Characterization and identification of (CT)n microsatellites in soybean using sheared genomic libraries.

Abstract

Three small insert (300 to approximately 600 bp) sheared genomic libraries were constructed by pipetting and DNase I treatment of soybean DNA. About 15,000 clones from each library were screened for CT- simple sequence repeats (CT-SSRs). The CT-SSRs were abundant in the soybean genome at an estimated frequency of approximately one SSR per 110 kb of genomic DNA. Following the sequencing of 129 positive clones, the repeat types and frequency of CT repeats among the positive clones were characterized. Forty-nine primer pairs were designed and preliminarily evaluated for their ability to amplify genomic DNA from a set of six varieties, including parents of a mapping family. Amplified products were analyzed by 10% PAGE. Eighty-eight percent of the designed primers were able to amplify all these genomic DNAs using a single PCR profile of 53 degrees C annealing temperature, of which 22 (45%) were polymorphic in the six varieties, and 14 of them were polymorphic in the parents of the mapping family. The polymorphic primer sets were further assessed for allelic information using DNA from 16 soybean cultivars. The average number of alleles was 4, ranging from 2 to 7 with the highest polymorphism information content value 0.84. Fourteen of these SSRs were mapped, using an existing soybean RFLP map. The findings presented here will advance our understanding of the soybean genome, and assist in the mapping genome and discrimination of closely related varieties of this species.