Genetic Variability in Nicotianatabacum and Nicotiana Species as Revealed by RAPD Markers: 1. Development of the RAPD Procedure

Abstract

At present there is no information about the level of genetic variability in N. tabacum and in the Nicotiana genus as revealed by random amplified polymorphic DNA (RAPD). Such knowledge could be useful for taxonomic and breeding purposes. The aim of this paper is to assess the potential application of the DNA polymorphisms generated by RAPD markers within this genus and in tobacco. As rigorously standardized reaction conditions are required to obtain a reproducible RAPD marker, four rapid DNA extraction methods were compared and several parameters of the reaction conditions for the random polymorphic DNA amplification were analysed and optimized. The DNA of six-week-old leaves of N. tabacum var. Samsun was obtained with the following methods differing in the strategy of purification: the cetyltrimethylammonium bromide (CTAB) method, that of Edwards, nucleon phytopure system and the method of Goring. Reproducible amplification profiles were obtained with all the methods except for Edwards'. As regards amplification conditions, the effects of primer-template annealing temperature, of a final extension step, of the number of cycles and of the length of extension time in each cycle were analysed. Moreover, the effects on amplification reaction of the DNA amount, of MgCl2, primer and deoxynucleotide triphosphate (dNTP) concentration were evaluated. Then DNA of 12 Nicotiana species and Nicotianatabacum was amplified with primers OPA-01 and OPA-13 which revealed a considerable polymorphism. The same primers used to analyse 36 var. of N. tabacum belonging to different types, showed identical amplification profiles. Further amplification experiments were carried out with only 12 of the tobacco lines; three primers among the 12 assayed revealed one polymorphic fragment each.

Genetic Variability of Macedonian Tobacco Varieties Determined by Microsatellite Marker Analysis

“…Restriction fragment length polymorphism (RFLP) markers were among the first molecular markers applied in tobacco research and were used to study the function of particular cloned genes [6]. PCR based random amplified polymorphic DNA (RAPD) markers have been applied to identify tobacco chromosome regions carrying disease resistance genes [7,8] and for variety identification purposes [9][10][11][12]. Amplified fragment length polymorphism (AFLP) markers were also used to distinguish among related varieties of cultivated tobacco [13,10].…”

Genetic diversity in Indian FCV and burley tobacco cultivars

“…High degree of polymorphism was also observed among the Nicotiana species using two RAPD primers (Del Piano et al 2000) and AFLP markers (Ren and Timko 2001), whereas Del Piano et al (2000) reported very low level of polymorphism in 12 lines of tobacco using RAPD.…”

“…Ten primers produced 100% polymorphic bands. By comparing with the previous studies, Del Piano et al (2000) reported complete monomorphic pattern in 36 tobacco lines belonging to different tobacco types, using RAPD primers, whereas Zhang et al (2005) obtained 63% polymorphic bands with 24 tobacco cultivars using 17 RAPD primers.…”

“…In tobacco, the RAPD technique has been mainly used to identify markers linked to genes for resistance to pathogens (Bai et al 1995;Yi et al 1998). Previously, a few studies have assessed the genetic diversity in tobacco cultivars using RAPD and found a low degree of polymorphism (Xu et al 1998;Del Piano et al 2000;Zhang et al 2005).…”

Molecular diversity in the genus Nicotiana as revealed by randomly amplified polymorphic DNA

“…In tobacco, RAPD has been used mainly to identify markers linked to genes for resistance to pathogens (Bai et al, 1995;Rufty et al, 1997;Yi et al, 1998). RAPD procedure was developed for tobacco by using 12 species of Nicotiana and 12 varieties of N. tabacum using 3 random primers (Del Piano et al, 2000). Evolutionary relationship among seven species of Nicotiana was analyzed by Amplified fragment length polymorphism (AFLP) markers was reported earlier (Ren and Timko, 2002).…”