Summary Melilotus species are used as green manure and rotation crops worldwide and contain abundant pharmacologically active coumarins. However, there is a paucity of information on its genome and coumarin production and function. Here, we reported a chromosome‐scale assembly of Melilotus albus genome with 1.04 Gb in eight chromosomes, containing 71.42% repetitive elements. Long terminal repeat retrotransposon bursts coincided with declining of population sizes during the Quaternary glaciation. Resequencing of 94 accessions enabled insights into genetic diversity, population structure, and introgression. Melilotus officinalis had relatively larger genetic diversity than that of M. albus. The introgression existed between M. officinalis group and M. albus group, and gene flows was from M. albus to M. officinalis. Selection sweep analysis identified candidate genes associated with flower colour and coumarin biosynthesis. Combining genomics, BSA, transcriptomics, metabolomics, and biochemistry, we identified a β‐glucosidase (BGLU) gene cluster contributing to coumarin biosynthesis. MaBGLU1 function was verified by overexpression in M. albus, heterologous expression in Escherichia coli, and substrate feeding, revealing its role in scopoletin (coumarin derivative) production and showing that nonsynonymous variation drives BGLU enzyme activity divergence in Melilotus. Our work will accelerate the understanding of biologically active coumarins and their biosynthetic pathways, and contribute to genomics‐enabled Melilotus breeding.

From the viewpoint of a seeds business, classification of locations into homogeneous groups based on genotype × environment interaction (GE) facilitates selection of testing sites and proper placement hybrids or cultivars. Our principal objective was to develop methods to cluster ICI Seeds' strip‐test locations into homogeneous groups with respect to GE. Difficulties encountered in these cluster analyses included missing values in the distance matrix used for cluster analysis (caused by unbalanced data), selection of a proper distance measure, and graphical presentation of clusters. We propose use of a modified distance measure to resolve the first two difficulties. Dendograms are used to present clusters, and bar charts are used to show relative effects of individual hybrids or cultivars within clusters. Classification of counties in Iowa is into four groups: northern, central, southeastern, and southwestern regions. The results of the GE analyses compare favorably with breeder and agronomist observations on hybrid adaptation and show some similar groupings with divisions of the state specified in the Iowa State University yield test report.

Melilotus is an important genus of legumes with industrial and medicinal value, partly due to the production of coumarin. To explore the genetic diversity and population structure of Melilotus, 40 accessions were analyzed using long terminal repeat (LTR) retrotransposon-based markers. A total of 585,894,349 bp of LTR retrotransposon sequences, accounting for 55.28% of the Melilotus genome, were identified using bioinformatics tools. A total of 181,040 LTR retrotransposons were identified and classified as Gypsy, Copia, or another type. A total of 350 pairs of primers were designed for assessing polymorphisms in 15 Melilotus albus accessions. Overall, 47 polymorphic primer pairs were screened for their availability and transferability in 18 Melilotus species. All the primer pairs were transferable, and 292 alleles were detected at 47 LTR retrotransposon loci. The average polymorphism information content (PIC) value was 0.66, which indicated that these markers were highly informative. Based on unweighted pair group method with arithmetic mean (UPGMA) dendrogram cluster analysis, the 18 Melilotus species were classified into three clusters. This study provides important data for future breeding programs and for implementing genetic improvements in the Melilotus genus.