Research Article |
Corresponding author: Runglawan Sudmoon ( rsudmoon@yahoo.com ) Academic editor: Pavel Stoev
© 2016 Arunrat Chaveerach, Tawatchai Tanee, Arisa Sanubol, Pansa Monkheang, Runglawan Sudmoon.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Chaveerach A, Tanee T, Sanubol A, Monkheang P, Sudmoon R (2016) Efficient DNA barcode regions for classifying Piper species (Piperaceae). PhytoKeys 70: 1-10. https://doi.org/10.3897/phytokeys.70.6766
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Piper species are used for spices, in traditional and processed forms of medicines, in cosmetic compounds, in cultural activities and insecticides. Here barcode analysis was performed for identification of plant parts, young plants and modified forms of plants. Thirty-six Piper species were collected and the three barcode regions, matK, rbcL and psbA-trnH spacer, were amplified, sequenced and aligned to determine their genetic distances. For intraspecific genetic distances, the most effective values for the species identification ranged from no difference to very low distance values. However, P. betle had the highest values at 0.386 for the matK region. This finding may be due to P. betle being an economic and cultivated species, and thus is supported with growth factors, which may have affected its genetic distance. The interspecific genetic distances that were most effective for identification of different species were from the matK region and ranged from a low of 0.002 in 27 paired species to a high of 0.486. Eight species pairs, P. kraense and P. dominantinervium, P. magnibaccum and P. kraense, P. phuwuaense and P. dominantinervium, P. phuwuaense and P. kraense, P. pilobracteatum and P. dominantinervium, P. pilobracteatum and P. kraense, P. pilobracteatum and P. phuwuaense and P. sylvestre and P. polysyphonum, that presented a genetic distance of 0.000 and were identified by independently using each of the other two regions. Concisely, these three barcode regions are powerful for further efficient identification of the 36 Piper species.
DNA barcoding, matK gene, Piper species, psbA-trnH spacer, rbcL gene
Plants in the genus Piper have been used since prehistoric times for a variety of human activities. They are used as spices, in traditional and processed forms of medicines, in cosmetic compounds, in cultural activities and as insecticides (
For plants, however, it is more of a challenge. Currently, several research groups are seeking a suitable genome region, and this effort has led to the identification of appropriate regions for DNA barcoding in some plant groups, such as the matK gene (Siripiyasing et al. 2012, Tanee et al. 2012), the rbcL gene (Tanee et al. 2012,
The standard barcodes used for most investigations of plants are the three plastid barcodes, which include matK gene, rbcL gene and psbA-trnH spacer, and one nuclear (ITS) regions identified by the
The aim of this research was to construct barcodes for Piper species in Thailand using matK, rbcL and the psbA-trnH spacer regions, as these species are important medicinal plants that have not been fully explored for barcode identification. Here we initiate the development of reference barcodes for plant parts, young plants and plant products.
Species and sites of Piper recently reported in Thailand (
Whole genomic DNA was extracted using a Plant Genomic DNA Extraction Kit (RBC Bioscience) following the kit protocols.
Polymerase chain reaction (PCR) analyses were performed with primer pairs (5'–3') ATCCATCTGGAAATCTTAGTTC and GTTCTAGCACAAGAAAGTCG (
The specific fragments amplified were sequenced at the DNA Sequencing Unit, Faculty of Medicine, Ramathibodi Hospital, Bangkok, Thailand. The sequences were then analyzed using Blast tools (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences were aligned for each genome region amplified to determine genetic distance values by MEGA6 (
Thirty-six Piper species were collected to construct barcodes. Because most of the Piper species that we investigated were wild, it was difficult to collect a sufficient amount of samples from all 36 Piper species to adequately construct barcodes. Sufficient samples were obtained for four species, P. nigrum, P. betle, P. sarmentosum and P. retrofractum, which are all economic plants.
The amplification of barcode bands from the matK region was not successful in two species, including P. montium and P. rubroglandulosum (♀). This may be because the DNAs were fragmented at the primer regions. Table
GenBank accession numbers of DNA barcoding from three regions of Piper species.
The intraspecific genetic distances for each region were the following: 1) for the matK region, the lowest value of 0.000 was observed in P. dominantinervium, P. hongkongense, P. kraense and P. longum, while the highest value of 0.386 was observed for P. betle; 2) for the rbcL region, the lowest value of 0.000 was observed in P. dominantinervium, P. hongkongense, P. longum, P. pedicellatum, P. pilobracteatum, P. polysyphonum, P. sarmentosum, P. sylvestre and P. wallichii, while the highest value of 0.166 was observed in P. betle; 3) for the psbA-trnH spacer region, the lowest value of 0.000 was observed in P. dominantinervium, P. khasianum, P. kraense, P. longum, P. montium, P. mutabile, P. nigrum, P. pilobracteatum, P. polysyphonum and P. sarmentosum while the highest value of 0.117 was observed in P. boehmeriifolium.
The interspecific genetic distances for each region were the following: 1) for the matK region the lowest value of 0.000 was observed in the paired species P. kraense and P. dominantinervium, P. magnibaccum and P. kraense, P. phuwuaense and P. dominantinervium, P. phuwuaense and P. kraense, P. pilobracteatum and P. dominantinervium, P. pilobracteatum and P. kraense, P. pilobracteatum and P. phuwuaense and P. sylvestre and P. polysyphonum, while the highest value of 0.486 was observed between P. ribesioides and P. pilobracteatum; 2) for the rbcL region, the lowest value of 0.000 was observed between pairs P. dominantinervium and P. caninum, P. kraense and P. boehmeriifolium, P. maculaphyllum and P. khasianum, P. magnibaccum and P. khasianum, P. magnibaccum and P. caninum, P. magnibaccum and P. dominantinervium, P. montium and P. khasianum, P. montium and P. magnibaccum, P. mutabile and P. caninum, P. mutabile and P. dominantinervium, P. mutabile and P. magnibaccum, P. nigrum and P. caninum, P. nigrum and P. dominantinervium, P. nigrum and P. magnibaccum, P. nigrum and P. mutabile, P. pedicellatum and P. khasianum, P. pedicellatum and P. magnibaccum, P. pedicellatum and P. montium, P. pedicellatum and P. pendulispicum, P. pendulispicum and P. caninum, P. pendulispicum and P. dominantinervium, P. pendulispicum and P. magnibaccum, P. pendulispicum and P. mutabile, P. pendulispicum and P. nigrum, P. phuwuaense and P. caninum, P. phuwuaense and P. dominantinervium, P. phuwuaense and P. magnibaccum, P. phuwuaense and P. mutabile, P. phuwuaense and P. nigrum, P. phuwuaense and P. pedicellatum, P. pilobracteatum and P. caninum, P. pilobracteatum and P. mutabile, P. polysyphonum and P. khasianum, P. polysyphonum and P. magnibaccum, P. polysyphonum and P. montium, P. sarmentosum and P. longum, P. sylvestre and P. khasianum, P. sylvestre and P. magnibaccum, P. sylvestre and P. montium, P. thomsonii and P. nigrum, P. pilobracteatum and P. phuwuaense, P. polysyphonum and P. pendulispicum, P. polysyphonum and P. pedicellatum, P. sylvestre and P. pendulispicum, P. sylvestre and P. pedicellatum, P. sylvestre and P. polysyphonum, P. wallichii and P. umbellatum, P. protrusum and P. phuwuaense, and P. protrusum and P. pilobracteatum, while the highest value of 0.213 was observed in the P. betle and P. argyritis pair; 3) for the psbA-trnH spacer region the lowest value of 0.000 was observed in the pairs of P. montium and P. magnibaccum, P. pilobracteatum and P. caninum, P. polysyphonum and P. pedicellatum, P. ribesioides and P. pedicellatum, P. sarmentosum and P. longum, P. sylvestre and P. pedicellatum, P. wallichii and P. khasianum, P. wallichii and P. pedicellatum, P. protrusum and P. magnibaccum, P. sylvestre and P. polysyphonum, P. sylvestre and P. ribesioides, P. wallichii and P. polysyphonum, P. wallichii and ribesioides, P. wallichii and P. sylvestre, and P. yinkiangense and P. betle, while the highest value of 0.228 was observed between P. semiimmersum and P. umbellatum.
The genetic distance of the matK region in Table
Genetic distance values of the matK region for Piper identification, a representative example.
P. argyritis | P. betle | P. betle | P. betle | P. betloides | P. boehmeriifolium | P. boehmeriifolium | P. caninum | P. caninum | P. colubrinum | P. colubrinum | P. crocatum | P. dominantinervium | P. dominantinervium | P. hongkongense | P. hongkongense | P. khasianum | P. khasianum | P. kraense | P. kraense | P. longum | P. longum | P. maculaphyllum | P. maculaphyllum | P. magnibaccum | P. magnibaccum | P. nigrum | P. nigrum | P. pedicellatum | P. pedicellatum | P. pendulispicum | |
P. argyritis | .000 | ||||||||||||||||||||||||||||||
P. betle | .323 | .000 | |||||||||||||||||||||||||||||
P. betle | .076 | .362 | .000 | ||||||||||||||||||||||||||||
P. betle | .126 | .386 | .106 | .000 | |||||||||||||||||||||||||||
P. betloides | .249 | .423 | .262 | .260 | .000 | ||||||||||||||||||||||||||
P. boehmeriifolium | .004 | .323 | .074 | .124 | .252 | .000 | |||||||||||||||||||||||||
P. boehmeriifolium | .011 | .325 | .074 | .126 | .258 | .009 | .000 | ||||||||||||||||||||||||
P. caninum | .013 | .328 | .080 | .132 | .249 | .009 | .017 | .000 | |||||||||||||||||||||||
P. caninum | .013 | .328 | .080 | .132 | .249 | .009 | .017 | .000 | .000 | ||||||||||||||||||||||
P. colubrinum | .020 | .332 | .087 | .134 | .267 | .017 | .022 | .026 | .026 | .000 | |||||||||||||||||||||
P. colubrinum | .089 | .375 | .143 | .195 | .310 | .085 | .093 | .093 | .093 | .072 | .000 | ||||||||||||||||||||
P. crocatum | .054 | .358 | .106 | .145 | .267 | .052 | .056 | .061 | .061 | .065 | .130 | .000 | |||||||||||||||||||
P. dominantinervium | .004 | .323 | .072 | .121 | .252 | .002 | .007 | .011 | .011 | .015 | .087 | .050 | .000 | ||||||||||||||||||
P. dominantinervium | .004 | .323 | .072 | .121 | .252 | .002 | .007 | .011 | .011 | .015 | .087 | .050 | .000 | .000 | |||||||||||||||||
P. hongkongense | .013 | .321 | .080 | .130 | .260 | .011 | .011 | .020 | .020 | .024 | .095 | .059 | .009 | .009 | .000 | ||||||||||||||||
P. hongkongense | .013 | .321 | .080 | .130 | .260 | .011 | .011 | .020 | .020 | .024 | .095 | .059 | .009 | .009 | .000 | .000 | |||||||||||||||
P. khasianum | .007 | .325 | .076 | .126 | .249 | .002 | .011 | .011 | .011 | .020 | .087 | .054 | .004 | .004 | .013 | .013 | .000 | ||||||||||||||
P. khasianum | .007 | .325 | .074 | .124 | .249 | .004 | .009 | .013 | .013 | .017 | .089 | .052 | .002 | .002 | .011 | .011 | .002 | .000 | |||||||||||||
P. kraense | .004 | .323 | .072 | .121 | .252 | .002 | .007 | .011 | .011 | .015 | .087 | .050 | .000 | .000 | .009 | .009 | .004 | .002 | .000 | ||||||||||||
P. kraense | .004 | .323 | .072 | .121 | .252 | .002 | .007 | .011 | .011 | .015 | .087 | .050 | .000 | .000 | .009 | .009 | .004 | .002 | .000 | .000 | |||||||||||
P. longum | .013 | .325 | .080 | .130 | .258 | .011 | .015 | .020 | .020 | .024 | .091 | .054 | .009 | .009 | .017 | .017 | .013 | .011 | .009 | .009 | .000 | ||||||||||
P. longum | .013 | .325 | .080 | .130 | .258 | .011 | .015 | .020 | .020 | .024 | .091 | .054 | .009 | .009 | .017 | .017 | .013 | .011 | .009 | .009 | .000 | .000 | |||||||||
P. maculaphyllum | .065 | .369 | .130 | .171 | .282 | .065 | .072 | .074 | .074 | .080 | .141 | .111 | .065 | .065 | .074 | .074 | .063 | .063 | .065 | .065 | .072 | .072 | .000 | ||||||||
P. maculaphyllum | .054 | .341 | .072 | .113 | .249 | .052 | .054 | .061 | .061 | .065 | .130 | .085 | .050 | .050 | .059 | .059 | .054 | .052 | .050 | .050 | .054 | .054 | .111 | .000 | |||||||
P. magnibaccum | .007 | .325 | .074 | .124 | .249 | .004 | .009 | .013 | .013 | .017 | .089 | .052 | .002 | .002 | .011 | .011 | .002 | .000 | .002 | .002 | .011 | .011 | .063 | .052 | .000 | ||||||
P. magnibaccum | .009 | .323 | .076 | .124 | .256 | .007 | .011 | .015 | .015 | .015 | .087 | .054 | .004 | .004 | .013 | .013 | .009 | .007 | .004 | .004 | .013 | .013 | .069 | .054 | .007 | .000 | |||||
P. nigrum | .015 | .334 | .087 | .130 | .262 | .017 | .022 | .026 | .026 | .030 | .102 | .061 | .015 | .015 | .024 | .024 | .020 | .017 | .015 | .015 | .022 | .022 | .072 | .063 | .017 | .020 | .000 | ||||
P. nigrum | .009 | .325 | .080 | .121 | .249 | .011 | .015 | .020 | .020 | .024 | .095 | .059 | .009 | .009 | .017 | .017 | .013 | .011 | .009 | .009 | .017 | .017 | .072 | .054 | .011 | .013 | .020 | .000 | |||
P. pedicellatum | .011 | .328 | .080 | .126 | .252 | .007 | .015 | .015 | .015 | .024 | .091 | .054 | .009 | .009 | .017 | .017 | .009 | .011 | .009 | .009 | .013 | .013 | .067 | .046 | .011 | .013 | .022 | .013 | .000 | ||
P. pedicellatum | .011 | .325 | .074 | .124 | .258 | .009 | .004 | .017 | .017 | .022 | .093 | .056 | .007 | .007 | .011 | .011 | .011 | .009 | .007 | .007 | .015 | .015 | .072 | .054 | .009 | .007 | .022 | .015 | .015 | .000 | |
P. pendulispicum | .004 | .325 | .076 | .126 | .252 | .007 | .011 | .015 | .015 | .020 | .091 | .050 | .004 | .004 | .013 | .013 | .009 | .007 | .004 | .004 | .009 | .009 | .067 | .050 | .007 | .009 | .013 | .009 | .009 | .011 | .000 |
Most of the 43 species of wild Piper in Thailand have many functional uses. Only four species, P. betle, P. retrofractum, P. nigrum and P. sarmentosum are economic and cultivated species, and all of these species are also used as ingredients in the products mentioned above in the introduction. Piper betle is a well-known species that is important for its chemical substances, including essential oils, chavicol, cineol and eugenol, which can be used for medicinal and insecticidal purposes. Because these plants are widely used, and used in several forms, which include plant parts, powdered preparations, capsule formulations and other preparations, their authenticity should be verified using DNA barcodes to establish the worthiness of these products for medicinal, cosmetics and house-hold use. To overcome the problems associated with identifying species based on morphological characters, DNA barcoding has been employed. For flowering plants in Thailand, the psbA-trnH spacer region was suggested as an efficient DNA barcode marker in Senna species (
The results from DNA barcoding 36 Piper species using three different marker regions support a previous hypothesis of genetic distance values (
Interspecific genetic distance values for identification of the eight pairs Piper species by rbcL and psbA-trnH spacer sequences.
Pairs of species | matK region | rbcL region | psbA-trnH spacer region |
---|---|---|---|
P. kraense and P. dominantinervium | 0.000 | 0.005-0.008 | 0.111-0.117 |
P. magnibaccum and P. kraense | 0.000 | 0.008 | 0.1110-0.123 |
P. phuwuaense and P. dominantinervium | 0.000 | 0.000-0.003 | 0.021-0.026 |
P. phuwuaense and P. kraense | 0.000 | 0.005-0.008 | 0.021-0.129 |
P. pilobracteatum and P. dominantinervium | 0.000 | 0.003 | 0.021 |
P. pilobracteatum and P. kraense | 0.000 | 0.003 | 0.010-0.123 |
P. pilobracteatum and P. phuwuaense | 0.000 | 0.003 | 0.016-0.021 |
P. sylvestre and P. polysyphonum | 0.000 | 0.000 | 0.000-0.010 |
The results presented here support those of
The authors are grateful for the financial support provided by the Thailand Research Fund through the Research Career Development Grant (Grant No. RSA5580054). The authors confirm that there is no conflict of interest.