Investigating male sterility in potato: a multi-omics landscape of anther development and developing Dominant Gene Male Sterile (DGMS) lines by gene editing

Daisy Li

Male sterility is an important process in plant breeding and is used to generate hybrid seed and increased yield through heterosis (hybrid vigour). Recent advances in understanding the molecular mechanisms of male sterility systems have enabled the development of more efficient hybrid breeding strategies. A range of genes controlling male fertility have been identified, enabling the development of novel hybridization systems in crops like maize, rice and wheat. However, few male sterile genes or lines have been reported for potato. With a push to develop propagation by seed rather than by tubers for the potato industry, male sterile lines are needed to produce vigorous and uniform F1 hybrids. • The first phase of this project was to study anther development in potato in detail. This revealed four major developmental stages involving 14 stages (S1-14), similar to the developmental stages in rice and Arabidopsis. • The second phase was to identify novel target genes and lipid metabolism-related male fertile genes by integrating data from transcriptomics and lipid metabolism. Through weighted gene co-expression network analysis (WGCNA), 235 core genes were identified. To further investigate the role of the core genes, two target genes (StNIP7 and StSKIP23-like) thought to affect pollen viability in developing anthers were selected for functional validation using CRISPR-Cas9 gene editing. • The third phase aimed to develop DGMS (Dominant Gene Male Sterility) lines using CRISPR-Cas9 to edit upstream open reading frames (uORFs). Analysis of 2,879 anther-specific genes identified 430 containing uORFs with AUG start codons and Kozak consensus sequences, 32 of which exhibited high expression at the S8 stage. Based on uORF length the StSKIP23-like gene (not previously reported in potato) was selected for functional validation. CRISPR-Cas9-mediated uORF knockout resulted in reduced pollen viability and germination, and these phenotypes were stably inherited in the T1 generation. These results demonstrate the potential of editing uORFs of anther developmental genes as a new approach for generating DGMS lines in potato breeding. Conclusions: This study provides new insights into the regulation of anther development in potato and establishes a foundation for further genetic improvement of male sterility traits. It advances the understanding of male sterility by identifying key regulatory genes and metabolic pathways involved in anther development. Successful implementation of uORF-targeted gene-editing offers a promising strategy for developing stable DGMS lines to facilitate commercial production of hybrid potato seeds.

Investigating male sterility in potato: a multi-omics landscape of anther development and developing Dominant Gene Male Sterile (DGMS) lines by gene editing

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