# Genomic resources for Australian alfalfa (Medicago sativa L.) genomics: reformatted reference genome, annotated variants, gene presence-absence and diversity analysis from genome re-sequencing

## TL;DR

Researchers improved genomic resources for alfalfa, a key forage crop, by reformating its genome and analyzing genetic diversity and gene presence-absence variation.

## Contribution

The study provides a reformatted allele-aware reference genome and population-level gene presence-absence analysis for alfalfa breeding.

## Key findings

- Little genetic differentiation exists between alfalfa cultivars, with higher diversity within cultivars.
- Gene presence-absence variation was identified at the population level in alfalfa.
- RNA sequencing expanded the predicted gene set, enhancing genomic resources for alfalfa improvement.

## Abstract

Alfalfa (Medicago sativa L.), a globally significant forage crop, has faced limited breeding progress in recent decades. Several challenges hinder genetic gain in alfalfa, including its status as an outbreeding tetraploid species with pronounced inbreeding depression, high parent numbers in synthetic crosses resulting in limited genetic differentiation between cultivars and a lack of genomic resources to advance genomic breeding techniques in the species.

We aim to address some of these limitations by generating genomic resources for alfalfa improvement, including reformatting an allele-aware reference genome to remove duplicate haplotypes while retaining presence absence variation, genome annotation identifying genes and functional elements, SNP discovery and SNP variant effect prediction. The predicted gene set was expanded by the inclusion of RNA sequencing from multiple tissue types and stress treatments. Genetic diversity of 316 samples from seven commercially available cultivars relevant to Australian grazing systems was examined, including a population level analysis of gene presence-absence variation. There is little genetic differentiation between cultivars, with higher diversity within than between cultivars. Several genes were found to display presence-absence at the population level.

These findings provide insights for alfalfa breeding programs and underscore the need for continued efforts in developing genomic tools to unlock the crop’s full potential.

The online version contains supplementary material available at 10.1186/s12870-025-07941-5.