Background Medicago ruthenica, a superior leguminous forage, is strongly drought-resistant and has wide adaptability. It is closely related to Medicago sativa (a commonly cultivated forage). Characterized by its high tolerance to environmental stress, M. ruthenica is a valuable genetic resource. However, its low yield limits its large-scale promotion and utilization. Leaf morphology, an important agronomic trait, is closely related to forage yield and photosynthetic efficiency. Results In this study, leaf width and leaf area were found significantly positively correlated with forage yield in M. ruthenica. Specifically, the cultivar M. ruthenica ‘Mengnong No.1’ (MN No.1) with a larger leaf area had higher photosynthetic efficiency. Furthermore, the key miRNA-target gene networks associated with high photosynthetic efficiency of M. ruthenica were identified using small RNAs and transcriptome sequencing. Totally, 63 differentially expressed miRNAs (DEMs) were identified, including some known miRNAs (miR408, miR171, and miR398, etc.). These known miRNAs corresponded to 55 target genes; 6 miRNA-target gene pairs had negatively correlated expression patterns. Degradome sequencing analysis further confirmed the authenticity of miR408-targeted cleavage of LAC5; namely, the miR408-LAC5 module may mediate the mechanism of high photosynthetic efficiency formation in M. ruthenica. Conclusions This study for the first time proposed that leaf morphology as a key trait affecting the high-yielding plant type of M. ruthenica. The key gene modules screened in this study may provide new insights into the elucidation of photosynthetic efficiency mechanism and the bio-design breeding of legume forages in the future.

Medicago ruthenica L. could be used as the crossbreeding material in forage crops to provide rich resistance gene resources based on its strong resistance to adversity stress. This study aimed to identify the authenticity and analyze heterosis of the intraspecific hybrids acquired from two Medicago ruthenica L. materials. The results showed 85 true hybrids in 118 F1 progenies identified by one SSR marker and five SRAP markers. Besides, the SRAP markers (13.89%) indicated higher identification efficiency than SSR markers (7.69%), and the rate of true hybrids in HZ population (100%) was higher than that in ZH population (36.54%). The six agronomic traits varied to different degrees, and their variation coefficients ranged from 18.53% to 45.72% in 13 hybrids of ZH population in 2019 and 2020. Moreover, ZH7 and ZH8 presented excellent agronomic performance, which could be used as candidate materials for further research. The mid-parent heterosis (Hm) of all agronomic traits was between -20.55% and 36.46%, and heterobeltiosis (Hh) showed negative values of 13 hybrids.