OPTIMASI METODE ANALISIS MOLEKULER DELAPAN AKSESI SORGUM (Sorghum bicolor (L.) Moench) DAERAH JAWA TIMUR MENGGUNAKAN PENANDA RAPD
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Institusion
Universitas Muhammadiyah Malang
Author
Rohman, Syaiur
Subject
S Agriculture (General)
Datestamp
2018-10-29 08:06:02
Abstract :
Sorghum is one of cereal crops that is potentially developed in Indonesia. Food needs and main energy resources in Indonesia tend to increase. Sorghum is a dry land crop that is suitable for food diversification and bioenergy resource. There needs to be development of sorghum cultivars through plant breeding. Genetic analysis is one of important steps either morphological or molecular in plant breeding. Genetic analysis of sorghum can be implemented using RAPD markers. This study was aimed to optimize the method of molecular analysis of sorghum accessions from East Java using RAPD markers. This study was held at Plant Molecular Laboratory, Biotechnology Development Center/Pusat Pengembangan Bioteknologi (PUSBANG BIOTEK), University of Muhammadiyah Malang. Eight sorghum accessions were obtained from Pasuruan, Sampang, Jombang, Lamongan, Tuban, and Tulungagung. DNA was extracted from young leaf using three CTAB methods. The extracted DNA was qualitatively analyzed through electrophoresis and quantitatively analyzed based on purity level of DNA concentration. PCR-RAPD analysis used 11 kinds of primers from Operon Kit A (OPA 2, OPA 6, OPA 8, OPA 10, OPA 11, OPA 12, OPA 13, OPA 16, OPA 18, OPA 19, OPA 20) with 33 cycles of PCR and the temperatures were set to 95oC (denaturation), 36oC (annealing), and 72oC (extension). Primers effectivity were analyzed base on number of bands and polymorphic percentage on each primer using software GelAnalyzer 2010. The result of this study showed clear bands on each primer after electrophoresis of extracted DNA from three modified combination methods. The purity number of DNA were between 1,8-2,0, and DNA concentration were between 0,65-1,03 µg/µl. The most optimum times and temperatures of PCR in this study were 95oC for pre-denaturation (5 minutes), 95oC denaturation (1 minute), 36oC annealing (1 minute), 72oC extension (1,5 minutes), and 72oC post extension (5 minutes) with 33 cycles of PCR. 11 randomized primers that were used in this study delivered 5 primers that were able to amplified the DNA of eight sorghum accessions. The 5 primers were OPA 02, OPA 10, OPA, 13, OPA 19, and OPA 20. The highest polymorphic percentage were obtained from OPA 10 and OPA 20.
Sorghum is one of cereal crops that is potentially developed in Indonesia. Food needs and main energy resources in Indonesia tend to increase. Sorghum is a dry land crop that is suitable for food diversification and bioenergy resource. There needs to be development of sorghum cultivars through plant breeding. Genetic analysis is one of important steps either morphological or molecular in plant breeding. Genetic analysis of sorghum can be implemented using RAPD markers. This study was aimed to optimize the method of molecular analysis of sorghum accessions from East Java using RAPD markers. This study was held at Plant Molecular Laboratory, Biotechnology Development Center/Pusat Pengembangan Bioteknologi (PUSBANG BIOTEK), University of Muhammadiyah Malang. Eight sorghum accessions were obtained from Pasuruan, Sampang, Jombang, Lamongan, Tuban, and Tulungagung. DNA was extracted from young leaf using three CTAB methods. The extracted DNA was qualitatively analyzed through electrophoresis and quantitatively analyzed based on purity level of DNA concentration. PCR-RAPD analysis used 11 kinds of primers from Operon Kit A (OPA 2, OPA 6, OPA 8, OPA 10, OPA 11, OPA 12, OPA 13, OPA 16, OPA 18, OPA 19, OPA 20) with 33 cycles of PCR and the temperatures were set to 95oC (denaturation), 36oC (annealing), and 72oC (extension). Primers effectivity were analyzed base on number of bands and polymorphic percentage on each primer using software GelAnalyzer 2010. The result of this study showed clear bands on each primer after electrophoresis of extracted DNA from three modified combination methods. The purity number of DNA were between 1,8-2,0, and DNA concentration were between 0,65-1,03 µg/µl. The most optimum times and temperatures of PCR in this study were 95oC for pre-denaturation (5 minutes), 95oC denaturation (1 minute), 36oC annealing (1 minute), 72oC extension (1,5 minutes), and 72oC post extension (5 minutes) with 33 cycles of PCR. 11 randomized primers that were used in this study delivered 5 primers that were able to amplified the DNA of eight sorghum accessions. The 5 primers were OPA 02, OPA 10, OPA, 13, OPA 19, and OPA 20. The highest polymorphic percentage were obtained from OPA 10 and OPA 20.
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