Cross-species amplification of mitochondrial DNA sequence-tagged-site markers in conifers: the nature of polymorphism and variation within and among species in Picea

Abstract

Primers previously developed to amplify specific non-coding regions of the mitochondrial genome in Angiosperms, and new primers for additional non-coding mtDNA regions, were tested for their ability to direct DNA amplification in 12 conifer taxa and to detect sequence-tagged-site (STS) polymorphisms within and among eight species in Picea. Out of 12 primer pairs, nine were successful at amplifying mtDNA in most of the taxa surveyed. In conifers, indels and substitutions were observed for several loci, allowing them to distinguish between families, genera and, in some cases, between species within genera. In Picea, interspecific polymorphism was detected for four loci, while intraspecific variation was observed for three of the mtDNA regions studied. One of these (SSU rRNA V1 region) exhibited indel polymorphisms, and the two others ( nad1 intron b/c and nad5 intron1) revealed restriction differences after digestion with Sau3AI (PCR-RFLP). A fourth locus, the nad4L- orf25 intergenic region, showed a multibanding pattern for most of the spruce species, suggesting a possible gene duplication. Maternal inheritance, expected for mtDNA in conifers, was observed for all polymorphic markers except the intergenic region nad4L- orf25. Pooling of the variation observed with the remaining three markers resulted in two to six different mtDNA haplotypes within the different species of Picea. Evidence for intra-genomic recombination was observed in at least two taxa. Thus, these mitotypes are likely to be more informative than single-locus haplotypes. They should be particularly useful for the study of biogeography and the dynamics of hybrid zones.

Chloroplast and mitochondrial molecular tests identify European×Japanese larch hybrids

“…Nevertheless, their application in a single taxonomic unit, as in Larix species, was not completely successful. Jaramillo- Correa et al (2003) have also observed difficulties for applying universal mitochondrial primers in gymnosperms.…”

Species-diagnostic markers in Larix spp. based on RAPDs and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications

“…In most conifers including Larix, the chloroplast genome is paternally inherited, whereas the mitochondrial genome is maternally inherited (Szmidt et al 1987;Mogensen 1996;Whittle and Johnson 2002;Jaramillo-Correa et al 2003). Five mtDNA introns [cox1-intron1 (Lu et al 1998), matR-intron1 (Qiu et al 1999), nad1-intron b/c , nad3-intron1 (Soranzo et al 1999), and nad5-intron1 Table 4), and 1.0 U of Platinum Taq DNA polymerase (Invitrogen).…”

Identification of native Dendrobium species in Thailand by PCR-RFLP of rDNA-ITS and chloroplast DNA

“…Notably, ITS sequences have been extensively used to determine genetic diversity and to classify several plants species because they are highly variable 5,6 . Besides the ITS sequences, organelle DNA, chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA), are the other targets of genetic diversity and classification studies due to their maternal inheritance, smaller-sized molecule compared to genomic DNA, and low level of recombination, which make them easier and more reliable to examine the phylogenetic applications 7,8 . The rDNA-ITS or organelle DNAs has been widely used for the above-mentioned applications not only by sequences analysis but also by comparison of their PCR-amplified fragments.…”