Dataset Information

Genome-wide identification of microsatellites in white clover (Trifolium repens L.) using FIASCO and phpSSRMiner.

ABSTRACT: BACKGROUND:Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover. RESULTS:Genomic libraries containing simple sequence repeat (SSR) sequences were constructed using a modified Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) procedure and phpSSRMiner was used to develop the microsatellite markers. SSR motifs were isolated using two biotin-labeled probes, (CA)17 and (ATG)12. The sequences of 6,816 clones were assembled into 1,698 contigs, 32% of which represented novel sequences based on BLASTN searches. Approximately 32%, 28%, and 16% of these SSRs contained hexa-, tri-, and di-nucleotide repeats, respectively. The most frequent motifs were the CA and ATG complementary repeats and the associated compound sequences. Primer pairs were designed for 859 SSR loci based on sequences from these genomic libraries and from GenBank white clover nucleotide sequences. A total of 191 SSR primers developed from the two libraries were tested for polymorphism in individual clones from the parental genotypes GA43 ('Durana'), 'SRVR' and six F1 progeny from a mapping population. Ninety two percent produced amplicons and 66% of these were polymorphic. CONCLUSION:The combined approach of identifying SSR-enriched fragments by FIASCO coupled with the primer design and in silico amplification using phpSSRMiner represents an efficient and low cost pipeline for the large-scale development of microsatellite markers in plants.The approach described here could be readily adapted and utilized in other non-related species with none or limited genomic resources.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC2517061 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Genome-wide identification of microsatellites in white clover (Trifolium repens L.) using FIASCO and phpSSRMiner.

Zhang Yan Y He Ji J Zhao Patrick X PX Bouton Joseph H JH Monteros Maria J MJ

Plant methods 20080716

<h4>Background</h4>Allotetraploid white clover (Trifolium repens L.) is an important forage legume widely cultivated in most temperate regions. Only a small number of microsatellite markers are publicly available and can be utilized in white clover breeding programs. The objectives of this study were to develop an integrated approach for microsatellite development and to evaluate the approach for the development of new SSR markers for white clover.<h4>Results</h4>Genomic libraries containing sim ...[more]

PMID: 18631390

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Project description:BACKGROUND: White clover (Trifolium repens L.) is a temperate forage legume with an allotetraploid genome (2n=4×=32) estimated at 1093 Mb. Several linkage maps of various sizes, marker sources and completeness are available, however, no integrated map and marker set has explored consistency of linkage analysis among unrelated mapping populations. Such integrative analysis requires tools for homoeologue matching among populations. Development of these tools provides for a consistent framework map of the white clover genome, and facilitates in silico alignment with the model forage legume, Medicago truncatula. RESULTS: This is the first report of integration of independent linkage maps in white clover, and adds to the literature on methyl filtered GeneThresher®-derived microsatellite (simple sequence repeat; SSR) markers for linkage mapping. Gene-targeted SSR markers were discovered in a GeneThresher® (TrGT) methyl-filtered database of 364,539 sequences, which yielded 15,647 SSR arrays. Primers were designed for 4,038 arrays and of these, 465 TrGT-SSR markers were used for parental consensus genetic linkage analysis in an F1 mapping population (MP2). This was merged with an EST-SSR consensus genetic map of an independent population (MP1), using markers to match homoeologues and develop a multi-population integrated map of the white clover genome. This integrated map (IM) includes 1109 loci based on 804 SSRs over 1274 cM, covering 97% of the genome at a moderate density of one locus per 1.2 cM. Eighteen candidate genes and one morphological marker were also placed on the IM. Despite being derived from disparate populations and marker sources, the component maps and the derived IM had consistent representations of the white clover genome for marker order and genetic length. In silico analysis at an E-value threshold of 1e-20 revealed substantial co-linearity with the Medicago truncatula genome, and indicates a translocation between T. repens groups 2 and 6 relative to M. truncatula. CONCLUSIONS: This integrated genetic linkage analysis provides a consistent and comprehensive linkage analysis of the white clover genome, with alignment to a model forage legume. Associated marker locus information, particularly the homoeologue-specific markers, offers a new resource for forage legume research to enable genetic analysis and improvement of this forage and grassland species.

Dataset Information

Genome-wide identification of microsatellites in white clover (Trifolium repens L.) using FIASCO and phpSSRMiner.

Publications

Genome-wide identification of microsatellites in white clover (Trifolium repens L.) using FIASCO and phpSSRMiner.

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