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

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.

Genome-Wide Mapping of Targets of Maize Histone Deacetylase HDA101 Reveals Its Function and Regulatory Mechanism during Seed Development

“…Previous studies identified 34 genes that show cell type-specific expression during endosperm differentiation (Gómez et al, 2009;Li et al, 2014;Muñiz et al, 2014) (Figure 3A). Consistent with the morphological abnormalities observed in seeds of the hda101-1 mutant, our results from the RNA-seq analysis, performed using three biological replications, showed that 26 out of these 34 genes exhibited a statistically significant decrease of their transcript level in 4-DAP seeds of the hda101-1 mutant compared with B73 ( Figure 3A).…”

“…BETL-specific genes are often expressed in a dynamic pattern during endosperm development, characterized by significant upregulation at 4 to 8 DAP, when rapid cell differentiation and dramatic reprogramming of the kernel transcriptome take place (Gómez et al, 2009;Li et al, 2014;Muñiz et al, 2014). We therefore used RT-qPCR to assess whether loss of HDA101 protein in early seed development affects the temporal expression pattern of six BETL-specific genes ( Figure 3D).…”

Transfer cells

“…Most of the identified proteins showing decreased glycosylation in mn 1 were involved in post-translational modification, protein turnover, chaperone function, carbohydrate metabolism and cell wall biosynthesis, suggesting links to endoplasmic reticulum stress and the unfolded protein response in the mutant due to compromised glycosylation levels. Muñiz et al ( 2014 ) describe establishment of a PCR-based forward genetic screen using expression domain-specific markers to identify new mutant lines showing altered TC development. Application of this forward genetics approach using genes known to be tissue or developmental-stage specific offers new tools to extend the use of maize as a model for TC research.…”