Freezing stress is a major limiting environmental factor that affects the productivity and distribution of alfalfa (Medicago sativa L.). There is growing evidence that enhancing freezing tolerance through resistance-related genes is one of the most efficient methods for solving this problem, whereas little is known about the complex regulatory mechanism of freezing stress. Herein, we performed transcriptome profiling of the leaves from two genotypes of alfalfa, freezing tolerance “Gannong NO.3” and freezing-sensitive “WL326GZ” exposure to −10°C to investigate which resistance-related genes could improve the freezing tolerance. Our results showed that a total of 121,366 genes were identified, and there were 7,245 differentially expressed genes (DEGs) between the control and treated leaves. In particular, the DEGs in “Gannong NO.3” were mainly enriched in the metabolic pathways and biosynthesis of secondary metabolites, and most of the DEGs in “WL326GZ” were enriched in the metabolic pathways, the biosynthesis of secondary metabolites, and plant-pathogen interactions. Moreover, the weighted gene co-expression network analysis (WGCNA) showed that ATP-binding cassette (ABC) C subfamily genes were strongly impacted by freezing stress, indicating that ABCC8 and ABCC3 are critical to develop the freezing tolerance. Moreover, our data revealed that numerous Ca2+ signal transduction and CBF/DREB1 pathway-related genes were severely impacted by the freezing resistance, which is believed to alleviate the damage caused by freezing stress. Altogether, these findings contribute the comprehensive information to understand the molecular mechanism of alfalfa adaptation to freezing stress and further provide functional candidate genes that can adapt to abiotic stress.

Background Medicago sativa L. ‘Qingshui’ is a valuable rhizomatous forage germplasm resource. We previously crossed Qingshui with the high-yielding Medicago sativa L. ‘WL168’ and obtained novel rhizomatous hybrid strains (RSA-01, RSA-02, and RSA-03). Telomere dynamics are more accurate predictors of survival and mortality than chronological age. Based on telomere analyses, we aimed to identify alfalfa varieties with increased stamina and longevity for the establishment of artificial grazing grasslands. Methods In this study, we performed longitudinal analysis of telomerase activity and relative telomere length in five alfalfa varieties (Qingshui, WL168, RSA-01, RSA-02, and RSA-03) at the age of 1 year and 5 years to examine the relationship among telomerase activity, rate of change in relative telomere length, and longevity. We further aimed to evaluate the longevity of the examined varieties. Telomerase activity and relative telomere length were measured using enzyme-linked immunosorbent assay and real-time polymerase chain reaction, respectively. Results We observed significant differences in telomerase activity between plants aged 1 year and those aged 5 years in all varieties except WL168, and the rate of change in telomerase activity does not differ reliably with age. As telomerase activity and relative telomere length are complex phenomena, further studies examining the molecular mechanisms of telomere-related proteins are needed. Relative telomere lengths of Qingshui, WL168, RSA-01, RSA-02, and RSA-03 in plants aged 5 years were higher than those aged 1 year by 11.41, 11.24, 9.21, 10.23, and 11.41, respectively. Relative telomere length of alfalfa tended to increase with age. Accordingly, alfalfa varieties can be classified according to rate of change in relative telomere length as long-lived (Qingshui, WL168, and RSA-03), medium-lived (RSA-02) and short-lived (RSA-01). The differences in relative telomere length distances of Qingshui, WL168, RSA-01, RSA-02, and RSA-03 between plants aged 1 and 5 years were 10.40, 13.02, 12.22, 11.22, and 13.25, respectively. The largest difference in relative telomere length was found between Qingshui and RSA-02 at 2.20. Our findings demonstrated that relative telomere length in alfalfa is influenced by genetic variation and age, with age exerting a greater effect.

Freezing stress is one of the most detrimental environmental factors that can seriously impact the growth, development, and distribution of alfalfa (Medicago sativa L.). Exogenous salicylic acid (SA) has been revealed as a cost-effective method of improving defense against freezing stress due to its predominant role in biotic and abiotic stress resistance. However, how the molecular mechanisms of SA improve freezing stress resistance in alfalfa is still unclear. Therefore, in this study, we used leaf samples of alfalfa seedlings pretreatment with 200 μM and 0 μM SA, which were exposed to freezing stress (-10°C) for 0, 0.5, 1, and 2h and allowed to recover at normal temperature in a growth chamber for 2 days, after which we detect the changes in the phenotypical, physiological, hormone content, and performed a transcriptome analysis to explain SA influence alfalfa in freezing stress. The results demonstrated that exogenous SA could improve the accumulation of free SA in alfalfa leaves primarily through the phenylalanine ammonia-lyase pathway. Moreover, the results of transcriptome analysis revealed that the mitogen-activated protein kinase (MAPK) signaling pathway-plant play a critical role in SA alleviating freezing stress. In addition, the weighted gene co-expression network analysis (WGCNA) found that MPK3, MPK9, WRKY22 (downstream target gene of MPK3), and TGACG-binding factor 1 (TGA1) are candidate hub genes involved in freezing stress defense, all of which are involved in the SA signaling pathway. Therefore, we conclude that SA could possibly induce MPK3 to regulate WRKY22 to participate in freezing stress to induced gene expression related to SA signaling pathway (NPR1-dependent pathway and NPR1-independent pathway), including the genes of non-expresser of pathogenesis-related gene 1 (NPR1), TGA1, pathogenesis-related 1 (PR1), superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), glutathione-S-transferase (GST), and heat shock protein (HSP). This enhanced the production of antioxidant enzymes such as SOD, POD, and APX, which increases the freezing stress tolerance of alfalfa plants.

Rhizome-rooted Medicago sativa L. “Qingshui” is an excellent germplasm for establishing grazing and ecological grasslands but inferior in yield, in which both high production and ecological values can be achieved by cross-breeding. We have obtained valuable rhizome-rooted hybrid strains (RSA-01, RSA-02, and RSA-03) by crossing of Qingshui and the high-yielding Medicago sativa L. “WL168.” In this study, the Qingshui plants with low production performance were crossed for improvement, and progenies with better production and higher quality than those of Qingshui were selected. The results reveal that the branch number, crude protein (CP) content, and relative feed value (RFV) of RSA-01; the stem thickness, CP content, and ether extract (EE) content of RSA-02; and the plant height, stem thickness, branch number, and dry hay yield of RSA-03 were higher than those of Qingshui. Except for the leaf/stem ratio and plant height of RSA-01, leaf/stem ratio of RSA-02, and plant height of RSA-03, the coefficient of variation (CV) of yield traits of the hybrid strains was lower than those of Qingshui, ranging from 0.1% to 4.28%. In addition to the lignin and acid detergent fiber content of RSA-01 as well as EE content of RSA-02 and RSA-03, the CV of the nutritional traits of the hybrid strains was low, ranging from 0.60% to 3.43%. The tested samples were ranked as follows based on yield performance and nutritional values: WL 168 > RSA − 03 > RSA − 01 > RSA − 02 > Qingshui and RSA − 01 > WL 168 > RSA − 03 > Qingshui > RSA − 02 , respectively. Compared with parental Qingshui, RSA-01, RSA-02, and RSA-03 show better yield performance; meanwhile, RSA-01 and RSA-03 had higher nutritional traits. RSA-01 shows heterosis in branch number, CP content, and RFV; RSA-02 shows heterosis in stem thickness and RSA-03 in plant height, stem thickness, branch number, fresh yield, dry hay yield, and CP content. Notably, the low production performance of Qingshui was improved after crossing it with WL168, substantially resulting in an abundant rhizome-rooted germplasm resource for the establishment of grazing grasslands.

Rhizome-rooted alfalfa (Medicago sativa L.) is an excellent forage for establishing grazing and ecological grasslands, requiring a high and stable yield. Studying the genetic and physiological basis of stable expression of biomass traits is essential for improving production performance in rhizome-rooted alfalfa. We analyzed forage mass and photosynthetic physiological indices of the improved progenies (RSA−01, RSA−02, and RSA−03), parental “Qingshui” (CK1), and “WL168” (CK2) at ages one and five years and their relationships, then revealed heterotic stability. Moreover, we explored the effects of interannual dynamics and genetic differences on tested indices. The results revealed compared with the forage mass of CK1, RSA−03 at ages one and five years increased by 22.17% and 19.72%, respectively, while RSA−01 and RSA−02 varied from 1.40% to 8.65%, indicating obvious heterosis in forage mass of RSA−03. At one year of age, Gs value, Car content and SS content of RSA−03 were higher than those of CK1; SS content of RSA−03 were higher than those of CK2 and RSA−02; Ci, Gs and Tr values of RSA−03 were higher than those of RSA−01. At five years of age, Pn, Gs, and WUE values, and Sta content of RSA−03 were higher than those of CK1; Ci value and Suc content of RSA−03 were higher than those of CK2; Car content and Gs value of RSA−03 were higher than those of other progenies. The forage mass; Chl(a/b) ratio; Pn, Gs, and WUE values; Suc content of RSA−03 at age five years were higher than those at age one year by 9.99%–44.24%. Through path analysis, Gs and NSC were direct factors affecting forage mass at age one year, and both Pn and SP affected forage mass indirectly through Gs; Gs and Chl(a+b) were direct factors affecting forage mass at age five years, and SS affected forage mass indirectly through Gs. Interestingly, Chlb, Chl(a/b), Pn, Tr, Gs, Ci, Suc, SP, and SS were more influenced by age than genetics, while the opposite was true for Car and Sta. Accordingly, RSA−03 showed obvious and stable heterosis in forage mass and photosynthetic physiology, recommending the establishment of grazing pastures and ecological vegetation.