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Projects
Gh: Identification of 700 New Microsatellite Loci from Cotton (G. hirsutum)
- Contributors
Steven M. Hoffman, Daniel Grum, Alan E. Pepper
Department of Biology, Texas A&M University,
College Station, TX, 77843
John Z. Yu, Russel J. Kohel
USDA-ARS Southern Plains Research Center
College Station, TX 77843
Jin-Hua Xiao
Department of Molecular Breeding,
Monsanto Inc.
- Project Description
Microsatellite libraries were generated from genomic DNA of Gossypium hirsutum cultivar Tamcot Sphinx (El-Zik and Thaxton, 1996). Reference genotypes Gossypium barbadense cultivar 3-79 and Gossipium hirsutum cultiver TM-1 have been described previously (Kohel et al. 1970, Kohel et al 2001), as have recombinant inbred lines (RILs) from a TM-1 x 3-79 interspecific cross (Kohel and Yu, 2004). We generated two new microsatellite-enriched libraries using the biotinylated oligonucleotide capture method described by Reddy et al. (2001) with the minor modifications described below. The capture oligonucleotides employed were b(CA)20, b(GA)20, b(AGA)15 , where “b” denotes 5' biotin group. The two final washes of biotinylated-DNA duplex bound to streptavidin-coated paramagenetic beads were carried out in 3X SSC at 60°C in order to provide a high level of stringency, with the goal of maximizing efficiency of microsatellite capture. Captured fragments were cloned into the plasmid vector pCR4-TOPO using a Topo-TA cloning kit (Invitrogen). Clones were unidirectionally sequenced using the M13(-21) forward primer. Sequence quality was determined by phred (Ewing et al. 1998, Ewing and Green 1998). Quality scores were Q10 = 713 and Q20 = 582. Sequences were trimmed based on quality (phred cut-off of 10) and to remove vector and adaptor-primer AP11 (employed in the capture). BLAST (Altschul et al. 1990) was used to identify redundant sequences within the collection and to identify those sequences that are redundant with other publically available microsatellite marker loci in the CottonDB database and the Cotton Microsatellite Database (Blenda et al. 2006). Using sequences from non-redundant clones, PCR primers flanking each microsatellite motif were designed manually to achieve a salt-adjusted (50mM Na 2+) T m of 62-64°C, and to produce theoretical minimum amplicons (i.e. hypothetical amplicons with a single repeat unit) in the 60-120bp size range. Primers were evaluated using publicly available web-based applications (Sigma-Genosys, Woodlands, TX) for internal structure, and potential for homodimer and heterodimer formation.
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