Development of RAPD-SCAR markers for Lonicera japonica Thunb. (Caprifolicaceae) variety authentication by improved RAPD and DNA cloning

Abstract

<p>Genetic diversity within a species is a common feature, which plays a vital role in its survival and adaptability, and is important for the identification and authentication of a species. <em>Lonicera japonica</em> is a traditionally used medicinal plant, which have been recently genetically characterized by an improved random amplified polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific <em>L. japonica</em> variety. The DNAs were extracted from fresh young leaves of different samples of <em>L. japonica</em> collected from Shenzhen, Yichang, Leshan, Emei and Loudi, China. The DNA materials were amplified using improved RAPD PCR. Different RAPD bands were excised, cloned and developed for stable sequence-characterized amplified region (SCAR) markers with different species. Two SCAR markers, JYH3-3 and JYH4-3, have been successfully cloned from improved RAPD fragments. The SCAR marker JYH3-3 was found specific for all of the <em>L. japonica</em> samples collected from the different regions, and another marker JYH 4-3 was strictly specific to the Shenzhen sample from Guangdong province, which is geographically distant from Hubei, Sichuan and Hunan Provinces (source of other <em>L. japonica</em> samples). The marker JYH3-3 was found as specific molecular marker for the identification of <em>L. japonica</em>, while JYH4-3 was found as molecular marker strictly specific for the Shenzhen sample. The developed SCAR markers might serve as more specific molecular markers for <em>L. japonica</em> variety authentication. The combination of improved RAPD analysis and SCAR marker development have resulted useful tools to study the genetic variety of any organism, which we have successfully applied here in <em>L. japonica</em>.</p><p>de cualquier organismo, que hemos aplicado con éxito en <em>L. japonica</em>.<strong></strong></p>

Development of two novel specific SCAR markers by cloning improved RAPD fragments from the medicinal mushroom Ganoderma lucidium (Leysser: Fr) Karst

“…A number of recent studies have reported the potential of traditional RAPD and improved RAPD for the genetic characterization and identification of different plant or animal species (Bhat et al, 2012;Kumla et al, 2012;Fu et al, 2013;Noormohammadi et al, 2013;Shakeel et al, 2013;Mei et al, 2014). When the RAPD technique is combined with the SCAR marker technology, specificity and stability is significantly improved, which makes genetic identification and authentication more convenient and efficient for testing different alleles (Rajesh et al, 2013;Yang et al, 2013Yang et al, , 2014Mei et al, 2015).…”

“…White colonies were screened out by blue/white screening. The presence of the correct insert was verified by PCR amplification using the T7/SP6 primer pair (T7 primer: 5'-TAATACGACTCACTATAGGG-3', SP6 primer: 5'-ATTTAGGTGACACTATAGAA-3'), and by EcoRI digestion (Fu, 2012;Yang et al, 2013Yang et al, , 2014Fu et al, 2015). The cloned DNA fragments of the correct size were then sequenced by the Sanger method.…”

Short Communication: Rapid and accurate genetic authentication of Penthorum chinense by improved RAPD-derived species-specific SCAR markers

“…When RAPD is combined with SCAR, it can improve the stability and specificity, making genetic identification and authentication more efficient in studying different alleles. In our lab, we have successfully characterized a number of plant or fungal species by developing SCAR markers based on RAPD amplicons; namely Dimocarpus longan , Acorus species (Ryuk et al 2014), Lonicera japonica (Yang et al 2014), Trichoderma cf. harzianum (Pérez et al 2014), Cordyceps sinensis (Lam et al 2015), Litchi chinensis , Angelica sinensis , and Ganoderma lucidum (Khan et al 2016).…”

Development of novel SCAR markers for genetic characterization of Lonicera japonica from high GC-RAMP-PCR and DNA cloning

“…A number of compounds that have been identified and isolated from Lonicera species, including biflavonoids, quercetin, phenolic acids, and dicaffeoylquinic acid, are supposed to be responsible for these various pharmacological and biological activities (Shang et al, 2011;Fu et al, 2013). L. confusa is known as Shan Yin Hua in markets, but its pharmacological components and biological activities might be different, and it is considered a substitute for L. japonica (Shang et al, 2011;Yang et al, 2014). In addition, L. japonica is more expensive than L. confusa.…”