Maize Genomics and Genetics 2025, Vol.16, No.5, 251-257 http://cropscipublisher.com/index.php/mgg 255 regulatory factor. Such results were achieved through high-density marker analysis combined with functional genomics techniques, and the accuracy was obviously much higher than that of traditional QTL mapping (Figure 2) (Zhang et al., 2019). Figure 2 Down-regulation of nac7 in maize increased gain yield in two-year field trials (Adopted from Zhang et al., 2019) Image caption: (a) nac7 RNAi plants driven by an Ubi promoter showed stay-green phenotype in a yield trial under normal nitrogen condition. Pictures were taken postanthesis. (b) Stay-green score of two hybrid lines expressing nac7 RNAi in Iowa and Tennessee with 2 plots per location. Scores ranged from 1 to 9 with ‘9’ being a fully green canopy and ‘1’ being completely senesced with no green. Stay-green score was analysed by linear unbiased prediction model. Significance between transgenic events and null comparator was determined at the P < 0.1 level shown with *. (c) nac7 was down-regulated in three nac7 RNAi events under field conditions as measured by qPCR (n = 8). Transcript level of nac7 relative to the endogenous reference eIF4g, a maize eukaryotic translation initiation factor, was calculated by the ∆Ct method. Significant difference between each transgenic event and its null was determined using the t-test, *P < 0.001. (d) Yield tests in two years demonstrated that down-regulation of nac7 increased yield in three hybrid lines. Yield was analysed by linear unbiased prediction model and shown as the best linear unbiased predictions (BLUPs). Summary table shows the yield and yield difference between nulls and the transgenic events in megagram/hectare (Mg/ha) at multilocations under normal nitrogen and well-watered conditions. Statistical significance was determined at the P < 0.05 level shown with * (Adopted from Zhang et al., 2019) 6.2 Multi-location trials of NILs for stable QTL expression analysis Molecular verification in the laboratory alone is not enough; the true value can only be seen in the fields. In multi-site and multi-year field trials, the research team selected parents with nac7 RNAi backgrounds to hybridize with some superior inbred lines, and the test species were evaluated in multiple environments. The results were quite consistent: the green leaves were retained for a longer time, and the yield also increased, with an average increase of 0.29 tons per hectare. Converted, this is approximately an increase of 4.6 bushels per acre. The significance of such multi-point experiments lies in that they not only verify the stability of QTL but also directly reflect the breeding potential of this trait (Zhang et al., 2019). 6.3 Functional verification and biological significance of candidate genes such as Stay-Green1 and SAG12 When it comes to the functions of candidate genes, some have already provided initial clues. Like nac7, research shows that it may regulate a whole set of genes involved in photosynthesis, chlorophyll degradation and protein renewal, and these processes are also closely related to the green-retaining trait. As for genes like Stay-Green1 and SAG12, although there is currently a lack of direct evidence in corn, they have been proven to be related to aging
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