Identifying the rose varieties and natural forms using ISSR-markers

Identifying among varieties and natural forms of roses were carried out using ISSR thechnics to determine kinship. It was studied 6 foreign varieties, 16 varieties bred in Nikitsky Botanical Garden, as well as 2 natural forms (Rosa canina and Rosa chinensis). Despite some samples breaking into three clades, many of them don’t belong to any of these groups due to the lack of relative ties, that confirms the high genetic diversity in the studied material. The revealed molecular genetic differences in rose forms and varieties can be used to identify and certify cultivars promising for their complex resistance to the continental climate conditions.


Introduction
Roses, or rather various species and hybrids of representatives of the genus Rosa L., are the leading culture in the world decorative gardening. The Central Siberian Botanical Garden of SB RAS (CSBG SB RAS) has been long studying the complex resistance to the continental climate conditions among different rose varieties of Nikitsky Botanical Garden (NBG) selection. It carries out biomorphological studies of formation shoot systems (FSS), as well as a histochemical research of the seasonal dynamics of starch, sugars, proteins, lipids and shoot lignification processes.
As the representatives of the Rosa L. genus complex are a part of the CSBG's bioresource collection, to identify related forms the estimation of rose verities using molecular genetic technics has begun. Among molecular genetic tools, the most common and informative method is the analysis of electrophoretic spectra of inter-microsatellite DNA sequences (ISSR -Inter Simple Sequence Repeat). ISSR markers are used in genetics, phylogenetics, and breeding to study genetic diversity and differentiation of genotypes [1][2][3]. Nowadays, the genetic identification is considered an urgent task of modern breeding.
Since representatives of the complex of the genus Rosa L. are part of the CSBG collection of biological resources, the evaluation of rose varieties using the molecular genetic method has begun. ISSR marker molecular genetic technique, widely spread to differentiate breeding material in various plant species [4][5][6], was used in the work. Molecular markers are characterized by a high level of polymorphism between varieties and can be effectively applied to assess general genetic characteristics.
The work objective is to determine the relationship between two natural forms and 22 varieties of foreign and NBG breeding from the CSBG SB RAS' collection.

Method
The study was carried out on dried leaves (herbarium material) of rose forms and varieties. DNA was isolated using the CTAB technique developed by J. Doyle [7] with some modifications. The method is based on cell lysis with cetyltrimethylammonium bromide buffer, deprotinization with chloroform, and DNA precipitation with isopropanol.
Amplification was carried out according to the following program: primary denaturation at 95°С -2 min; 38 cycles -denaturation 94°С -20 sec, primer annealing -45 sec (Taannealing temperatures and nucleotide sequences are shown in Table 2), elongation -1.5 min; final elongation -7 min. 8 primers were used for amplification. SYBR-Green I, an intercalating dye specific for double-stranded DNA, was applied as dye. Electrophoretic separation of amplification products was done in a 1-1.5% agarose gel with 1 × TBEbuffer.
The polymorphism quantitative assessment of applied markers and divergence level determination between the studied simples were based on state matrices of binary characters. The presence or absence of peptide components or PCR fragments of the same size were designated as states 1 or 0.
To process the obtained data statistically, TREECON software package (version 1.3b) was used [8]. Genetic distances were calculated according to the formula: where Nxy -the number of common fragments for x and y samples, Nx and Ny -the number of fragments for x and y samples respectively [9]. Dendrograms were created using the Neighbor-Joining (NJ) algorithm with 100 bootstrap pseudo-replicas. The polymorphism percentage (P, %) of each primer was calculated with the formula where Np -the polymorphic fragment number, N -the total fragment number.

Results and discution
Microsatellite DNA regions of the studied samples are analyzed using eight ISSR primers (Table 1). Each DNA band (amplified fragments) was treated as an independent character, being scored as present (1) or absent (0), resulting a binary data matrix which was used to compute primer banding characteristics such as: total number of bands (TNB), number of polymorphic bands (NPB), percentage of polymorphic bands (PPB). The studied primers have generated 175 amplified fragments (bands) from 250 to 2200 bp in length, from which 164 were polymorphic. The number of fragments amplified by a primer range from 19 (UBC834) to 26 (HB-12) ( Table 1). The polymorphism level detected by a single primer vary from 84.6 (amplified with HB-10) to 96% (with UBC-857), and averaged is 90.5%. Fig. 1 shows the ISSR profile of the samples obtained by amplification with HB12 primer. The electrophoregram demonstrates a high polymorphism of electrophoretic spectra confirming the wide genetic diversity of the studied rose forms and varieties.  On the NJ-dendrogram (Fig. 2) the studied samples were divided into three clades, wich confirms the genetic diversity high degree. The first clade includes two natural forms of Rosa canina, Rosa chinensis and eight varieties bred in NBG (Krymskiy Fakel, Malchik-spalchik, Krymskiy Samotsvet, Ayu-Dag, Lezginka, Chatyr-Dag, Zolotaya Osen, Krymskoye Ozherelye). . It should be noted that these varieties belong to different types of roses: Excelsa is included to the group of hybrids with Rosa wichuriana (currently Rosa lucieae), Popcorn is a miniature rose. Relative similarity is revealed between Rosa chinensis and the variety Krymskiy Fakel. It's not possible to establish relative ties for the remaining this group varieties, that indicates a wide genetic diversity of the selection material used to create them.
Despite some samples breaking into groups, many of them don't belong to any of these groups due to the lack of relative ties, that confirms the genetic diversity high degree of the studied material. In the first group they are Rosa canina natural form, Ayu-Dag and Krymskiy Samocvet varieties. The second and third groups mainly consist of varieties genetically diverse, although some of them form related pairs within the group.
Eight ISSR primers are tested on 22 varieties and 2 natural forms of roses in this work. The studies confirm that ISSR marker tool is quite suitable to investigate the origin of rose cultivars, and determine their genetic diversity degree. The revealed molecular genetic differences in garden rose forms and varieties can be used to identify and certify varieties promising for their complex resistance to the continental climate conditions. This work was supported by the project of АААА-А17-117012610053-9 "Identification of ways to adapt plants to contrasting habitat conditions at the population and organismic levels"