Development and significance of RAPD-SCAR markers for the identification of Litchi chinensis Sonn. by improved RAPD amplification and molecular cloning

Jingliang Cheng,Md Asaduzzaman Khan,Chunli Wei,Shelly Fu,Yan Long,Junjiang Fu

doi:10.1016/j.ejbt.2014.11.004

Abstract

article i nfo Background: Analysis of genetic diversity is important for the authentication of a species. Litchi (Litchi chinensis Sonn.) is a subtropical evergreen tree. Recently, L. chinensis has been characterized by an improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis. The goal of this study was to develop sequence-characterized amplified region (SCAR) markers from the improved RAPD fragments for the genetic analysis of L. chinensis. Results: The improved RAPD fragments from L. chinensis were cloned, sequenced and converted into stable SCAR markers. Sequencing of three cloned RAPD fragments revealed that the clone L7-16 consisted of 222 nucleotides (GenBank accession number KM235222), clone L9-6 consisted of 648 nucleotides (GenBank accession number KM235223), and clone L11-26 consisted of 369 nucleotides (GenBank accession number KM235224). Then, specific primers for SCAR markers L7-16, L9-6, and L11-26 were designed and synthesized. PCR amplification was performed using DNA templates from 24 different samples, including 6 samples of L. chinensis and other plants. The SCAR marker L9-6 was specific for all of the L. chinensis samples, the SCAR marker L11-26 specific for five L. chinensis samples, and the SCAR marker L7-16 only specific for the samples from Luzhou. Conclusions: This study developed stable SCAR markers for the identification of L. chinensis by the cloning of the improved RAPD fragments. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species.

Highlights

Analysis of genetic diversity is important for the identification and authentication of a species
The selected positive clones L7-16, L9-6 and L11-26 were identified by extraction of plasmid and DNA digestion by using EcoR I enzyme (Fig. 2d)
Clones L7-16, L9-6 and L11-26 were selected for Sanger sequencing

Summary

Introduction

Analysis of genetic diversity is important for the identification and authentication of a species. For the analysis of genetic diversity, a number of molecular marker techniques have been developed over the last thirty years These include random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), inter-retrotransposon amplified polymorphism (IRAP), inter-simple sequence repeat (ISSR), internal transcribed spacer (ITS), restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) analysis. These analyses have all been used for the genetic characterization and authentication of unicellular and multicellular organisms [1,2,3,4]. Combining RAPD and SCAR markers provides a simple and reliable tool for the genetic characterization of plant species

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Conclusion