The Low Copy Nuclear Gene Region, Granule Bound Starch Synthase (GBSS1), as a Novel Mini-DNA Barcode for the Identification of Different Sage (Salvia) Species

Abstract

Morphological similarity within species makes the identification and authentication of Salvia species challenging, especially in dietary supplements that contain processed root or leaf powder of different sage species. In the present study, the species discriminatory power of 2 potential DNA barcode regions from the nuclear genome was evaluated in 7 medicinally important Salvia species from the family Lamiaceae. The nuclear internal transcribed spacer 2 and the exon 9 – 14 region of low copy nuclear gene WAXY coding for granule-bound starch synthase 1 were tested for their species discrimination ability using distance, phylogenetic, and BLAST-based methods. A novel 2-step PCR method with 2 different annealing temperatures was developed to achieve maximum amplification from genomic DNA. The granule-bound starch synthase 1 region showed higher amplification and sequencing success rates, higher interspecific distances, and a perfect barcode gap for the tested species compared to the nuclear internal transcribed spacer 2. Hence, these novel mini-barcodes generated from low copy nuclear gene regions (granule-bound starch synthase) that were proven to be effective barcodes for identifying 7 Salvia species have potential for identification and authentication of other Salvia species.

DNA barcoding in herbal medicine: Retrospective and prospective

“…Nucleotide signatures are developed for particular species or genus groups and are highly conserved and free of variation within the corresponding taxon. The application of DNA mini-barcodes in the identification of Chinese medicinal materials in the last 10 years was overviewed in Table 1 [ [77] , [78] , [79] , [80] , [81] , [82] , [83] , [84] , [85] , [86] , [87] , [88] , [89] , [90] , [91] , [92] , [93] ]. With the advantages of a high success rate of amplification and sequencing, DNA mini-barcoding undoubtedly has bright application prospects in the detection and identification of biological samples.…”

“… DNA mini-barcode Length (bp) Object of identification Results or conclusions Refs. Nucleotide signature in ITS2 27 24 batches of CPM labelled Panax quinquefolius 5 batches being counterfeits, and 2 batches containing adulterants [ 77 ] Nucleotide signature in ITS2 23 7 batches of CPM labelled Ephedra Successful retrieve of the nucleotide signature in seven CPM [ 78 ] P6 loop of trnL (UAA) intron region 34–61 15 medicinal plants in 45 processed Chinese medicinal materials A candidate mini-barcode in medicinal materials control [ 79 ] Ycf1a , yaf1b , rps16 60, 100, 280 Medicinal materials of Panax notoginseng Discrimination power of ycf1 at 91.67%, ycf1b at 100%, and rps16 at 83.33% [ 80 ] MatK 151, 200 Senna obtusifolia and Senna occidentalis Successful detection in experimental mixtures [ 81 ] Granule bound starch synthase ( GBSS1 ) 200 Salvia species Higher identification competence than ITS2 [ 82 ] Nucleotide signature in ITS2 37 43 Angelicae sinensis radix products including 15 extracts and 28 CPM 7/15 of products and 9/28 of CPM being adulterants [ 83 ] Short region in ITS2 …”

“…In such cases, other new loci are needed [ 80 , 81 ]. Mini-barcodes located in the hypervariable regions of organelle genomes, such as cp-genomes and mitochondrial genomes, have been developed as auxiliary tools [ 82 ]. Dong et al.…”

Successful development of molecular diagnostic technology combining mini-barcoding and high-resolution melting for traditional Chinese medicine agarwood species based on single-nucleotide polymorphism in the chloroplast genome

“…DNA mini-barcoding is the development of shorter molecular markers (usually 100-300 bp) based on the nucleotide profile of a specific species or genus group, effectively solving the problem of identifying herbs with varying degrees of DNA degradation (Yeo et al, 2020;Parveen et al, 2022). High-resolution melting (HRM) is a classical melting curve analysis method based on PCR fragments, which, compared to other molecular identification methods, allows the sample to be identified, even if a single-base variation occurs in the DNA sequence, facilitating the quantitative determination of the rate of adulteration (Wu et al, 2008;Ganopoulos et al, 2011;Duan et al, 2018;Buddhachat et al, 2021).…”