Estimation of genetic distance among genotypes of caraway ( Carum carvi L . ) using RAPD-PCR

In order to estimate genetic diversity among starting materials and breeding strains of caraway, a collection of 17 accessions from botanical gardens in Europe, two cultivars ‘Rekord’ and ‘Kończewicki’, and four own breeding strains were analyzed by RAPD-PCR. The representative samples, each of five individual plants of accession, cultivar or strain, were taken from young rosette leaves. Forty of Genset Oligos RAPD primers were used for analysis and eight of them produced clear and reproducible banding patterns. In total, 62 banding patterns were obtained revealing 23 polymorphic bands, whereas the number of polymorphic bands ranged from two to four for one primer. The GS12 and GS43 primers generated two polymorphic bands, while each of the GS8, GS21, GS22, GS41 and GS53 primers generated three bands. The GS53 primer was the most informative one, revealing 60% of the estimated polymorphism. The estimated value of genetic distance ranged from 0.22 to 0.67. The lowest genetic distance was found between accessions from Cluj and Lousanne (0.22). The highest genetic distance was estimated between accession from Berlin and the strain no. 6 of cultivar ‘Kończewicki’ (0.67). UPGMA cluster analysis, based on eight RAPD primers, categorized the analyzed genotypes into four groups.


Introduction
Caraway (Carum carvi L.), a biennial herb of the family Apiaceae is widely cultivated in northern Europe, Russia, India, Canada and the United States and the world production of fruit may reach 15-20 thousand tones a year.In Poland caraway is the one of the most important cultivated medicinal plant which is cultivated on the area of 5000 ha and the fruit production is five thousand tones a year.The dried fruits of caraway (Carvi Fructus) are used mainly as a spice in food industry (SEIDLER-ŁOŻYKOWSKA;BOCIANOWSKI, 2012).In medicine caraway is used for its digestive, carminative, spasmolytic and stimulant properties.Limonene and carvone are the most important compounds of essential oil which is the main active substance of caraway fruit.Caraway is also the source of essential oil for cosmetic industry.Moreover, caraway oil is used as an alternative sprouting inhibitor during potatoes or flower bulb storage (HARTMANS et al., 1995).Moreover, caraway could be a component of animal forage to enhance their well-being (SADOWSKA; OBIDOWSKA, 1998).Development of high yielding cultivars characterized by high content of active substances and better adapt to abiotic and biotic stress is the main aim of caraway breeding programs.The detailed evaluation of starting materials with respect to their genetic diversity is necessary for identifying the sources of variation.The assessment of genetic diversity with the use of molecular markers enables to define specific markers associated with, or linked to important agronomic and phenotypic traits which could be further used for selection.
Only a few species of medicinal plants were applied for analysis with respect to determination of genetic diversity using molecular markers, i.e., marjoram (Origamum majorana L.), St. John's Wort (Hypricum perforatum L.), artichoke (Cynara scolymus L.), sweet basil (Origanum basilicum L.), chamomile (Chamomila recutita (L.) Rausch.)(ARNHOLDT-SCHMITT, 2002;KLÖCKE et al., 2002;MESSMER et al., 2002;WETZEL et al., 2002).Frequently, the selected molecular markers (random amplified polymorphic DNA, RAPD) are used for the detection of plant adulterants in some spice i.e. black pepper, chili or turmeric (DHANYA; SASIKUMER, 2010).Within the family of Apiaceae, genotypes of carrot (Daucus carrota L.) lines and accessions were characterized with the use of molecular markers.Bradeen et al. (2002) analyzed 124 carrot genotypes with 140 AFLP markers revealing high genetic diversity among wild type and population varieties.Grzebelus et al. (2001) used 33 RAPD and 88 AFLP markers for evaluation of genetic diversity among 31 carrot lines and accessions, as well as heterosis effect of three F 1 hybrids, in comparison with their parental lines.Based on the polymorphism level revealed by RAPD and AFLP analyzes, dendrograms showing genetic distance among the tested lines and accessions were constructed (GRZEBELUS et al., 2002;BARAŃSKI et al., 2004).As to our knowledge no results on molecular analysis of caraway genetic diversity were published yet.Although there were done some research in caraway doubled haploid (DH) lines by Ferrie et al. (2011) who investigated agronomic performance of 25 caraway DH lines in the field trials.
Here we present our results concerning assessment of genetic diversity among starting materials and breeding strains of caraway.

Plant material
A collection of 24 caraway accessions (Table 1) was established at the Institute of Medicinal Plants in Poznan.In 2007 and 2008 seeds of the collected accessions were sown in a greenhouse and 5-8-leaves plantlets were planted into a field at the beginning of May.In autumn, every year young rosette leaves of five plants from each accession were taken.Then these five leaves were mixed to make the bulk samples for DNA isolation.
Amplification products were resolved by 1.5% agarose gel electrophoresis at 100 V by 2.5h.The 100-bp DNA Ladder Plus (GeneRuller TM ) was used as DNA fragment length determination.

Statistical analysis
The coefficients of genetic distance (D) for all caraway collection accessions were calculated according to the formula given by Nei (1972): where: N A is the number of bands present in accession A, N B is the number of bands present in accession B, N AB is the number of bands present in accessions A and B.
Based on calculated coefficients the accessions were grouped hierarchically using the unweighted pair group method of arithmetic means (UPGMA).The relationship among the accessions was presented as a dendrogram.The reliability of the similarity tree was assessed by the bootstrap method (FELSENSTEIN, 1985) with 1000 replications.We used 95% as the statistically significant values.Bootstrap analysis was performed by using the program MEGA 3.1.
The genetic distance value estimated on the basis of RAPD markers ranged from 0.22 to 0.67 (Table 3).The lowest genetic distance was found between accessions from Cluj and Lousanne (0.22).Also low genetic distance values were found for accessions from Cluj and Bayreuth (0.26), Wroclaw and Jena (0.26) and Cluj and Prague (0.27).The highest genetic distance was revealed between accession from Berlin and strain no.6 of cultivar 'Kończewicki' (0.67).High genetic distances were found between accession from Berlin and strain no.9/13 (0.66) and between accession from Warsaw and strain no.9/12 (0.66).
Up to now, no results have been published on caraway DNA analysis by RAPD-PCR, as well as by other molecular markers.Nevertheless, some medicinal plants species were analyzed by this method.Arnholdt-Schmitt (2002) determined genetic variability among St. John's wort (Hypericum perforatum L.) populations, using 44 RAPD primers.The 11 of St. John's wort populations in which determined 23 polymorphic bands were used for genetic distance evaluation (from 0.1054 to 1.0986).Moreover, RAPD markers linked to hypericine and flavonols content were identified.Sixteen RAPD primers were used for determining DNA polymorphism among marjoram (Origanum majorana L.) accessions (KLÖCKE et al., 2002) and the detected 128 polymorphic bands were applied for evaluation of genetic distance among the analyzed accessions.Twenty of Iranian black cumin (Bunium persicum Boiss.)accessions were analyzed with the use of 15 RAPD and 17 AFLP primers revealing 192 polymorphic bands by RAPD.The genetic distance ranged from 0.40 to 0.82 and it did not correlate with geographical distance of places of genotype origin (PEZHMANMEHR et al., 2009).Pirkhezri et al. (2010) reported the RAPD analysis of chamomile (Chamomilla recutita (L.) Rausch.)populations and cultivars determining 205 polymorphic bands by the selected 18 primers.The range of similarity varied from 0.15 to 0.78 and the populations were divided into two main groups.Lately, genetic variability of 32 cumin (Nigella sativa L.) genotypes was detected with the use of 58 RAPD primers, generating 164 (66%) polymorphic fragments.UPGMA cluster analysis indicated seven distinct groups (IQBAL et al., 2011).All the authors mentioned above pointed out that their studies provided important information for management of germplasm resources and could be used in breeding programs for crop improvement.

Conclusion
The presented in this work evaluation of genetic variation of the caraway collection will be useful for selection of appropriate plant materials in breeding programs and will be applied for future research on designing genetic markers linked to the important quality traits, as: carvone and limonene content.