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Background  Fall dormancy and freezing tolerance characterized as two important phenotypic traits, have great effects on productivity and persistence of alfalfa ( Medicago sativa  L.). Despite the fact that one of the most limiting traits for alfalfa freezing tolerance in winter is fall dormancy, the interplay between fall dormancy and cold acclimation processes of alfalfa remains largely unknown. We compared the plant regrowth, winter survival, raffinose and amino acids accumulation, and genome-wide differentially expressed genes of fall-dormant cultivar with non-dormant cultivar under cold acclimation.    Results  Averaged over both years, the non-dormant alfalfa exhibited largely rapid regrowth compared with fall dormant alfalfa after last cutting in autumn, but the winter survival rate of fall dormant alfalfa was about 34-fold higher than that of non-dormant alfalfa. The accumulation of raffinose and amino acids were significantly increased in fall dormant alfalfa, whereas were decreased in non-dormant alfalfa under cold acclimation. Expressions of candidate genes encoding raffinose biosynthesis genes were highly up-regulated in fall dormant alfalfa, but down-regulated in non-dormant alfalfa under cold acclimation. In fall dormant alfalfa, there was a significantly down-regulated expression of candidate genes encoding the glutamine synthase, which is indirectly involved in the proline metabolism. A total of eight significantly differentially expressed transcription factors (TFs) related to CBF and ABRE-BFs were identified. The most up-regulated TFs in fall dormant alfalfa cultivar were ABF4 and DREB1C.    Conclusions  Fall dormant alfalfa drastically increased raffinose and amino acids accumulation under cold acclimation. Raffinose-associated and amino acid-associated genes involved in metabolic pathways were more highly expressed in fall dormant alfalfa than non-dormant alfalfa under cold acclimation. This global survey of transcriptome profiles provides new insights into the interplay between fall dormancy and cold acclimation in alfalfa.    Electronic supplementary material  The online version of this article (10.1186/s12870-019-1773-3) contains supplementary material, which is available to authorized users.

How fall dormancy benefits alfalfa winter-survival? Physiologic and transcriptomic analyses of dormancy process