The development and evaluation of consensus chloroplast primer pairs that possess highly variable sequence regions in a diverse array of plant taxa

Abstract

Although universal or consensus chloroplast primers are available, they are limited by their number and genomic distribution. Therefore, a set of consensus chloroplast primer pairs for simple sequence repeats (ccSSRs) analysis was constructed from tobacco (Nicotiana tabacum L.) chloroplast sequences. These were then tested for their general utility in the genetic analysis of a diverse array of plant taxa. In order to increase the number of ccSSRs beyond that previously reported, the target sequences for SSR motifs was set at A or T ( n >/= 7) mononucleotide repeats. Each SSR sequence motif, along with +/-200-bp flanking sequences from the first of each mononucleotide base repeat, was screened for homologies with chloroplast DNA sequences of other plant species in GenBank databases using BLAST search procedures. Twenty three putative marker loci that possessed conserved flanking sequence spans were selected for consensus primer pair construction using commercially available computer algorithms. All primer pairs produced amplicons after PCR employing genomic DNA from members of the Cucurbitaceae (six species) and Solanaceae (four species). Sixteen, 22 and 19 of the initial 23 primer pairs were successively amplified by PCR using template DNA from species of the Apiaceae (two species), Brassicaceae (one species) and Fabaceae (two species), respectively. Twenty of 23 primer pairs were also functional in three monocot species of the Liliaceae [onion (Allium cepa L.) and garlic (Allium sativum L.)], and the Poaceae [oat (Avena sativa L.)]. Sequence analysis of selected ccSSR fragments suggests that ccSSR length and sequence variation could be useful as a tool for investigating the genetic relationships within a genus or closely related taxa (i.e., tribal level). In order to provide for a marker system having significant coverage of the cucumber chloroplast genome, ccSSR primers were strategically "recombined" and named recombined consensus chloroplast primers (RCCP) for PCR analysis. Successful amplification after extended-length PCR of 16 RCCP primer pairs from cucumber ( Cucumis sativus L.) DNA suggested that the amplicons detected are representative of the cucumber chloroplast genome. These RCCP pairs, therefore, could be useful as an initial molecular tool for investigation of traits related to a chloroplast gene(s) in cucumber, and other closely related species.

Molecular Analysis of the Genetic Diversity of Chinese Hami Melon and Its Relationship to the Melon Germplasm from Central and South Asia

“…Another marker of the chloroplast genome, the consensus chloroplast SSR marker, ccSSR7 (Chung and Staub, 2003), was used in this study because Tanaka et al (2006) reported that ccSSR7 was polymorphic by deletion of 5 bp (ATATT). The nucleotide sequence of each primer was: ccSSR7-F-5' CGGGAAGGGCTCG KGCAG 3' (K = T or G) and ccSSR7-R-5' GTTCGAAT CCCTCTCTCTCCTTTT 3'.…”

Chloroplast diversity of Casearia grandiflora in the Cerrado of Piauí State

“…For chloroplast microsatellite analyses, we employed universal primer pairs described to dicotyledonous angiosperms by Weising and Gardner (1999) and Chung and Staub (2003).…”

Methods for Development of Microsatellite Markers: An Overview

“…The identification and development of cpSSRs is achieved principally by utilizing the nucleotide sequence information retrieved from public databases (Weising and Gardner, 1999;Chung and Staub, 2003) and also by sequencing PCR products generated by "consensus/universal primers", capable of amplifying cpDNA regions in several species.…”

“…Sequencing of amplicons generated by these universal cpDNA-PCR primers provide basic information about mononucleotide tracts and flanking sequences in the amplicons. Since the flanking regions of cpSSR loci are highly conserved, 'universal cpSSR primers' (Weising and Gardner, 1999;Chung and Staub, 2003;Ebert and Peakall, 2009) that enable the amplification of cpSSRs across species/taxa have been identified. Most of the molecular studies involving chloroplast microsatellites now rely on testing these universal cpSSRs in the target species.…”