AUTHOR=He Shengnan , An Tai , A Runa , Liu Shouan 

TITLE=Validation of Reliable Reference Genes for RT-qPCR Studies of Target Gene Expression in Colletotrichum camelliae During Spore Germination and Mycelial Growth and Interaction With Host Plants

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.02055

DOI=10.3389/fmicb.2019.02055

ISSN=1664-302X

ABSTRACT=<p>The tea plant [<italic>Camellia sinensis</italic> (L.) O. Kuntze] is one of the most important leaf crops, and it is widely used for the production of non-alcoholic beverages worldwide. Tea also has a long history of medicinal use. <italic>Colletotrichum camelliae</italic> Massee is one of the dominant fungal pathogens that infects tea leaves and causes severe tea anthracnose disease. To analyze the molecular biology of <italic>C. camelliae</italic>, the quantification of pathogen gene expression by the RT-qPCR method is necessary. Reliable RT-qPCR results require the use of stable reference genes for data normalization. However, suitable reference genes have not been reported in <italic>C. camelliae</italic> thus far. In this study, 12 candidate genes (i.e., <italic>CcSPAC6B12.04c</italic>, <italic>CcWDR83</italic>, <italic>Cchp11</italic>, <italic>Ccnew1</italic>, <italic>CcHplo</italic>, <italic>CcRNF5</italic>, <italic>CcHpcob</italic>, <italic>CcfaeB-2</italic>, <italic>CcYER010C</italic>, <italic>CcRNM1</italic>, <italic>CcUP18</italic>, and <italic>CcACT</italic>) were isolated from <italic>C. camelliae</italic> and assessed as potential reference genes. The expression stability of these genes in <italic>C. camelliae</italic> during spore germination and mycelial growth and interaction with host plants was first evaluated using several statistical algorithms, such as geNorm, NormFinder, and Bestkeeper. A web-based analysis program, Refinder, was then used to find the most suitable reference genes. Our results indicated that <italic>Cenew1</italic>, <italic>CcHplo</italic>, and <italic>CcSPAC6B12.04c</italic> were the most stable reference genes in <italic>C. camelliae</italic> under all conditions. Our work provided the most suitable reference genes for future studies performed to quantify the target gene expression levels of <italic>C. camelliae.</italic></p>