# Genome-wide identification of raffinose synthase gene family in barley (Hordeum vulgare L.) reveals its role in multiple abiotic stress tolerance

## TL;DR

This study identifies and characterizes raffinose synthase genes in barley, revealing their role in helping the plant tolerate environmental stresses like drought and salt.

## Contribution

The first genome-wide characterization of the raffinose synthase gene family in barley, identifying key stress-responsive genes.

## Key findings

- Eight HvRS genes were identified in the barley genome, distributed across four chromosomes.
- HvRS2 and HvRS5 showed significant upregulation under salt, drought, and ABA treatments, indicating their role in stress response.
- Pan-genome haplotype analysis revealed high nucleotide diversity and geographic divergence in HvRS2 and HvRS5 haplotypes.

## Abstract

Raffinose synthase (RS) catalyzes the biosynthesis of raffinose from galactinol and sucrose, playing an important role in plant stress tolerance and seed development. However, the RS gene family has not been systematically characterized in barley. In this study, we performed a comprehensive genome-wide identification of RS gene family in barley. A total of eight HvRS genes were identified from the Morex V3 genome, which are distributed across four chromosomes. Phylogenetic analysis of RS homologs from other plants showed that the 79 RS proteins were classified into five distinct clades, revealing close evolutionary relationships among barley, wheat, and Brachypodium distachyon. Promoter cis-element analysis revealed an abundance of hormone- and stress-responsive elements, suggesting roles of HvRS genes in adaptive responses. Quantitative real-time PCR (qRT-PCR) analysis revealed that HvRS2 and HvRS5 were markedly upregulated in roots and/or shoots under salt, drought (simulated by polyethylene glycol, PEG), and abscisic acid (ABA) treatments, indicating their involvement in the early-phase abiotic stress response. Relative to time-matched controls, the HvRS2 expression levels in roots increased to 9.90-fold at 24 h under salt stress and to 49.1-fold at 6 h under ABA treatment. Meanwhile, HvRS5 expression levels in shoots reached 40.0-fold at 6 h under salt stress and to 26.6-fold at 12 h ABA treatment relative to controls. Pan-genome haplotype analysis of the two stress-responsive genes, HvRS2 and HvRS5, revealed substantial nucleotide diversity, identifying 19 and 25 distinct haplotypes, respectively. Notably, the geographic distribution of haplotypes in domesticated barley exhibited a pronounced East-West divergence, suggesting that they may experience divergent selection during domestication. Collectively, this study offers a comprehensive systematic characterization of the RS gene family in barley, and highlights HvRS2 and HvRS5 as promising candidates for improving abiotic stress tolerance. These findings lay a solid foundation for subsequent functional validation and stress-resilience breeding in barley.