Plastid genome characterisation in Brassica and Brassicaceae using a new set of nine SSRs

Abstract

We report a new set of nine primer pairs specifically developed for amplification of Brassica plastid SSR markers. The wide utility of these markers is demonstrated for haplotype identification and detection of polymorphism in B. napus, B. nigra, B. oleracea, B. rapa and in related genera Arabidopsis, Camelina, Raphanus and Sinapis. Eleven gene regions (ndhB-rps7 spacer, rbcL-accD spacer, rpl16 intron, rps16 intron, atpB-rbcL spacer, trnE-trnT spacer, trnL intron, trnL-trnF spacer, trnM-atpE spacer, trnR-rpoC2 spacer, ycf3-psaA spacer) were sequenced from a range of Brassica and related genera for SSR detection and primer design. Other sequences were obtained from GenBank/EMBL. Eight out of nine selected SSR loci showed polymorphism when amplified using the new primers and a combined analysis detected variation within and between Brassica species, with the number of alleles detected per locus ranging from 5 (loci MF-6, MF-1) to 11 (locus MF-7). The combined SSR data were used in a neighbour-joining analysis (SMM, D (DM) distances) to group the samples based on the presence and absence of alleles. The analysis was generally able to separate plastid types into taxon-specific groups. Multi-allelic haplotypes were plotted onto the neighbour joining tree. A total number of 28 haplotypes were detected and these differentiated 22 of the 41 accessions screened from all other accessions. None of these haplotypes was shared by more than one species and some were not characteristic of their predicted type. We interpret our results with respect to taxon differentiation, hybridisation and introgression patterns relating to the 'Triangle of U'.

Extremely high cytoplasmic diversity in natural and breeding populations of Lolium (Poaceae)

“…These unusual groupings could be explained by high homoplasy in the data set caused by rapid molecular evolution at the loci studied. Parallel evolution at these loci would therefore be expected to be high and this would obscure phylogenetic signal of the markers (Flannery et al, 2006). They are thus potentially more useful for assessing variation within species than among species.…”

“…Distances based on a stepwise mutation model, such as the dm 2 -distance (Goldstein et al, 1995;Flannery et al, 2006), were not used because the size variation of alleles could be attributed to both SSR length variation and other types of substitutions (non-SSR indels). From this matrix, a dendrogram showing the similarities between populations was constructed using the unweighted pair group method with arithmetic means (UPGMA) method (Sneath and Sokal, 1973) as implemented in POPGENE (Yeh and Boyle, 1997).…”

“…Although cpDNA generally has lower variability than nuclear DNA (Wolfe et al, 1987), chloroplast simple sequence repeat (cpSSR) loci have been shown to be polymorphic particularly at mononucleotide repeat loci (Powell et al, 1995) and can be used to investigate plant population structure, diversity and differentiation (reviewed by Provan et al, 2001). They can be used to monitor the transmission of chloroplast genomes during hybridization and introgression in wild or breeding populations or to characterize plastid genome type for breeding purposes (Flannery et al, 2006). cpSSRs can be used in breeding programmes as cultivar identifiers as an addition to nuclear DNA markers (Joshi et al, 1999).…”

“…Genetic studies are required for L. perenne to better understand the extent of infraspecific variation, how this diversity is partitioned within and among populations, and to help infer phylogeographic patterns. Chloroplast microsatellite (cpSSR) markers have been previously used successfully to assess variation and chloroplast DNA (cpDNA) diversity in a range of other plant species (Provan et al, 2001;Flannery et al, 2006). cpDNA restriction fragment length polymorphism (RFLP) markers and DNA sequencing have been used to assess the phylogenetic relationships of L. perenne to other Lolium and Festuca species (Darbyshire and Warwick, 1992;Charmet et al, 1997;Catalan et al, 1997Catalan et al, , 2004Torrecilla et al, 2004).…”

Cytoplasmic-nuclear variation in a diversity-fixed foundation set ofBrassica juncea (L.) Czern & Coss.

“…These included 73 SSRs for the A genome (Kim et al, 2009), 48 SSR markers for the B genome, and 64 SSRs constructed from genomic libraries of B. nigra (Lowe et al, 2004). In addition, 9 chloroplastspecific SSR primers were used to document plastid variability in the diverse Brassica gene pool (Flannery et al, 2006). The SSR polymorphism survey, genotyping, and scoring were conducted following Kim et al (2009).…”