Year 2015, Volume 16 , Issue 1, Pages 45 - 49 2016-03-16

Buğdayda Kahverengi Pas Hastalığına Dayanıklılığı Sağlayan Bazı Lr Genlerinin Multipleks PZR Metodu ile Taranması
SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD

Mehmet AYBEKE [1]


Kahverengi pas buğdaylarda önemli bir fungal hastalık olup bütün dünyada önemli verim kayıplarına neden olmaktadır. Türkiye’de yapılan çalışmalarda Lr9, Lr19, Lr24 ve Lr28 gibi çeşitli Lr genlerinin bu hastalığa dayanıklılığı sağladığı tespit edilmiştir. Bu genlerin çeşitlerde ve örneklerde ıslah süresince başarılı bir şekilde taranması önemlidir. Bu nedenle; çalışmada esasen bu 4 geni içine alacak şekilde multipleks polimeraz zincir reaksiyonu (PZR) metodolojisinin başarılı bir şekilde tek seferde uygulanabileceği bir yöntemin oluşturulması amaçlanmıştır. Çalışmada her bir gen için referans oluşturacak şekilde pas kapan nörseri örnekleri kullanılmış ve ön denemelerdeki PZR şartları şu şekildedir: başlangıç denatürasyon initial 94°C’de 3 dk, denatürasyon 95°C’de (35 siklus, 30’ar sn), bağlanma (annealing) 58°C’de 30 s, uzama (elongation) 72°C’de 60 s ve son uzatma (final extension) 72°C’de 30 dk. olmak üzeredir. Primerler: Lr9F: TCCTTTTATTCCGCACGCCGG, Lr9R: CCACACTACCCCAAAGAGACG; Lr19F: CATCCTTGGGGACCTC, Lr19R: CCAGCTCGCATACATCCA; Lr24F: TCTAGTCTGTACATGGGGGC, Lr24R: TGGCACATGAACTCCATACG; Lr28F: CCCGGCATAAGTCTATGGTT, Lr28R: CAATGAATGAGATACGTGAA. Genelde tüm genler için ortak olmak üzere optimum bağlanma sıcaklığının, 61°C, uzatma sıcaklığının 62°C or 64°C olduğu tespit edilmiştir. Sonuç olarak, yeni PCR metodu ile tek gel taşıyan örneklerde başarılı bir şekilde taranma yapılmıştır. Optimal Multipleks PCR koşulları: denatürasyon 94°C’da 1 dk., 35 siklus [94°C’da 30 s., 61°C’da 30 s annealing ve 64–68°C’de 2 dk. extention] ve final extension 72°C’de 30 dk. olarak tespit edilmiştir. İlaveten; optimize multipleks PCR testleri ile Lr19, Lr24 ve Lr28 taşıyan çok genli örneklerde pozitif sonuçlar da alınmıştır. Çalışmanın bundan sonraki diğer Lr genleri ile büyütülmesi hedeflenmektedir. 

Leaf rust is a fungal disease in wheat that causes significant decrease in yield around the world. In Turkey, several genes, including leaf rust-resistant (Lr) Lr9, Lr19, Lr24 and Lr28, have been found to induce disease resistance. To obtain resistant cultivars during the breeding process, screening of these genes in various specimens is crucial. Thus, we aimed in the present study primarily to improve the multiplex polymerase chain reaction (PCR) methodology by which four Lr genes could be simultaneously screened in plant samples carrying these genes. Serial PCR experiments were carried out for determination of optimal PCR conditions for each Lr gene and in all studies nursery lines were used. PCR conditions were determined as follows: 35 cycles of 95°C for denaturation (30 s), 58°C for annealing (30 s) and 72°C for elongation (60 s), with an initial 94°C denaturation (3 min) and a 72°C extension (30 min). The primers used in the PCR runs were as follows: Lr9F: TCCTTTTATTCCGCACGCCGG, Lr9R: CCACACTACCCCAAAGAGACG; Lr19F: CATCCTTGGGGACCTC, Lr19R: CCAGCTCGCATACATCCA; Lr24F: TCTAGTCTGTACATGGGGGC, Lr24R: TGGCACATGAACTCCATACG; Lr28F: CCCGGCATAAGTCTATGGTT, Lr28R: CAATGAATGAGATACGTGAA. We found that the optimum annealing temperature for all four genes was 61°C and extension temperatures were 62°C or 64°C. Finally, using this new PCR method, we successfully screened these genes in specimens carrying only one single Lr gene. Optimal multiplex PCR conditions were; denaturation at 94°C for 1 min, 35 extension cycles [94°C for 30 s, 57–61ºC (ideal 61°C for 30 s), and 64–68°C for 2 min] and final extension at 72°C for 30 min. In addition, we achieved positive results when running the optimised multiplex PCR tests on Lr19, Lr24 and Lr28. Future studies are planned to expand new wide multiplex PCR method to include all other Lr genes.
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Subjects Biology
Journal Section Research Article/Araştırma Makalesi
Authors

Author: Mehmet AYBEKE

Dates

Application Date : September 29, 2015
Acceptance Date : November 15, 2019
Publication Date : March 16, 2016

Bibtex @ { trkjnat267862, journal = {Trakya University Journal of Natural Sciences}, issn = {2147-0294}, eissn = {2528-9691}, address = {}, publisher = {Trakya University}, year = {2016}, volume = {16}, pages = {45 - 49}, doi = {}, title = {SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD}, key = {cite}, author = {AYBEKE, Mehmet} }
APA AYBEKE, M . (2016). SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD. Trakya University Journal of Natural Sciences , 16 (1) , 45-49 . Retrieved from https://dergipark.org.tr/en/pub/trkjnat/issue/25380/267862
MLA AYBEKE, M . "SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD". Trakya University Journal of Natural Sciences 16 (2016 ): 45-49 <https://dergipark.org.tr/en/pub/trkjnat/issue/25380/267862>
Chicago AYBEKE, M . "SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD". Trakya University Journal of Natural Sciences 16 (2016 ): 45-49
RIS TY - JOUR T1 - SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD AU - Mehmet AYBEKE Y1 - 2016 PY - 2016 N1 - DO - T2 - Trakya University Journal of Natural Sciences JF - Journal JO - JOR SP - 45 EP - 49 VL - 16 IS - 1 SN - 2147-0294-2528-9691 M3 - UR - Y2 - 2019 ER -
EndNote %0 Trakya University Journal of Natural Sciences SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD %A Mehmet AYBEKE %T SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD %D 2016 %J Trakya University Journal of Natural Sciences %P 2147-0294-2528-9691 %V 16 %N 1 %R %U
ISNAD AYBEKE, Mehmet . "SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD". Trakya University Journal of Natural Sciences 16 / 1 (March 2016): 45-49 .
AMA AYBEKE M . SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD. Trakya Univ J Nat Sci. 2016; 16(1): 45-49.
Vancouver AYBEKE M . SCREENING OF Lr GENES PROVIDING RESISTANCE TO LEAF RUST IN WHEATH USING MULTIPLEX PCR METHOD. Trakya University Journal of Natural Sciences. 2016; 16(1): 49-45.