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Plant invertases comprise a family of enzymes that catalyze the irreversible hydrolysis of sucrose into glucose and fructose, which plays a crucial role in carbohydrate partitioning and regulating plant growth and development. However, currently, there is little information on the roles of invertases in the development of pear trees. In this study, we performed genome-wide identification of a total of 23 invertases from the predicted genome data of the pear plant (Pyrus bretschneideri R.). Based on phylogenetic analysis, the pear invertases could be clustered into 13 acid invertase genes and 10 alkaline/neutral invertase genes. Chromosome location showed that tandem duplication and segmental duplication have played an important role in the expansion of acid and neutral/alkaline invertases in pear plants. Evolutionary analysis of invertases from pear and two model plant species (Arabidopsis thaliana and Oryza sativa) indicated that the evolutionary divergence of invertases occurred before the common ancestor of dicots and monocots. Tissue-specific expression analysis showed that the transcripts of all the identified invertase genes were detectable in various tissues. We hypothesize that the identified invertase genes all participate in regulating pear growth and development.

Training systems generally alter tree architecture, which modulates light microclimate within the canopy, for the purpose of improving photosynthetic efficiency and fruit quality. Gene expression quantification is one of the most important methods for exploring the molecular mechanisms underlying the influence of training systems on pear photosynthesis, and suitable reference genes for gene expression normalization are a prerequisite for this method. In this study, the expression stability of nine common and four novel candidate genes were evaluated in 14 different pear leaf samples in two training systems, including those at four developmental stages (training_period) and from different parts of the trees (training_space), using two distinct algorithms, geNorm and NormFinder. Our results revealed that SKD1 (Suppressor of K+ Transport Growth Defect1)/ YLS8 (Yellow Leaf Specific 8) and ARM (Armadillo) were the most stable single reference genes for the 'training_period' and 'training_space' subsets, respectively, although these single genes were not as stable as the optimal pairs of reference genes, SKD1+YLS8 and ARM+YLS8, respectively. Furthermore, the expression levels of the PpsAPX (Ascorbate peroxidase) gene showed that the arbitrary use of reference genes without previous testing could lead to misinterpretation of data. This work constitutes the first systematic analysis regarding the selection of superior reference genes in training system studies, facilitating the elucidation of gene function in pear and providing valuable information for similar studies in other higher plants.