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

Luquan Yang,Li Zhu,Zhiqiang Mei,Junjiang Fu,Weichan Yang,Yan Long,Chunli Wei,Md Asaduzzaman Khan,Manman Yang,Tiandan Zhang

doi:10.15517/rbt.v62i4.13493

Abstract

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. Lonicera japonica is a traditionally used medicinal plant, which have been recently genetically characterized by an improved ran- dom amplified polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific L. japonica variety. The DNAs were extracted from fresh young leaves of different samples of L. japonica 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 differ- ent 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 L. japonica 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 L. japonica samples). The marker JYH3-3 was found as specific molecular marker for the identification of L. japonica, 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 L. japonica 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 L. japonica.

Highlights

Genetic differences within a species are a common feature in the living world, and a result of the adaptation to changing environments
When random amplified polymorphic DNA (RAPD) is combined with Sequence-characterized amplified region (SCAR) markers, the analytical procedure becomes a simple PCR analysis, using PCR primers designed from the sequence of RAPD amplicons (Kumla et al, 2012; Rajesh et al 2013), as we have indicated earlier (Fu et al, 2013)
Cloning of RAPD fragments: Two RAPD primers, SBC-Q2 (Q2) and SBC-I10 (I10), were used to improve RAPD amplification from five DNA samples of L. japonica, which were collected from Shenzhen City of Guangdong Province, Yichang City of Hubei Province, Leshan and Emei City of Sichuan Province and Loudi City of Hunan Province, respectively (Fu et al, 2013)

Summary

Introduction

Genetic differences within a species are a common feature in the living world, and a result of the adaptation to changing environments. Considering the analysis of the genetic diversity, a number of molecular marker techniques have been developed over the last 30 years: random amplified polymorphic DNA (RAPD), simple sequence repeat. (SSR), inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) analysis (Agarwal, Shrivastava, & Padh, 2008; Liu, Li, Khan, & Zhu, 2012; Fu, Yang, Khan, & Mei, 2013). These techniques are currently being used for the genetic characterization and identification of different unicellular and multicellular organisms. We have developed SCAR markers from previously established RAPD fragments for the genetic characterization, authentication and validation of L. japonica

Methods

Results

Conclusion