Alfalfa is an important legume forage grown worldwide and its productivity is affected by drought and salt stresses. In this work, three alfalfa germplasms with contrasting tolerances were used for unraveling the transcriptomic responses to drought and salt stresses. Twenty-one different RNA samples were extracted and sequenced using the PacBio and the Illumina platforms to obtain full-length transcriptomic profiles in response to germplasm, stress condition or tissue source (leaf, stem and root). A total of 1,124,275 and 91,378 unique isoforms and genes were obtained, respectively. Comparative analysis identified multiple transcriptional and post-transcriptional regulation mechanism, including alternative splicing events, long non-coding RNAs (lncRNAs), transcription factors, and nonsense-mediated mRNA decay events. This is the first report of a genome-wide profiling of non-coding RNAs in alfalfa. Finally, a weighted gene regulation network was generated allowing to identify master genes including transcription factors and other regulators such lncRNAs in regulation of plant response to drought and salt stresses. This research provides new insight to understand the molecular mechanisms by which drought and high salinity affect alfalfa growth and production and improves and refine the underestimated transcriptional landscape in M. sativa.

Yield is one of the most important agronomic traits in alfalfa breeding; however, this is a complex trait affected by genetic and environmental factors. In this study, we used multi-environment trials to test yield-related traits in a diverse panel composed of 200 alfalfa accessions and varieties. Phenotypic data of maturity (MSC), dry matter, plant height (PH), yield (Yi), and fall dormancy (FD) were collected in three locations: Idaho, Oregon, and Washington from 2018 to 2020. Single-trial and stagewise analyses within each environment were used to obtain estimated means by genotype by trait by environment. Genome-wide association studies identified a total of 84 non-redundant markers associated with the traits analyzed. Of those, 55 markers were located at 44 different loci. Ten significant SNPs at the same locus were associated with FD and they were linked to a gene annotated as a nuclear fusion defective 4-like (NFD4) protein. SNPs linked to transcription factors such as Cysteine3Histidine (C3H), Hap3/NF-YB family, and serine/threonine-protein phosphatase 7 proteins were associated with MSC, PH, and Yi, respectively. Our results provide insight into the genetic factors that influence alfalfa maturity, yield, and dormancy, which is helpful to speed up the genetic gain of alfalfa yield improvement.