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Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome

M Vukosavljev¹, G D Esselink, W P C van 't Westende

¹Wageningen UR Plant Breeding, Wageningen University & Research Centre, P.O. Box 386, NL-6700AJ, Wageningen, the Netherlands; C.T. de Wit Graduate School for Production Ecology and Resource Conservation (PE&RC), Wageningen, the Netherlands.

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|June 5, 2014

Summary

Developing highly polymorphic microsatellite markers is now more efficient. Our new strategy screens for length variants in next-generation sequencing reads, significantly improving marker informativeness for genetic studies.

Keywords:

RNA-seq microsatellite marker next-generation sequencing simple sequence repeat

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Area of Science:

Genetics
Molecular Biology
Bioinformatics

Background:

Developing microsatellite markers is crucial for genetic studies but faces challenges in identifying informative polymorphic loci.
Next-generation sequencing has simplified marker cloning, but efficient screening for polymorphic and informative markers remains difficult.
Current methods for differentiating polymorphic from non-polymorphic loci and selecting markers with a high effective number of alleles are laborious and manual.

Purpose of the Study:

To develop an efficient strategy for identifying and developing highly polymorphic microsatellite markers.
To overcome the hurdles of differentiating polymorphic loci and ensuring markers are sufficiently informative for broad deployment.
To refine marker development by screening for length variants in next-generation sequencing data.

Main Methods:

A strategy was developed to screen next-generation sequencing reads from multiple genotypes for repeats exhibiting the most length variants.
These selected variants were then developed into microsatellite markers.
The strategy was validated using Illumina paired-end transcriptome sequences from 11 tetraploid garden roses.

Main Results:

Of 48 tested markers, 46 were polymorphic, with 10 amplifying more than one locus.
An initial refinement step identified replicate markers, which were subsequently filtered out.
The refined approach yielded 18 highly polymorphic markers, averaging 11.7 alleles per marker in 11 tetraploid roses, surpassing previous benchmarks.

Conclusions:

The developed strategy significantly advances the efficient development of highly polymorphic microsatellite markers.
This approach streamlines the selection of informative markers, reducing labor and improving marker quality for genetic research.
The method effectively identifies markers with a high effective number of alleles, suitable for diverse applications.