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Screening for quantitative real-time PCR reference genes with high stable expression using the mRNA-sequencing data for pear

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Abstract

Housekeeping genes were considered to have a constant expression without spatio-temporal difference. They are commonly used as the internal reference for PCR-based gene expression analysis. However, increasing evidence revealed that the expression of housekeeping genes also varied with tissues and treatments. Previous studies for reference gene identification were usually limited to a small number of housekeeping genes based on their expression stability comparison. In this study, reference genes for pear fruit were initially screened on the transcriptome level with 10 messenger RNA (mRNA)-seq datasets from the fruit of two pear species across important developmental stages. Ten potential reference genes were chosen for validation using the mRNA-seq data from fruit skin libraries. Five potential reference genes are proposed that can be used as suitable controls for PCR-based expression studies in pear fruit. Four of the selected genes had homologous annotation of important roles in the basic biological processes. Sulfhydryl oxidase 2 (SOX2) and protein phosphatase (PP) 2A also showed high stable expression in both pear fruit and other tissues and should be more widely applicable as reference genes in different tissues or species. This study provided a comprehensive view for transcriptional analysis in pear fruit, which will help researchers to select reference genes for normalization against target genes by qRT-PCR across different developmental stages of the fruit as well as for different tissues and species.

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Data archiving statement

Pear gene sequences and annotation information presented in this report are available in the pear genome database (Pear Genome Project, http://peargenome.njau.edu.cn/) and NCBI database (https://www.ncbi.nlm.nih.gov/). The apple genes are available in Malus all species | GDR (https://www.rosaceae.org/species/malus/all).

Funding

This research was partially supported by the Special Funds for China Agriculture Research System (CARS-28-03); the Key Project for New Agricultural Cultivar Breeding in Zhejiang Province, China (2016C02052); and Zhejiang Provincial Natural Science Foundation of China (LY15C150003).

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Authors and Affiliations

  1. Institute of Horticulture, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province, China

    Yuezhi Wang, Meisong Dai, Danying Cai & Zebin Shi

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  1. Yuezhi Wang
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  2. Meisong Dai
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  3. Danying Cai
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  4. Zebin Shi
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Correspondence to Zebin Shi.

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Communicated by D. Chagné

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Supplemental Figure S1

Quality detection of different total RNA samples by agarose gel electrophoresis. CK1, water control. CK2, total RNA control. S1~S9, different total RNA samples for the qRT-PCR analysis. (PDF 30 kb)

Supplemental Figure S2

The quality testing results of part of total RNA samples by Agilent 2100 bioanalyzer. (PDF 358 kb)

Supplemental Figure S3

Ct values of the six candidate reference genes tested in the eight leaf samples of different pear genotypes. (A) Ct values of the six candidate reference genes with three replicates. (B) The mean Ct values of the six candidate reference genes in all pear samples. The bars indicate the maximum and minimum Ct values. (PDF 52 kb)

Supplemental Table S1

Analysis of the expression stability of Actin genes using the RNA-seq data in ten different samples of pear fruit. (XLSX 12 kb)

Supplemental Table S2

Expression stability of the candidate reference genes in leaf tissues of the eight pear genotypes calculated by BestKeeper and coefficient of variation. (DOCX 14 kb)

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Wang, Y., Dai, M., Cai, D. et al. Screening for quantitative real-time PCR reference genes with high stable expression using the mRNA-sequencing data for pear. Tree Genetics & Genomes 15, 54 (2019). https://doi.org/10.1007/s11295-019-1361-6

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  • DOI: https://doi.org/10.1007/s11295-019-1361-6

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