Kentucky State University
A Comparison of Agarose, Metaphor Agarose, and Polyacrylamide Gel Electrophoresis Systems in Resolving Pawpaw Molecular Markers
Institution
Kentucky State University
Faculty Advisor/ Mentor
Kirk Pomper; Jeremiah Lowe
Abstract
Pawpaw (Asimina triloba) is a tree fruit native to Kentucky with potential as a new crop for small farmers. The fruit pulp can be used to make an excellent ice cream. Since 1994, Kentucky State University (KSU) has served as the USDA National Clonal Germplasm Repository, or gene bank, for pawpaw; therefore, the assessment of genetic diversity with molecular markers in pawpaw is an important research priority at KSU. Using the polymerase chain reaction (PCR) with simple sequence repeat (SSR) primers and pawpaw template DNA, products between 150 to 500 bp are usually amplified and visualized via electrophoresis. The objective of this study was to determine if SSR-PCR products separated on agarose, metaphor agarose, and polyacrylamide gel electrophoresis systems display unique scoring patterns that result in different genetic separation upon analysis for nine pawpaw cultivars. Using a DNAmite kit, DNA was extracted from young leaves collected from the pawpaw cultivars: Cales Creek, Davis, Middletown, NC-1, Overleese, Rebecca’s Gold, Taytwo, Wilson, and Zimmerman. SSR primers B129 and C104 were developed by Genetic Information Services (Chatsworth, CA) and were used to amplify the pawpaw template DNA. PCR products were separated by electrophoresis on 3% agarose, 3% metaphor agarose, and 5% polyacrylamide gels. Following electrophoresis, gels were stained with ethidium bromide and photographed. Gel analysis was carried out using Kodak 1D software. Dendrograms were produced from the marker scoring data using NTSYSpc v2.11T. Each electrophoresis system produced a unique separation of the pawpaw cultivars, although some groupings were similar using the different systems.
A Comparison of Agarose, Metaphor Agarose, and Polyacrylamide Gel Electrophoresis Systems in Resolving Pawpaw Molecular Markers
Pawpaw (Asimina triloba) is a tree fruit native to Kentucky with potential as a new crop for small farmers. The fruit pulp can be used to make an excellent ice cream. Since 1994, Kentucky State University (KSU) has served as the USDA National Clonal Germplasm Repository, or gene bank, for pawpaw; therefore, the assessment of genetic diversity with molecular markers in pawpaw is an important research priority at KSU. Using the polymerase chain reaction (PCR) with simple sequence repeat (SSR) primers and pawpaw template DNA, products between 150 to 500 bp are usually amplified and visualized via electrophoresis. The objective of this study was to determine if SSR-PCR products separated on agarose, metaphor agarose, and polyacrylamide gel electrophoresis systems display unique scoring patterns that result in different genetic separation upon analysis for nine pawpaw cultivars. Using a DNAmite kit, DNA was extracted from young leaves collected from the pawpaw cultivars: Cales Creek, Davis, Middletown, NC-1, Overleese, Rebecca’s Gold, Taytwo, Wilson, and Zimmerman. SSR primers B129 and C104 were developed by Genetic Information Services (Chatsworth, CA) and were used to amplify the pawpaw template DNA. PCR products were separated by electrophoresis on 3% agarose, 3% metaphor agarose, and 5% polyacrylamide gels. Following electrophoresis, gels were stained with ethidium bromide and photographed. Gel analysis was carried out using Kodak 1D software. Dendrograms were produced from the marker scoring data using NTSYSpc v2.11T. Each electrophoresis system produced a unique separation of the pawpaw cultivars, although some groupings were similar using the different systems.