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United States Department of Agriculture

Agricultural Research Service

Title: A Novel Method for Screening Cotton Cdnas Using Fluorochrome-Labeled Ssr-Specific Primers

Authors
item Saha, Sukumar
item Karaca, M - MISSISSIPPI STATE UNIV
item Jenkins, Johnie
item Zipf, A - ALABAMA A&M
item Reddy, U - TEXAS A&M UNIVERSITY
item Pepper, A - TEXAS A&M UNIVERSITY

Submitted to: Euphytica
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 14, 2002
Publication Date: May 1, 2003
Citation: SAHA, S., KARACA, M., JENKINS, J.N., ZIPF, A., REDDY, U., PEPPER, A. Simple sequence repeats as useful resources to study transcribed genes of cotton. Euphytica. 2003. v.130.p.355-364.

Interpretive Summary: Identification of differentially expressed genes at the molecular level is important to understand any biological process. Microsatellites, also known as SSR, are one of the most variable types of tandem repetitive DNA found in plants and animals. SSRs are considered as marker of choice for genome mapping in many crop species. The characteristics that make SSR attractive for DNA analysis includes its simple PCR based method, abundance, presence throughout the genome and highly polymorphic nature. The conventional idea is that SSRs are normally located in non-functional genomic regions. Here we describe a simple PCR based procedure for detecting differences in functional genes ( cDNAs) between cotton samples using SSR specific primers. We observed that 52% of the SSR primer pairs amplified cotton cDNA templates. DNA sequencing of the functional genes (ESTs) further revealed the presence of the repeat motif like genomic DNAs on the ESTs. There has been no report on the use of SSR- specific primers in the differential screening of functional plant genes, although the genomic databases indicated the presence of an SSR motif in many functional plant genes. The merit of this method strongly supported by many recent published information about the presence of repeat motifs on the DNA sequences of ESTs.

Technical Abstract: The analysis of differential gene expression in cotton has been hindered by the paucity of suitable methods. Here we describe novel detection of cDNAs by automated capillary electrophoresis (CE) using fluorescent labeled sequence repeat(SSR) specific primers. Fluorescent signals enabled detection of polymorphis bands differing by only one or two base pairs between the cDNA samples. We observed that 52% of the SSR primer pairs amplified cotton cDNA templates,contrasting with the conventional idea that SSRs are located in non-functional genomic regions. The presence of SSR on cDNAs were confirmed based on DNA sequencing of the ESTs revealing the presence of the repeat motif on ESTs. There has been no report on the use of SSR-specific primers in the differential screening of functional plant genes, although the genomic databases indicated the presence of an SSR motif in many functional plant genes. SSRs are commonly used as marker of choice in genome mapping in many crop species because they are PCR based markers, highly polymoprhic, abundant and widely distributed throughout the genome. This method demonstrated SSR can be used to integrate functional and structural genome mapping information. Identification of SSR motif in open reading frames of cDNAs further demonstrated effective use of SSR-specific primers for screening cDNAs in cotton. Sequence information also revealed some ESTs did not have the repeat motif, however, contained the flanking primer sequence. This could indicate the repeat motif was located in the intron region of the ESTs.

Last Modified: 10/24/2014
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