Selection and Validation of Reference Genes Desirable for Gene Expression Analysis by qRT-PCR on Seed Germination of Castanea henryi

Seed germination is the beginning of the plants life cycle, and seed biology is one of the most extensively researched areas in plant physiology, however, Castanea henryi as an important seed plant, the stable internal reference gene during germination is not clear. In this study, seven candidate genes (TUA, TUB, TIF, UBC, RPL21, RPL30, RPL34) were screened out from transcriptome data, we analyzed the expression of seven candidate reference genes in C. henryi at different germination stages with RT-qPCR, and using common algorithms including NormFinder, geNorm and BestKeeper to evaluate the candidate genes stability. The results showed that RPL34 and RPL30 were selected as the most stable genes by NormFinder; TIF was the most stable gene identified by BestKeeper; RPL34 and RPL21 were the most stable genes ranked by geNorm, and TUB was the most unstable gene identified by all of the three software. The RPL34 gene was used as the reference gene, to detected the expression trend of two starch synthetase genes SS1 and SS2 during germination by RT-qPCR, the results of RT-qPCR and transcriptome sequencing were basically consistent, which verified the stability of RPL34 candidate gene. Our result is not only showed functional genes for germination of C. henryi seeds and provide useful guidelines for the selection of reliable reference genes for the normalization of RT- qPCR data for germination of seed plants.