Differentiation of Populusspecies by chloroplast SNP markers for barcoding and breeding approaches

About 30 species within the genus Populus are classified in six sections. Several species belonging to different sections are cross-compatible, resulting in a high number of naturally occurring interspecific hybrids. Additionally, an even higher number of hybrids has been produced in huge breeding programs during the last 100 years. Hence, determination of poplar species used for the production of "multi-species-hybrids" is often difficult, and a challenge for the development of molecular markers aimed at species identification. Moreover, 14 of the 30 poplar species known are often used for the production of artificial hybrids and clones. In this study, over 20 regions of the chloroplast genome of poplars were analyzed by the use of 23 primer combinations already established for "barcoding" purposes, and 17 new primer combinations previously designed, with the aim of testing their suitability to differentiate 14 poplar species. Only about half of the established barcoding primer combinations yielded PCR amplification products, while new primer combinations had a much higher amplification success. Species-specific SNPs or Indels were detected in 11 out of the 14 poplar species analyzed. In order to set up a low-cost and fast method for species identification, we developed PCR-RFLPs applicable to seven of the species-specific SNPs. Overall, a high variability was found in the chloroplast intergenic spacers, as much as four primer combinations were needed to differentiate 11 species. Based on our results, the use of multi-locus combinations is recommended in barcoding analyses.