Abstract
BackgroundSingle-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.ResultsIn 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2743-x) contains supplementary material, which is available to authorized users.
Highlights
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
The length of the scaffolds varied from 200 bp to 576,627 bp, with an average of 1,693 bp; 360,557 scaffolds were longer than 2 Kbp and 9,448 scaffolds were longer than 10 Kbp (Table 2)
We investigated whether the motif type, repeat length and repeat number influence the polymorphism rate of single-locus simple sequence repeats (SSRs) markers
Summary
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. The peanut holds an important status and substantial efforts have been made to develop various types of molecular markers in recent years, such as restriction fragment length polymorphisms (RFLPs) [1, 2], random amplified polymorphic DNAs (RAPDs) [3,4,5], amplified fragment length polymorphisms (AFLPs) [6, 7], simple sequence repeats (SSRs) [8, 9], insertions/deletions (INDELs) [10], and single nucleotide polymorphisms (SNPs) [11, 12] These markers were developed for genetic linkage mapping [13, 14], genetic diversity studies [9, 15, 16], and for use in plant breeding programs [10, 17]. Lack of the tools for ideal molecular markers and genomic resources are important factors hampering the development of genetic research and molecular breeding of peanut
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