Gene expression patterns were widely used to better analyze gene expression levels and understand their biological functions. Recognized as an effective tool for performing accurate and rapid quantification of target gene expression, qRT-PCR was commonly performed in bioresearch. Generally, traditional housekeeping genes were wildly used to standardize the transcriptional accumulations of target genes such as TUB, ACT, UBQ, EF1-α and GAPDH. Nevertheless, the common reference genes are not consistently stably expressed and thus can’t apply to all species [11, 12]. Based on previous studies, reference genes for fruit development are varying among different genera and cultivars, such as TEF2, UBQ10 and RP II in peach , EF1-α, CKL and WD40 in apple , BPS1 and ICDH1 in pear , RPT6A and RPN5A in strawberry . Therefore, the selection of optimal reference genes for data normalization was critical in qRT-PCR assays. In this study, we identified the appropriate reference genes for fruit development and ripening in three pear cultivars of Cuiguan, Hosui and Xueqing.
The transcriptome sequencing analysis was a high-throughput sequencing technology, which provide unbiased test transcripts and increased test specificity [22, 36, 37]. Therefore, the transcriptome sequencing data provides a new resource for screening reference genes at the genome level. The candidate reference genes had been identified via transcriptome data in diverse plant species, such as Oryza sativa , Fagopyrum esculentum , Brassica napus , and Euscaphis konishii . In this study, we selected 10 relatively stable candidate genes for reference genes based on transcriptome data of three pear cultivars in four different developmental stages (Table 1).
The studies on pear, which is identified as the third largest temperate fruit tree, are obtaining progresses with the release of the pear (Pyrus bretschneideri) genome datasets . In previous studies, several traditional or novel reference genes were identified and evaluated under various biotic or abiotic stresses and at each stage of developmental in different tissues of diverse pear cultivars. Ubiquitous housekeeping genes may exhibit inconsistent stability in different conditions [24, 42, 43]. For instance, EF1α and TUB-b2 were the most stable in different pear cultivars, GAPDH and EF1α were the most suitable in different tissues, while TUB-b2 and GAPDH were the most stable in developmental stage . In addition, it has been reported that different groups of pear tissues have their own best reference genes for different experimental purposes. In pollen, the PbrGAPDH, PbrPP2A and PbrUBI were suitable reference genes for low temperature, NaCl treatment and CuCl2 treatment, respectively. And PbrEF1α was stable reference genes for all abiotic stresses . In leaf, SKD1and ARM were the most apropos single reference genes for leave of the ‘training_period’ and ‘training_space’, respectively . In pear peel, ACT6/7/8/9 and NAP1 were recommended as the optimal reference genes . Reference genes in pear fruit were studied relatively more, PbPDI.F1 presented the highest expression stability during pear fruit developmental , the housekeeping gene EF1α members showed an obviously unstable expression in pear fruits at different developmental stages , SOX2 and PP2A showed most highly and stably expression in pear fruit , and BPS1 and ICDH1 were the high stable expressed genes . In this study, we identified stably and novel reference genes in the fruits of three different pear varieties. Therefore, reliable and accurate reference genes were urgently needed to been identified according to different experimental requirements.
We used three analysis algorithms (geNorm, NormFinder and BestKeeper) to evaluate the expression stability of 10 candidate genes in different stages samples of 10 Cuiguan, 13 Hosui and 13 Xueqing, respectively. Although different statistical algorithms and analytical procedures may lead to divergent stability ranking, most results were consistent according to these three algorithms. Finally, an online tool RefFinder was used to comprehensively integrate all the ranking (Table 2). The result showed that Pbr028511, Pbr038418 and Pbr041114 were the most stable reference genes in Cuiguan, Housui and Xueqing fruit, respectively. In additiong, each of them possesses more stable expression than PbrTUB in pear fruits during all the stages of fruit development and ripening.