Development of mitochondrial SNP markers in different Populus species

Abstract

Key message We aim to develop, for the first time, poplar species-specific mitochondrial SNP markers which can be employed as diagnostic tool, e.g., to identify mitochondria belonging to different poplar species used in protoplast fusion experiments and for population genetics approaches. Abstract In most higher plant species, the inheritance of chloroplasts and mitochondria is maternal. In contrast to chloroplast DNA, however, the mitochondrial genome, especially for woody plants including poplar, has seldom been the object of molecular biological research. Published studies on mitochondrial DNA variation in poplar have dealt with genetic relationships using RFLP analyses for population genetic purposes, but so far not for species identification. In this study, we for the first time aim to develop poplar species-specific mitochondrial SNP markers which can be employed as a diagnostic tool, e.g., to identify mitochondria belonging to different poplar species used in protoplast fusion experiments. In total, seven mitochondrial regions were PCR amplified, sequenced and screened for SNPs differentiating clones belonging to six different poplar species from three sections. Two regions (Sc438, contig42) revealed to be monomorphic in the six Populus species. For the five other regions, SNPs could be identified that are suitable for distinguishing between Populus species, either by a SNP-related CAPS (cleaved amplified polymorphic site) marker to be employed in robust PCR-RFLPs, or simply on the basis of the sequence to be screened by PCR and subsequent Sanger sequencing. As a proof of concept, the developed mitochondrial SNP markers confirmed results obtained with chloroplast SNP markers in interspecific poplar hybrids, either with respect to the direction of the cross or the reclassification of putative pure lines as hybrid lines.

Genome Sequences of Populus tremula Chloroplast and Mitochondrion: Implications for Holistic Poplar Breeding

“…the P. tremula -specific marker rpoC2-rpoC1 ( Mls I) and the P. alba -specific marker trnH-psbA (Alw26I) [13,14], the maternal part in the 717–1B4 clone could be without doubt classified as P. tremula (S2 appendix; 717–1B4 provided a positive result with the P. tremula-specific marker and a negative result with the P. alba -specific marker). Our results were reinforced by recently developed mitochondrial molecular markers [12].…”

“…Due to the comparatively low mutation rate in mitochondrial genomes, barcoding attempts based on mtDNA are rare in land plants. The first mitochondrial markers for species-differentiation in Populus based on SNPs were developed recently [12] and the described P. tremula-specific SNPs were also obvious in the P. tremula mitochondrial genomes sequenced in this study. An attempt to also differentiate between cultivars by mitochondrial SNPs has been made recently in date palm [75].…”

“…Such high-coverage regions and also low coverage-regions (see also Materials and Methods) were validated by Sanger-sequencing of PCR amplicons including (overlapping parts of) these regions (related primer sequences in S1 appendix). The 38 new Sanger sequences were added to an existing dbGSS library of mtDNA Sanger sequences of 717–1B4 at GenBank [12] (dbGSS library accession number LIBGSS_039210; GenBank accession numbers of the new sequences within the library: KS308288 ff.). The mtDNA sequence of 717–1B4 (KT337313) was validated with the Sanger sequences as well as P. tremula mitochondrial NGS-scaffolds that were independently generated in the scope of the P. tremula v1.0-UPSC genome assembly which is available since recently at PopGenie v3 [76,77].…”

“…For genetic differentiation of vascular plants, molecular markers are used mainly based on DNA variations in two chloroplast regions ( rbcL and matK ) as a two-locus barcode [10,11]. In addition, chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) molecular markers can be applied in inter-specific hybrids to determine the maternal cross partner and to unravel the direction of the cross [12–14]. Chloroplast markers are also largely used to study the phylogeography of many plant species including trees ( e.g ., [15,16]).…”

Development of Multiplexed Marker Sets to Identify the Most Relevant Poplar Species for Breeding

“…The identification of poplar species is necessary for breeding activities; nevertheless, most authors concentrate on smaller species complexes, such as Chinese poplars [27,31] or on a smaller number of species to be differentiated [13]. After starting with seven species in 2012 [26] and increasing the number to 14 in 2014 [28], we are now able to identify/differentiate 19 different poplar species.…”