Haplotype-resolved genome of autotetraploid alfalfa (Medicago sativa) Regen-SY27x uncovers large scale structural variation and resistance gene dynamics

Authors: KAUR, HARPREET - University Of Minnesota; CONNOR, CAMERON - National Center For Genome Resources; GOMEZ, ADAM - National Center For Genome Resources; MUDGE, JOANN - National Center For Genome Resources; FARMER, ANDREW - National Center For Genome Resources; SHANNON, LAURA - University Of Minnesota; Samac, Deborah

Submitted to: bioRxiv
Publication Type: Pre-print Publication
Publication Acceptance Date: 5/11/2026
Publication Date: 5/11/2026
Citation: Kaur, H., Connor, C., Gomez, A., Mudge, J., Farmer, A., Shannon, L.M., Samac, D.A. 2026. Haplotype-resolved genome of autotetraploid alfalfa (Medicago sativa) Regen-SY27x uncovers large scale structural variation and resistance gene dynamics. bioRxiv. https://doi.org/10.64898/2026.05.01.722254.
DOI: https://doi.org/10.64898/2026.05.01.722254

Interpretive Summary: Alfalfa is a cornerstone feed resource for dairy and beef herds and an integral resource in diverse cropping systems, offering improved yields, reducing erosion, and building soil health and fertility. However, progress in breeding alfalfa has stagnated due to the lack of modern plant breeding tools such as a reference genome sequence, a high quality and continuous representation of the genetic material. Advanced DNA sequencing techniques were used to decipher a complete reference genome for for Regen-SY27x, an alfalfa plant that is widely used in molecular biology research. The assembled genome consists of 3.2 billion base pairs of DNA with highly contiguous sequences near the length of each chromosome, separating the four copies of each chromosome. A total of 226,191 genes were identified, of which 178,985 have an assigned function. The Regen-SY27x genome assembly sets a new benchmark in gene content and annotation quality with a total gene count that exceeds all previously published alfalfa genome assemblies and achieves the highest completeness to date, establishing it as the most comprehensive and high-quality alfalfa genome currently available. The Regen-SY27x reference genome will provide a stable foundation for analysis of DNA variation in alfalfa cultivars, accelerate alfalfa improvement, and will serve as a model for assembly of other complex genomes.

Technical Abstract: Alfalfa is a cornerstone feed resource for dairy and beef herds and an integral resource in diverse cropping systems, offering improved yields, reducing erosion, and building soil health and fertility. However, progress in breeding alfalfa has stagnated due to the lack of modern plant breeding tools such as a reference genome sequence, a high quality and continuous representation of the genetic material. Advanced DNA sequencing techniques were used to decipher a complete reference genome for for Regen-SY27x, an alfalfa plant that is widely used in molecular biology research. The assembled genome consists of 3.2 billion base pairs of DNA with highly contiguous sequences near the length of each chromosome, separating the four copies of each chromosome. A total of 226,191 genes were identified, of which 178,985 have an assigned function. The Regen-SY27x genome assembly sets a new benchmark in gene content and annotation quality with a total gene count that exceeds all previously published alfalfa genome assemblies and achieves the highest completeness to date, establishing it as the most comprehensive and high-quality alfalfa genome currently available. The Regen-SY27x reference genome will provide a stable foundation for analysis of DNA variation in alfalfa cultivars, accelerate alfalfa improvement, and will serve as a model for assembly of other complex genomes.