The chromosome-level assembly of the wild diploid alfalfa genome provides insights into the full landscape of genomic variations between cultivated and wild alfalfa

Abstract

Alfalfa (Medicago sativa L.) is one of the most important forage legumes in the world, including autotetraploid (M. sativa ssp. sativa) and diploid alfalfa (M. sativa ssp. caerulea, progenitor of autotetraploid alfalfa). Here, we reported a high-quality genome of ZW0012 (diploid alfalfa, 769 Mb, contig N50 = 5.5 Mb), which was grouped into the Northern group in population structure analysis, suggesting that our genome assembly filled a major gap among the members of M. sativa complex. During polyploidization, large phenotypic differences occurred between diploids and tetraploids, and the genetic information underlying its massive phenotypic variations remains largely unexplored. Extensive structural variations (SVs) were identified between ZW0012 and XinJiangDaYe (an autotetraploid alfalfa with released genome). We identified 71 ZW0012-specific PAV genes and 1296 XinJiangDaYe-specific PAV genes, mainly involved in defence response, cell growth, and photosynthesis. We have verified the positive roles of MsNCR1 (a XinJiangDaYe-specific PAV gene) in nodulation using an Agrobacterium rhizobia-mediated transgenic method. We also demonstrated that MsSKIP23_1 and MsFBL23_1 (two XinJiangDaYe-specific PAV genes) regulated leaf size by transient overexpression and virus-induced gene silencing analysis. Our study provides a high-quality reference genome of an important diploid alfalfa germplasm and a valuable resource of variation landscape between diploid and autotetraploid, which will facilitate the functional gene discovery and molecular-based breeding for the cultivars in the future.

Keywords: alfalfa; genome assembly; nodule‐specific cysteine‐rich peptides; population structure; structural variants.

Figure 1

Distribution of genomic features with the alfalfa ZW0012 genome. The circular tracks representation of the (a) gene density, (b) GC content, (c) density of Copia retrotransposons, and (d) density of Gypsy retrotransposons.

Figure 2

Population genetic structure of 64 ssp. caerulea; (a) ADMIXTURE plot of 64 ssp. caerulea accessions shows two subpopulations (K = 2), the information of all ssp. caerulea accessions was list in Table S17. (b) Geographical distribution of 64 ssp. caerulea accessions, which are represented by triangles on the map based on ADMIXTURE groups. The green represents the group north and the blue represents the group south. (c) Phylogenetic tree of 64 ssp. caerulea accessions. (d) 3D diagram of principal component analysis (PCA) of the variation among all 64 ssp. caerulea accessions. X, Y, and Z axis coordinates represent principal component 1, principal component 2 and principal component 3, respectively. (e) LD decay of all, the group north and the group south within 300 kb. (f) Eight genes present in PI464715 but absent in ZW0012. (g) Eight genes present in ZW0012 but absent in PI464715.

Figure 3

Phylogenetic and evolutionary analysis of the ZW0012 genome. (a) Venn diagram of shared orthologous gene families in ZW0012 (ssp. caerulea), XinJiangDaYe (ssp. sativa), and M. truncatula. (b) Distribution of synonymous nucleotide substitution rates (Ks) of orthologous gene pairs of three Medicago species. (c) Phylogenetic tree of 15 species and the evolution of gene families. The numeric value (blue) beside each node shows the estimated divergence time of each node (MYA, million years ago) and the number of expanded (red) and contracted (green) gene families is also marked.

Figure 4

Gene synteny analysis between ZW0012 and M. truncatula (A17), ZW0012 and XinJiangDaYe genomes.

Figure 5

Characterization of selected CNV genes and PAV segments in ZW0012 and XinJiangDaYe. (a) CNV genes displaying low expressed in ZW0012 but highly expressed in XinJiangDaYe. (b) Eight genes present in XinJiangDaYe (left of red lines) but absent in ZW0012 (right of red lines). (c) PAV genes display low expressed or silent in ZW0012 but highly expressed in XinJiangDaYe.

Figure 6

Phenotypic characterization of MsNCR1 and control plants under nodulation conditions. (a) Photographs of the transgenic root and control root. (b) Morphology of nodules. (c) The number of nodules. (d) Nitrogenase activity. Alfalfa plants were inoculated with Sm1021 after 7 days of nitrogen deficiency treatment. Photographs were taken at 28 DPI. The experiment was repeated three times, and one representative set of results is shown. An asterisk indicates a significant difference (**P < 0.01, *P < 0.05).

Figure 7

Phenotypic characterization of two F‐box family genes (MsSKIP23_1 and MsFBL23_1) for leaves development. (a) Photographs of leaves of the transgenic and control plants. (b, c) length and width of leaves in XinJiangDaYe and transgenic XinJiangDaYe plants. (d, e) length and width of leaves in ZW0012 and transgenic ZW0012 plants. The experiment was repeated three times, and one representative set of results is shown. An asterisk indicates a significant difference (***P < 0.001, **P < 0.01, *P < 0.05).