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United States Department of Agriculture

Agricultural Research Service

Title: Improved Methods for Screening Cotton Cdnas Using Fluorochrome-Labeled Aflp and Ssr-Specific Primers

Authors
item Saha, Sukumar
item Karaca, M - MISSISSIPPI STATE UNIV
item Jenkins, Johnie
item Lang, D - MISSISSIPPI STATE UNIV

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: June 12, 2000
Publication Date: N/A

Interpretive Summary: Gene discovery, i.e. detection of genes whose expression affects biological processes, is a frequent goal of modern genetic research. Cotton genetic research has ben hindered by the lack of effective methods to identify and isolate differentially expressed genes. A high throughput, fluorescent differential expression method on an automated capillary electrophoresis unit to study differentially expressed genes has proven to be very efficient, giving significant cost reduction and time saving compared to similar radioactive methods in other organisms. We describe here two novel PCR based methods to isolate and identify functional genes in cotton with fluorescent-labeled amplified fragment length polymorphic (AFLP) and simple sequence of short repeat (SSR) primers. AFLP- and SSR-based genetic markers have been widely used in many crops including cotton for genome mapping due to their highly polymorphic nature. There are only a few reports in crops other than cotton on AFLP marker-based studies and no reports on an SSR-based method to study differentially expressed genes. Our results demonstrated that the number of visible AFLP markers, ranging from 3 to 25 from cDNA, the functional genome, in comparison to 40 to 90 AFLP markers from genomic DNA, made the cDNA sample analyses easier. We observed that 40% of the SSR primer pairs amplified cotton cDNAs contrasting with the conventional idea that SSRs are located in the non-functional genomic regions. We isolated a 420 bp cDNA fragment following our method. After determining the sequence of the fragment, a BLAST DNA database search revealed strong homology (>70%) to methionine synthase genes from Arabidopsis thailiana, Cantharanus roseus, Mesembryanthemum crystallinum, and Coleus blumei.

Technical Abstract: The analysis of differential gene expression in cotton has been hindered by the paucity of suitable methods. Here we describe the detection of cDNAs using fluorescent labeled AFLP- and SSR-specific primers by automated capillary electrophoresis. The fluorescent signals enabled detection of polymorphic bands differing by only one of two base pairs between the cDNA samples. We observed that 90% of the AFLP primers amplified both cDNAs and genomic DNAs. The reduced number of visible AFLP bands, ranging from 3 to 25 in cDNA in comparison to 40 to 90 bands from genomic DNAs, made the cDNA sample analyses easier. We observed that 40% of the SSR primer pairs amplified cotton cDNAs contrasting with the conventional idea that SSRs are located in the non-functional genomic regions. There has been no report on the use of SSR-specific primers in the identification of functional plant genes, although the genomic databases indicated the presence of an SSR motif in many functional plant genes. We used automated GENESCAN software as a diagnostic tool to identify the bands. We isolated a 420 bp EST fragment using agarose gel electrophoresis that was sequenced in both directions using the forward and reverse primers. A BLAST search found that the 420 bp cDNA has strong homology (>70%) to methionine synthase genes from Arabidopsis thaliana, Cantharanus roseus, Mesembryanthemum crystallinum, and Coleus blumei. Our results demonstrated the effective use of both AFLP- and SSR-specific primers for screening cDNAs in cotton.

Last Modified: 10/30/2014
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