Ciljevi ovog istraživanja su sljedeći: utvrditi razinu varijabilnosti unutar i između populacija divlje ruže iz različitih regija na osnovi morfoloških, pomoloških i genetih analiza; na tri generativna potomstva analizirati morfološku, pomološku i genetsku varijabilnost između i unutar potomstava.
U ovom istraživanju, radi procjene morfološke, pomološke i genetske varijabilnosti, korišteno je 57 autohtonih primki - genotipova divlje ruže (Rosa spp.). Za dio istraživanja in situ analizirane su 24 primke iz tri regije: brdsko planinska, kontinentalna i mediteranska. Kao dio istraživanja ex situ, analizirane su 33 primke triju elitnih biljaka porijeklom iz Korčule (P1), Malešnice (P2) i Prigorja Brdovečkog (P3).
Morfološke analize za mjerenje trnovitosti izvršene su uzorkovanjem izbojaka krajem kolovoza ili početkom rujna 2008. i 2010. godine (sa svake primke po dva izbojka, sa svakog izbojka po dva uzorka dužine 10 cm na gornjoj i donjoj trećini). Morfološka analiza promjera cvijeta izvedena je u vrijeme cvatnje 2013. godine pri čemu je sa svake biljke uzorkovano po 10 cvjetova.
Za pomološke analize su 2010. i 2012. godine slučajnim odabirom unutar populacija izdvojene 1, 2 ili 3 primke, a sa svake primke oko 80 plodova (uzimanje uzoraka plodova provedeno je krajem kolovoza ili početkom rujna u vrijeme dozrijevanja). Od pomoloških svojstava mjerena su: dužina ploda (mm), širina ploda (mm), masa ploda (g), masa mesa ploda (g), randman mesa (%), indeks oblika ploda i ukupna suha tvar (%).
Genetska varijabilnost in situ i ex situ analizirana je primjenom 6 polimorfnih SSR (Simple Sequence Repeats) markera. Uzorkovanje biljnog materijala za genetske analize provedeno je u kolovozu 2008., istovremeno s uzorkovanjem biljnog materijala za morfološke i pomološke analize. Na svim genotipovima za ekstrakciju DNA uzimani su mladi listovi s vrhova mladica i čuvani do postupka liofilizacije te pohranjeni do izolacije DNA na -80°C. Izolacija i pročišćavanje genomske DNA divljih ruža izvršena je prema protokolu po modificiranoj metodi prema Doyle & Doyle-u (1990.). Za identifikaciju genotipova i utvrđivanje genetske raznolikosti korišteni su mikrosateliti odnosno SSR (Simple Sequence Repeat) markeri.
Morfološke i pomološke analize otkrile su značajnu varijabilnost in situ i ex situ populacija za važna agronomska svojstva. Velika fenotipska plastičnost morfoloških i pomoloških svojstava rezultat je utjecaja ekoloških čimbenika i nasljednih svojstava.
U istraživanju genotipova in situ, najveća varijabilnost svojstava utvrđena je u kontinentalnom području. Većina agronomski značajnih svojstava ističe se kod genotipova RC35 i RC52. Analiza pomoloških karakteristika ex situ populacija generativnih potomaka omogućila je potpuno odvajanje samo P1 populacije mediteranskog porijekla. Najveća varijabilnost kao i najpoželjnija agronomska svojstva zabilježeni su u populaciji P3.
Genetska analiza dokazala je taksonomski status genotipova divljih ruža i utvrdila razinu polimorfizma i stupanj genetske udaljenosti između genotipova divljih ruža u Hrvatskoj. Dobiveni rezultati u skladu su s istraživanjima ove i srodnih vrsta i mogu se koristiti kao podloga za daljnja istraživanja za razvoj i uzgoj novih sorti i hibrida divlje ruže.
|Abstract (english)|| |
Morphological, pomological and genetic variability of dog rose (rose hip) genotypes (Rosa canina L.)
Wild or dog rose (Rosa canina L.) is widespread in the form of spontaneous populations and ecotypes throughout Croatia. It is also known as šipak, pasja drača, šipurak etc. It is taxonomically classified into the genus Rosa, the subgenus Rosa and the section Caninae which is the largest and most complex in Europe. Relations within this genus remain unclear due to the difficulty in marking species boundaries and taxonomy which is extremely complex and is still being researched in depth. These differences are mainly the result of difficulties in identifying roses due to heterogamous canina meiosis, intra-species variability, predominant matroclinal inheritance, polyploidy, and interspecific hybridization.
Dog rose is evolutionarily young, and in agriculture a relatively new fruit species of great potential whose cultivation, as well as selection work, is still in its infancy. The adaptability to different ecological conditions and the ability to hybridize within and with related species with the creation of new genetic combinations and polyploidy have allowed it to spread in temperate climates of the northern hemisphere. Its phylogenetic development is not yet complete, so there are difficulties in marking species boundaries. Due to its nutritional value, it has the potential to be used in medicine, cosmetics and the food industry. Because of this, there is interest in developing varieties and hybrids and increasing production in conventional and organic farming.
In dog rose breeding, the greatest attention is paid to the quality of the fruit which is the main key to greater market expansion. In addition, the lack of prickles, processing potential, adaptability and resistance to pests are important.
It is possible to isolate genotypes of agronomically desirable properties (time and length of ripening, amount of antioxidants, fruit size and firmness, fruit flesh ratio, vitamin C content, etc.) based on the knowledge about the variability of biological and pomological properties and create varieties suitable for plantation cultivation by methods of classical selection.
So far, no systematic research of botanical and agro-biological properties has been conducted in Croatia, nor has agro-biodiversity of this species been used for breeding.
The objectives of this study are the following: determine the level of variability within and between dog rose populations from different regions based on morphological, pomological and genetic analyses; analyze morphological, pomological and genetic variability between and within offspring on three generative offsprings.
In this study, 57 autochthonous wild rose accessions (Rosa spp.) were used to evaluate morphological, pomological and genetic variability. For part of the in situ study, 24 accessions from three regions were analyzed: mountainous, continental and Mediterranean. As part of the ex situ study, 33 accessions of three elite plants originating from Korčula (P1), Malešnica (P2) and Prigorje Brdovečko (P3) were analyzed.
Materials and methods
To assess the phenotypic and genotypic divergence, dog rose (R. canina L.) accessions representing natural populations of dog rose collected from 15 localities in different geographical areas were analyzed.
Morphological analyses to measure prickliness were performed by sampling shoots in late August or early September 2008 and 2010.
For pomological analyses fruits were collected by random selection within the populations from the selected accessions at the end of August or the beginning of September 2010 and 2012. The following pomological traits were measured: fruit length (mm), fruit width (mm), fruit weight (g), fruit flesh weight (g), fruit flesh ratio (%), fruit shape index and total dry matter (%).
Sampling of shoots, flowers and fruits was carried out on all accessions except those that were destroyed in the meantime in the original locations which is why there are variations in the number of samples.
Sampling of plant material for genetic analyses was carried out in August 2008, at the same time as sampling of plant material for morphological and pomological analyses.
Young leaves were taken from the tips of the shoots on all DNA extraction genotypes and stored until lyophilization. Lyophilization was carried out by cold drying in a lyophilizer to prevent the degradation of the DNA of the plant material. Storage of liofilized samples until DNA isolation was performed at -80 °C.
Isolation and purification of genomic DNA of dog roses was carried out under a protocol according to the Doyle & Doyle modified method (1990). The concentration and purity of the isolated DNA was determined by agarose gel electrophoresis and by comparison with standards of known concentrations.
Microsatellites and SSR (Simple Sequence Repeat) markers developed and assigned for the purposes of this research by the „Stichting Dienst Landbouwkundig Onderzoek Foundation (DLO)“ - Praktijkonderzoek Plant & Omgeving/Plant Research International (PPO/PRI), Plant Breeding, Wageningen, The Netherlands, were used to identify genotypes and determine genetic diversity and were previously used for similar research. Twenty-three SSR primers were tested preliminary, and six were selected for the final analysis (RhO517, RhAB15, RhD201, RhEO506, RhP507, Rh1402).
The method developed by M. Schuelke (2000) with the universal M13(-21) primer according to published temperature conditions was used to amplify the markers. The PCR reaction was performed according to the protocol of Esselink et al. (2003). A capillary sequencer and Gene Mapper 4.0 software were used to visualize and separate the amplified fragments.
Results and Conclusion
Morphological and pomological analyses revealed significant variability of in situ and ex situ populations for important agronomic properties. Great phenotypic plasticity of morphological and pomological properties is the result of the influence of ecological factors and hereditary characteristics.
In the in situ genotype study, the greatest variability of properties was determined in the continental region. Most of the agronomically important properties stand out for the RC35 and RC52 genotypes. The analysis of the pomological characteristics of ex situ populations of generative offsprings only allowed complete separation of the P1 population of the Mediterranean origin. The highest variability as well as the most desirable agronomic properties was determined within the P3 population.
Genetic analysis has proven the taxonomic status of Croatian dog rose genotypes and determined the level of polymorphism and the degree of genetic distance between Croatian genotypes. The results obtained are consistent with studies of this and related species and can be used as a basis for further breeding research for the development and cultivation of new dog rose varieties and hybrids.