GENETIC PURITY ASSESSMENT OF COMMON BUCKWHEAT VARIETY ‘ DARJA ’ WITH THE USE OF SSR MOLECULAR MARKERS

In order to assess the genetic purity of common buckwheat variety ‘Darja’ which is the most commonly produced variety of this crop in Bosnia and Herzegovina, 10 SSR markers have been used. Five samples have been collected from different production regions in B&H (Breza, Nisici Plateau, Ustikolina, Bihac and Bosanska Krupa) and compared to the reference ‘Darja’ sample obtained from an ex situ seed collection from Slovenia. Seven out of ten primer pairs used managed to amplify SSR alleles. Analyses of molecular variance (AMOVA) showed a significant differentiation between the reference and all analyzed ‘Darja’ samples. Furthermore, the factorial correspondence analysis revealed a clear differentiation between the reference and ‘Darja’ samples from the most known production regions of common buckwheat in B&H clustering four out of five analyzed samples very close together. The most divergent one among the analyzed samples was the one from Ustikolina. Genetic purity of varieties of all of cross pollinated species produced in Bosnia and Herzegovina is questionable due to the general use of farm-saved seeds.


Introduction
Common buckwheat (Fagopyrum esculentum Moench), usually a diploid (2n = 16) annual crop plant, originating from southwestern China (Ohnishi, 1998a), is widely cultivated in Asia, Europe and America.It has a narrow gene pool (Ma et al., 2009) and its limited distribution contributes to the fact that most of the varieties grown are local populations adapted to their environmental conditions through cultivation (Song et al., 2011).High genetic diversity among and within common buckwheat varieties is a result of complete allogamy due to the heterostylous self-incompatibility system (Iwata et al., 2005).The mentioned diversity has been studied using allozyme analysis (Ohnishi, 1988b), RAPDs (Iwata et al., 2005;Sharma & Jana, 2002;Murai & Ohnishi, 1996), AFLPs (Iwata et al., 2005;Konishi et al., 2005) and SSRs (Song et al., 2011;Ma et al., 2009;Konishi et al., 2006;Iwata et al., 2005).All of these approaches proved to be effective in revealing genetic differences between samples of common buckwheat.
In terms of field crop production, alternative cereals, such as common buckwheat, are of an immense importance for European agriculture (Đikić et al., 2013).The common buckwheat variety 'Darja' is presumed to be the one of the most cultivated varieties in the countries of Southeastern Europe (Gadžo et al., 2016;Grahić et al., 2016a;Nikolić et al., 2010;Bavec et al., 2002).Besides 'Darja', buckwheat producers from Southeastern Europe tend to use local common buckwheat varieties, such us 'Čebelica' and 'Goluba' (Gadžo et al., 2016;Nikolić et al., 2010), and even local varieties of another species from the Fagopyrum genus -Fagopyrum tataricum (L.) Gaertn (Tartary buckwheat).It is important to mention that producers generally use farm-saved seeds as sowing material, not taking into account the disturbance of varieties' genetic integrity due to cross-pollination of common buckwheat (Grahić et al., 2016b), nor the specific requirements of those populations regarding some agro technical measures, such as fertilization (Grahić et al., 2016a).
Until now, SSR molecular markers have not been used to assess genetic purity of common buckwheat varieties that are produced in Bosnia and Herzegovina (B&H).In fact, diversity studies of field crops in Western Balkan countries are more frequently conducted through morphological evaluations (Savić et al., 2014;Grahić et al., 2013;Mladenović et al., 2012).
The specific objective of this study is to assess the genetic purity of five different samples of 'Darja' which were collected from the most known production regions of this crop in B&H.

SSR analyses
Seeds from six samples of 'Darja', analyzed in this study, are currently maintained at the Gene bank of the Faculty of Agriculture and Food Sciences in Sarajevo.The first sample, that served as 'Darja' reference, was obtained from an ex situ seed collection from Slovenia.Remaining five samples were collected from the best known production regions of common buckwheat in B&H (Breza, Nisici Plateau, Ustikolina, Bihac and Bosanska Krupa); all of them were produced under the name 'Darja'.Seeds were sown in pots, and 16 seedlings were taken from each sample for the purposes of genetic analyses.
Genomic DNA was extracted from green leaves of the collected buckwheat seedlings using peqGOLD plant DNA kit-a (Peqlab) according to the manufacturer's instructions.Ten primer pairs (Table 1), previously published by Kishore et al. (2012), Ma et al. (2009) and Iwata et al. (2005) were used for SSR amplifications.
The M13F-tail PCR method (Schuelke, 2000) was used to measure the size of PCR products.PCR amplification was carried out in the total volume of code and DNA sequences of 10 primer pairs used in the assessment of genetic purity of 'Darja', a common buckwheat variety

SSR polymorphism
Of ten primer pairs used, seven managed to generate scorable SSR alleles.The remaining three primer pairs were discarded from further analyses.The total of 35 and 50 alleles were detected with the set of seven SSRs for the reference and five analyzed 'Darja' samples respectively, resulting in an average of 5.0 and 7.1 alleles per locus (Table 2).Lower values were reported by Ma et al. (2009) (5.90), who analyzed 41 common buckwheat populations from the National Agrobiodiversity Center in Korea.Song et al. (2011), who analyzed 179 accessions of common buckwheat obtained somewhat higher number of alleles per locus (7.90).Much higher values have been reported by Iwata et al. (2005) (40.60) for buckwheat germplasm from Japan.All calculated parameters (observed and expected heterozygosity and the PIC value) had higher average values in 'Darja' referent sample.
Seven SSRs identified 3 specific alleles in the referent sample and 18 specific alleles in the analyzed samples which indicates that the uncontrolled production of common buckwheat in B&H, mostly in regards to the use of farm-saved seeds as sowing material, led to the loss of genetic purity of the most commonly used common buckwheat variety in Western Balkans (Table 3).
(2012) showed that 83.49% of the total genetic variation was attributed to genetic diversity among populations and 16.51% occurred within the populations.The high percentage of variance between populations, in that study, can be explained by the fact that Tartary buckwheat is a selfpollinating species.There are still many uncertainties regarding the history of buckwheat production in B&H, leaving enough room for the assumption that the same local populations are present in the mentioned regions.
The most divergent one among the analyzed samples was the one from Ustikolina, but the reasons for that are still unclear.

Figure 1 .
Figure 1.Multivariate analysis (Factorial CorrespondenceAnalysis -FCA) of simple sequence repeat data for the reference and analyzed samples of 'Darja'-sample centroids (above) and individuals (beneath) shown

Table 2 .
Characterization of the 7 microsatellite loci used on six common buckwheat samples (16 samples per sample)

Table 3 .
Specific alleles found in the referent and the analyzed buckwheat samples

Table 4 .
Analysis of molecular variance (AMOVA) based on the 7 simple sequence repeat loci for each combination of reference and analyzed samples 'Darja' and 'Darja (Breza)'