An Improved Method for Efficient DNA Extraction from Grapevine

Öz

Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops worldwide which has been the subject of extensive genetic studies including gene mapping, genetic transformation, and DNA fingerprinting. Grapevines are rich in polysaccharides, polyphenolic compounds, and various secondary metabolites, many of which have significant importance in food, agrochemical, and pharmaceutical industries. While metabolites are one of the indicators of quality of grapevines, the presence of them makes grapevine one of the most difficult plants to extract DNA from. These metabolites not only affect DNA extraction procedures but also downstream reactions such as restriction digestion and PCR. Development of new genotyping techniques based on sequencing such as genotyping by sequencing (GBS) requires high-quality DNA for digestion and sequencing. To date, several protocols have been developed for DNA extraction from grapevine. In this study, three different protocols with modifications were compared for DNA extraction performance from grapevine leaves from four different cultivars. Efficiencies of these methods were determined by extracted DNA’s quantity and quality. To confirm the suitability for GBS, extracted DNA was digested with restriction enzymes. Although all protocols were based on the traditional CTAB method, they resulted in different DNA yield and restriction digestion efficiency. The modified protocol including PVP-40 and ß-mercaptoethanol was found to be the most efficient method to obtain high quality and quantity grapevine DNA that is amenable to restriction digestion.

Anahtar Kelimeler

• grapevine
• DNA extraction
• restriction digestion
• GBS

Asmada Verimli DNA Ekstraksiyonu için Geliştirilmiş Bir Yöntem

Öz

Asma (Vitis vinifera L.), dünya çapında en eski ve en önemli çok yıllık bitkilerden biridir ve gen haritalaması, genetik transformasyon ve DNA parmak izi gibi kapsamlı genetik çalışmalara konu olmuştur. Asmalar, polisakkaritler, polifenolik bileşikler ve birçoğu gıda, zirai kimya ve ilaç endüstrilerinde önemli öneme sahip çeşitli ikincil metabolitler bakımından zengindir. Asma kalitesinin göstergelerinden biri metabolitler olsa da, bunların varlığı asmayı DNA ekstraksiyonu en zor bitkilerden biri haline getirir. Bu metabolitler sadece DNA ekstraksiyon prosedürlerini etkilemekle kalmaz, aynı zamanda restriksiyon enzimleri ile kesimi ve PCR gibi reaksiyonları da etkiler. Genotyping by sequencing (GBS) gibi dizilemeye dayalı yeni genotipleme teknikleri, restriksiyon ve dizileme için yüksek kaliteli DNA gerektirir. Bugüne kadar asmadan DNA ekstraksiyonu için çeşitli protokoller geliştirilmiştir. Bu çalışmada, dört farklı çeşide ait asma yapraklarından DNA ekstraksiyon performansı için üç farklı protokol karşılaştırılmıştır. Bu yöntemlerin etkinlikleri, ekstrakte edilen DNA'nın miktarı ve kalitesi ile belirlenmiştir. GBS'ye uygunluğu doğrulamak için, ekstrakte edilen DNA, restriksiyon enzimleri ile kesilmiştir. Tüm protokoller geleneksel CTAB yöntemine dayanmasına rağmen, farklı DNA verimi ve restriksiyon kesimi verimliliği ile sonuçlanmıştır. PVP-40 ve ß-merkaptoetanol içeren modifiye protokolün, restriksiyona uygun yüksek kalite ve miktarda asma DNA'sı elde etmek için en etkili yöntem olduğu tespit edilmiştir.

Anahtar Kelimeler

• asma
• DNA
• restriksiyon
• GBS

Kaynakça

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