SCAR Markers for Correct Identification of Phyllanthus amarus, P. fraternus, P. debilis and P. urinaria used in Scientific Investigations and Dry Leaf Bulk Herb Trade

Abstract

The trade in Phyllanthus material as bulk herb is rampant and mainly involves herbaceous species such as Phyllanthus amarus, P. fraternus, P. debilis and P. urinaria. These species are very important in herbal medicines and have varied activities. In India these species grow sympatrically and there are chances of deliberate or ignorant adulteration of crude drugs, lowering the efficiency of the medication for its intended purpose. Secondly, incorrect identification may also lead to erroneous reports on activities/molecules. To overcome this problem in crude drug (dry leaf power) and compliment morphological identification in live plant, we have developed SCAR markers for all four species. In each species, we selected one fragment as being monomorphic between accessions but differing in size between species. These species-specific fragments were selected, cloned and sequenced. Based on the sequences, primer pairs were designed and amplification conditions standardized. SCAR markers were isolated from population DNA amplification profiles and validated by sequencing. The species-specific SCAR primers could retrieve the same size and sequence of fragments as in the RAPD profile. These fragments are 1150 bp, 317 bp, 980 bp and 550 bp in size for P. amarus, P. fraternus, P. debilis and P. urinaria, respectively. Additional fragments in P. debilis and P. urinaria indicate different alleles. The retrieval of same size and sequence of species-specific unique SCAR markers from the respective accessions (mixed DNA sample of same accessions) indicates the usefulness to study natural hybridization between the species in addition to adulteration.

Key words

Euphorbiaceae - Phyllanthus amarus - Phyllanthus debilis - Phyllanthus fraternus - Phyllanthus urinaria - SCAR marker

References

• 1 Chaudhary L B, Rao R R. Taxonomic study of herbaceous species of Phyllanthus L.(Euphorbiaceae) in India.  Phytotaxonomy. 2002;  2 143-62
• 2 Shin M S, Kang E H, Lee Y I. A flavonoid from medicinal plants blocks hepatitis B virus-e antigen secretion in HBV-infected hepatocytes.  Antiviral Res. 2005;  67 163-8
• 3 Hout S, Chea A, Bun S, Elias R, Gasquet M, Timon-David P. Screening of selected indigenous plants of Cambodia for antiplasmodial activity.  J Ethnopharmacol. 2006;  107 12-8
• 4 Webster G L. A monographic study of the West Indian species of Phyllanthus. .  J Arnold Arbor. 1957;  38 295-373
• 5 Mitra R L, Jain S K. Concept of Phyllanthus niruri (Euphorbiaceae) in Indian floras.  Bull Bot Surv India. 1985;  27 161-76
• 6 Martin G B, Williams J GK, Tanksley S D. Rapid identification of markers linked to a Pseudomonas resistance gene in tomato by using random primers and near-isogenic lines.  Proc Natl Acad Sci USA. 1991;  88 2336-40
• 7 Paran I, Michelmore R W. Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce.  Theor Appl Genet. 1993;  85 985-93
• 8 Jung G, Skroch P W, Nienhuis J, Coyne D P, Arnold-Santangia E, Ariyarathne H M. et al . Confirmation of QTL associated with common bacterial blight resistance in 4 different genetic backgrounds in common bean.  Crop Sci. 1999;  39 1448-55
• 9 Hernandez P, Martin A, Dorado G. Development of SCARs by direct sequencing of RAPD products: a practical tool for the introgression and marker- assisted selection of wheat.  Mol Breed. 1999;  5 245-53
• 10 Ohmori T, Murata M, Motoyoshi F. Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato.  Theor Appl Genet. 1996;  92 151-6
• 11 Vidal J R, Delavault P, Coarer M, Defontaine A. Design of grapevine (Vitis vinifera L.) cultivar-specific SCAR primers for PCR fingerprinting.  Theor Appl Genet. 2000;  101 1194-201
• 12 Scheef E A, Casler M D, Geunhwa J. Development of species-specific SCAR markers in bentgrass.  Crop Sci. 2003;  43 345-9
• 13 Dnyaneshwar W, Preeti C, Kalpana J, Bhushan P. Development and application of RAPD SCAR marker for identification of Phyllanthus emblica LINN.  Biol Pharm Bull. 2006;  29 2313-6
• 14 Das M, Bhattacharya S, Pal A. Generation and characterization of SCARs by cloning and sequencing of RAPD products: a strategy for species-specific marker development in bamboo.  Ann Bot (Lond). 2005;  95 835-41
• 15 Naqvi N I, Chattoo B B. Development of a sequence characterized amplified region (SCAR) based indirect selection method for a dominant blast-resistance gene in rice.  Genome. 1996;  39 26-30
• 16 Jang I, Moon J H, Yoon J B, Yoo J H, Yang T J, Kim Y J. et al . Application of RAPD and SCAR markers for purity testing of F1 hybrid seed in chili pepper (Capsicum annuum).  Mol Cells. 2004;  18 295-9
• 17 Kim D H, Kim B D. Development of SCAR markers for early identification of cytoplasmic male sterility genotype in chili pepper (Capsicum annuum L.).  Mol Cells. 2005;  20 416-22
• 18 Hagen A R, Giese H, Brochmann C. Trans-Atlantic dispersal and phylogeography of Cerastium arcticum (Caryophyllaceae) inferred from RAPD and SCAR markers.  Am J Bot. 2001;  88 103-12
• 19 Khanuja S PS, Shasany A K, Darokar M P, Kumar S. Rapid isolation of DNA from dry and fresh samples of plants producing large amounts of secondary metabolites and essential oils.  Plant Mol Biol. Rep1999;  17 1-7
• 20 Shasany A K, Darokar M P, Dhawan S, Gupta A K, Gupta S, Shukla A K. et al . Use of RAPD and AFLP markers to identify inter-and intraspecific hybrids of Mentha.  J Hered. 2005;  96 542-9
• 21 Webster G L. A revision of Phyllanthus (Euphorbiaceae) in the continental United States.  Brittonia. 1970;  22 44-76
• 22 Webster G L. Studies on the Euphorbiaceae, Phyllanthoideae I. The American species of Phyllanthus described by Linneaus.  J Arnold Arbor. 1956;  37 1-14
• 23 Kim C S, Lee C H, Han D H, Ryu K H, Lee C H. SCARs markers derived from RAPD for cultivar identification in Pyrus pyrifolia. .  J Korean Soc Hort Sci. 2000;  41 125-8
• 24 Lee G P, Lee C H, Kim C S. Molecular markers derived from RAPD, SCAR and the conserved 18S rDNA sequences for classification and identification in Pyrus pyrifolia and P. communis. .  Theor Appl Genet. 2004;  108 1487-91
• 25 Olson M, Hood L, Cantor C, Botstsin D. A common language for physical mapping of the human genome.  Science. 1989;  245 1434-5
• 26 Adam-Blondon A F, Sévignac M, Bannerot H, Dron M. SCAR, RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean.  Theor Appl Genet. 2005;  88 865-70