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United States Department of Agriculture

Agricultural Research Service

Title: Gel Based DNA Marker Technologies in Cotton

Authors
item Saha, Sukumar
item Tan, H - MISSISSIPPI STATE UNIV
item Karaca, M - MISSISSIPPI STATE UNIV
item Jenkins, Johnie

Submitted to: International Cotton Advisory Committee Recorder
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 1, 1999
Publication Date: N/A

Interpretive Summary: This paper presents a summarized report on DNA markers in cotton. Our results showed that DNA extracted using a modified method of Dneasy Plant Mini Kit (Qiagen, Santa Clarita, CA) from crushed freeze-dried cotton leaf tissues yielded high molecular weight DNA which was readily digestible with restriction enzymes and served as a template for PCR amplification. We discussed the advantages and disadvantages of RFLP, RAPD, SSR and AFLP marker methods in cotton. We reported about fluorescently-labeled amplified DNA markers in cotton using SSR and AFLP primers. These DNA markers are visualized as peaks on electropherograms using an automated capillary electrophoretic (CE) system. Automatic sample loading, digitized output of peak position, automated data collection and analysis and no need for gel preparation makes this automated method suitable to analyze large numbers of samples within a very short time. This method is sensitive enough to detect one/two base pair DNA fragment size differences between alleles of a DNA marker. We observed about 3-4 times more cotton SSR markers in CE in comparison to detection on agarose gel. However, this automated method can only efficiently detect DNA fragment size differences of small base sizes ranging from 40 to 500 bp. The preliminary results using this method detected about 25 polymorphic AFLP polymorphic markers, 7 polymorphic SSR markers per primer combination at the interspecific level and 16 polymorphic AFLP markers and 5 polymorphic SSR markers per primer combination at the intraspecific level. Our results demonstrated that the AFLP method could be very useful in detecting polymorphic markers in comparison to SSR markers in cotton.

Technical Abstract: Using a modified method of Dneasy Plant Mini Kit (Qiagen, Santa Clarita, CA) from crushed freeze-dried cotton leaf tissues yielded high molecular weight DNA which was readily digestible with restriction enzymes and served as a template for PCR amplification. We discussed the advantages and disadvantages of RFLP, RAPD, SSR and AFLP marker methods in cotton. We reported fluorescently-labeled amplified DNA markers in cotton using SSR and AFLP primers. These DNA markers were observed as peaks on electropherograms, using an automated capillary electrophoretic (CE) system. Automatic sample loading, digitized output of peak position, automated data collection and analysis and no need for gel preparation makes this automated method suitable to analyze large numbers of samples within a very short time. The preliminary results using this method detected about 25 polymorphic AFLP polymorphic markers, 7 polymorphic SSR markers per primer combination at the interspecific level and 16 polymorphic AFLP markers and 5 polymorphic SSR markers per primer combination at the intraspecific level. Our results demonstrated that the AFLP method was more useful in detecting polymorphic markers in comparison to SSR markers in cotton.

Last Modified: 7/25/2014
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