DNA Extraction and Application of SSR Markers in Genetic Identification of Grapevine Cultivars

Microsatellite markers (SSR markers) are widely used in grapevine genetic research for identification of cultivars, parentage analysis, and genetic characterization of germplasm. Aim of this work was extraction of total DNA, primer selection and design, PCR protocols and analysis of DNA sequences with special emphasize on variability between collected samples of different grapevine cultivars. The material used in this study were samples of grapevine leaves of different autochthonous and introduced cultivars from grapevine collection on Experimental field “Radmilovac” at the Faculty of Agriculture, University in Belgrade and from the National fruit collection “Brogdale” from UK. Standard set of nine primers for grapevine was used. Analyses were performed in Molecular Genetics Laboratory, School of Agriculture, Policy and Development, University of Reading, Reading, UK. Extraction and purification of total DNA from fresh and frozen plant material (grapevine leaves) was performed using a DNeasy ® Plant Mini (Qiagen Inc.) kit. The concentration of extracted DNA was measured by NanoDrop spectrophotometer and stored on -20°C until use. In the study, we utilized the protocol for Type-it Microsatellite How to cite this book chapter: Ranković-Vasić, Z. and Nikolić, D. 2019. DNA Extraction and Application of SSR Markers in Genetic Identification of Grapevine Cultivars. In: Vucelić Radović, B., Lazić, D. and Nikšić, M. (eds.) Application of Molecular Methods and Raman Microscopy/ Spectroscopy in Agricultural Sciences and Food Technology, Pp. 23–43. London: Ubiquity Press. DOI: https://doi.org/10.5334/bbj.c. License: CC-BY 4.0 24 Application of Molecular Methods and Raman Microscopy PCR Kit, optimized for fluorescent primers, and subsequent high-resolution fragment analysis by capillary sequencing instruments, following the Typeit Microsatellite PCR Handbook (Qiagen Inc.). The results of DNA analyses should be combined with ampelographic descriptors in identification of cultivars and planning the selection of grapevine varieties with desirable viticultural and enological values.


Introduction
Grapevine (Vitis vinifera L.) is one of the most valuable horticultural species. Currently, there are a large but imprecise number of grapevine cultivars in the world. In many regions have the synonyms (different names for the same cultivar) as well as homonyms (different cultivars identified under the same name). This number could likely be reduced once all cultivars are properly genotyped and compared. Identification of grapevine cultivars based on morphological differences between plants may be incorrect due to the influence of ecological factors. Therefore, methods for analysis at the cultivar genotype level have been developed. In the last twenty years, various techniques for the characterization of cultivars at the level of DNA (RFLP, RAPD, AFLP, SCAR and SSR markers) and isoenzymes have been established. The most appropriate for genotyping are those, using microsatellite markers (Jakše et al. 2013). In the past decade, the application of methods for molecular characterization has been significantly enhanced, particularly, DNA technology in ampelography, helping to identify varieties and their origin. Microsatellite markers (SSR markers) are widely used in grapevine genetic research for identification of cultivars, parentage analysis, and genetic characterization of germplasm. Microsatellites or simple sequence repeats (SSRs) have proved to be the most effective markers for grapevine genotyping (Sanchez-Escribano et al. 1999;Laucou et al. 2011). Thomas and Scott (1993) first used microsatellites for the identification of grapevine cultivars and demonstrated that microsatellite sequences are often represented in the grapevine genome and are very informative for the identification of Vitis vinifera cultivars. Hundreds of microsatellite markers for grapevines have been developed and most of them are publicly available ( Bowers et al. 1996;Arroyo-Garcia & Martinez-Zapater, 2004;Adam-Blondon et al. 2004;Merdinoglu et al. 2005;Cipriani et al. 2008). A set of six (VVS2, VVMD5, VVMD7, VVMD27, VrZag62, VrZAG79) or nine (previous six, combined with the following three: VVMD32, VVMD36, VVMD25) microsatellite markers has been used in grapevine genotyping studies, mostly for determining genetic variability among European grapevine cultivars, which are highly polymorphic (Sefc et al. 2001;This et al. 2004;Žulj et al. 2013). Aim of this research was extraction total DNA, primer selection and design, PCR protocols and analysis of DNA sequences with special emphasize on variability between collected samples of different grapevine cultivars.

Plant material
The material used in this study, were the samples of grapevine leaves of different grapevine cultivars. The source of the material was either the developed leaves from vines in the vineyard from collection "Brogdale", UK (leaves should be the size of a few centimeters, Fig. 1a, b, c), and leaves obtained from cuttings in the laboratory (the method of "provocation"), from collection "Radmilovac", Serbia (Fig. 2a, b).

Note:
• You can not use partially developed or fully developed buds (Fig. 3).
• Buds have a high concentration of protein.
• Isolation of DNA will fail (will be very difficult) if extraction is carried out from the buds (if used Kit); would not provide adequate DNA concentration.  • The leaves can be kept in the freezer (in paper bags) until the beginning of DNA isolation (Fig. 4) • The samples can be kept in the 1.5 or 2 ml tubes (-20°C) (Fig. 5).
• The samples can be lyophilized (weight about 20 mg), but the extracted DNK is not of desirable quality (Fig. 6).   • Working with lyophilized samples is more difficult (weight measurement of samples is complicated). • Each sample must have a code.

DNA extraction
Extraction and purification of total DNA from fresh or frozen plant material (grapevine leaves) was performed using a DNeasy ® Plant Mini Kit following the standard protocol for isolation of DNA from plant leaf tissue outlined in the DNeasy Plant protocol handbook (Qiagen Inc.).
• If necessary, redissolve any precipitates in buffer AP1 and buffer AP3/E concentrates. • Add ethanol to buffer AW and buffer AP3/E concentrates. • Preheat a water bath or heating block to 65°C.

Extraction protocol:
1. Plant leaves (about 150-170 mg fresh material) (Fig. 7) are grinded under liquid nitrogen (Fig. 8) to a fine powder using a mortar and pestle ( Fig. 9) or Tissue Lyser (Fig. 10). The tissue powder and liquid nitrogen were transferred to 1.5 ml tube and allowed the liquid nitrogen to evaporate ( Fig. 11).      3. The tubes were incubated at 65°C for 10 min on Termomixer that was set up to shake from 450 to 500 rpm (Fig. 13). During this step cells were lysed. 4. 130 μl of Buffer AP2 (P3) were added to the lysate, mixed, and tubes were incubated for 5 min on ice. During this step detergent, proteins and polysaccharides were precipitated. The tubes with lysate were centrifuged for 5 min at 14 000 rpm speed (or on max 14 680 rpm) in order to remove the precipitates (Fig. 14). The samples in the centrifuge must be uniformly distributed (in equilibrium) (Fig. 15).
5. The obtained lysate ( Fig. 16) was applied to the QIAshredder spin column placed in a 2 ml collection tube (with pink cover) and centrifuged for 2 min at 14 000 rpm speed (or max speed on 14 680 rpm). 6. The flow-through fraction (400-450 µl) from step 5 was transferred to a new tube without disturbing the cell-debris pellet. 7. 600 µl of Buffer AW1 were added to the cleared lysate and mixed by pipetting. 8. 650 μl of the mixture from step 7 were transferred to the DNeasy mini spin column sitting in a 2 ml collection tube and centrifuged for 1 min at 8000 rpm. After that flow-through were discarded. 9. The centrifugation at 8 000 rpm for 1 min was repeated. 10. DNeasy column were placed in a new 2 ml collection tube and 500 μl Buffer AW2 (AW) were added to the DNeasy column and centrifuged for 1 min at 8 000 rpm. After that flow-through were discarded.  11. 500 μl Buffer AW2 (AW) were added to the DNeasy column and centrifuged for 2 min at 14 000 rpm (or max 14680 rpm) to dry the membrane. It is important to dry the membrane of the DNeasy column since residual ethanol may interfere with subsequent reactions. This spin ensures that no residual ethanol will be carried over during elution. After centrifugation flow-through were discarded. 12. The DNeasy column were transferred to a 1.5 ml tubes and pipeted 60 μl of preheated (65°C) Buffer AE directly onto the DNeasy membrane. The column were incubated for 5 min (may be up to 15 minutes) at room temperature and then centrifuged for 1 min at 8 000 rpm to elute DNA. 13.
Step 12 was repeated. 14. The filter is removed. Tube with the DNA sample should be closed and placed on ice. Note: • Always mark the tubes used in the extraction.
• Take care of the cleanliness of the desk.
• The working surface has to be cleansed several times during the procedure • Always wear clean gloves (change the gloves several times during the work).
• For each pipetting must be put new sequel to the pipette.

Measuring the DNA concentration
Spectrophotometry is used to determine the concentration of DNA in the sample. The concentration of extracted DNA was measured by NanoDrop spectrophotometer (Fig. 17) and storage on -20°C until use.
• When the blank is read, should not get any spectrum on the screen (without spectrum). • Good concentration of DNA is shown in Table 1.
• The ration of 260/280 and 260/230 must be higher of 1.7, for extracted DNA, to be considered as high-quality material, suitable for further analyses. • In addition to estimate the quality of purified total DNA by calculating the ration 260/280 and 260/230, we also analyzed all samples by gel electrophoresis (see 2.6.1).

Note:
The DNA analyses should be combined with ampelographic descriptions, IPGRI, UPOV, OIV (1997) in planning the selection of grapevine varieties with desirable viticultural and enological value.
• A volume of master mix components is multiplied by the number of samples (for bigger number of samples).   (Fig. 19).

Note:
• PCR machine typically works about 1.30 hours.
• Depending on primers and overall experimental design, the different temperature and time settings could be used (Fig. 20). Different temperature is shown in Table 3. • PCR reactions in a Veriti TM Thermal Cycler (Applied Biosystems, Foster City, California, USA) using the following conditions: 94°C for 2 min, 35 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C, with a final extension of 30 min at 72°C. • PCR reactions in a GeneAmp PCR 9700 thermocycler (Applied Biosystems, Foster City, CA) using the following conditions: 94°C for 5 min, 30 cycles of 1 min at 94°C, 1 min at 51°C or 49°C (for multiplex or singleplex PCR, respectively), and 1 min at 72°C, with a final extension of 30 min at 72°C. • Can be used singleplex and multiplex reactions. Two multiplex PCR reactions were carried out for five (VVS2, VVMD7, VVMD27, VrZAG62, VrZAG79) and three (VVMD25, VVMD28, VVMD32) of the analyzed SSR a singleplex for VVMD5 (Vilanova et al. 2009).

Preparing for sequencing analysis (Protocol for Purification of PCR Products):
Transfer PCR reaction mixture (whole quantity, about 35 μl) to a 1.5 ml microfuge tube (blue cover) and add 3 volumes of Binding buffer 1 (105 μl). Then, according to the protocol described in DNA Cleanup Handbook at http://www.nbsbio.co.uk/downloads/DNA_Cleanup_Handbook.pdf 1. Transfer the above mixture solution to the spin column and keep it at room temperature for 2 minutes. Centrifuge at 10 000 rpm for 2 minutes. Discharge the eluate from the tube. 2. Add 750 μl of Wash solution to the column and spin at 10 000 rpm for 2 minutes. 3. Repeat the previous washing procedure using the same conditions. In order to remove any residual wash solution, extend spinning duration for 1 minute. 4. Place the column into a clean 1.5 ml tube and add 30-50 μl (usually 30 μl) of Elution buffer exactly into center of the column. Leave it at room temperature for 2 minutes (can also stand for 5 minutes). Centrifuge at 10 000 rpm for 2 minutes to elute the DNA. Note: • The incubation of the column, with the Elution buffer, at higher temperatures (up to 50°C) may slightly increase the yield especially for large DNA.

Electrophoresis
Electrophoresis is a method that is used to separate DNA fragments and determine their sizes by comparing them to the sizes of known fragment lengths. In our protocol, we applied this meted to test purified total DNA (2.2.) and the PCR products (2.4.)

Gel Electrophoresis
Procedure for making agarose gel: 1. Measure 0.25 g of the agarose and 25 ml × 1 of TAE buffer (100 ml TAE × 10 add 1000 ml distilled water). Note: Agarose gels: Commonly are used concentrations of agarose gel from 0.7% to 2% depending on the size of bands needed to be separated. Measure 0.60 g of the agarose and 60 ml × 1 of TAE buffer for biggest size electrophoresis box. Simply adjust the amount of starting agarose to %g/100 mL TAE (i.e. 2g/100mL will give you 2%). 2. Dissolve agarose by heating in microwave oven (20-30 sec).
Note: Caution HOT! Be careful stirring, eruptive boiling can occur. 3. Let agarose solution cool down for 5 min. 4. According to https://www.addgene.org/protocols/gel-electrophoresis/ add ethidium bromide (EtBr) to a final concentration of approximately 1.0 μg/ml. Ethidium bromide binds to the DNA and allows visualization of the DNA when the gel is exposed to ultraviolet (UV) light.

Note:
• EXTREME CAUTION! It is known that ethidium bromide is a mutagen. It is necceassary to wear gloves, a laboratory coat and safety glasses when using this techique. • Mildori Green Nucleic Acid Staning Solution is a safe alternative to traditional EtBr stain for detecting DNA in agarose gels. 5. Position the well comb in place and pour the agarose into a gel tray.
Note: Avoid formation of air bubbles which will disrupt the gel network by pouring agarose solution slowly. If bubbles are formed use a pipette tip to push them away towards the edges of the gel. 6. Cool newly poured gel for 10-15 minutes in a refrigarator or let it cool at room temperature for 20-30 minutes, until it completely solidifies.

Visualization of DNA fragments
1. By using any device that has a source of UV light your DNA fragments will visualize and look like bands on the gel. According to https:// www.addgene.org/protocols/gel-electrophoresis/ pay attention to the following: • UV light is very harmfool to your eyes and skin. Wearing protective glasses, gloves and laboratory coat is necessary. • Using long-wavelength UV and the shortest possible exposition time will minimize damage to the DNA in the case that further analysis of DNA is planned. 2. Visualize DNA fragments by WP (GelDoc -It TS2 /Imager Benchop UV Transilluminator) (Fig. 26). 3. Gel in Transilluminator (Fig. 27). 4. Software: UVP TS2 (Fig. 28). 5. Printing by digital graphic printer UP-D897.

Analyzing Gel:
Use the DNA ladder, in the first lane, as a guide (the manufacturer's instruction will indicate the size of each band) to determine the length of the bands detected in the sample lanes and visualize purified total DNA (Fig. 29) and also visualize the PCR products (Fig. 30).
Note (according to https://www.addgene.org/protocols/gel-electrophoresis/): • In order to get better resolution (crispness) of samples DNA bands lower voltage in a longer duration of a run can be used. Alternative is to choose a wider gel comb or to load lesser amount of DNA into the well. • In order to get better separation of bands in the case of similarly sized fragments a higher percentage of agarose gel can be used to better separate smaller bands, and a lower percentage of agarose gel to separate larger bands.