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Methods Mol Biol. 2018;1676:97-108. doi: 10.1007/978-1-4939-7315-6_5.

Deletion Mutagenesis and Identification of Causative Mutations in Maize.

Author information

1
Department of Agronomy and Horticulture, Center for Plant Science Innovation, Beadle Center for Biotechnology, University of Nebraska - Lincoln, 1901 Vine Street, P.O. Box 880665, Lincoln, NE, 68588-0665, USA.
2
School of Biological Sciences, Center for Plant Science and Innovation, Beadle Center for Biotechnology, University of Nebraska - Lincoln, 1901 Vine Street, P.O. Box 880665, Lincoln, NE, 68588-0665, USA.
3
Department of Agronomy and Horticulture, Center for Plant Science Innovation, Beadle Center for Biotechnology, University of Nebraska - Lincoln, 1901 Vine Street, P.O. Box 880665, Lincoln, NE, 68588-0665, USA. dholding2@unl.edu.

Abstract

We describe a method for gamma-irradiation of mature maize seeds to generate mutants with opaque endosperm and reduced kernel fill phenotypes. We also describe methods for mapping mutants and identifying causal gene mutations. Using this method, a population of 1788M2 families and 47 Mo17 × F2s showing stable, segregating, and viable kernel phenotypes was developed. For molecular characterization of the mutants, we utilized a novel functional genomics platform that combines separate Bulked Segregant RNA and exome sequencing data sets (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. We also describe the use of exome capture sequencing of F2 mutant and normal pools to perform mapping and candidate gene identification without the need for separate RNA-seq (BSEx-seq). To exemplify the utility of the deletion mutants for functional genomics and provide proof-of-concept for the bioinformatics platform, we summarize the identification of the causative deletion in two mutants. Mutant 937, which was characterized by BSREx-seq, harbors a 6203-bp in-frame deletion covering six exons within the Opaque-1 gene on chromosome 4. Preliminary investigation of opaque mutant 1486 with BSEx-seq shows a tight mapping interval and associated deletion on chromosome 10.

KEYWORDS:

BSR-seq; Causative mutation; Exome-seq; Maize functional genomics; Opaque

PMID:
28986905
DOI:
10.1007/978-1-4939-7315-6_5
[Indexed for MEDLINE]

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