Separation of DNA for molecular markers analysis from leaves of the Vitis vinifera.

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).

Polymorphism of some native Sistan grapes assessed by long and short primers for RAPD markers.

Grapevines have Bronze ages archive in Sistan area of Iran. In order to study the genetic variation and taxonomic relationships between 6 cultivars of the Sistan grapevines (Vitis vinifera L.) at molecular level, random amplified polymorphic DNA (RAPD) markers were used. The data were subjected to statistical analyses and genetic resemblance was calculated using Dice similarity index. The grapevines related to the different geographic areas of Sistan were assessed by 50 short (10 mer) and long (15-21 mer) primers. Out of 50 primers which were tested, 21 primers gave reproducible results. Selected primers created 497 bands. Resulting profiles showed that the produced bands varied in size from 300 to 3500 base pairs. The numbers of reliable polymorphic fragments for short and long primers were 86 and 334 bands, respectively. In multiplication reaction the items in the size area of 564 to 1904 base pair resulted for short primers and 564 to 4277 base pair for long primers. From the bands calculated a matrix that was analyzed by the unweighted pair group method on arithmetic averages to draw a dendrogram. The population was classified in 4 main groups in which Red Yaghooti and White Yaghooti had the maximum and Red Yaghooti and Laal had the minimum similarity coefficients. In our study, by comparing the results gained from technique long and short primers in RAPD, the potential value of long primers for the production ofpolymorphism in grapes was identified.