Low freezing tolerance reduces the persistence of red clover under northern climate. The incidence of winter damages in perennial crops could increase in the future due to the adverse effects of the predicted warmer fall temperature on plant cold acclimation. To accelerate breeding progress, two cultivars of red clover Christie (C-TF0) and Endure (E-TF0) were exposed to a recurrent selection protocol for freezing tolerance performed indoor. New populations were obtained after five (C-TF5 and E-TF5), six (C-TF6 and E-TF6), and seven (C-TF7 and E-TF7) cycles of recurrent selection. These populations were overwintered under natural conditions and monitored for freezing tolerance and cold-induced molecular traits. Freezing tolerance was improved by up to 6 °C in recurrently selected populations when compared to initial cultivars confirming that further progress are achieved with advanced cycles of selection. Monthly analysis of biochemical changes shows that higher starch concentrations at the onset of the fall hardening period are contributing to the acquisition of superior freezing tolerance through its impact on sucrose accumulation. They also contribute to the vigor of spring regrowth by sustaining more pinitol and proline synthesis. Larger concentrations of these metabolites in populations with higher levels of freezing tolerance (TF7) hint at their involvement in winter survival of red clover. Among genes differentially expressed in response to both cold acclimation and recurrent selection, a concomitant cold induction of APPR9 and cold repression of 1-aminocyclopropane-carboxylate synthase suggests a link between the repression of a pathway regulated by ethylene and the improvement of freezing tolerance in red clover.

中文翻译：

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反复选择红三叶草种群越冬期间的生化和分子反应以提高抗冻性

低耐寒性降低了红三叶草在北方气候下的持久性。由于预测的较暖秋季温度对植物冷驯化的不利影响，未来多年生作物冬季损害的发生率可能会增加。为了加快育种进程，红三叶草 Christie (C-TF0) 和 Endure (E-TF0) 的两个栽培品种接受了室内进行的抗冻性循环选择方案。在五次（C-TF5 和 E-TF5）、六次（C-TF6 和 E-TF6）和七次（C-TF7 和 E-TF7）循环选择后获得新种群。这些种群在自然条件下越冬，并监测耐寒性和冷诱导的分子特征。与初始栽培品种相比，反复选择的种群的抗冻性提高了 6°C，这证实了通过先进的选择周期取得了进一步的进展。生化变化的月度分析表明，秋季硬化期开始时较高的淀粉浓度通过其对蔗糖积累的影响有助于获得优异的抗冻性。它们还通过维持更多的松醇和脯氨酸合成来促进春季再生的活力。在具有较高抗冻性 (TF7) 水平的人群中，这些代谢物的浓度越高，暗示它们与红三叶草的冬季存活有关。在响应冷驯化和循环选择而差异表达的基因中，