Nighttime Fluorescence Phenotyping Reduces Environmental Variability for Photosynthetic Traits and Enables the Identification of Candidate Loci in Maize

Fangyi Li^1,2,3Marcin Grzybowski^2,3,4Rebecca L Roston^1,3*James C Schnable^2,3*

• ¹Department of Biochemistry, College of Arts and Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States
• ²Department of Agronomy and Horticulture, College of Agricultural Sciences and Natural Resources, University of Nebraska-Lincoln, Lincoln, Nebraska, United States
• ³Center for Plant Science Innovation, University of Nebraska, Lincoln, Illinois, United States
• ⁴Faculty of Biology, University of Warsaw, Warsaw, Masovian, Poland

High-throughput phenotyping is crucial for unraveling the genetic basis of variation in photosynthetic activity. However, the heritability of chlorophyll fluorescence parameters measured during the day is often low as a result of high levels of variation introduced by environmental fluctuations. To address these limitations, we measured fluorescence phenotypes at night, leveraging natural dark adaptation to minimize environmental noise. This significantly increased the heritability of fluorescence traits compared to daytime measurements, with the maximum quantum yield of photosystem II (Fv/Fm) showing an increase in heritability from 0.32 to 0.72. Genome-wide association studies (GWAS) conducted using three photosynthetic fluorescence traits measured at night across two growing seasons identified several significant single nucleotide polymorphisms (SNPs). Notably, two candidate genes near SNPs linked to multiple fluorescence traits, Zm00001eb271820 and Zm00001eb012130, have known roles in photosynthesis regulation. Four of the significant signal nucleotide polymorphisms identified in GWAS conducted using nighttime collected data also exhibited statistically significant associations with the same phenotypes during the day. In a majority of other cases, direction of effect was consistent but greater variance in day measured data relative to night measured data resulted in the differences not being statistically significant. These results highlight the effectiveness of phenotyping photosynthetic traits at night in reducing environmental noise and enhancing the discovery of genomic intervals related to photosynthesis. While nighttime data collection may not be applicable for all photosynthetic traits, it offers a promising avenue for advancing our understanding of the genetic variation of photosynthesis in modern crop species.