Calcium-dependent protein kinases (CDPKs), play multiple roles in plant development, growth and response to bio- or abiotic stresses. Calmodulin-like domains typically contain four EF-hand motifs for Ca²⁺ binding. The CDPK gene family can be divided into four subgroups in Arabidopsis, and it has been identified in many plants, such as rice, tomato, but has not been investigated in alfalfa (Medicago sativa subsp. sativa) yet. In our study, 38 non-redundant MsCDPK genes were identified from the “XinJiangDaYe” alfalfa genome. They can be divided into four subgroups which is the same as in Arabidopsis and Medicago truncatula, and there were 15, 12,10 and 1 in CDPK I, II, III and IV, respectively. RNA-seq analysis revealed tissue-specificity of 38 MsCDPK genes. After researching the transcriptome data, we found these 38 MsCDPK members responsive to drought, salt, and cold stress treatments. Further analysis showed that the expression of almost all the MsCDPKs is regulated by abiotic stresses. In addition, we chose MsCDPK03, MsCDPK26, MsCDPK31 and MsCDPK36 for RT-qPCR validation which was from CDPK I-IV subgroups respectively. The result showed that the expression of these four genes was significantly induced by drought, salt and cold treatments. The subcellular location experiment showed that these four proteins were all located in nucleus. In our study, we identified 38 distinct MsCDPK genes within the alfalfa genome, which were classified into four groups. We conducted a comprehensive analysis of various gene features, including physicochemical properties, phylogenetic relationships, exon-intron structures, conserved motifs, chromosomal locations, gene duplication events, cis-regulatory elements, 3D structures, and tissue-specific expression patterns, as well as responses to drought, salt, and cold stresses. These results also provide a solid foundation for further investigations into the functions of MsCDPKs aimed at improving drought tolerance in autotetraploid cultivated alfalfa through genetic engineering.

Germplasm resources within the Medicago genus are highly regarded for their value as forage crops and their critical roles in nitrogen cycling, ecosystem restoration, and soil structure improvement. Therefore, understanding the diversity of seed morphology in this genus is essential for advancing its development and utilization. This study analyzed seed samples from 587 germplasm accessions representing 77 species within Medicago genus, as well as 32 accessions from 21 species within the closely related genus Trigonella. A statistical analysis was conducted on twelve traits, including seven quantitative traits-straight length (SL), straight width (SW), width-to-length ratio (WL), perimeter (PE), radicle length (RL), hilum length (HL), and 100-seed weight (SY)-and five qualitative traits, including seed coat condition, radicle characteristics, seed size, shape, and color. The results revealed that: (1) there was significant diversity (P < 0.05) in SL, SW, WL, PE, RL, HL, and SY across Medicago species; (2) principal component analysis of the 587 Medicago accessions identified SL, SW, PE, HL, RL, and SY as the primary contributors to morphological diversity; and (3) high-resolution images of seeds from various accessions were captured for future research. This study provides a solid foundation for the establishment of seed banks and the enhancement of germplasm resources through the systematic analysis of these morphological traits.

The GRAS gene family encodes a group of plant-specific transcription factors essential for regulating plant growth, development and stress responses. While the GRAS gene family has been extensively studied in various plant species, a comprehensive characterization of the GRAS gene family in Medicago ruthenica has not yet been conducted. In this study, a total of 62 MrGRAS gene family members were identified through a comprehensive whole-genome analysis of M. ruthenica, and phylogenetic analysis categorized these 62 genes into 13 distinct groups. Gene structure and conserved domain analysis showed that MrGRAS genes from the same evolutionary branch share similar exon–intron architecture and conserved motifs. A large number of hormone-responsive, growth and development and stress-responsive cis-regulatory elements were detected in the upstream sequences of MrGRAS genes. RT-qPCR analysis showed that drought stress significantly induced the expression of nine selected MrGRAS genes. Overall, this study analyzed the phylogenetic relationships, conserved domains, cis-regulatory elements and expression patterns of the GRAS gene family in M. ruthenica, filling the gap in the identification of the MrGRAS gene family and laying the foundation for functional analysis of the MrGRAS gene family.