Development of mPing-based activation tags for crop insertional mutagenesis

Abstract

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

Keywords: activation tagging; gene discovery; soybean; transposable element.

FIGURE 1

Structure of mPing‐based activation tags and yeast transposition assays. Diagram indicating the structure of the constructs (a). TIR sequences are indicated by green arrows, red indicates mutations in the mmPing20 element, and light green boxes indicate enhancer sequences. The images were made using Geneious version 2020.0 created by Biomatters. Yeast transposition frequency of the mPing, mmPing20, and mPing‐based activation tags (NL60, 2xE, 4xE, mmPing20F, and mmPing20B) (b). Error bars indicate the standard error of at least 6 replicates normalized to mPing. Statistical differences from multiple comparisons with a Tukey's test (one‐way ANOVA) are indicated by different letters (p ranging from .0439 to <.001)

FIGURE 2

Plasmid maps. Diagrams depicting the T‐DNA portions of pEarleyGate 103 mmPing20F (a) and pWMD23 (b). Black wedges indicate the position of the primers used to detect excision of the mmPing20F element. The image was made using Geneious version 2020.0 created by Biomatters

FIGURE 3

Development and analysis of the mmPing20F soybean population. Diagram showing the crosses that were made between the transgenic lines carrying the mmPing20F construct and the pWMD23 (TPase) construct (a). Colors indicate the amount of transposition detected in the F1 generation from each respective cross. PCR analysis of the 13 plants from the F2 generation of the 16‐28 line (b). In the PCR with mmPing20F flanking primers, the upper band (960 bp) indicates that the element is still located in the transgene, while the lower band (317 bp) indicates mmPing20F has excised. Pong TPase primers indicate if the TPase expression construct pWMD23 is present

FIGURE 4

Genomic features associated with mmPing20F insertions identified by sequencing. The insertion site corresponds to the base immediately flanking the mmPing20F tag. Distances of zero indicate the insertion is within the annotated gene model. Negative distances denote upstream insertions. Shaded cells with the “+” sign indicate the insertion is in the gene feature

FIGURE 5

mmPing20F insertions detected in multiple generations. Each column indicates the absence “−” or insertion “+” at specific loci. The original progenitor (yellow cell), three first‐generation progeny (white), and 12 s‐generation progeny (grey cells) were analyzed

FIGURE 6

Expression changes in local genes associated with the mPing‐based activation tag. The magenta box represents the mmPing20F insertion position (box not to scale). The data points correspond to annotated gene models with the arrowhead indicating transcriptional orientation (arrows are not drawn to scale). The green data points represent significantly upregulated genes (padj < .05) using a Wald test and Benjamini‐Hochberg multiple testing correction