Our studies aim to monitor molecular biological markers of lutein biosynthesis by targeted gene expression tests in three sweet maize hybrids, Dessert R78, Messenger and Honey, during the generative phase of the plants. We found that quantitative real-time PCR (qPCR) is an effective tool for measuring gene expression levels and that accurate, reproducible results depend on the correct choice of reference genes used to normalize the data.

The leaf and fruit samples for our studies were collected from the Demonstration Garden of the Böszörményi Road Campus of the University of Debrecen from the beginning of July to the end of July 2021, at five different time points. During sampling, replicates were collected and field frozen in liquid nitrogen to preserve RNA and stored at -80 °C until analysis. Samples were homogenized and RNA isolated under liquid nitrogen, after which RNA quality was assessed by electrophoresis on 1% agarose gel followed by spectrophotometry using A260/A280 and A260/A230 ratios. Based on preliminary studies, we selected four reference genes for our studies, encoding tubulin (TUB), ubiquitin (UBI), actin (ACT) and a thioredoxin-like gene (TLG). Of these four candidate reference genes, three (TUB, UBI and ACT) gave satisfactory results and were selected for further downstream testing. PCR products were confirmed by sequencing and sequence alignment to the corresponding genes. In a second step, known molecular biological lutein biosynthesis markers were verified and validated. Seven target genes and eight pairs of primers (PSY, HYD, CYP97C, PDS, ZDS, LCYB, LCYE) were selected for lutein biosynthesis gene expression tests. These results showed that real-time PCR reactions are efficient in the selection of sweet maize hybrids and are suitable for mass screening of samples.

Keywords: sweet maize, carotenoids, lutein, gene expression, housekeeping gene