Pyrethrin is a specialized metabolite of Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.), Asteraceae, known worldwide as an effective bioinsecticide. It consists of six active compounds: Pyrethrin I and II, cinerin I and II, and jasmolin I and II. Pyrethrin accumulates mainly in the flower heads and its content depends on numerous factors, such as the flower developmental stage. This study aims to investigate the accumulation patterns of six pyrethrin compounds in the flower heads of Dalmatian pyrethrum over the eight developmental stages (FS1 to FS8), and to make a comparison in six natural populations. Ultrasound assisted extraction was used to extract the pyrethrin, while qualitative and quantitative analysis was performed by High performance liquid chromatography. The accumulation patterns of different pyrethrin compounds were generally similar and also synchronous between different populations, while the pyrethrin I/pyrethrin II ratio showed irregular patterns. In all populations studied, the highest increase of all compounds was observed from FS1 to FS2. Their concentration continuously increased, reaching the highest values at FS4 stage (2–5 rows of open disc flowers), and generally decreased gradually towards FS6 or FS7 and then stagnated until the FS8 stage. Despite the very similar accumulation pattern of pyrethrin compounds in the different populations, the significant differences in their content suggest a different genetic background. Knowledge of the dynamics of pyrethrin compounds accumulation across flower development stages is valuable for determining the optimal harvest time of pyrethrum flower heads.
Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevis.) Sch. Bip.) is an outcrossing plant species (2n = 18) endemic to the eastern Adriatic coast and source of the natural insecticide pyrethrin. Due to the high repeatability and large genome (1C-value = 9.58 pg) our previous attempts to develop microsatellite markers using the traditional method were unsuccessful. Now we have used Illumina paired-end whole genome sequencing and developed a specific procedure to obtain useful microsatellite markers. A total of 796,130,142 high-quality reads (approx. 12.5× coverage) were assembled into 6,909,675 contigs using two approaches (de novo assembly and joining of overlapped pair-end reads). A total of 31,380 contigs contained one or more microsatellite sequences, of which di-(59.7%) and trinucleotide (25.9%) repeats were the most abundant. Contigs containing microsatellites were filtered according to various criteria to achieve better yield of functional markers. After two rounds of testing, 17 microsatellite markers were developed and characterized in one natural population. Twelve loci were selected for preliminary genetic diversity analysis of three natural populations. Neighbor-joining tree, based on the proportion of shared alleles distances, grouped individuals into clusters according to population affiliation. The availability of codominant SSR markers will allow analysis of genetic diversity and structure of natural Dalmatian pyrethrum populations as well as identification of breeding lines and cultivars.
Teucrium montanum L. and T. capitatum L. are two plant species with overlapping distribution in the Balkan Peninsula, especially in Croatia, where several occurrences of their putative hybrid species T. × rohlenae have been recorded. Next-generation sequencing of both species and de novo assembly was carried out resulting in 120 contigs for T. montanum and 1685 contigs for T. capitatum assembled. The Dig-up primers pipeline was used for SSR mining of both assemblies, applying different criteria that resulted in 112 SSR candidates for testing. A subset of 41 SSRs was selected and after two rounds of testing, twelve SSRs were developed and characterized. A total of 232 alleles were detected with 5 to 29 alleles per locus, based on the test sample. The genetic diversity analysis of three Teucrium taxa from a single location revealed a higher level of diversity in T. montanum than in T. capitatum with intermediate values for the hybrid species. The NeighborNet diagram and genetic structure analysis grouped the taxa into two separate clusters, the first of which consisted exclusively of T. montanum, while the other was composed of intermixed T. capitatum and T. × rohlenae individuals. The availability of SSR markers for two Teucrium taxa will allow in-depth analysis of genetic diversity and structure, as well as molecular identification of their putative hybrids in the future.