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ARS Home » Midwest Area » St. Paul, Minnesota » Plant Science Research » Research » Publications at this Location » Publication #410330

Research Project: Genetic Improvement and Cropping Systems of Alfalfa for Livestock Utilization, Environmental Protection and Soil Health

Location: Plant Science Research

Title: Construction of a SNP-based genetic linkage map in alfalfa (Medicago sativa L.) using three different reference genomes

item KAUR, HARPEET - University Of Minnesota
item Dornbusch, Melinda - Mindy
item SHANNON, LAURA - University Of Minnesota
item Samac, Deborah - Debby

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 12/15/2023
Publication Date: 1/10/2024
Citation: Kaur, H., Dornbusch, M.R., Shannon, L.M., Samac, D.A. 2024. Construction of a SNP-based genetic linkage map in alfalfa (Medicago sativa L.) using three different reference genomes. Tools for Polyploids Workshop. San Diego, California. January 10, 2024.

Interpretive Summary:

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) with genome size of approximately 800 MB. The objective of this study was to compare high-density genetic maps developed using GBS-based SNP markers called using three different reference genomes: 1) the ZhongmuNo.1 monoploid genome assembly, 2) the first homolog of allele-aware XinJiangDaYe genome assembly, and 3) the stable FASTA format of a graph-based pangenome developed using ZhongmuNo.1 as reference with four additional assemblies. A biparental F1 population of 166 plants was made by crossing RegenSY27x and UMN3988 genotype 5. After trimming low quality bases and removing low quality reads from raw GBS data, 1.4 billion single-end 100 bp size reads were used to develop three SNP marker datasets. The percent heterozygosity and missing data in each individual were similar for all three SNP datasets. We called genotypes and imputed missing data using ‘updog’. The final SNP datasets consisted of 10,652, 12,392 and 9,809 SNP markers in 164 individuals for the three reference genomes, respectively, which were used to produce three linkage maps. To reduce the inflation in map distance, gaps between markers, and increase the marker density mappoly was run again after removing SNPs with more than a 10 cM gap between them. The final phased linkage map with four haplotypes consisted of 2,482, 2,635, and 2,618 SNP markers spanning 1743.66, 2576.59, and 2701.13 cM in 8 linkage groups for ZhongmuNo.1, XinJiangDaYe, and the graph-based pangenome, respectively. The number and order of SNP markers in pangenome, with only 277 additional SNPs included from four other genomes, has significantly increased the linkage map length and average gap size, in comparison with monoploid ZhongmuNo.1 reference genome. However, the proportion of SNP markers mapped to the total number of markers used for linkage mapping was improved in pangenome (0.27) as compared to the ZhongmuNo.1 (0.23) and XinJiangDaYe (0.21) assemblies. 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 in a population. These results increase the available molecular marker resources and will be used for scaffolding the reference genome sequences for U.S. alfalfa germplasm.