Selection and validation of suitable reference genes for qRT-PCR analysis in pear leaf tissues under distinct training systems

Abstract

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.

Comparative physiological and transcriptomic analysis of pear leaves under distinct training systems

“…The trees in this experimental orchard received fertilizers, irrigation, and chemical thinners in accordance with the local recommendations. Trees have been trained to SP or DP system as described previously 9 . Briefly, the SP system consists of one central stem and 3–5 primary branches that are upright and located at about 0.5–2 m from the ground.…”

“…Two reference genes, i.e. PpSKD1 and PpYLS8 , which proved to be stably expressed in leaves during different growth stages under distinct pear training systems, were used as suitable internal controls to normalize the qRT-PCR data 9 . Relative quantification was calculated based on the Ct method (2 −△△ C T ).…”

“…It is therefore not surprising that plants have evolved several strategies to optimize the photosynthetic machinery to light changes 5 , 6 . In the field of fruit tree cultivation and management, training systems generally determine tree architecture, which represent spatial strategy for light interception and distribution within the canopy, and therefore influence photosynthetic efficiency and productivity 7 – 9 . The net photosynthetic rate ( P N ) is an important parameter that can be used to analyze canopy receptive competence of photosynthetically active radiation (PAR), and help selection what is the optimal canopy architecture through pruning and thinning 10 .…”

“…Training systems exhibit significant spatio-temporal variability due to architectural traits such as canopy position, crop load and canopy sides (sunny/shady side), and therefore lead to photosynthetic variability. A good training system can optimize canopy structure, which could improve microclimate conditions and affect overall canopy photosynthetic productivity 9 , 11 . In most natural canopies, the light availability decreases from the exterior to the interior of the canopy 12 .…”

“…However, the high labor requirements of traditional training systems are driving innovation in orchard systems. Hence, flat-type trellis systems, such as ‘Joint Tree’ and ‘Double Primary Branches Along the Row’, have the advantage of reduced labor and thus been developed for pear cultivation 9 , 22 . These arrangements have also been found to allow trees to receive sunlight uniformly and achieve relatively uniform fruit quality.…”