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

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.

Anahtar Kelimeler:

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

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

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,

PDF

___

Alberio C, Izquierdo NG, Galella T, Zuil S, Reid R, Zambelli A, Aguirrezabal LA (2016) A new sunflower high oleic mutation confers stable oil grain fatty acid composition across environments. European Journal of Agronomy 73: 25-33.
Baydar H, Erbas S (2005) Influence of seed development and seed position on oil, fatty acids and total tacopherol contents in sunflower (Helianthus annuus L.). Turkish Journal of Agriculture and Forestry 29: 179-186.
Berville A, Lacombe S, Veillet S, Granier C, Leger S, Jouve P (2009) Method of selecting sunflower genotypes with high oleic acid content in seed oil, The Patent Cooperation Treaty (PCT), WO 2005/106022 A2.
Bilgen BB (2016) Characterization of sunflower inbred lines with high oleic acid content by DNA markers. In Kaya Y and Hasancebi S (Eds) Proceedings of the 19th international sunflower conference. ISA, Edirne, Turkey, pp. 662-668.
Broun P, Gettner S, Somerville C (1999) Genetic engineering of plant lipids. Annual Review of Nutrition 19: 197-216.
CETIOM (2002). Technical Center for Oilseed Crops and Industrial Hemp.
Cvejic S, Jocic S, Dimitrijevic A, Imerovski I, Miladinovic D, Jockovic M, Miklic V (2016) An EMS mutation altering oil quality in sunflower inbred line. In Kaya Y and Hasancebi S (Eds) Proceedings of the 19th international sunflower conference. ISA, Edirne, Turkey, pp. 422-430.
Dehmer KJ, Friedt W (1998) Development of molecular markers for high oleic acid content in sunflower (Helianthus annuus L.). Industrial Crops and Products 7: 311-315.
Delplanque B, Le Roy B, Senault C, Lemort N (1997) Reduced capacity of cholesterol efflux, delayed postprandial lipid response, and abnormal Apo-CIII distribution in normolipemic sujects with premature coronary heart disease. Atherosclerosis 134(1,2): 338-4, pp. 200.
Demurin Y and Borisenko O (2011) Genetic collection of oleic acid content in sunflower seed oil. Helia 34: 69-74.
Dimitrijevic A, Imerovski I, Dragana M, Cvejic S, Jocic S, Zeremski T, Sakac Z (2017) Oleic acid variation and marker-assisted detection of Pervenets mutation in high- and low-oleic sunflower cross. Crop Breeding and Applied Biotechnology 17: 235-241.
Evci G, Pekcan V, Yilmaz MI, Cıtak N, Tuna N, Ay O, Pilaslı A, Kaya Y (2016) Determination of yield performances of oleic type sunflower (Helianthus annuus L.) hybrids resistant to broomrape and downy mildew. Ekin Journal of Crop Breeding and Genetics 2(1): 45-50.
Fernandez Martinez JM, Perez Vich B and Velasco L (2009) Sunflower. In: Oil Crops, Handbook of Plant Breeding, V.4, Vollmann, J. and Rajcan, I. (eds.), Springer, 155-232.
Garcia-Diaz MT, Martinez-Rivas JM, Mancha M (2002) Temperature and oxygen regulation of oleate desaturation in developing sunflower (Helianthus annuus) seeds. Physiologia Plantarum 114: 13-20.
Grandon NG, Moreno MV, Scorcione MC, Gieco JO, Alvarez D, Paniego N, Heinz R (2012) Characterization of sunflower inbred lines (Helianthus annuus L.) for high oleic acid content using SSR markers. p. 217. In: Proc. 18th Int. Sunfl. Conf., Mar del Plata, Argentina. Int. Sunfl. Assoc., Paris, France.
Harwood JL (1996) Recent advances in the biosynthesis of plant fatty acids. Biochimica et Biophysica Acta 130: 7-56.
Hongtrakul V, Slabaugh MB and Knapp SJ (1998) A seed specific D12 oleate desaturase gene is duplicated, rearranged, and weakly expressed in high oleic acid sunflower lines. Crop Science 38: 1245-1249.
Izquierdo NG, Aguirrezabal LA (2008) Genetic variability in the response of fatty acid composition to minimum night temperature during grain filling in sunflower. Field Crops Research 106: 116-125. Lacombe S, Kaan F, Griveau Y, Berville A (2004) The Pervenets high oleic mutation: Methodological studies. Helia 40: 41-54.
Lacombe S, Souyris I, Berville AJ (2009) An insertion of oleate desaturase homologous sequence silences via siRNA the functional gene leading to high oleic acid content in sunflower seed oil. Molecular Genetics and Genomics 281: 43-54.
Martinez-Rivas JM, Garcia-Diaz MT, Mancha M (2000) Temperature and oxygen regulation of microsomal oleate desaturase (FAD2) from sunflower. Biochemical Society Transactions 28: 890-2.
Martinez-Rivas JM, Sperling P, Luehs W, Heinz E (2001) Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high oleic varieties of sunflower (Helianthus annuus L.). Molecular Breeding 8: 159-168.
Nagarathna TK, Shadakshari YG, Ramanappa TM (2011) Molecular analysis of sunflower (Helianthus annuus L.) genotypes for high oleic acid using microsatellite markers. Helia 34: 63-68.
Neto AR, Miguel AMRO, Mourad AL, Henriques EA, Alves RMV (2016) Environmental effect on sunflower oil quality. Crop Breeding and Applied Biotechnology 16: 197-204.
Rauf S, Jamil N, Tariq SA, Khan M, Kausar M, Kaya Y (2017) Progress in modification of sunflower oil to expand its industrial value. Journal of the Science of Food and Agriculture 97(7): 1997-2006.
Schuppert GF, Tang S, Slabaugh MB and Knapp SJ (2006) The sunflower high-oleic mutant Ol carries variable tandem repeats of FAD2-1, a seed-specific oleoyl-phosphatidyl choline desaturase. Molecular Breeding 17: 241-256.
Singchai A, Muangsan N, Machikowa T (2013) Evaluation of SSR markers associated with high oleic acid in sunflower. International Journal of Biological, Food, Veterinary and Agricultural Engineering 7: 631-634.
Skoric D, Jocic S, Lecic N, Sakac Z (2007) Development of sunflower hybrids with different oil quality. Helia 30: 205-212.
Soldatov KI (1976) Chemical mutagenesis in sunflower breeding. Proc. 7th International Sunflower Conference, 27 June - 3 July, Krasnodar, Russia, pp. 352-357.
van der Merwe, Labuschagne MT, Herselman L, Hugo A (2015) Effect of heat stress on seed yield components and oil composition in high- and mid-oleic sunflower hybrids. South African Journal of Plant and Soil 32: 121-128.
Vannozzi GP (2006) The perspectives of use of high oleic sunflower for oleochemistry and energy raws. Helia 29: 1-24.
Varshney RK, Graner A, Sorrels ME (2005) Genic microsatellite markers in plants: features and applications. Trends in Biotechnology 23(1): 48-55.