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Research Project: Expanding Resiliency and Utility of Alfalfa in Agroecosystems

Location: Plant Science Research

Title: Impact of increased population size and three reference genomes on the development of linkage maps in alfalfa

Author
item KAUR, HARPREET - University Of Minnesota
item Dornbusch, Melinda
item SHANNON, LAURA - University Of Minnesota
item Samac, Deborah

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/4/2026
Publication Date: 9/6/2026
Citation: Kaur, H., Dornbusch, M.R., Shannon, L.M., Samac, D.A. 2026. Impact of increased population size and three reference genomes on the development of linkage maps in alfalfa. Crop Science. 66(5). Article e70361. https://doi.org/10.1002/csc2.70361.
DOI: https://doi.org/10.1002/csc2.70361

Interpretive Summary: Modern tools for improving crop plants require accurate complete DNA sequences and inexpensive DNA markers systems for identifying and tracking key agronomic traits such as for yield and disease resistance. Such tools have been difficult to develop for crops with complex genetics such as alfalfa that has four sets of chromosomes. Creating gene maps of each chromosome with DNA markers regularly spaced along each chromosome is a key step for assembling whole genome sequences for alfalfa. In this study, methods for building genetic maps for alfalfa were developed using a high density of markers and different reference genome sequences. The maps differed by the reference sequence, demonstrating the high amount of variability present in different alfalfa varieties. Maps built on the larger number of reference sequences had a higher number of DNA markers, improving the accuracy of genetic analyses. These markers will be used for assembling the reference genome sequences for U.S. alfalfa, leading to more accurate and representative genome assemblies, identification of gene regions associated with important agronomic traits, and breeding of more productive alfalfa for U.S. farmers.

Technical Abstract: Alfalfa (Medicago sativa L.) is an important perennial forage legume grown worldwide. It is an outcrossing, highly heterozygous autotetraploid species (2n=4x=32). The objective of this study was to compare high-density genetic maps developed using genotyping-by-sequencing-based SNP markers called using three different reference genomes: the ZhongmuNo.1 monoploid genome assembly (Map 1), the first homolog of the allele-aware XinJiangDaYe genome assembly (Map 2), and the stable FASTA format of a graph-based pangenome developed using ZhongmuNo.1 as reference with four additional assemblies (Map 3). The mapping population consisted of 165 F1 individuals and all final maps were resolved into four haplotypes with eight linkage groups. Map 1 was built based on 10,652 markers, 2482 of which made it into the final 1743.66 cM map. Map 2 used 12,392 markers, 2635 of which are included in the final 2635 cM map. Map 3 was built based on 9809 markers, 2618 of which appear in the final 2701.13 cM map. The four additional reference genomes used for Map 3 as compared to Map 1 only resulted in the addition of 277 markers but significantly increased both the linkage map length and the average gap size. However, the proportion of SNP markers mapped as compared to the total number of markers used for linkage mapping was higher in Map 3 (0.27) than Map1 (0.23) or Map 2 (0.21). These results demonstrated that high within-species genomic variability is present in alfalfa and SNP calling using reference genomes of different cultivars can produce different genetic maps for the same population.