An optimized PCR protocol with newly designed primers for reliable molecular selection of high oleic type sunflower

Abstract

High oleic sunflower is one of the most significant oilseed crops due to the stability of its oil in processing and desirable characteristics for health. Determination of high oleic sunflower by standard methods such as gas chromatography is time consuming and expensive. On the other hand, marker-assisted selection analysis with molecular markers associated with high oleic acid trait is a useful and powerful tool in order to facilitate sunflower breeding programs. In this study, we compared three molecular markers which have been used for selection of the high oleic sunflower varieties. We also describe an optimized PCR protocol with newly designed two primer pairs targeting normal sequence of FAD2 gene as internal control and direct analysis of the inserted DNA sequences which is known to be closely linked to the Pervenets mutation. According to results of our study, showing the insertion site which is linked to the Pervenets mutation by the insertion specific PCR protocols is more reliable than the SSR marker for selection of the high oleic sunflower varieties. Because we have not been able to get successful results with the available PCR protocols described for the insertion site, we report here a novel multiplex PCR protocol with newly designed primers enabling reliable discrimination of the high oleic and low oleic sunflower genotypes in a single PCR tube, which offers some advantages to the breeders by means of saving money and time.

Keywords

• Helianthus annuus L.,Marker-assisted selection,Molecular marker,Oleic acid

Yüksek oleik tip ayçiçeğinin güvenilir moleküler seçiminde yeni tasarlanmış primerler ile optimize PCR protokolü

Abstract

Yüksek oleik tip ayçiçeği, işlenme sürecindeki kararlı yapısı ve sağlık açısından istenen özelliklere sahip olması nedeniyle en önemli yağ bitkilerinden birisidir. Yüksek oleik içeriğine sahip ayçiçeğinin gaz kromatografisi gibi standart metotlarla tayini zaman alıcı ve pahalıdır. Diğer yandan, yüksek oleik asit özelliği ile ilişkili moleküler belirteçlerle yapılan seleksiyon ayçiçeği yetiştiriciliğini kolaylaştırmak açısından kullanışlı ve güçlü bir araçtır. Bu çalışmada, yüksek oleik tip ayçiçeği çeşitlerinin seçiminde kullanılan üç moleküler belirteç karşılaştırılmıştır. Bu çalışma ile ayrıca, kontrol olarak FAD2 geninin normal dizisini ve Pervenets mutasyonuyla yakından ilişkili olduğu bilinen ilave DNA dizilerinin doğrudan analizini hedefleyen yeni tasarladığımız primerler ile optimize ettiğimiz yeni bir PCR protokolü tanımlanmıştır. Çalışmamızın sonuçlarına göre, yüksek oleik tip ayçiçeği çeşitlerinin seçiminde Pervenets mutasyonuyla yakın ilişkili olan ilave bölgeyi özgün PCR protokolleri ile göstermek SSR belirtecinden daha güvenilirdir. İlave bölgeye yönelik mevcut PCR protokolleri ile başarılı sonuçlar elde edemediğimiz için, bu çalışmada yetiştiricilere/ıslahçılara para ve zamandan tasarruf etmek gibi avantajlar sunan, yüksek ve düşük oleik tip ayçiçeği genotiplerinin tek bir PCR tüpünde güvenilir şekilde ayrımını sağlayan, yeni primer çiftlerinin kullanıldığı yeni bir multipleks PCR protokolü bildirilmektedir.

Keywords

• Helianthus annuus L.,Markıra dayalı seleksiyon,Oleik asit,Moleküler markır

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