Results

Results 2013

• the populations used in the analysis were mapped according to their origins, using GIS (Geographic information System).

Fig 1: the geographical origins of the Onobrychis transsilvanica and O. montana populations analysed

(A - Alps and Jura; W - Western Carpathians; AD- Dinaric Alps; Sw - South-Western Carpathians; E+Se - Eastern - South-Eastern Carpathians).

• DNA extraction from leaves stored in silicagel.

Fig 2: DNA extraction verification on 1% agarose gel.

• Testing multiple chloroplastic regions.

- 23 chloroplastic markers were tested.

Fig 3: Verification of the PCR amplicons.

- 3 chloroplastic markers selected.

• NJ tree based on the concatenated sequences of the selected markers was constructed.

(images with NJ analysis and the obtained sequences will be elaborated after the

publication of the data).

Main conclusions so far:

• a subspecies rank for Onobrychis transsilvanica should be considered appropriate.

• the current survey of Carpathian populations of O. transsilvanica demonstrated the existance of a genetic structure correlated to distribution, with two major phylogeographical groups formed by South-Western (Retezat Mts) and East - South-Eastern Carpathian.

For a comprehensive report of Phase 1 (2013) please send us an email in order to grant you permission to access it.

Results 2014

• a first set of 8 SSR primer pairs was tested for polymorphism and optimized (i.e. annealing T, elongation time, MgCl2 concentration, etc) for further microsatellite analysis of the Onobrychis populations.

• a second set of 6 SSR primer pairs from different sources was tested.

• a third set of 9 SSR primer pairs was tested.

• after PCR testing and electrophoretic migration, 10 SSR markers were chosen and were considered suitable for achieving the intended purpose.

Table 1: Name, sequence and annealing temperature of the selected SSR markers:

• PCR conditions were the following:

Table 2: PCR amplification steps (X- annealing temperature varies slightly between primers).

• Six of the primer pairs tested and the obtained amplifications.

Fig 1: Eg. of six SSR markers amplification.

• few examples of polymorphism identified as the result of the electrophoretic migration:

Ex 1: primer pair MtBA04CO8

Ex 2: primer pair MtBA27D09

Ex 3: primer pair BG178

Ex 4: primer pair AG81

Ex 5: primer pair MTIC272

• PCR amplification of all the populations analysed with the specific primers selected.

- Example of amplification in the case of primer MtBB22G10 (random samples were verified on the agarose gel).

-Example of amplification in the case of primer MTIC272 (random samples were verified on the agarose gel).

• • all the analysed populations were electrophoretically migrated on a Genetic Analyzer using all the selected SSR.

Main conclusions so far:

• 23 SSR markers tested for polymorphism.

• 10 SSR markers selected.

• succesful migration for all the analysed populations.

For a comprehensive report of Phase 1 (2014) please send us an email in order to grant you permission to access it.

Results 2015

- According to the project plan in this phase were followed and completed the following objectives and activities:

O1) Data analysis and processing the results obtained previously.

A1) Alleles scoring based on genetic profiles previously.

A2) Data analysis and results interpretation using specific programs.

A3) Final scientific report.

A4) Scientific publications.

A1) Alleles scoring based on genetic profiles previously.

- we used 16 populations of Onobrychis montana and O. transsilvanica (Table 1).

Table 1. Sampled populations of Onobrychis montana and O. transsilvanica: numbering, taxon, acronym, geographic origin (Country: Ro – Romania; Fr – France; Po – Poland; Sk – Slovakia; Mne – Montenegro, Sampling locality,), coordinates, Nei’s (1973) gene diversity.

* data will be available after the publication of the submited manuscript

- seven selected SSR primer pairs fluorescently dyed (6-FAM) (Table 2) were selected and used in reactions.

Table 2. Number of alleles, PIC value, Gene diversity of seven SSR loci analysed in 16 Onobrychis populations.

* data will be available after the publication of the submited manuscript

A2) Data analysis and results interpretation using specific programs.

- PowerMarker v.3.25 was used to calculate the total number of alleles, gene diversity and polymorphism information content (PIC).

- The UPGMA tree was built using with SplitsTree v.4.10.

- The relationships among individuals were analysed using Principal Coordinates Analysis (PCoA) based on the between-individual Jaccard similarities computed with the PAST v.2.17 software.

- A spatial analysis of molecular variance was performed using the program SAMOVA v.1.0 to define groups of populations (K) that are geographically homogeneous and genetically differentiated from each other.

- STRUCTURE v.2.3.4 software was used to detect genetic groups and to inference a population structure.

Results

- The Neighbor-joining tree (Fig. 1) of the 16 populations of Onobrychis montana and O. transsilvanica, plus the outgroup, revealed a bootstrap supported division between eight clusters.

Figure 2. Neighbor-joining consensus tree of the Onobrychis montana and O. transsilvanica populations. Bootstrap values above 50% are shown on major branches. Acronyms of populations as in Table 1. Details of the figure will be available after the publication of the submited manuscript.

- The SAMOVA indicated that the genetic differentiation among groups was the highest (FCT = 0.9941; P < 0.01) when samples were pooled into eight groups (Fig. 2).

Figure 2: SAMOVA results for Onobrychis montana and O. transsilvanica. Determination of best K (number of groups) based on FCT and populations grouping at best K value (K=8). Acronyms of the populations as in Table 1. Details of the figure will be available after the publication of the submited manuscript.

-The STRUCTURE analysis (outgroup excluded) exhibited some interesting results. The separation between the Alps and the Carpathians was indicated as the major split in the data set at K = 2. The highest mean likelihood was obtained for K = 6 (Figure 3, b).

Figure 3: (a) Population structure. The clusters obtained based on STRUCTURE analysis performed on all individuals from the populations. Acronyms of the populations as in Table 1; (b) Inference of best K. The best number of clusters (K) to describe population structure is inferred using the output data from STRUCTURE and the ad hoc criterion proposed by Evano et al, 2005. Details of the figure will be available after the publication of the submited manuscript.

- a PCoA analysis based on the SSR data was performed for all of the individuals (Fig. 4).

Figure 4: Principal Coordinates Analysis (PCoA) of Onobrychis montana and O. transsilvanica individuals based on Jaccard’s similarity coefficient. The symbols from the chart represents the following populations: * explanation of the PCoA figure will be available after the publication of the submited manuscript.

Detailes about discutions and conclusions of the performed study will be available after the publication of the submited manuscript.

A3) Final scientific report.

- Considering the genetic data from cpDNA (optionally AFLP technique) and SSR we composed un sintetic scientific final report for the entire project.

A4) Scientific publications.

- we prepared two manuscript fo publication:

„Genetic divergence and phylogeography of the alpine plant taxon Onobychis transsilvanica (Fabaceae)”, 2015, Authors: Ioan Băcilă, Şuteu Dana and Gheorghe Coldea, Botany, 93: 1-10, dx.doi.org/10.1139/cjb-2014-0175.

„Molecular taxonomy of Onobrychis transsilvanica Simk. (Fabaceae) based on genomic SSR fingerprinting”, 2015, Authors: Ioan Băcilă, Şuteu Dana and Gheorghe Coldea, Biologia, under review.