Screening of optimal reference genes for qRT-PCR and preliminary exploration of cold resistance mechanisms in Prunus mume and Prunus sibirica varieties.

Prunus sibirica and Prunus mume are closely related plant species that differ in cold tolerance. Hybrids of P. sibirica and true mume, belonging to the apricot mei group, inherited strong cold resistance from P. sibirica. These materials are favourable for research on the molecular mechanisms of cold resistance. However, no suitable reference genes have been identified for analysing gene expression patterns between P. sibirica and P. mume. Ten candidate reference genes were assessed, namely, actins (ACT2-1, ACT2-2, ACT2-3, ACT2-4), protein phosphatase 2A-1 (PP2A-1), ubiquitins (UBQ2, UBQ3), ubiquitin extension protein (UBQ1) and tubulins (TUB1, TUB2), with four distinct algorithms (geNorm, NormFinder, BestKeeper and RefFinder). UBQ2 was recognized as the best reference gene in stems and buds across materials (P. sibirica; 'Xiaohong Zhusha', 'Beijing Yudie', and 'Xiao Lve' for true mume; and 'Dan Fenghou', 'Fenghou', and 'Yanxing' for apricot mei) under cold stress. In addition, the temporal and spatial expression patterns of PmCBF6 and PmLEA10 among seven varieties during winter periods were analysed using UBQ2 as a reference gene. The expression differed significantly among cultivars, which may contribute to their differences in cold tolerance. This paper confirmed the strong cold tolerance of apricot mei. And the best internal reference gene suitable for seven varieties was selected: UBQ2. Based on the above results, the expression of PmCBF6 and PmLEA10 genes during wintering in seven varieties was analysed. The molecular mechanisms of cold resistance were found to be possibly different in different varieties of P. sibirica and P. mume.