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.

Omics-Facilitated Crop Improvement for Climate Resilience and Superior Nutritive Value

“…et al, 2020 ; Gogolev et al, 2021 ; Kumar R. et al, 2021 ). Such modern plant breeding technologies include double-haploid (DH) breeding ( Yan et al, 2017 ), induced mutagenesis ( Kharkwal and Shu, 2009 ), CRISPR-Cas based gene editing technologies (see Ahmar et al, 2020 ; Steinwand and Ronald, 2020 ; Fiaz et al, 2021 ; Gao, 2021 ; Kumar A. et al, 2021 ; Marsh et al, 2021 ; Sinha et al, 2021 ), and the single seed chipping (SSC) facilitated marker-based early generation selection (MEGS) technique ( Parmar et al, 2021 ), among others. For instance, the SSC facilitated MEGS protocol could be used to successfully advance 3.5 breeding generations in groundnuts, and could significantly cut the time required to complete the entire breeding cycle by approximately 6-8 months.…”

“…For instance, the SSC facilitated MEGS protocol could be used to successfully advance 3.5 breeding generations in groundnuts, and could significantly cut the time required to complete the entire breeding cycle by approximately 6-8 months. Additionally, the SSC technique did not significantly affect germination percentage (as it remained high, 95-99%) ( Parmar et al, 2021 ). Therefore, this technique could be an indispensible tool to promote high-throughput genotyping and speed breeding of climate-smart groundnut (and possibly other legume) crop cultivars.…”

Breeding More Crops in Less Time: A Perspective on Speed Breeding

“…A germination rate of 95–99% was observed from the chipped seeds. The study led to a time saving of 6–8 months following the implementation of this integrated approach in groundnut research and breeding [ 65 ]. Since 2016, SB has been integrated within all cool-season legume public breeding programs in Australia, with more than 45,000 individuals processed through an aSSD platform at the University of Western Australia.…”

Phenotypic and genotypic diversity of Bambara groundnut (Vigna subterranea L. Verdc) germplasm in Malawi

“…Qualitative data were presented using gures taken from the eld and percent variation was calculated. Leaf samples were sampled using a procedure as described by Intertek (Parmar et al, 2021). Brie y, the two leaf discs were collected using a leaf puncher from a fresh clean leaf and placed in the 96 well Abgene plates until the 94 wells were lled while two wells were left blank as control.…”