM and used Phyllanthus amarus with its allies including P. urinaria, P. debilis and P. virgatus as a case study to test the performance of MFin-HRM in aiding species taxonomy/authentication. Preliminarily, out of ten ISSR primers, three primers, UBC807, UBC825 and UBC827, gave independently different melting fingerprints of each species. The number of melting peaks appeared to relate to the major bands (high intensity in gel) of DNA fingerprint, however, it did not exceed three melting peaks for each primer despite the DNA fingerprint having more than three fragments. As we noted 5
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Fig. 4. Adulterant detection by MFin-HRM using the three ISSR primers. The admixture between P. amarus and P. urinaria in various concentration (ng/μl) ratios as follows: 10:0, 5:5, 2.5:7.5, 1.2:8.8, 0.6:9.4, 0.3:9.7, 0.1:9.9 and 0:10. The admixtures were created for melting fingerprint by HRM using UBC807 (A, B), UBC825 (C, D) and UBC827 (E, F).
as the presence of 3% P. amarus in P. urinaria rendered a dramatic change in the normalisation plot. These results indicated that the sensitivity of a suitable primer and melting peak was also specific to the plant species; thus, we suggest that the use of MFin-HRM to detect adulteration needs further study for specific cases. More importantly, known species involved in adulterant detection need to be co-tested each time testing is conducted, as a reference for pure samples. Our previous study employed Bar-HRM using the trnL region for detecting adulteration between P. amarus and P. urinaria and it seemed to be more straightforward than MFin-HRM (Buddhachat et al., 2015). This method needs to be improved for adulterant determination. Apart from the optimal final concentration of DNA template and adulterant detection ability, we exhibited the reliability of MFin-HRM using UBC807 for several individuals of each species, generating constant melting fingerprints. Yet, there were two samples of P. urinaria (PU8 and PU9) that showed divergent melting fingerprints from the others, and the ISSR fingerprints of these samples contained an additional DNA fragment at approximately 500 bp. This result indicated that the MFin-HRM method might also allow differentiation/authentication of varieties and strains of plant species. Moreover, we employed MFinHRM using UBC807 for testing various forms (capsule, tablet, tea infusion, dried materials) of herbal products labelled as P. amarus to
et al., 2012). In addition, we observed that changes in the concentration of the DNA template can shift the melting fingerprint. The appropriate final concentration of DNA recommended as the template for generating a melting fingerprint by MFin-HRM is more than 100 pg/μl. A small amount of DNA template can be used to give a constant DNA pattern but some DNA fragments in the DNA fingerprint seemed to fade out or disappear, resulting in the alteration of the melting fingerprint due to the interaction of different sequences of DNA molecules within a reaction. Moreover, we found that the kinds of primers and species can alter the sensitivity and consistency of making the melting fingerprint at different concentrations of DNA template. Adulterant detection by MFin-HRM was possible using UBC807 and UBC825 because a transformation of the melting fingerprints in melting profile plots and an obvious, gradual change of the relative signal of P. amarus to P. urinaria in normalisation plots were seen, especially with UBC807. Within a melting fingerprint, there were a few melting peaks for each species, but some peaks could be effectively used for monitoring the adulterant level, for instance, a melting peak of 82 °C derived from UBC807. Furthermore, the sensitivity in detecting the level of adulterant differed depending on the primer used. For example, UBC807 seemed to be sensitive to the amount of P. amarus in P. urinaria 6
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Fig. 5. The consistency of MFin-HRM using UBC807 for four Phyllanthus species. Genomic DNA of (A) P. amarus; n = 6, (B) P. urinaria; n = 8, (C) P. debilis; n = 5 and (D) P. virgatus; n = 6 were used as template for reproducibility of MFin-HRM to differentiate herbaceous Phyllanthus species. The normalisation (E) and difference (F) plots of the four species are illustrated.
fingerprint pattern of each single species, whereas Bar-HRM can produce an amplicon from a locus using a primer pair. The melting peaks of a species obtained from Bar-HRM can be similar to or overlapping that of another species, as reported in a study by Osathanunkul et al. (2016). Although increasing the number of barcode regions augmented the species resolution for a Thai medicinal plant, with the highest of 99%, such resolution required at least four barcodes including rpoC1 + trnL +rbcLB+rbcLC or rpoC1 + rbcLB+rbcLC+matK. Meanwhile, MFinHRM uses a single primer but can generate data for multiple loci (Osathanunkul et al., 2016). However, what we have one important concern about MFin-HRM is to likely occur the variation among population (between individuals) within same species as ISSR primers would produce the variation of DNA bands between individual. Therefore, the suitable ISSR primers or other DNA fingerprint methods
determine whether they were made of authentic P. amarus or not. For seven out of ten products, we obtained a melting fingerprint, and all but one of these (C10) agreed with that of P. amarus. Although C3 had a similar melting fingerprint to that of P. amarus, its ISSR fingerprint differed from P. amarus's ISSR fingerprint. We postulated that the C3 product may have had other constituents admixed in a small amount, leading to the presence of extra DNA bands in the agarose gel. There were three products in the form of tea infusions/dried materials that were unable to yield any melting fingerprints. We would need to improve DNA extraction from these forms. What makes MFin-HRM more compelling than Bar-HRM as a choice for species identification is that the MFin-HRM method uses only a single primer to generate a melting fingerprint resulting from multiloci as a consequence of producing a possible large number of melting 7
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Fig. 6. Species authentication of commercial products labelled as Phyllanthus amarus using MFin-HRM with UBC807. (A) Melting fingerprint and (B) DNA fingerprint. PA, PU, PD and PV represent P. amarus, P. urinaria, P. debilis and P. virgatus, respectively. M indicates 100 bp DNA marker. The numbers indicate the code of commercial products C1-C11 and N represents the negative control.
species diagnosis, a wide range of relevant plant species needs to be analysed to create a reference database. Further, this method can be utilised for the quality control of medicinal plant products in the industrial scale. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.plgene.2019.100203.
(e.g. start codon targeted (SCoT) or sequence related amplified polymorphism (SRAP)) that enable to generate the different DNA fingerprint only between species but same within species are investigated for further studies. We suggest the protocol for species identification of other medicinal plant species using MFin-HRM as the follow. (i) DNA used for species identification using MFin-HRM should be good quality and quantity (more than 1 ng/μl in concentration). (ii) DNA is used for screening the suitable primer for creating DNA fingerprint by agarose gel electrophoresis that are different among species and high percentage of polymorphic band. (iii) the proper primers would be extended to create the melting fingerprint by MFin-HRM and the melting peak observed should have a negative derivative of fluorescence (F) over temperature (T) (-dF/dt) of more than 4 in order to facilitate for observation. (iv) Reproducibility test of MFin-HRM for species identification would be assessed using several individual of each species.
Author contributions Kittisak Buddhachat (KB) conducted and designed all experiments. Phanupong Changtor (PC) and Sunatcha Ninket (SN) performed part of the experiments. KB and SN analysed the data. KB wrote the manuscript. KB, PC and SN have read the manuscript.
Declaration of Competing Interest 5. Conclusions
The authors declare that there was no conflict of interest regarding the publication of this paper.
Taken together, our findings revealed that a melting curved-based genotyping method, MFin-HRM, provides a novel and rapid technique with high reliability and reproducibility to aid species identification or authentication for four Phyllanthus species in a closed tube and can be expanded for a large number of species in further studies. Before MFinHRM can be applied for further practical use as a routine method for
Acknowledgements This research was financially supported by research from the Faculty of Science, Naresuan University, 2018 (R2561E001). 8
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