Evidence for maternal control of seed size in maize from phenotypic and transcriptional analysis

Abstract

Seed size is an important component of grain yield and a key determinant trait for crop domestication. The Krug Yellow Dent long-term selection experiment for large and small seed provides a valuable resource to dissect genetic and phenotypic changes affecting seed size within a common genetic background. In this study, inbred lines derived from Krug Large Seed (KLS) and Krug Small Seed (KSS) populations and reciprocal F1 crosses were used to investigate developmental and molecular mechanisms governing seed size. Seed morphological characteristics showed striking differences between KLS and KSS inbred lines, and the reciprocal cross experiment revealed a strong maternal influence on both seed weight and seed size. Quantification of endosperm area, starchy endosperm cell size, and kernel dry mass accumulation indicated a positive correlation between seed size, endosperm cell number, and grain filling rate, and patterns of grain filling in reciprocal crosses mirrored that of the maternal parent. Consistent with the maternal contribution to seed weight, transcriptome profiling of reciprocal F1 hybrids showed substantial similarities to the maternal parent. A set of differentially expressed genes between KLS and KSS inbreds were found, which fell into a broad number of functional categories including DNA methylation, nucleosome assembly, and heat stress response. In addition, gene co-expression network analysis of parental inbreds and reciprocal F1 hybrids identified co-expression modules enriched in ovule development and DNA methylation, implicating these two processes in seed size determination. These results expand our understanding of seed size regulation and help to uncover the developmental and molecular basis underlying maternal control of seed size in maize.

Keywords: Endosperm; gene expression; maize; maternal effect; seed development; seed size..

Fig. 1.

Analysis of seed weight and seed size in KLS30- and KSS30-derived inbred lines and their reciprocal F₁ hybrids. (A) Mature seeds of inbreds and F₁ reciprocal hybrids. S1S1, S3S3, L1L1, and L3L3 are inbred lines derived from KSS30 and KLS30, respectively. F₁ reciprocal hybrids (e.g. S1L1) are designated with the maternal parent on the left and the paternal parent on the right. Scale bar=10mm. (B) Seed weight per 100 seeds (mean ±SD, n=3). (C) Parental effects on seed weights. Seeds were divided into four groups based on the common maternal parent (upper panel) and paternal parent (lower panel). The letters indicate significantly different statistical groups according to ANOVA (Tukey tests, P<0.05). (D) Average seed width and seed depth quantified by image analysis from 100 seeds (error bars=SD of 100 seeds). (E) Density plot of the distribution of seed area between maternal groups. Measurements were from image analysis of 100 seeds. L1, L3, S1, and S3 refer to the maternal parents shared in the group.

Fig. 2.

Comparison of endosperm area and cell size of KLS and KSS inbreds as well as grain filling rate in inbreds and hybrids. (A) Representative endosperms of KLS and KSS inbred lines at 17 days after pollination (DAP). Endosperm zones for measuring cell size are highlighted by the yellow rectangles. (B) Endosperm area of KLS and KSS inbreds at 17 DAP. Measurements are taken for each genotype on 33–54 developing kernels from three ears (means ±SD). Means with different letters are significantly different from each other (Tukey’s multiple pairwise comparisons test, P<0.05). (C) Endosperm cells of KLS and KSS inbred lines. Kernel sections dissected as described in the Materials and methods were stained with berberine sulfate and observed with a confocal microscope. Representative images are shown at ×20 magnification. Scale bars=100 µm. (D) Cell size in the endosperms of KLS and KSS inbreds at 17 DAP. Each data point represents 300–500 measurements (means ±SE). Different letters indicate significant differences according to ANOVA (Tukey’s test, P<0.05). (E) Grain filling rate in KLS and KSS inbreds and the corresponding hybrids. For clear demonstration, shown here is a time course of kernel dry weight collected from one KLS inbred (L1L1) and one KSS inbred (S3S3) and their crosses with two other inbred lines (S3L1/S3L3 and L1S1/L1S3). Values are the mean ±SD for n=6 replicates from three ears per date, 10 kernels per replicate. Trend line equations are shown for the inbred lines. S1S1, S3S3, L1L1, and L3L3 are inbred lines derived from KSS30 and KLS30, respectively. F₁ reciprocal hybrids (e.g. S3L1) are designated with the maternal parent on the left and the paternal parent on the right.

Fig. 3.

Transcriptome profile of inbred lines derived from KSS30 and KLS30 and their reciprocal hybrids at 14 and 17 days after pollination (DAP). Hierarchical clustering of log₂ transformation of the expressed genes (fragments per kilobase of exon model per million fragments mapped >1) was used to generate the tree. _14 and _17 indicate samples collected at 14 DAP and 17 DAP, respectively. S1S1, S3S3, L1L1, and L3L3 are inbred lines derived from KSS30 and KLS30, respectively. F₁ reciprocal hybrids (e.g. S1L1) are designated with the maternal parent on the left and the paternal parent on the right. S1, S3, L3, and L1 refer to the common maternal parent shared in the group samples.

Fig. 4.

Differentially expressed genes (DEGs) between inbred lines derived from the KSS30 and KLS30 populations. (A) Heatmap of DEGs with 2-fold up- or down-regulation between KLS and KSS inbreds (KLS versus KSS) at 14 DAP and 17 DAP. (B) Significantly enriched Gene Ontology (GO) categories with the up-regulated genes in KLS inbred lines. (C) Distribution of transcription factor families among DEGs. The percentage on the x-axis was plotted as the number of differentially expressed transcription factors in each genotype out of all the differential transcription factors identified in the family. _14 and _17 indicate samples collected at 14 DAP and 17 DAP, respectively. S1S1, S3S3, L1L1, and L3L3 are inbred lines derived from KSS30 and KLS30, respectively.

Fig. 5.

Identification of co-expression module genes associated with seed weight using the WGCNA method. (A) Module–trait relationship showing the significance of the module eigengene correlation with seed weight. Shown are correlations and P-values; cell color denotes correlation (white, positive correlation; red, negative correlation according to the color key). (B) Distribution of gene number including non-transcription factors and transcription factors among the modules that were significantly associated with seed weight. (C) Heatmap of M12 gene expression (upper panel) and expression levels of the corresponding eigengene across the samples in the M12 module (lower panel). The heatmap (upper panel) and barplot of eigengene expression (lower panel) have the same samples (x-axis). Rows of the heatmap correspond to genes, columns to samples; red in the color key denotes overexpression, green underexpression. (D) Differential expression of ZAG2 and ZMM1 in all samples grouped by maternal genotypes at 14 DAP (_14 in axis) and 17 DAP (_17 in axis). S1, S3, L1, and L3 signify the maternal parents that the samples have in common. _14 and _17 indicate samples collected at 14 DAP and 17 DAP, respectively. S1S1, S3S3, L1L1, and L3L3 are inbred lines derived from KSS30 and KLS30, respectively. F₁ reciprocal hybrids (e.g. S1L1) are designated with the maternal parent on the left and the paternal parent on the right.