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Background: Single-locus markers have many advantages compared with multi-locus markers in genetic and breeding studies because their alleles can be assigned to particular genomic loci in diversity analyses. However, there is little research on single-locus SSR markers in peanut. Through the de novo assembly of DNA sequencing reads of A. hypogaea, we developed single-locus SSR markers in a genomic survey for better application in genetic and breeding studies of peanut. Results: In this study, DNA libraries with four different insert sizes were used for sequencing with 150 bp paired-end reads. Approximately 237 gigabases of clean data containing 1,675,631,984 reads were obtained after filtering. These reads were assembled into 2,102,446 contigs with an N50 length of 1,782 bp, and the contigs were further assembled into 1,176,527 scaffolds with an N50 of 3,920 bp. The total length of the assembled scaffold sequences was 2.0 Gbp, and 134,652 single-locus SSRs were identified from 375,180 SSRs. Among these developed single-locus SSRs, trinucleotide motifs were the most abundant, followed by tetra-, di-, mono-, penta- and hexanucleotide motifs. The most common motif repeats for the various types of single-locus SSRs have a tendency to be A/T rich. A total of 1,790 developed in silico single-locus SSR markers were chosen and used in PCR experiments to confirm amplification patterns. Of them, 1,637 markers that produced single amplicons in twelve inbred lines were considered putative single-locus markers, and 290 (17.7 %) showed polymorphisms. A further F2 population study showed that the segregation ratios of the 97 developed SSR markers, which showed polymorphisms between the parents, were consistent with the Mendelian inheritance law for single loci (1:2:1). Finally, 89 markers were assigned to an A. hypogaea linkage map. A subset of 100 single-locus SSR markers was shown to be highly stable and universal in a collection of 96 peanut accessions. A neighbor-joining tree of this natural population showed that genotypes have obviously correlation with botanical varieties. Conclusions: We have shown that the detection of single-locus SSR markers from a de novo genomic assembly of a combination of different-insert-size libraries is highly efficient. This is the first report of the development of genome-wide single-locus markers for A. hypogaea, and the markers developed in this study will be useful for gene tagging, sequence scaffold assignment, linkage map construction, diversity analysis, variety identification and association mapping in peanut. [ABSTRACT FROM AUTHOR]