Molecular approaches in the analysis of red clover rhizobium symbiosis

Authors: Dinkins, Randy; Koch, Lisa; Sullivan, Michael; ZHU, HONGYAN - University Of Kentucky

Submitted to: International Grasslands Congress
Publication Type: Proceedings
Publication Acceptance Date: 3/3/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary: Red clover (Trifolium pratense L.) is an important forage and pasture legume grown throughout temperate regions and is a major contributor of biological fixed nitrogen because of its ability to fix atmospheric nitrogen. Red clover has not been a model legume primarily due to self-incompatibility and the associated high heterozygosity despite its relatively small genome. Thus, it has not been a significant contributor in molecular or genetic studies and basic information on red clover legume/rhizobium symbiosis is lacking. Recent advances red clover genome assemblies outlined in this presentation, has allowed for significant progress in the identification of genes, chromosomal regions and pathways involved in characterizing red clover genotype diversity and persistence, mapping of 2,4-D tolerance, isoflavone production, identification of red clover genotypes differing in their nitrogen fixation potential, nutritional quality, as well as genes that are regulated during rhizobium symbiosis further discussed in the current presentation. As the genomic reference sequences are further refined and the annotations clarified, the use of these genomic resources will allow for the characterization of the genes involved in rhizobium, interaction and genes and pathways affecting persistence, forage yield and quality, all of which will facilitate genetic improvement and red clover cultivar development.

Technical Abstract: Red clover (Trifolium pratense L.) is an important forage and pasture legume grown throughout temperate regions. It is a major contributor of biological fixed nitrogen because of its ability to fix atmospheric nitrogen. Due to the high cost of nitrogen fertilizers pasture legumes have been increasingly important in forage production settings. Red clover has not been a model legume primarily due to self-incompatibility and the associated high heterozygosity, therefore it has not been a significant contributor in molecular or genetic studies and basic information on red clover legume/rhizobium symbiosis is lacking. Using recent annotated genomic resources, RNA-seq expression analysis and CRISPR/Cas9 mutagenesis, we characterized a number of genes that are expressed only in nodule forming roots. These include genes that encode proteins with homology to nodule-specific cysteine rich proteins (NCRs) and nodule-specific Polycystin-1, Lipoxygenase, Alpha toxic (PLAT) domain proteins (NPDs) that are postulated to be involved in plant rhizobium interactions. Our results indicate that red clover has one of the highest numbers of expressed NCR, and ATS3-like/NPD, peptides currently known in the Inverted-Repeat Lacking Clade (IRLC) of legumes. The knowledge of the expression of these genes and the continued analysis of the genetic variation in red clover should aid in breeding of genotypes with increased rhizobium selection specificity and increased nitrogen fixation efficiency.