Flowchart showing comparison of seed-based genotyping (orange arrows) and leaf-based genotyping (green arrow).

ICRISAT optimizes seed-chip genotyping in groundnut

Chips away time and cost from groundnut breeding.

Flowchart showing comparison of seed-based genotyping (orange arrows) and leaf-based genotyping (green arrow).

Flowchart showing comparison of seed-based genotyping (orange arrows) and leaf-based genotyping (green arrow).

Researchers at ICRISAT have established seed-chip genotyping for groundnut as a significantly faster and cheaper method for genotyping than the oft-used leaf-disc technique. In a recent research publication, they have also shown that the former can be easily integrated with rapid generation advancement (RGA) to hasten groundnut research by six to eight months.

In single seed chipping method, about 20 mg is chipped off from the posterior of a seed cotyledon (embryonic leaf) using a scalpel. The DNA in this chip is comparable in quality and quantity to the DNA from punched leaf samples. The results of genotyping are clear by 15 days as against 40 days that it takes through the leaf-disc technique. It was also found that chipping does not affect seed germination as both chipped and non-chipped seeds showed similar germination percentage after 30 days of storage at 4˚C. Following the efficiency gains the ICRISAT team has witnessed, an optimized seed-chip based genotyping (shown in the image) has replaced the leaf-disc-based genotyping in ICRISAT’s groundnut breeding program.

Rationale for single chip genotyping at ICRISAT

Genotyping (determining gene-level differences in an individual compared to rest of the population) is used for various purposes in breeding programs, such as the selection of desirable lines in large segregation population, identification of duplicates in germplasm, estimation of genetic purity of available varieties and confirmation of hybrids derived from two-parent or multi-parent crosses.

At ICRISAT, the groundnut breeding program has, for a decade, been using marker-assisted selection (MAS) for developing high-oleic, rust and late leaf spot diseases-resistant groundnuts.

The existing process using leaf-disc genotyping involves planting seeds in pots or in the field, label preparation and printing, tagging individual plants with labels, collecting the punched leaf discs from the individual plants, discarding the plants based on the genotype result and harvesting the genotype-confirmed plants while following the results sheet with the labels on the plants. This is a laborious and expensive process.  Genotyping using leaf-punching costs US$7.76 per sample and takes about 40 days, which can be reduced to US$2.5 with a 15 days’ time duration by deploying the new seed-chipping based genotyping.

“Seed-chip-based genotyping allows selection without the need for growing plants; which reduces the expenses required during maintenance of undesired and desired lines/accessions in the field during leaf-based genotyping methods,” said Dr Manish Pandey, Senior Scientist -Groundnut Genomics and corresponding author of the paper published in Agronomy.

Single seed chipping proves most advantageous when the proportion of selectable candidates is about 10-20% of the total candidates from which selections need to be made.

Researchers say that when combined with RGA, single seed-chip genotyping can significantly reduce research duration and increase genetic gain. At ICRISAT’s semi-controlled RGA greenhouse in Hyderabad, India, three-and-a-half generations of groundnut has been achieved per year. Researchers are now working on designing a fully controlled RGA facility to optimize and scale-up RGA to churn out at least 50% of groundnut breeding populations.

“The application of seed chipping technology complements low cost rapid generation advancement for cost effective development of high oleic groundnut line breeding and cultivar improvement with an enhanced rate of genetic gain. The Breeding Schema for groundnut integrates single seed-based genotyping in F4 and rapid generation advancement (RGA) to advance F2 to F3 and F3 to F4 Single Seed Descents are advanced,” said Dr P Janila, Principal Scientist-Groundnut Breeding and co-corresponding author of the study.

According to Dr Rajeev Varshney, Global Program Director, Accelerated Crop Improvement, seed-chip genotyping can be used for several purposes in breeding programs and can help significantly help accelerate crop improvement.

“Reducing the time taken for every breeding cycle is a significant way of enhancing genetic gain in crop research. If we are to continue feeding a growing world and meet the nutritional demands while besting the climate crisis, all means to accelerate genetic gains have to be embraced. Seed-chip genotyping and RGA are two of the most potent tools available,” said Dr Varshney.

The authors of the paper, Single Seed-Based High-Throughput Genotyping and Rapid Generation Advancement for Accelerated Groundnut Genetics and Breeding Research, are Parmar S, Deshmukh DB, Kumar R, Manohar SS, Joshi P, Sharma V, Chaudhari S, Variath MT, Gangurde SS, Bohar R, Singam P, Varshney RK, Janila P and Pandey MK (2021).

The scientific work described here has been supported by the National Agricultural Science Fund (NASF) of Indian Council of Agricultural Research (ICAR), Department of Biotechnology (DBT), India, and Bill & Melinda Gates Foundation (High Throughput Genotyping Project—HTPG (OPP1130244), and OPEC Fund for International Development (OFID) and CGIAR Research Program on Grain Legumes and Dryland Cereals.

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