Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping

Area of Science:

• Plant genetics and genomics
• Molecular biology techniques
• Bioinformatics and computational biology

Background:

• Next-generation sequencing (NGS) library construction typically requires purified DNA to avoid inhibition by plant impurities like polyphenols.
• Traditional methods involving restriction enzymes for genome complexity reduction are hindered by DNA purification steps.

Purpose of the Study:

• To develop a streamlined and efficient NGS library preparation protocol for plants that bypasses the need for DNA purification.
• To enhance the flexibility of polymorphism detection in various crop species.

Main Methods:

• Development of degenerate oligonucleotide primer MIG-seq (dpMIG-seq) by modifying the MIG-seq primer set with degenerate oligonucleotides.
• Application of the dpMIG-seq protocol to construct libraries from unpurified DNA in diverse crop species, including fruit trees.
• Utilizing dpMIG-seq for lineage selection in wheat and for linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean.

Main Results:

• The dpMIG-seq protocol successfully generated libraries from unpurified plant DNA across multiple species.
• dpMIG-seq demonstrated flexibility in detecting polymorphisms and enabled stable implementation in various crops.
• The method facilitated efficient genetic analyses, including lineage selection and QTL mapping, without DNA concentration adjustments.

Conclusions:

• The dpMIG-seq protocol offers a robust and versatile alternative for NGS library preparation in plants, especially when dealing with low-quality or unpurified DNA.
• This method significantly simplifies the workflow for genetic diversity studies and marker-assisted selection in a wide range of plant species.
• dpMIG-seq holds considerable potential for advancing genetic research and crop improvement programs globally.