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Pan-genomic analysis highlights genes associated with agronomic traits and enhances genomics-assisted breeding in alfalfa

Nature Genetics volume 57, pages 1262–1273 (2025)Cite this article

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Abstract

Alfalfa (Medicago sativa L.), a globally important forage crop, is valued for its high nutritional quality and nitrogen-fixing capacity. Here, we present a high-quality pan-genome constructed from 24 diverse alfalfa accessions, encompassing a wide range of genetic backgrounds. This comprehensive analysis identified 433,765 structural variations and characterized 54,002 pan-gene families, highlighting the pivotal role of genomic diversity in alfalfa domestication and adaptation. Key structural variations associated with salt tolerance and quality traits were discovered, with functional analysis implicating genes such as MsMAP65 and MsGA3ox1. Notably, overexpression of MsGA3ox1 led to a reduced stem–leaf ratio and enhanced forage quality. The integration of genomic selection and marker-assisted breeding strategies improved genomic estimated breeding values across multiple traits, offering valuable genomic resources for advancing alfalfa breeding. These findings provide insights into the genetic basis of important agronomic traits and establish a solid foundation for future crop improvement.

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Fig. 1: Layout of the alfalfa graph pan-genome study.
Fig. 2: Distribution and diversity of representative alfalfa accessions.
Fig. 3: Detection of SVs and construction of the pan-genome based on the 24 de novo assembled alfalfa genomes.
Fig. 4: Functional impact of SV in alfalfa leaf morphology under salt stress.
Fig. 5: Functional validation of a key gene identified by pan-genomic analysis of SLR phenotype using SNP-GWAS and SV-GWAS.
Fig. 6: GWAS and genomic prediction accuracies using SV and SNP markers across 54 phenotypic traits.

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Data availability

The sequencing raw data have been deposited in the NCBI database under accession code BioProject PRJNA1197171. The haploid reference genome is derived from a previously published study5. The assembled data have been deposited in the NCBI database under the BioProject accession code PRJNA1220045. Additionally, the data are available via Zenodo at https://doi.org/10.5281/zenodo.14118213 (ref. 85) and via Figshare at https://figshare.com/articles/dataset/Alfalfa/28426967 (ref. 86). Resequencing data used in this study were obtained from Zhang’s research, and the relevant data have been provided in his published article45. The RNA sequence data from this study have been deposited in the NCBI database under accession code BioProject PRJNA1083622. The phenotypes used in GWAS and GS studies are available via Zenodo at https://doi.org/10.5281/zenodo.14869063 (ref. 87).

Code availability

All codes associated with this project are available via GitHub at https://github.com/hefei0609-afk/Alfalfa and via Zenodo at https://doi.org/10.5281/zenodo.14800545 (ref. 88).

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Acknowledgements

This work was supported by China Agriculture Research System of MOF and MARA (grant no. CARS-34 to Q.Y.), the Biological Breeding-National Science and Technology Major Project (grant no. 2022ZD04011 to R.L.), the Key Projects in Science and Technology of Inner Mongolia (grant no. 2021ZD0031 to R.L.) and Agricultural Science and Technology Innovation Program of CAAS (grant no. ASTIP-IAS14 to Q.Y.).

Author information

Author notes

  1. These authors contributed equally: Fei He, Shuai Chen, Yangyang Zhang.

Authors and Affiliations

  1. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China

    Fei He, Yangyang Zhang, Lin Chen, Fan Zhang, Mingna Li, Xue Wang, Huigang Lv, Yajing Li, Xianyang Li, Xueqian Jiang, Ming Xu, Bilig Sod, Junmei Kang, Ruicai Long & Qingchuan Yang

  2. National Key Laboratory for Tropical Crop Breeding, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China

    Shuai Chen, Kun Chai, Weilong Kong, Shenyang Qu & Xingtan Zhang

  3. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Agriculture, Guangxi Key Laboratory of Sugarcane Biology, Guangxi University, Nanning, China

    Qing Zhang

  4. School of Grassland Science, Beijing Forestry University, Beijing, China

    Tiejun Zhang, Xiaofan He & Xiao Li

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Contributions

Q.Y., R.L. and X.Z. designed this project and coordinated the research activities. F.Z., J.K., H.L., L.C., Xianyang Li, M.L., X.W., X.J., B.S., M.X. and Y.L. collected and provided plant materials. F.Z., R.L. and X.Z. participated in the genome sequencing and resequencing. S.C., S.Q. and K.C. assembled the genomes. S.C., W.K., Q.Z., K.C. and S.Q. performed the gene annotation. S.C. and F.H. analyzed RNA-seq data. F.H. constructed the sequence and gene-based pan-genome. F.Z., S.C. and F.H. contributed to population GWAS analysis. Y.Z. performed functional verification. X.H., Xiao Li and T.Z. conducted a whole-genome selection analysis. F.H., S.C., X.Z., R.L. and Q.Y. interpreted the data and contributed to the manuscript writing.

Corresponding authors

Correspondence to Xingtan Zhang, Ruicai Long or Qingchuan Yang.

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Extended data

Extended Data Fig. 1 Population structure and Fixation Index of the global alfalfa diversity panel.

a. Population structure of the alfalfa panel was inferred by assuming three subpopulations (K). Each color represents a different subpopulation. b. Word cloud of the primary origin countries for alfalfa varieties in Group1, Group2, and Group3. Font size represents the relative proportion of varieties from each country. Group1 is predominantly from the United States, Group2 from China, and Group3 from Turkey, with contributions from other countries as well. c. The PCA scatter plot shows the distribution of PC1 and PC2, with different colors representing different groups (Group1, Group2, Group3). d. Fixation Index (FST) values among Group1, Group2, and Group3 alfalfa accessions.

Extended Data Fig. 2 The genome structure variations (SVs) between species of alfalfa.

a, Chromosome. b–h, means the distribution of repeat density, gene density, SNP/Indel density, deletions, insertion, duplication and inversion.

Extended Data Fig. 3 Genome-wide association study (GWAS) for monosaccharide content, In Vitro True Dry Matter Degradability at 24 h (IVTDMD24), and In Vitro True Dry Matter Degradability at 30 h (IVTDMD30).

a, c, e, present the Manhattan and QQ plots of the GWAS results for monosaccharide, IVTDMD24, and IVTDMD30, respectively, using structural variation (SV) markers. b, d, f, show the Manhattan and QQ plots for the same traits using single nucleotide polymorphism (SNP) markers. The red dashed line indicates the Bonferroni-corrected genome-wide significance threshold (α = 0.05/n, where 'n' is the total number of independent SNPs and effective SVs). g, i, k, depict scatter plots of the peak structural variations in chromosome 1 for the three traits, with the horizontal line marking the Bonferroni-corrected genome-wide significance threshold. h, j, l, display boxplots of the three traits across different accessions, categorized by the alleles they carry. The sample sizes for the REF and ALT groups are 171 and 5, respectively. In boxplots, the 25% and 75% quartiles are shown as lower and upper edges of boxes, respectively, and central lines denote the median. The whiskers extend to 1.5 times the inter-quartile range. P-values were computed from two-tailed Student’ s t-test.

Extended Data Fig. 4 Impact of MsGA3ox1 overexpression on alfalfa morphology traits.

a, Comparison between WT alfalfa plants and overexpression lines (OE3, OE7, and OE12). b-e, Quantitative measurements of MsGA3ox1 expression levels, plant height, SLR, and biomass. f, Photographs of leaves from WT, OE3, OE7, and OE12 lines at the 3rd, 4th, and 5th stem nodes. g-i, Comparative assessments of leaf area, leaf length, and leaf width between WT and MsGA3ox1 overexpression lines as shown in f. j, Comparison of WT and MsGA3ox1 overexpression lines in the number of trifoliolate leaves. The scale bar represents 5 cm. Asterisks denote statistical significance with ‘*’ ‘**’ and ‘***’ indicating P < 0.05, P < 0.01and P < 0.001, respectively. Data are presented as means ± SEM, with three independent experimental replicates for panel b, six independent experimental replicates for panels c, d, e, and j, and nine independent experimental replicates for panels g, h, and i. The control group (WT) is the Zhongmu No.1 variety of Medicago sativa L.

Extended Data Fig. 5 Phenotypic characterization of alfalfa quality traits in MsGA3ox overexpression lines.

The bar graphs depict a comparative analysis of crude protein (CP) (a), acid detergent fiber (ADF) (b), neutral detergent fiber (NDF) (c), lignin content (d), total digestible nutrients (TDN) (e), and net energy for gain (NEg) (f) between WT and overexpressed lines OE1 and OE3. Asterisks denote levels of statistical significance compared to WT (*P < 0.05, **P < 0.01, ***P < 0.001). Data are presented as means ± SEM, with four biological replicates per group. The control group (WT) is the Zhongmu No.1 variety of Medicago sativa L.

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He, F., Chen, S., Zhang, Y. et al. Pan-genomic analysis highlights genes associated with agronomic traits and enhances genomics-assisted breeding in alfalfa. Nat Genet 57, 1262–1273 (2025). https://doi.org/10.1038/s41588-025-02164-8

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