Table 2. The differential expression of miRNA genes in alfalfa in response to cold and/or freezing stresses.

miRNA	Fold Change		Statistical Significance
	Cold	Freezing	Cold	Freezing
miR156c-3p	−0.62	−1.02	**	**
miR156i-5p	−0.78	−1.43	**	**
miR159a	−1.85	−1.15	**	**
miR159b	0.00	9.80		**
miR160e	0.11	1.50		**
miR166e-5p	−0.58	−1.64		**
miR166f	1.52	2.81	**	**
miR166g-5p	−0.75	−1.97	**	**
miR167a	−1.11	−1.53	**	**
miR167b-5p	−0.94	−1.99	**	**
miR168c-3p	−0.19	−1.04		**
miR171a	−0.99	1.43		**
miR172a	−1.77	−2.30	**	**
miR172c-3p	−1.21	−1.82	**	**
miR172d-3p	−1.31	−0.69	**	**
miR2119	−0.90	2.08		**
miR396a-5p	−1.74	−2.31	**	**
miR396b-3p	−0.71	−1.16	*	**
miR398a-5p	−2.28	0.17	**
miR5037c	2.01	1.63	**	*
miR5231	−0.93	−1.94	**	**
miR5232	0.10	−1.01		*
miR5234	−0.63	−1.37	*	**
miR5239	−0.85	−1.58	**	**
miR5287b	−0.30	−1.83		**
NmiR0018	−1.01	−0.28	**	*
NmiR0019	−0.62	−3.60	**	**
NmiR0026	−0.69	−1.37	**	**
NmiR0028	−0.84	−1.07	**	**
NmiR0029	−0.98	−1.85	**	**
NmiR0043	−0.51	−1.92		**
NmiR0047	−0.45	−2.18	*	**
NmiR0049	−1.79	−2.58	**	**
NmiR0051	−2.04	−2.21	**	**
NmiR0053	−0.52	−2.20		*

The miRNA abundance was evaluated and normalized using the TPM method, and the expression values of miRNAs whose expression was zero were adjusted to 0.01. The fold changes were computed from cold [log₂(Cold/Control)] and freezing [log₂(Freezing/Control)] based expression. Meanwhile, chi-squared tests were also performed to test the significance of each comparison, using the R software. A p-value no more than 0.01 was set as the extremely significant level (**), while a p-value greater than 0.01 and no more than 0.05 was defined as significant (*).