Using Seed Chipping to Genotype Maize Kernels

Mills, Alison M; Allsman, Lindy A.; Leon, Sareen; Rasmussen, Carolyn G.

doi:10.21769/bioprotoc.3553

Cited by 6 publications

(5 citation statements)

References 7 publications

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“…Another approach is to preselect plants to be phenotyped on the basis of their genetic constitution by predicting gene effects with statistical models in the same fashion as for genomic selection. Such predictions can be combined with the use of non-destructive seed chipping (Mills et al, 2020) to pick specific gene combinations before sowing and therefore reduce the number of plants to be tested. Once the gene space is lowered, the BREEDIT pipeline can be followed again to design validation constructs by engineering a vector containing gRNAs targeting only the genes retained in the selected subset.…”

Section: Discussionmentioning

confidence: 99%

BREEDIT: A novel multiplex genome editing strategy to improve complex quantitative traits in maize (Zea mays L.)

Lorenzo

Debray

Herwegh

et al. 2022

Preprint

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Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Though new technologies are being applied to tackle the problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in gene engineering via the CRISPR/Cas technology pave the way to accelerate plant breeding and meet this increasing demand. Here, we present a gene discovery pipeline named 'BREEDIT' that combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought resistance. We induced gene knockouts in 48 growth-related genes using CRISPR/Cas9 and generated a collection of over 1000 gene-edited maize plants. Edited populations displayed, on average, significant increases of up to 10% for leaf length and 20% for leaf width compared with controls. For each gene family, edits in subsets of genes could be associated with increased traits, allowing us to reduce the gene space needed to focus on for trait improvement. We propose BREEDIT as a pipeline which can be rapidly applied to generate a diverse collection of mutants to identify subsets of promising candidates that could be later incorporated in breeding programs.

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Section: Discussionmentioning

confidence: 99%

BREEDIT: A novel multiplex genome editing strategy to improve complex quantitative traits in maize (Zea mays L.)

Lorenzo

Debray

Herwegh

et al. 2022

Preprint

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Section: [Key Features]mentioning

confidence: 99%

“…Seed-based DNA genotyping methods have been implemented in small-scale maize breeding programs and genotyping laboratories but are still ineffective based on safety and scalability (Gao et al, 2008; Mills et al, 2020). One seed-based DNA genotyping method is based on soaking maize kernels in water before endosperm chipping (Gao et al, 2008).…”

Section: [Background]mentioning

confidence: 99%

“…The caveats of this procedure include susceptibility to fungal contamination and low seed germination if kernels are not dried and stored well. Another common method uses a razor blade for seed chipping (Mills et al, 2020), which presents injury hazards and is time-consuming, thus making it impractical to chip a large population quickly. In this study, we used clipping pliers, a safe alternative to previous chipping methods that is scalable to high throughput applications.…”

Section: [Background]mentioning

confidence: 99%

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An effective and safe maize seed chipping protocol using clipping pliers with applications in small-scale genotyping and marker-assisted breeding

Zebosi,

Ssengo,

Geadelmann

et al. 2024

Preprint

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In applications such as marker-assisted breeding and positional cloning, tissue sampling and plant tracking are vital steps in the genotyping pipeline. They enable the identification of desirable seedlings, saving time and reducing the cost, space, and handling required for growing adult plants, especially for greenhouses and winter nurseries. Small-scale marker-assisted selection laboratories rely heavily on leaf-based genotyping, which involves over-planting large, segregating populations followed by leaf sampling, genotyping, and backtracking to identify desired individuals, which is costly and laborious. Thus, there is a need to adopt seed-based genotyping to reduce costs and save time. Therefore, we developed a safe and cheap seed-chipping protocol using clipping pliers to chip seeds to genotype before planting. To identify a cost-effective and high throughput DNA extraction method, we tested four extraction methods and assessed the quality of the seed DNA using PCR. For three of the methods, seed-based DNA was of comparable quality to DNA extracted from leaf punches. We also compared seed- and leaf-derived DNA from the same individuals in a segregating population to test for genotyping miscalls that could arise due to the presence of maternally derived pericarp in the seed samples and found zero miscalls of 43 potential instances. Germination rates of chipped and unchipped seeds were the same for the inbreds tested, B73 and Mo17. However, chipped seeds grew slower until ~14 days after sowing. Overall, seed sampling using clipping pliers provides a simple, reliable, and high-throughput method to identify specific genotypes before planting.

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“…This population is genotyped, with a very high number of (representative) individuals. Genotyping is conducted on seeds instead of plants (chipped seed, Mills et al, 2020) and genotypic data are used to predict line per se performance or varietal ability (see above). Selection is applied accordingly, between chipped seeds.…”

Section: Genomic Selectionmentioning

confidence: 99%

Conventional and Molecular Breeding Tools for Accelerating Genetic Gain in Faba Bean (Vicia Faba L.)

et al. 2021

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Faba bean is a cool-season grain legume crop, which is grown worldwide for food and feed. Despite a decrease in area under faba bean in the past, the interest in growing faba bean is increasing globally due to its high seed protein content and its excellent ecological service. The crop is, however, exposed to diverse biotic and abiotic stresses causing unstable, low grain yield. Although, sources of resistance to main diseases, such as ascochyta blight (Ascochyta fabae Speg.), rust (Uromyces viciae-fabae (Pers.) Schroet.), chocolate spot (Botrytis fabae Sard.) and gall disease (Physioderma viciae), have been identified, their resistance is only partial and cannot prevent grain yield losses without agronomical practices. Tightly associated DNA markers for host plant resistance genes are needed to enhance the level of resistance. Less progress has been made for abiotic stresses. Different breeding methods are proposed, but until now line breeding, based on the pedigree method, is the dominant practice in breeding programs. Nonetheless, the low seed multiplication coefficient and the requirement for growing under insect-proof enclosures to avoid outcrossing hampers breeding, along with the lack of tools such as double haploid system and cytoplasmic male sterility. This reduces breeding population size and speed of breeding hence the chances of capturing rare combinations of favorable alleles. Availability and use of the DNA markers such as vicine-convicine (vc−) and herbicide tolerance in breeding programs have encouraged breeders and given confidence in marker assisted selection. Closely linked QTL for several biotic and abiotic stress tolerance are available and their verification and conversion in breeder friendly platform will enhance the selection process. Recently, genomic selection and speed breeding techniques together with genomics have come within reach to accelerate the genetic gains in faba bean. Advancements in genomic resources with other breeding tools, methods and platforms will enable to accelerate the breeding process for enhancing genetic gain in this species.

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Using Seed Chipping to Genotype Maize Kernels

Cited by 6 publications

References 7 publications

BREEDIT: A novel multiplex genome editing strategy to improve complex quantitative traits in maize (Zea mays L.)

BREEDIT: A novel multiplex genome editing strategy to improve complex quantitative traits in maize (Zea mays L.)

An effective and safe maize seed chipping protocol using clipping pliers with applications in small-scale genotyping and marker-assisted breeding

Conventional and Molecular Breeding Tools for Accelerating Genetic Gain in Faba Bean (Vicia Faba L.)

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