Abstract
Climate change and environmental degradation are threatening agriculture. Recently, novel plant shoot and root architectures have been proposed to help improve stress resilience, for example by facilitating water or nutrients absorption. However, knowledge about genes, developmental programs and gene expression regulatory circuits is largely missing. In this project (RADICALS), we aim to identify genes and gene-expression regulatory modules affecting root and shoot branching in barley and cultivated cereals. Public engagement and outreach activities are planned in order to disseminate scientific knowledge. RADICALS will contribute to our understanding of molecular mechanisms underlying tolerance to stresses. Summary of results for RADICALS PRIN2022 The RADICALS project aimed to elucidate the genetic and molecular mechanisms controlling root system architecture and shoot branching in barley, using this species as both a model and a target crop for improving resilience and sustainability in cereal production. The project combined forward genetics, genome-wide association studies (GWAS), mapping-by-sequencing, reverse genetics, transcriptomics, comparative genomics and bioinformatics to identify genes regulating lateral root development, root gravitropism and tillering. Despite the limited effects observed for nitrogen treatments and the challenges associated with phenotyping root circumnutation, the project successfully achieved its major objectives by identifying novel loci and candidate genes, generating valuable phenotypic and genomic resources, and establishing experimental pipelines for future functional validation.
Results achieved
by UNIBO (Research Unit 1) UNIBO coordinated the large-scale phenotypic characterization of barley root architecture using the TILLMore mutant population and the WHEALBI diversity panel. Approximately 600 mutant lines were screened under controlled conditions, leading to the identification of mutants with altered lateral root development and gravitropic responses. In particular, mutant TM1354 displayed reduced lateral root number and a strong hypergravitropic phenotype, while TM213 was identified as a mutant affected in early lateral root development and subsequently demonstrated to be a new allele of PIN1A. Screening of the WHEALBI collection identified accessions with contrasting lateral root phenotypes and root growth behaviour, including WB-075 and WB-363. UNIBO performed mapping-by-sequencing and genetic analyses that led to the identification of the novel Egt3 locus on chromosome 6H controlling root gravitropism and lateral root development. Integration of independent mutant datasets further narrowed the interval to a single high-confidence candidate gene (HORVU.MOREX.r3.6HG0630640), providing strong evidence for the molecular basis of the phenotype. Genome-wide association analyses on the WHEALBI panel identified 12 QTLs for lateral root density and 16 QTLs for lateral root length, together with several promising candidate genes controlling root architecture. Comparative genomics analyses across barley, wheat, rice and maize further strengthened candidate gene prioritization. In parallel, CRISPR/Cas9 vectors targeting the Egt3 candidate gene were developed to support future functional validation.Results achieved
by UNIMI (Research Unit 2) UNIMI focused on the molecular characterization of genes regulating tillering and the coordination of root and shoot development through the strigolactone pathway. A major achievement was the functional analysis of the HvLBO gene, encoding a key enzyme in strigolactone biosynthesis. Gene-edited, Cas9-free barley lines carrying independent hvlbo mutations demonstrated prolonged tillering, increased tiller number and altered responses to nitrogen availability. Treatment with synthetic strigolactones restored the wild-type phenotype, confirming the central role of HvLBO in regulating shoot branching. In addition, mutant analyses demonstrated that HvLBO also contributes to seminal root development, revealing an important role for strigolactones in coordinating above- and below-ground plant architecture. These results culminated in a high-impact publication in the Journal of Experimental Botany. UNIMI also investigated the genetic basis of the high-tillering mutant HT5478 by combining exome sequencing, bulk-segregant analysis and whole-genome sequencing. Although the trait proved genetically complex and no single causal gene could be unequivocally identified, several genomic regions and candidate genes involved in plant architecture were prioritized. Furthermore, UNIMI generated comprehensive RNA-seq datasets from root and shoot tissues of hvlbo and d14 mutants, establishing a valuable resource for reconstructing transcriptional networks controlling root and shoot branching. Parallel bioinformatic analyses produced comparative orthogroup databases and novel computational approaches for identifying conserved regulatory modules across cereal species, laying the foundation for future evo-devo analyses of plant architecture. Overall, the RADICALS project substantially advanced our understanding of the genetic regulation of cereal root architecture and shoot branching. The integration of forward genetics, genomic mapping, transcriptomics and comparative genomics resulted in the identification of novel loci, candidate genes and regulatory pathways controlling traits directly relevant to crop adaptation and resource-use efficiency. The generated resources provide an important basis for future functional studies and breeding programmes aimed at developing more resilient and sustainable cereal cultivars. Main products and outputs of the project • Identification and genetic mapping of the novel Egt3 locus controlling lateral root gravitropism. • Identification of PIN1A as the causal gene underlying the TM213 lateral-root mutant. • Identification of 12 QTLs for lateral root density and 16 QTLs for lateral root length in the WHEALBI diversity panel. • Functional characterization of HvLBO as a regulator of strigolactone-mediated control of tillering and root development. • Generation of Cas9-free HvLBO gene-edited barley lines. • Development of CRISPR/Cas9 constructs for functional validation of the Egt3 candidate gene. • Generation of extensive phenotypic, genomic and transcriptomic datasets for root and shoot architecture. • Development of comparative genomics and bioinformatics resources for identifying conserved regulatory networks across cereals. • Scientific publications, including the Journal of Experimental Botany article on HvLBO function and the GWAS study identifying major QTLs controlling barley root traits. • Dissemination through presentations at major international conferences (ISRR, PAG, BGN, SIGA), public engagement initiatives and training of MSc and BSc students.Project details
Unibo Team Leader: Silvio Salvi
Unibo involved Department/s:
Dipartimento di Scienze e Tecnologie Agro-Alimentari
Coordinator:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Total Eu Contribution: Euro (EUR) 198.678,00
Total Unibo Contribution: Euro (EUR) 107.253,00
Project Duration in months: 24
Start Date:
12/10/2023
End Date:
28/02/2026
