Applied Barcoding: The Practicalities of DNA Testing for Herbals
Abstract
:1. Introduction
2. Results
2.1. A Scheme for Applied Barcoding
2.1.1. Nomenclature, Taxonomy and Adulteration
2.1.2. Selection of a Panel of Potential Adulterant Hypericum Species
2.1.3. Public Databases of DNA Barcode Sequences
2.1.4. Reference Samples for Pilot Studies and Assay Standards
2.1.5. Test Samples for Barcode Assay Validation
2.2. The nrITS Barcode Region
2.2.1. A Curated Database of ITS Sequences
2.2.2. ITS Barcoding of Reference Samples
2.3. The ITS2 Barcode Region
2.3.1. ITS2 Sequences
2.3.2. Secondary Structure of ITS2 Sequences
2.4. The rbcLa Barcode Region
2.4.1. A Curated Database of rbcLa Sequences
2.4.2. Haplotype Mapping of Reference Samples
2.5. The matK Barcode Region
2.6. The psbA-trnH Barcode Region
2.6.1. A Curated Database of psbA-trnH Sequences
2.6.2. Reference and Test psbA-trnH Sequences
2.7. Choosing the Optimal Barcode
2.7.1. ITS Barcoding of Test Samples
2.7.2. Supporting Evidence from rbcL Haplotype and psbA-trnH Stem-Loop
3. Discussion
3.1. Applied Barcoding for Herbals
3.2. Choosing a Barcode for Species Identification
3.2.1. Defining a Reference Barcode Sequence: Sensitivity and Specificity
3.2.2. Matching a Reference Barcode Sequence: Accuracy and Precision
3.3. Recommended Barcodes for H. perforatum Authentication
3.4. Current Trends in Applied Barcoding
4. Materials and Methods
4.1. A panel of Potential Adulterant Hypericum Species
- Close relatives of H. perforatum;
- Common in commercial trade as ornamental or medicinal plants;
- Reported as adulterants of H. perforatum.
4.2. Plant and DNA Materials
4.3. DNA Extraction and Amplification of DNA Barcode Regions
Region | Primer Sequence | Program |
---|---|---|
ITS | ITS1- TCCGTAGGTGAACCTGCGG ITS4-TCCTCCGCTTATTGATATGC [101] | 7 min at 95 °C initial denaturation step, 30 cycles consisting of 1 min at 95 °C, 30 s at 60 °C and 1 min at 72 °C, final extension period of 7 min at 72 °C. |
trnH-psbA | trnHf_05-CGCGCATGGTGGATTCACAATCC psbA3_f–GTTATGCATGAACGTAATGCTC [102] | 5 min at 95 °C initial denaturation step, 35 cycles consisting of 1 min at 95 °C, 30 s at touchdown temperature and 1 min at 72 °C, final extension period of 7 min at 72 °C. Touchdown temperature began at 58 °C, reduced by 1 °C per cycle until 48 °C, then continued at 48 °C for the remainder of the program. |
rbcL | rbcLa_f -ATGTCACCACAAACAGAAAC rbcLa_rev-GTAAAATCAAGTCCACCRCG [103] | 5 min 95 °C initial denaturation step, 35 cycles consisting of 30 s at 95 °C, 20 s at 52 °C and 50 s at 72 °C, with a final extension period of 5 min at 72 °C. |
matK | 390F-CGATCTATTCATTCAATATTTC 1326R–TCTAGCACACGAAAGTCGAAGT 2.1-CCTATCCATCTGGAAATCTTAG 2.1a–ATCCATCTGGAAATCTTAGTTC X F- TAATTTACGATCAATTCATTC 5-GTTCTAGCACAAGAAAGTCG 3.2–CTTCCTCTGTAAAGAATTC 3F_KIM f-CGTACAGTACTTTTGTGTTTACGAG 1R_KIM r -ACCCAGTCCATCTGGAAATCTTGGTTC [104,105,106] | Initial “touch-up” program, 5 min 94 °C initial denaturation step, 5 cycles consisting of 30 s at 94 °C, 40 s at 44 °C and 40 s at 72 °C, followed by 30 cycles consisting of 30 s at 94 °C, 40 s at 46 °C and 40 s at 72 °C, with a final extension period of 3 min at 72 °C. The second amplification contained 2 μL of the initial PCR product diluted 1:200 as the DNA template. Second matK program: 5 min 94 °C initial denaturation step, 35 cycles consisting of 30 s at 95 °C, 20 s at 46 °C and 40 s at 72 °C, with a final extension period of 3 min at 72 °C. |
4.4. DNA Sequence Analysis of Barcode Amplicons
4.5. Computer Analysis of Barcode Regions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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BOLD | GenBank | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Species | rbcL | matK | trnH-psbA | ITS | ITS2 | rbcL | matK | trnH-psbA | ITS | ITS2 |
H. acmosepalum | 1 | 1 | 5 | |||||||
H. androsaemum | 5 (6) | 1 | 1 | 7 | 1 | 1 | 7 | 4 | ||
H. ascyron | 1 (2) | 1 | 1 | 2 (4) | 5 | 3 | 1 | 16 | 3 | |
H. athoum | 1 | 1 | 4 | |||||||
H. attenuatum | 2 | 7 | ||||||||
H. barbatum | 1 | 2 | ||||||||
H. calycinum | 1 | 1 | 2 | 1 | 1 | 1 | 7 | |||
H. crux-andreae | 2 | 1 | 2 | |||||||
H. delphicum | 2 | 6 | ||||||||
H. elegans | 1 | |||||||||
H. hirsutum | 4 (5) | 1 | (1) | 1 (3) | 6 | 1 | 5 | 2 | ||
H. japonicum | 2 | 3 | 9 | 5 | 10 | 7 | ||||
H. kouytchense | 2 | 1 | 4 | |||||||
H. maculatum | 7 (8) | (1) | 1 | 1 (9) | 11 | 3 | 14 | 3 | ||
H. montanum | 2 (3) | 1 | 1 | 2 | 3 | 1 | 6 | 1 | ||
H. olympicum | 1 | 2 | 9 | |||||||
H. patulum | 2 | 1 | 8 | 2 | ||||||
H. perforatum | 22 (40) | 3 (6) | (3) | (1) | 9 (27) | 38 | 11 | 7 | 40 | 17 |
H. tetrapterum | 2 (4) | 1 (2) | 4 | 3 | 2 | 2 | 5 | 2 | ||
H. undulatum | 3 (4) | 1 | 1 | 4 | 1 | 1 | 9 | 4 |
Species | Section | Robson Clade [24] | Meseguer Clade [26] | CBC/Perf |
---|---|---|---|---|
H. delphicum | Adenosepalum | 27 | E | 0 |
H. montanum | Adenosepalum | 27 | E | 0 |
H. barbatum | Drosocarpium | 13 | E | 0 |
H. attenuatum | Hypericum | 9 | E | 0 |
H. maculatum | Hypericum | 9 | E | 0 |
H. perforatum | Hypericum | 9 | E | 0 |
H. tetrapterum | Hypericum | 9 | E | 0 |
H. undulatum | Hypericum | 9 | E | 0 |
H. hirsutum | Taenocarpium | 18 | E | 0 |
H. olympicum | Oligostema | 14 | E | 0 |
H. acmosepalum | Ascyreia | 3 | D | 1 |
H. calycinum | Ascyreia | 3 | D | 1 |
H. kouytchense | Ascyreia | 3 | D | 1 |
H. patulum | Ascyreia | 3 | D | 1 |
H. androsaemum | Androsaemum | 5 | C | 2 |
H. crux-andreae | Myriandra | 20 | B | 4 |
H. ascyron | Roscyna | 7 | D | 5 |
H. japonicum | Trignobrathys | 30 | B | 5 |
Species | Type | 66 | 180 | 263 | 300 | 372 | 378 | 492 |
---|---|---|---|---|---|---|---|---|
Consensus | C | C | A | A | A | A | A | |
H. perforatum | 1p | T | T | T | G | C | . | . |
H. maculatum | 1 | . | T | . | G | C | . | . |
H. tetrapterum | 1 | . | T | . | G | C | . | . |
H. undulatum | 1 | . | T | . | G | C | . | . |
H. montanum | 1 | . | T | . | G | C | . | . |
H. hirsutum | 1 | . | . | . | G | C | . | . |
H. androsaemum | 2 | . | . | . | . | . | G | T |
H. ascyron | 2 | . | . | . | . | . | G | T |
H. calycinum | 2 | . | . | . | . | . | G | T |
H. crux-andreae | 2 | . | . | . | . | . | G | T |
H. japonicum | 2 | . | . | . | . | . | G | T |
Reference | Type | 66 | 180 | 263 | 300 | 372 | 378 | 492 |
---|---|---|---|---|---|---|---|---|
Consensus | C | C | A | A | A | A | A | |
H. perforatum 13876 | 1p | T | T | T | G | C | . | . |
H. delphicum 13938 | 1 | . | T | . | G | C | . | . |
H. montanum A12F | 1 | . | T | . | G | C | G | . |
H. kouytchense 13866 | 1–2 | . | . | . | . | . | . | . |
H. patulum 13908 | 1–2 | . | . | . | . | . | . | . |
H. perforatum 13921 | 1–2 | . | . | . | . | . | . | . |
H. perforatum 13932 | 1–2 | . | . | . | . | . | . | . |
H. androsaemum 13854 | 1–2 | . | . | . | . | . | . | . |
H. ascyron 13993 | 2 | . | . | . | . | . | G | T |
H. athoum 13923 | 2 | . | . | . | . | . | G | T |
H. calycinum 13929 | 2 | . | . | . | . | . | G | T |
H. maculatum 13896 | 2 | . | . | . | . | . | G | T |
Sample No. | DNA No. | ITS Match to H. perforatum | trnH-psbA Stem | rbcL Type | ID |
---|---|---|---|---|---|
mac 01 | 007 | No | L | 1 | M |
mac 02 | 011 | No | L | 1(p) | M |
mac 03 | 012 | No | L | 1p | M |
mac 04 | 018 | No | L | 1p | M |
mac 05 | 030 | L | 1p | m | |
mac 06 | 045 | No | L | 1-2 | m |
mac 07 | 001 | L | 1 | m | |
mac 08 | 031 | No | L | 1p | M |
mac 09 | 027 | No | L | 1 | M |
mac 10 | 022 | L | 1(p) | m | |
mac 11 | 014 | S | 1p | p | |
mac 12 | 034 | No | L | 1p | M |
mac 13 | 025 | No | L | 1p | M |
mac 14 | 019 | No | L | 1 | M |
mac 15 | 036 | No | s | 1p | m |
mac 16 | 023 | No | L | 1 | M |
perf 01 | 005 | S | 1p | p | |
perf 02 | 043 | Yes | S | 1-2 | P |
perf 03 | 017 | No | Sa | 1 | U |
perf 04 | 026 | Yes | S | 1p | P |
perf 05 | 016 | S | 1p | p | |
perf 06 | 038 | L | 1p | U | |
perf 07 | 044 | Yes | S | 1p | P |
perf 08 | 035 | Yes | S | 1 | P |
perf 09 | 029 | No | S | 1p | U |
perf 10 | 015 | S | 1p | p | |
perf 11 | 032 | Yes | S | 1 | P |
perf 12 | 041 | S | 1p | p | |
perf 13 | 028 | No | Sa | 1p | U |
perf 14 | 024 | S | 1p | p | |
perf 15 | 013 | L | 1p | U | |
perf 16 | 037 | Yes | S | 1p | P |
perf 17 | 039 | Yes | S | 1p | P |
perf 18 | 020 | Yes | Sa | 1p | P |
perf 19 | 042 | Yes | Sa | 1p | P |
perf 20 | 040 | Yes | Sa | 1p | P |
perf 21 | 033 | Yes | S | 1p | P |
perf 22 | 021 | L | 1(p) | U |
Species | Attribution | Section | Rel | MPNS | Cult | Adult |
---|---|---|---|---|---|---|
H. acmosepalum | N.Robson | Ascyreia | - | Y | ||
H. androsaemum | L. | Androsaemum | Y | Y | Y | |
H. ascyron | L. | Roscyna | Y | Y | ||
H. athoum | Boiss. & Orph | Adenosepalum | - | Y | ||
H. attenuatum | Fisch. ex Choisy | Hypericum | Y | - | ||
H. barbatum | Jacq. | Drosocarpium | - | Y | ||
H. calycinum | L. | Ascyreia | Y | Y | ||
H. crux-andreae | (L.) Crantz | Myriandra | Y | Y | ||
H. delphicum | Boiss. & Heldr. | Adenosepalum | - | |||
H. elegans | Stephan ex Willd. | Hypericum | Y | Y | ||
H. hirsutum | L. | Taeniocarpium | - | Y | ||
H. japonicum | Thunb. | Trigynobrathys | Y | Y | ||
H. kouytchense | H.Lev | Ascyreia | - | Y | ||
H. maculatum | Crantz | Hypericum | Y | Y | Y | |
H. montanum | L. | Adenosepalum | Y | |||
H. olympicum | L. | Oligostema | - | Y | ||
H. patulum | Thunb. | Ascyreia | Y | Y | Y | |
H. perforatum | L. | Hypericum | Y | Y | ||
H. tetrapterum | Fr. | Hypericum | Y | Y | Y | Y |
H. undulatum | Schousb. ex Willd. | Hypericum | Y | Y |
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Howard, C.; Lockie-Williams, C.; Slater, A. Applied Barcoding: The Practicalities of DNA Testing for Herbals. Plants 2020, 9, 1150. https://doi.org/10.3390/plants9091150
Howard C, Lockie-Williams C, Slater A. Applied Barcoding: The Practicalities of DNA Testing for Herbals. Plants. 2020; 9(9):1150. https://doi.org/10.3390/plants9091150
Chicago/Turabian StyleHoward, Caroline, Claire Lockie-Williams, and Adrian Slater. 2020. "Applied Barcoding: The Practicalities of DNA Testing for Herbals" Plants 9, no. 9: 1150. https://doi.org/10.3390/plants9091150