Single seed-based high-throughput genotyping and rapid generation advancement for accelerated groundnut genetics and breeding research

Abstract

The groundnut breeding program at International Crops Research Institute for the Semi-Arid Tropics routinely performs marker-based early generation selection (MEGS) in thousands of segregating populations. The existing MEGS includes planting of segregating populations in fields or glasshouses, label tagging, and sample collection using leaf-punch from 20–25 day old plants followed by genotyping with 10 single nucleotide polymorphisms based early generation selection marker panels in a high throughput genotyping (HTPG) platform. The entire process is laborious, time consuming, and costly. Therefore, in order to save the time of the breeder and to reduce the cost during MEGS, we optimized a single seed chipping (SSC) process based MEGS protocol and deployed on large scale by genotyping >3000 samples from ongoing groundnut breeding program. In SSC-based MEGS, we used a small portion of cotyledon by slicing-off the posterior end of the single seed and transferred to the 96-deep well plate for DNA isolation and genotyping at HTPG platform. The chipped seeds were placed in 96-well seed-box in the same order of 96-well DNA sampling plate to enable tracking back to the selected individual seed. A high germination rate of 95–99% from the chipped seeds indicated that slicing of seeds from posterior end does not significantly affect germination percentage. In addition, we could successfully advance 3.5 generations in a year using a low-cost rapid generation turnover glass-house facility as compared to routine practice of two generations in field conditions. The integration of SSC based genotyping and rapid generation advancement (RGA) could significantly reduce the operational requirement of person-hours and expenses, and save a period of 6–8 months in groundnut genetics and breeding research.