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. 2020 Feb 25;11:132.
doi: 10.3389/fpls.2020.00132. eCollection 2020.

Developmental Analysis of Mimulus Seed Transcriptomes Reveals Functional Gene Expression Clusters and Four Imprinted, Endosperm-Expressed Genes

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Free PMC article

Developmental Analysis of Mimulus Seed Transcriptomes Reveals Functional Gene Expression Clusters and Four Imprinted, Endosperm-Expressed Genes

Miguel A Flores-Vergara et al. Front Plant Sci. .
Free PMC article

Abstract

The double fertilization of the female gametophyte initiates embryogenesis and endosperm development in seeds via the activation of genes involved in cell differentiation, organ patterning, and growth. A subset of genes expressed in endosperm exhibit imprinted expression, and the correct balance of gene expression between parental alleles is critical for proper endosperm and seed development. We use a transcriptional time series analysis to identify genes that are associated with key shifts in seed development, including genes associated with secondary cell wall synthesis, mitotic cell cycle, chromatin organization, auxin synthesis, fatty acid metabolism, and seed maturation. We relate these genes to morphological changes in Mimulus seeds. We also identify four endosperm-expressed transcripts that display imprinted (paternal) expression bias. The imprinted status of these four genes is conserved in other flowering plants, suggesting that they are functionally important in endosperm development. Our study explores gene regulatory dynamics in a species with ab initio cellular endosperm development, broadening the taxonomic focus of the literature on gene expression in seeds. Moreover, it is the first to validate genes with imprinted endosperm expression in Mimulus guttatus, and will inform future studies on the genetic causes of seed failure in this model system.

Keywords: K-means clustering; MADS-box genes; Mimulus guttatus; developmental time course analysis; endosperm; genomic imprinting; ribonucleic acid sequencing; seed development.

Figures

Figure 1
Figure 1
(A) Percentage of mature seed phenotypes (round, shriveled, and flat) recovered from fully developed fruits from selfed Mimulus pardalis and Mimulus guttatus and reciprocal crosses of M. pardalis x M. guttatus. There was no significant difference in the distribution of seed types between the selfed fruits and hybrid fruits (MANOVA F9,45 = 1.09, p > 0.1). (B) Germination rates of selfed M. pardalis and M. guttatus seed, as well as M. pardalis x M. guttatus, and M. guttatus x M. pardalis hybrid seeds. Mp, M. pardalis; Mg, M. guttatus. Bars indicate standard error. Germination rates were generally high (57.9–89.74%) (N=4 for each selfing and cross).
Figure 2
Figure 2
(A) Morphology of Mimulus pardalis x Mimulus guttatus developing seeds collected 2, 4, 6, and 8 days after pollination (DAP). Sections were obtained from LR-white embedded seeds (see Methods). (B) Frequency distribution of embryonic stages of seeds collected at 2, 4, 6, and 8 DAP. (C) Principle component analysis (PCA) plot of RNA libraries.
Figure 3
Figure 3
Examples of non-overlapping k-means clusters of co-expressed genes and their significant gene ontology (GO) enrichment terms.
Figure 4
Figure 4
A heatmap of MADS-box type I gene expression in ovules and seeds. The nearest Arabidopsis thaliana homologue for each Mimulus guttatus MADS-box gene is indicated in parentheses (as determined by BLASTx).
Figure 5
Figure 5
Neighbor joining tree of type I MADS-box genes from rice (Oryza sativa), Arabidopsis thaliana, tomato (Solanum lycopersicum), and Mimulus guttatus (reference line IM62). Bootstrap support is represented by color (green ≥ 90, blue 80–89). Nodes with less than 80% support are not marked. M. guttatus genes expressed in our data set are color-coded by time of peak expression. A. thaliana expression profiles are indicated by color-coded triangles.
Figure 6
Figure 6
Validation of four paternally expressed imprinted genes: Migut.E01117 (MBD13), Migut.H00744 (ATRX5), Migut.I00545 (DnaJ), and Migut.N01317 (BGAL11) via semi-quantitative PCR and Sanger sequencing. (A) Allele-specific real-time (RT)-PCR analyses of each gene in the endosperm of selfed and reciprocal crosses of Mimulus pardalis (Mp) and Mimulus guttatus (Mg) is given. For each gene, gel shows the sizes of restriction fragments after RT-PCR amplification and digestion with restriction endonucleases. (B) Paternal expression was confirmed by real-time (RT)-PCR sequencing chromatographs at selected SNP regions measuring allele-specific expression in reciprocal crosses.

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