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Activity 1: Field campaign and morphometric analysis – May - Mid September 2018
- 6 field trips in the Romanian Carpathians and 1 in the Alps encompassing Austria, Slovenia, Italy, France and Switzerland
- 20 populations of Eritrichium sp. sampled from the Carpathians and 13 from the Alps
- 5 populations from the Carpathians and 23 from the Alps were added due to our helpful collaborators
- 30 accessions from the Natural History Museum, University of Oslo (NHMO) DNA Bank, comprising other Eritrichium species were added: Eritrichium villosum ssp. villosum, Eritrichium villosum ssp. pulvinatum, Eritrichium sericeum, Eritrichium sericeum ssp. sericeum, Eritrichium caucasicum, Eritrichium splendens, Eritrichium cammissonis, Eritrichium aretioides var. aretioides
Morphometric analysis:
14 morpho-anatomical traits were considered for 5 individuals from 39 populations (195 individuals in total)
Morphometric analysis: distribution analysis, correlations, linear regressions
Activity 2: Extraction of genomic DNA – Mid September - October 2018
- CTAB protocol adapted from Doyle and Doyle (1990), followed by an additional phenol-chlorophorm purification
- 581 individuals of Eritrichium nanum were extracted
Activity 3: Testing for polymorphic cpDNA markers – November - December 2018
- 25 chloroplastic regions were tested: intergenic spacers -rpL32-trnL, rpS12-rpL20, atpB-rbcL, trnQ-rpS16, trnV-ndhC, psbJ-petA, atpI-atpH, petL-psbE, psaI-accD, ndhJ-trnF, rpoB-trnC, ycf6-trnC, psbD-trnT, rpL32-ndhF, trnD-trnE, trnH-psbA, trnL-trnF, trnS-trnG, trnT-trnL, trnS-trnfM, rpS16-trnK, introns - rpS16, rpL16, trnL and trnG
Activity 4: Sanger cpDNA sequencing for all sampled individuals – January - March 2019
- 4 polymorphic regions (trnV-ndhc, ycf-trnC, psbJ-petA și petL-psbE) were sequenced
- Cynoglossum officinale and Pulmonaria filarszkyana were included as outgroups
Activity 5: ddRADsequencing – April - August 2019; September - October 2021
- based on Peterson et al. (2012) protocol with modifications from Capblanq et al. (2015)
- NGS sequencing outsourced to Novogene Company Limited (UK)
- 7 libraries (each including 47 samples) were successfully sequenced
Activity 6: Bioinformatic analysis – November- December 2021
- STACKS pipeline was used
- the final matrix contained 1520 independent SNPs
Activity 7: Data integration – April 2022 in October 2022
- distribution maps of haplotypes for Eritrichium nanum in Europe
- designation of area important to preserve the species' genetic diversity
Activity 8: Project management and dissemination – April 2022 in October 2022
- 2 international conferences attended
- 1 manuscript submitted to an ISI rated journal (so far)
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