Activity 1: Conservation of plant genetic resources

ACTIVITY DESCRIPTION

(1) Acquisition of the accessions and filling the gap in the Dalmatian pyrethrum (Tanacetum cinerariifolium /Trevir./ Sch. Bip.) and sage species (Salvia officinalis L.; S. x auriculata Mill.; Salvia fruticosa Mill.; Salvia brachyodon Vandas) collections: plant material (seed and leaf tissue) of the natural populations will be collected covering the whole distribution area in Croatia, Bosnia and Herzegovina, Montenegro and Albania.

(2) Establishment of the field trial: the collected seed will be reproduced and regenerated in the field trial.

(3) Preparation of Dalmatian pyrethrum samples: during the field trial plant material for the extraction of DNA for further genetic analyses will be sampled, while flower heads will be sampled for further biochemical analyses.

(4) Preparation of sage samples: leaf tissue of the sage species will be sampled in their natural habitat and stored in a bag filled with silica gel until the genetic analyses. For the biochemical analyses fresh above-ground parts of the plants will be collected.

The activity is connected to the objective 1 in the part concerning the establishment of the base and field collections of plant genetic resources of Dalmatian pyrethrum and sage species with the purpose of providing plant material for further analyses at the chemical and genetic level as well as conservation of these natural populations.

Activity 2: Phenotyping

ACTIVITY DESCRIPTION

(1)  Determination of the content and composition of pyrethrins in the flower heads of Dalmatian pyrethrum: sampling of the Dalmatian pyrethrum flower heads for biochemical analyses will be carried out at a stage when ¾ tubular flowers are opened. The flower heads will be dried naturally, in a dark and ventilated room, to a final moisture content of 10 to 12%. For the extraction of pyrethrins from the flower heads, the Ultrasonic Extraction procedure using acetone as a solvent will be applied. Identification and quantification of active pyrethrin components and total pyrethrin will be determined by High Performance Liquid Chromatography (HPLC).

(2) Determination of essential oil content and composition of sage samples: Microwave Extraction procedure will be used to isolate essential oils from the sage species, while the identification and quantification of the major active components will be carried out using Gas Chromatography coupled with Mass Spectrometry and FID detector (GC-MS / FID).

The activity is related to objective 1 in the part concerning the implementation of Dalmatian pyrethrum and sage collections phenotyping with the purpose of determining biochemical diversity of the sampled natural populations.

Activity 3: Genotyping 

ACTIVITY DESCRIPTION

(1) DNA and RNA extraction from plant material: DNA and RNA extraction from samples of Dalmatian pyrethrum and sage species will be conducted using extraction kits and automated extraction systems.

(2) Genome and transcriptome sequencing of Dalmatian pyrethrum and sage species: Genome and transcriptome sequencing will be conducted using Next Generation Sequencing (NGS) technology with special emphasis on ‘nanopores’ approach. The advantage of this approach is in reading of long DNA/RNA sequences, which will in combination with other NGS approaches, greatly facilitate the assembly of the whole genome/exon de novo. Since sages are an example of plant species with a small genome, and Dalmatian pyrethrum an example of a plant species with extremely large genome, NGS will be uniquely tested considering such extreme differences in genome size.

(3) Development of new SNP molecular markers: Using NGS technology (Sequencing-Based Diversity Array Technology /DarTSeq/) a considerable number of SNPs (Single Nucleotide Polymorphisms) will be developed and linked to genes for different biosynthetic pathways (especially those connected to the synthesis of pyrethrins and essential oils) detected by NGS of genome and transcriptome of Dalmatian pyrethrum and sage species.

 (4) Analysis using SNPs: The developed SNPs will be used to analyse genetic diversity and structure of sampled natural populations.

(5) Association mapping: Based on the results of genetic and biochemical analyses, Association Mapping will be conducted to identify the markers linked to the biosynthesis of pyrethrins in Dalmatian pyrethrum and essential oils in sage species.

The activity is connected to objective 1 in the part concerning the implementation of genotyping of the collection for identification of candidate genes connected with the synthesis of pyrethrins and major components of essential oils of sage species, estimation of genetic structure and diversity of natural populations of Dalmatian pyrethrum and sage species as well as identification of DNA markers linked to genes that monitor pyrethrin synthesis and major components of essential oils of sage species.