Constructing the alfalfa pangenome: Insights into structural variation and genomic complexity in an autopolyploid crop

Authors: KAUR, HARPREET - University Of Minnesota; SHANNON, LAURA - University Of Minnesota; Samac, Deborah

Submitted to: Plant and Animal Genome Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 12/15/2024
Publication Date: 1/10/2025
Citation: Kaur, H., Shannon, L.M., Samac, D.A. 2025. Constructing the alfalfa pangenome: Insights into structural variation and genomic complexity in an autopolyploid crop. Plant and Animal Genome Conference Proceedings. Plant and Animal Genome Conference, January 10-15, 2025, San Diego, California.

Interpretive Summary:

Technical Abstract: Pangenomes reveal how different individuals within a species vary at the genomic scale, which cannot be represented fully in a single reference genome. Building a comprehensive pangenome for alfalfa, a polyploid, highly heterozygous, and outcrossing species, is particularly challenging due to its complex autotetraploid genome (2n'='4x'='32; ~800 Mbp). For this study, we used three publicly available chromosome-scale assemblies for Chinese alfalfa cultivars and one scaffold-scale genome assembly from U.S. germplasm, which represent only part of the species' genetic diversity. Both linear and graph-based methods were employed to analyze the alfalfa pangenome, integrating multiple assemblies using the ZhongmuNo.1 genome as a reference. Comparative analysis with MUMmer and Assemblytics revealed substantial structural variation (SV) among these assemblies, with repeat expansions comprising 63% of the identified SVs. According to TopGO annotation results, 50.1% of SNPs occur outside genic regions, while only 33.0% of SVs are located outside of these regions. Interestingly, 13.61% of SVs were classified as having a high impact on genes, compared to just 0.10% of SNPs. On average, there was one SNP per 17 bp and one SV per 19 Kbp region, with SVs showing a more pronounced impact on gene expression and protein function than SNPs and INDELs. In graph-based assemblies, incorporating additional genomes increased the assembly size by 1.5 to 3 times compared to the ZhongmuNo.1 reference genome. To illustrate the intricacies captured by pangenome graphs for a complex crop genome, we selected five distinct gene sequences and aligned them with three different graph-based pangenomes. Comparing these three graph-based methods highlighted notable differences in the genomic variation each pipeline captures. For a more comprehensive view of the alfalfa pangenome, it’s essential to analyze a wider range of germplasm that encompasses the full spectrum of genetic diversity.