A PCR-based forward genetics screening, using expression domain-specific markers, identifies mutants in endosperm transfer cell development.

Luis M Muñiz,Maribel López,Olivier Sellam,Virginie Guyon,Gregorio Hueros,Pascual Peréz,Bouchaib Khbaya,Elisa Gómez

doi:10.3389/fpls.2014.00158

Luis M Muñiz, Maribel López + Show 6 more

Open Access

https://doi.org/10.3389/fpls.2014.00158

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Abstract

Mutant collections are an invaluable source of material on which forward genetic approaches allow the identification of genes affecting a wide variety of biological processes. However, some particular developmental stages and morphological structures may resist analysis due to their physical inaccessibility or to deleterious effects associated to their modification. Furthermore, lethal mutations acting early in development may escape detection. We have approached the characterization of 101 maize seed mutants, selected from a collection of 27,500 visually screened Mu-insertion lines, using a molecular marker approach based on a set of genes previously ascribed to different tissue compartments within the early developing kernel. A streamlined combination of qRT-PCR assays has allowed us to preliminary pinpoint the affected compartment, establish developmental comparisons to WT siblings and select mutant lines with alterations in the different compartments. Furthermore, clusters of markers co-affected by the underlying mutation were identified. We have analyzed more extensively a set of lines presenting significant variation in transfer cell-associated expression markers, and have performed morphological observations, and immunolocalization experiments to confirm the results, validating this approach as an efficient mutant description tool.

Highlights

The angiosperm seed is a complex structure evolutionarily designed to attain protection of the forming embryo, resist desiccation, facilitate its dispersal and nurture the new plant before it obtains sufficient photosynthetic ability for self-support
F2 seeds from 101 mutant lines were planted in the field to produce the material used in the present work
Immature cobs were opened for visual inspection starting at 13 days after pollination (13 DAP)

Summary

Introduction

The angiosperm seed is a complex structure evolutionarily designed to attain protection of the forming embryo, resist desiccation, facilitate its dispersal and nurture the new plant before it obtains sufficient photosynthetic ability for self-support. The maize seed has received particular attention due to its economic value and widespread cultivation (Timmermans et al, 2004). It is the result of a double fertilization event which produces two isolated compartments differing in ploidy level but otherwise genetically identical, the embryo and the endosperm. Active cell division yields to a differentiation process that results in four specialized tissues within the endosperm (Olsen, 2001; Consonni et al, 2005; Scanlon and Takacs, 2008): the starchy endosperm, which accumulates nutrients for seedling nutrition after germination; the ESR, presumably involved in nutrient transfer from the mother plant to the embryo and its protection (Balandín et al, 2005); the basal endosperm transfer layer (BETL), located right under the starchy endosperm and opposite to the placento-chalaza, where it facilitates solute uptake and secretes defensive peptides onto the seed-mother plant interface (Serna et al, 2001; Olsen, 2004); and the aleurone, a peripheral layer of nearly cubical cells which participates in substrate mobilization upon germination of the embryo (Bommert and Werr, 2001) and covers the entire surface of the endosperm except for the BETL

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