@Article{phyton.2023.026752,
AUTHOR = {Shoukai Lin, Shichang Xu, Liyan Huang, Fuxiang Qiu, Yihong Zheng, Qionghao Liu, Shiwei Ma, Bisha Wu, Jincheng Wu},
TITLE = {Selection and Validation of Reference Genes for Normalization of RT-qPCR Analysis in Developing or Abiotic-Stressed Tissues of Loquat (<i>Eriobotrya japonica</i>)},
JOURNAL = {Phyton-International Journal of Experimental Botany},
VOLUME = {92},
YEAR = {2023},
NUMBER = {4},
PAGES = {1185--1201},
URL = {http://www.techscience.com/phyton/v92n4/51265},
ISSN = {1851-5657},
ABSTRACT = {Loquat (<i>Eriobotrya japonica</i> Lindl.) is a subtropical evergreen fruit tree that produces fruits with abundant nutrients and medicinal components. Confirming suitable reference genes for a set of loquat samples before qRT-PCR experiments is essential for the accurate quantification of gene expression. In this study, eight candidate reference genes were selected from our previously published RNA-seq data, and primers for each candidate reference gene were designed and evaluated. The Cq values of the candidate reference genes were calculated by RT-qPCR in 31 different loquat samples, including 12 subgroups of developing or abiotic-stressed tissues. Different combinations of stable reference genes were screened according to a comprehensive rank, which was synthesized from the results of four algorithms, including the geNorm, NormFinder, BestKeeper and ΔCt methods. The screened reference genes were verified by normalizing <i>EjLGA1</i> in each subgroup. The obtained suitable combinations of reference genes for accurate normalization were <i>GAPDH</i>, <i>EF1α</i> and <i>ACT</i> for floral development; <i>GAPDH</i>, <i>UBCE</i> and <i>ACT</i> for fruit setting; <i>EF1α</i>, <i>GAPDH</i> and <i>eIF2B</i> for fruit ripening; <i>ACT</i>, <i>EF1α</i> and <i>UBCE</i> for leaves under heat stress; <i>eIF2B, UBCE</i> and <i>EF1α</i> for leaves under freezing stress; <i>EF1α</i>, <i>TUA</i> and <i>UBCE</i> for leaves under salt stress; <i>ACT</i>, <i>EF1α</i> and <i>eIF2B</i> for immature pulp under freezing stress; <i>ACT</i>, <i>UBCE</i> and <i>eIF2B</i> for immature seeds under freezing stress; <i>EF1α</i>, <i>eIF2B</i> and <i>UBCE</i> for both immature pulp and seeds under freezing stress; <i>UBCE</i>, <i>TUB</i> and <i>TUA</i> for red-fleshed fruits under cold-storage stress; <i>eIF2B</i>, <i>RPS3</i> and <i>TUB</i> for white-fleshed fruits under cold-storage stress; and <i>eIF2B</i>, <i>UBCE</i> and <i>RPS3</i> for both red- and white-fleshed fruits under cold-storage stress. This study obtained different combinations of stable reference genes for accurate normalization in twelve subgroups of developing or abiotic-stressed tissues in loquat. To our knowledge, this is the first report to obtain stable reference genes for normalizing gene expression of abiotic-stressed tissues in <i>E. japonica</i>. The use of the three most stable reference genes could increase the reliability of future quantification experiments.},
DOI = {10.32604/phyton.2023.026752}
}