On January 30, 2024, Wang Zan's team from China Agricultural University published an online article entitled "The chromosome-level assembly of the wild diploid alfalfa genome provides insights into the full landscape of genomic variations" in Plant Biotechnology Journal between cultivated and wild alfalfa". A high-quality genome of diploid alfalfa (Medicago sativa ssp. caerulea, the ancestor species of homotetraploid alfalfa) was assembled, and copy number variation (CNV) and acquisition/ of diploid and tetraploid alfalfa were identified. Deletion variants (PAV) and other structural variations were constructed to construct the structural variation map of the first diploid ancestral species and tetraploid cultivated alfalfa, and the function of PAV gene MsNCR1 in root nodule formation and F-box protein gene (MsSKIP23_1 and MsFBL23_1) in leaf development were verified, which provided important genetic resources for the mining of genes related to important agronomic traits in alfalfa.
Alfalfa is the most important legume grass in the world and is known as the "king of forage". Cultivated alfalfa is homotetraploid, which hinders the genetic study of important agronomic traits in alfalfa due to its high heterozygosity and self-incompatibility. The assembly of the diploid alfalfa genome and the analysis of the structural variation of diploid and tetraploid alfalfa are helpful to the understanding of alfalfa polyploidy, and it is of great significance to explore the key genetic loci and genes related to important agronomic traits and accelerate the molecular breeding of alfalfa.
The researchers used three generations of PacBio, Bionano and HIC technologies combined with state-of-the-art assembly strategies to obtain a complete and continuous high-quality diploid alfalfa genome. The genome size was 769 Mb, the Contig N50 reached 5.5 Mb, the BUSCO assessment result reached 98.5%, the LAI score reached 21.26 points, and the genome quality reached the "gold" reference genome. Using it as a reference genome, 64 diploid alfalfa germplasms were resequenced, and population structure analysis divided the diploid alfalfa germplasm into Southern and Northern groups, which were highly correlated with geographical distribution.
Genome assembly and population genetic structure analysis of diploid alfalfa at the chromosome level
Through the analysis of the structural variation of the diploid and tetraploid alfalfa genomes, 16,292 CNV reduction genes and 10,042 CNV increasing genes were identified in tetraploid alfalfa, which were mainly enriched in biological pathways related to plant development and environmental adaptation, such as plant hormone signal transduction, endocytosis, plant-pathogen interaction, and phenylacetone biosynthesis. A total of 6928 diploid specific genome fragments and 72 diploid PAV genes, as well as 104,985 tetraploid specific genome fragments and 1296 tetraploid PAV genes were identified by PAV analysis. PCR and expression analysis were used to verify the accuracy of genomic and structural variation analysis.
Identification of CNV and PAV genes
Furthermore, Agrobacterium rhizogenes-mediated genetic transformation system was used to silence the expression of MsNCR1 in tetraploid alfalfa roots, which reduced the number of nodules, while the overexpression of MsNCR1 in diploid alfalfa significantly increased the number of nodules, indicating that MsNCR1 is an important regulator of alfalfa nodules. The overexpression of MsSKIP23_1 and MsFBL23_1 in diploid alfalfa resulted in larger leaves, while in tetraploid alfalfa, the leaves became smaller by silent MsSKIP23_1 and MsFBL23_1 expression, which verified the function of MsSKIP23_1 and MsFBL23_1 in regulating leaf development of alfalfa.
Phenotypic identification of MsNCR1 regulating nodule formation
Phenotypic identification of MsSKIP23_1 and MsFBL23_1 regulating leaf development
Shi Kun, a postdoctoral fellow at China Agricultural University, is the first author of the paper, and Professor Wang Zan is the corresponding author. Professor Du Huilong of Hebei University, Dr. Li Yuxian of North China University of Science and Technology, Liang Chengzhi, researcher of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences, Professor Muhammet Şakiroğlu of Türkeş Science and Technology University of Turkey, Dong Hongbin, a master's student at China Agricultural University, and Zhou Yi, a doctoral student, participated in the study. This study was supported by the National Natural Science Foundation of China, the Beijing Municipal Natural Science Foundation, the National Forestry and Grassland Germplasm Bank Project, and the Talent Fund of China Agricultural University.
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