The optimal reference genes for RT-qPCR in various tissues of Cyperus rotundus L.

Background:  Purple nutsedge (Cyperus rotundus L.), a perennial C4 plant from the Cyperaceae family, is one of the most invasive weed species worldwide and significantly affects crop yields.

Objective:  To identify and validate stable reference genes (RGs) for reliable gene expression normalization in RT-qPCR analyses across various tissues of purple nutsedge.

Methods:  The expression patterns of 11 candidate RGs were evaluated across three tissue types, including tubers at three developmental stages forming tubers (FT), swelling tubers (ST), and mature tubers (MT), along with buds and leaves. Expression stability was evaluated using three widely accepted algorithms: geNorm, NormFinder, and BestKeeper. A consensus ranking (RK) was then generated using the RankAggreg package to identify the most stable RGs overall.

Results:  The most stable RGs varied across tissues. CrEF1α was optimal for FT, CrActin for ST, and CrADF7 for MT. For bud and leaf tissues, CrRPL11 and CrCYC showed the highest stability, respectively.

Conclusions:  This study demonstrates that the stability of RGs is tissue-dependent in purple nutsedge. The identified RGs provide a reliable basis for normalizing RT-qPCR data, enabling precise gene expression analyses in specific tissues such as tubers, buds, and leaves.

Keywords:
Purple nutsedge; geNorm; NormFinder; BestKeeper; Gene expression