Harnessing GhGRDP1 natural variation for enhanced cotton seed yield: a rhamnose-dependent strategy in breeding

Breeding high-yield crops has long been a fundamental goal in global agriculture, with seeds serving as the essential vehicle to achieve this aim. Therefore, understanding the regulatory mechanisms of seed size is critical for ensuring food security.

Our study focused on the natural variation, molecular regulatory mechanism and functional evolution of yield trait-related gene GhGRDP1, identified through GWAS and eQTL analyses in upland cotton natural population.

Population resequencing and phenotypic data analysis were combined with Kompetitive Allele Specific PCR, degradome, GUS staining assays, and qPCR revealed the natural variation, evolutionary history and geographic origins of GhGRDP1. Using CRISPR-Cas9 gene editing and plant transformation techniques, GRDP1 transgenic plants of cotton, rice and Arabidopsis were generated to unravel its conserved role in seed regulation. Co-IP, pull down, BiFC, LCI and enzyme activity assays were used to explore the molecular mechanism of GhGRDP1. The evolution of GRDP1 was understood through phylogenetic analysis and protein structure prediction, segmental interaction and enzyme activity assays.

A 1-bp deletion in the eighth exon of GhGRDP1 initiates the nonsense-mediated mRNA decay (NMD) mechanism, causing variations in seed index (SI) and lint percent (LP) among cotton populations. This variant, unique to Gossypium hirsutum, originated from specific races and was strongly selected during domestication. GhGRDP1 positively regulates seed size and weight without compromising fiber quality, correlating with osmolyte accumulation in ovules. GhGRDP1 interacts with UDP-L-rhamnose synthase (GhRHM1) in vivo and in vitro, functioning as an activator of GhRHM1 enzymatic activity involved in cell wall composition. Notably, GRDP1 evolved a short glycine-rich domain (sGRD) on C-terminal throughout core angiosperms, playing a conserved role in seed development across both monocots and eudicots.

These findings significantly advance understanding the genetic basis of phenotypic variation in cotton seed yield, providing valuable targets for high-yield crop breeding.