Abstract: In the present study, three DNA extraction procedures were examined to determine which might yield DNA from Grape leaves suitable for molecular analysis for RAPD, SSR. AFLP and etc analysis. The three methods examined were: the miniprep procedure and the modified CTAB for difficult species and protocol CTAB. Only the modified CTAB method consistently yielded DNA suitable for Polymerase Chain Reaction (PCR) amplification, regardless of plant growing conditions or leaf age. The quality and quantity of extracted genomic DNA gained from these methods are deliberated by means UV biophotometer, electrophoresis in 1.2% agarose gel and PCR. In this regard, application chosen for young and mature leaves, the most value of qualified DNA, is extracted from fully expanded leave when PVP was added to the extraction buffer. This same procedure also yielded PCR-amplifiable DNA from various other perennial, woody species and from other fruit species such as apple (Malus domestica), cherry (Prunus avium), peach (Prunuspersica), plum (Prunus domestica). DNA yield from this procedure is high (up to 1 mg g-1 of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the Polymerase Chain Reaction (PCR).
INTRODUCTION
Vitis vinifera taxonomically belongs to the Ampelidaceae or Vitaceae family. Grapevine is an ancient fruit species in Iran and its domestication goes back to 5000 years old (McGovern, 2003). Recently raisins of seeded and seedless grapevine were found by Iranian and Italian archeologists in Shahre Sokhta (Sistan, Iran).
The long history of grapevine growth has determined a complex picture in which many biotypes or even cultivars are misidentified or called by different names in different areas. This often makes genetic identification difficult. So it takes for serious to collect the varieties of all its genotypes, to assay and classify them. For this movement extraction and preparation of proper DNA samples is certainly of importance. Classifying the genetic resources and molecular methods play significant roles to manage and hoard such precious priceless collections.
Vitis vinifera and related species has been the subject of extensive genetic studies due to their worldwide cultivation and importance. Recently this plant has been used for gene mapping (Akkurt et al., 2007; Kikkert et al., 2005; Troggio et al., 2007), genetic transformation (Wang et al., 2005; Baribault et al., 1990; Hébert et al., 1993) and DNA fingerprinting (Di Gaspero et al., 2007; Adam-Blondon et al., 2004) However, molecular markers require DNA of suitable purity for the enzymatic PCR and it is often difficult to separate DNA from naturally occurring plant cell contaminants.
The relatively small genome size of Vitis vinifera (0.50 pg C-1) compared to many other perennial plant species (Lodhi and Reisch, 1995; Arumuganathan and Earle, 1991) should facilitate molecular genetic studies of Vitis. However, DNA extraction from grapevine has been difficult due to the presence of contaminants such as polyphenols and polysaccharides. In particular, polysaccharides (Do and Adams, 1991; Murray and Thompson, 1980; Fang et al., 1992) and polyphenolic compounds (Newbury and Possingham, 1977; Katterman and Shattuck, 1983; Couch and Fritz, 1990; Howland et al., 1991; Collins and Symons, 1992) can form a complex with and become irreversibly bound to nucleic acids during extraction (Varadarajan and Prakash, 1991).
Some of these contaminants can inhibit the activity of DNA modifying enzymes (Draper and Scott, 1988). This study indicates that extraction of DNA is not always simple or routine and that published protocols are not necessarily reproducible for all species (Stein, 1993; Rogers, 1994). In this laboratory, simple extraction protocols such as suggested by Wang et al. (1993), did not yield DNA from mature grape leaves. Other published protocols included the use of ultracentrifugation with cesium chloride (Baker et al., 1990), protoplast isolation (Deragon and Landry, 1992), detergents such as SDS (Dellaporta et al., 1983; Varadarajan and Prakash, 1991) and CTAB (Murray and Thompson, 1980; Doyle and Doyle, 1990), or Modified CTAB (Lodhi et al., 1994). In addition, PVP have been used to remove phenolic compounds (Doyle and Doyle, 1990) and excess polysaccharides (Do and Adams, 1991).
Respectively Successful extraction of PCR-amplifiable DNA can lead to the establishment of DNA fingerprints for individual grape cultivars. These fingerprints will be used as a diagnostic tool for cultivar identification. Grape cultivars are vegetatively propagated; therefore, a requirement of the DNA extraction method is that it not be influenced by leaf age or growing conditions that generated the leaf. The objectives of this study were to determine which of the three common DNA extraction methods (SDS, CTAB, or modified CTAB) might yield PCR-suitable DNA from Grapevine leaves, to evaluate the need for PVP in the extraction process, to examine the effect of leaf age and growing conditions on DNA extraction and to examine the reproducibility of the amplification products.
MATERIALS AND METHODS
Plant material: The plant material used for comparison of DNA extraction methods included 20 grape cultivars (Table 1) which all belong to Vitis vinifera were hardly collected from the demolished vineyards of different area of Iran. Leaf material of these 20 cultivars was available from 6 to 7 year-old field-grown material, 1 to 2-year-old rooted greenhouse cuttings in the Zabol University glass house and micropropagated material that was sub cultured a regular basis in the Biocenter of Zabol University.
Culture media: The powder media (4.2 g L-1) of Murashige and Skoog (1962) obtained from Sigma, 20 g L-1 sucrose, 0.1 mg L-1 IAA for rooting and 1.5 mg L-1 BAP were used to produce enough shoots and leaves in vitro. The leaf materials from jars and plants in pots were utilized for DNA isolation during the study.
DNA extraction: Leaf material included the blade and petiole from field and greenhouse-grown material and stems and leaves of micropropagated shoots. The leaf material was washed to remove any source of foreign DNA, such as insects or insect eggs on the field- or greenhouse-grown leaves. 0.5 to 1 g of leaf material was then frozen in liquid nitrogen, ground with a mortar and pestle and subjected to one of the following extraction procedures.
| Table 1: | Comparison between means of DNA concentration from leaf samples of 20 cultivars that collected from different area of Iran |
| Mean separation within columns by Duncan`s Multiple Range Test. Mean with the same alphabets are nonsignificant but mean with the different alphabets have significant difference | |
| Table 2: | Analysis of variance for different methods of DNA extraction, leaf type (young, mature) and growing condition (field, greenhouse, tissue culture) from Vitis vinifera |
| *Significant at p = 0.05, **Significant at p = 0.01 | |
Three DNA extraction methods, referred to as the SDS, CTAB and CTAB modified methods, were examined. The first method was the miniprep procedure (= SDS) described by Dellaporta et al. (1983). The second method was described by Doyle and Doyle (1990). The third method was modified Protocol CTAB suggested by Lodhi et al. (1994). The soluble PVP was added (wt:vol) in to the extraction buffer (Table 2) and after that, DNA was solubilized in TE buffer (Tris 10 M, EDTA 1 mM and pH 8.0) and stored at -20°C.
Data analysis: Three replications of a 3x3x2 factorial, completely randomized design were used in the analysis of interaction between extraction methods, growing conditions and leaf age (Table 2). The effect of the insoluble PVP (0, 50 mg Polyvinyl Pyrrolidone) on the DNA yields and purity of 20 cultivars (Table 3) were analyzed using three replications of a factorial completely randomized design. Six cultivars were native and other cultivars were collected from other regions.
Estimating of DNA quantity and quality: Two DNA quantification methods were used. The first method was biophotometer (Eppendorf AG.Hamburg) measurement with UV absorption at a wavelength of 260 nm (as nucleic acids absorbing wavelength of light) and also in wavelength of 280 nm (as the proteins absorbing wavelength of light). The second method was the minigel method using ethidium bromide fluorescence and a lambda DNA standard, as described by Sambrook et al. (1989).
Measures of DNA purity were determined by the A260:A280 and A260:A230 ratios. These ratios provide indications of protein and polyphenol and carbohydrate contamination, respectively (Manning, 1991). Absorption ratios were determined using the UV biophotometer. The samples with optimal values ranging from 1.8-2.0 were used for the amplification and the DNA concentration of 10 ng was formed to be used in the laboratory.
The electrophoresis of DNA on 1.2% agarose gel, Showed quality of DNA bands for each sample. Each one contained 8 μL of extracted DNA and 2 μL loading buffer, that put into the agarose gel sumps and loaded off in TBE buffer condition. Agarose gel was under a steady voltage of 75 for 1 h and then stained by ethidium bromide. The results were visualized and photographed by Gel Documentation system (Vilber Lourmat-France) under UV lighting.
DNA amplification: The ability of amplification of extracted DNA strands was proved by 2 randomized 10-nucleotide primer (Sinagen). Genomic DNAs were amplified using 2 different primers (5´- CGGCCCACGT-3´ and 5´-CCCACTACTG-3´) after optimization of amplification reaction. Polymerase chain reaction (PCR) was performed using a Gradient Eppendorf Termal Cycler based of what reported by Solouki et al. (2007). Each PCR reaction was carried out more than three times to be observed the repeatability of amplification patterns.
The reaction mixture with total volume of 25 μL containing of 7xPCR buffer (10 mM Tris-HCl, pH 8.0, 50 mM KCl, Sinagene), 0.1% triton x 100, 2 mM MgCl2, 200 μM dNTPs, 2U of Taq DNA polymerase (Sinagene), 0.4 μM primer and 10 ng genomic DNA was used. The Termal Cycler with the following profile was programmed (i) 40 cycles of 30 sec at 94°C, 1 min at 36°C and 2 min ramp until 72°C. (ii) Final step of 72°C for 8 min and then cooled to 4°C. The PCR products were separated by
| Table 3: | Analysis of variance for the effect of insoluble PVP (0, 50 mg Polyvinyl Pirrolidone) on 20 grape cultivars |
| *Significant at p = 0.05, **Significant at p = 0.01 | |
electrophoresis on 1.4% agarose gel in 0.5 x TAE buffer (0.045 M Tris, 0.001 M EDTA, Acetic acid) and stained with ethidium bromide. EcoR1/HindIII digested lambda DNA was used as molecular size marker. The gels were visualized by Gel Documentation system (Vilber Lourmat-France) and image of gels were printed.
RESULTS AND DISCUSSION
The number of PCR-amplifiable DNA samples, resulting from the SDS, CTAB and modified CTAB extraction methods are given in Table 4 and 5.
The results has indicates that Only the CTAB modified (with PVP in buffer extraction) consistently yielded DNA suitable for PCR-amplification from all growing conditions. Generally these results are accordance with those of Alaey et al. (2005).
DNA extracted by the SDS and CTAB methods was considered unsuitable because: (1) DNA yields were extremely low and (2) only a few of these DNA samples were amplified by the PCR using 2 single primers. Quality and quantity of extracted DNA were tested in two biophotometer- electrophoresis methods and also PCR. In due to the fact, when the ratio of absorption 260/280 nm is between 1.8 and 2, it shows the most absorption is presented by nucleic acids and therefore extracted DNA is well-qualified and its purity is acceptable.
Many of DNA samples obtained using from two methods SDS and CTAB did not produce enough DNA for minigel quantification because of extremely low yields. In these cases, DNA was quantified by UV absorption and some of these samples were amplified by the PCR. The other SDS and CTAB extracted DNA samples were quantified using the minigel method, but all were not PCR amplifiable.
It seems, Non-amplification of these DNA samples could be due to the extremely low yields and residual contaminants which interfere with the PCR. In contrast, yields for all modified CTAB extracted samples were quantified using the minigel method and amplified by
| Table 4: | Number of sample amplified by PCR for DNA extracted by the SDS, CTAB and CTAB modified methods From field, greenhouse and micropropagation plant material |
| Table 5: | Mean DNA yield and number of samples amplified by PCR for CTAB extraction method with and without the inclusion of PVP purification column |
PCR. So these results confirm that extracted DNA executed by modified CTAB method from young and mature leaves possess better quality and quantity in compare with the other methods. Experiments using PVP in conjunction with the SDS extraction method did not increase the number of DNA samples which could be amplified by the PCR (data are not shown).
The addition of PVP on CTAB modified extracted DNA did not interfere with amplification of the DNA sample. The average yield for CTAB modified extracted DNA was 0.5±1.0 mg g-1 fresh weight of leaf material. Yields were determined using the minigel method and are similar to those using variety of DNA extraction procedures found in the literature (Lodhi et al., 1994; Dellaporta et al., 1983; Rogers and Bendich, 1985), including ultracentrifugation with CsCl (Murray and Thompson, 1980). This study also indicates the recovery of high molecular weight with less shearing and easy mobility. The procedure is fast and simple that 30 to 40 DNA samples may be processed in a single day. It is also evident that the uncut DNA exhibits little shearing and is suitable for Southern hybridization (Southern, 1975). The DNA is also amplifiable in PCR using the RAPD technique (Williams et al., 1990).
Comparison of the two methods of quantifying DNA indicates that estimation of yield by UV absorption was greater than indicated by the minigel method. Although, Doyle and Doyle (1990) suggested that CTAB interferes with UV absorption at 260 nm, there was interference in the SDS-extracted samples also. Interference by RNA is possible, but agarose gel electrophoresis of extracted DNA samples did not reveal contamination by RNA. Another possible source of UV interference could be cellular compounds which not removed during the extraction process. Both polysaccharide (Draper and Scott, 1988) and phenolic compounds (Manning, 1991) can interfere with DNA extraction and quantification by UV absorption. Many polyphenolic compounds absorb in the 260 to 280 nm range (Harborne, 1963) and therefore could interfere with DNA quantification. The use of PVP discussed above, did not reduce the absorption interfering contaminants (data are not shown). Consequently, because of UV interference by an unknown contaminant, the validity of absorption ratios were questioned and with the exceptions indicated above, DNA yields were determined by the minigel method. The reproducibility of amplification products were examined for CTAB modified extracted DNA. Amplification products from three replications of a single DNA sample and from leaf samples that differ in age and source.
Similarly, differences in product reproducibility occur among cultivars. The results showed that products generated for some cultivars are different than others. Using 1 g leaf material in CTAB modified method with 0.50 mg PVP gained the most DNA extraction (177.2-227 Ng μL-1) of Askari Kardinal in compare with native grape namely Yahghooti Ghermez (7.9 -17.8 Ng μL-1). The results showed in Table 1.
Proper choice of leaf tissue is very important for DNA extraction. The use of very young leaf tissues has resulted in poor yields. We found that partially expanded leaves are the best material. This is consistent with the results reported by Di Gaspero et al. (2007), in which the best results were obtained from rapidly expanding leaves, one to two nodes from the shoot tip. With fully expanded leaves the yield was low and the DNA was not completely digestible. However, we were able to get equally good results with fully expanded leaves when PVP was added to the extraction buffer. The results presented only for two cultivars that had the least DNA concentration (Table 6-11). PVP has been used to remove polyphenols from mature, damaged and improperly stored leaf tissues (Rogers and Bendich, 1985; Doyle and Doyle, 1987; Howland et al., 1991). PVP forms complex hydrogen bonds with polyphenolic compounds which can be separated from DNA by centrifugation (Maliyakal, 1992). The presence of polyphenolic compounds can be reduced by keeping plant material frozen before extraction and by using PVP in the DNA extraction procedure.
The developmental stage of the plant is also important. The optimal time for leaf collection was during the period of active shoot elongation following bud break. Later in the season DNA extraction was difficult and the DNA obtained was unstable for long term storage. Remnant polysaccharides are a group of chemicals which
| Table 6: | Comparison between quantity of extracted DNA from leaf samples of Sistan Yaghooti Ghermez cultivar |
| Mean separation within columns by Duncan`s Multiple Range Test. Mean with the same alphabets are nonsignificant but mean with the different alphabets have significant difference | |
| Table 7: | Comparison between quantity and qualities of extracted DNA from leaf samples of Shiraz Yahghooti Ghermez cultivar |
| Mean separation within columns by Duncan`s Multiple Range Test. Mean with the same alphabets are nonsignificant but mean with the different alphabets have significant difference | |
| Table 8: | Comparison between qualities of extracted DNA from leaf samples of Sistan Yaghooti Ghermez. cultivar in the present of PVP |
| Mean separation within columns by Duncan`s Multiple Range Test. Mean with the same alphabets are nonsignificant but mean with the different alphabets have significant difference | |
| Table 9: | Comparison between qualities of extracted DNA from leaf samples of Shiraz Yahghooti Ghermez cultivar in the present of PVP |
| Mean separation within columns by Duncan`s Multiple Range Test. Mean with the same alphabets are nonsignificant but mean with the different alphabets have significant difference | |
| Table 10: | Comparison between qualities of extracted DNA from leaf samples of Sistan Yaghooti Ghermez cultivar without the present of PVP |
| Mean separation within columns by Duncan`s Multiple Range Test. Mean with the same alphabets are nonsignificant but mean with the different alphabets have significant difference | |
decrease DNA quality. To omit these materials, NaCl 5 M is recommended (Zhang et al., 2003). In this procedure, β-mercaptoeyhanol works as an antioxidant agent and forbids oxidation in polyphenols (Porebski et al., 1991; Bushra et al., 1999) and this can also elevate the extraction process during the method of (Lodhi et al., 1994).
In conclusion, only the modified CTAB method used in this study consistently yielded DNA suitable for PCR amplification from Grape leaves, regardless of plant growing conditions or leaf age. In addition, the application of this method to various species yielded PCR-amplifiable DNA, which suggests this procedure could yield suitable DNA from many other species. These
| Table 11: | Comparison between qualities of extracted DNA from leaf samples of Shiraz Yahghooti Ghermez cultivar without the present of PVP |
| Mean separation within columns by Duncan`s Multiple Range Test. Mean with the same alphabets are nonsignificant but mean with the different alphabets have significant difference | |
results are concordance with those of Rogers (1994), who demonstrated the superiority of CTAB-based extraction methods over those containing SDS or SDS and CTAB, for a number of plant and fungal species. The addition of PVP was necessary to enhance the generation of reproducible molecular markers.
Distinguishing between 20 Grape cultivars is possible using the CTAB modified -extraction and amplification protocols presented in this study. The yield and molecular weight of the CTAB modified-extracted DNA are comparable with DNA extracted using other more complex procedures.
ACKNOWLEDGMENT
Thank are due to the Zabol University and Biocenter for financial assistance.