Ziyao Zhao

Neural interfaces play an important role in vital sign monitoring [1] , clinical practice [2] , and neuroscience frontier research [3] using animal subjects, such as rodents and nonhuman primates [4] , [5] . Recently, neuroscientists have shown increasing interest in behavioral analysis of social situations with a single or multiple freely moving animals, which has been listed as a key investment by the NIH in 2023 [6] . Among those popular laboratory animals, marmosets are increasingly favored by researchers because of their small size, short sexual maturity, complex social patterns, and similarity to human beings [7] , [8] . A number of marmoset social behaviors, such as gaze following [9] , antiphonal calling [10] , and scent marking [11] , have been studied with neural signal acquisition and/or stimulation to affect the behavior of the freely moving marmoset at the same time.

Key message Two round-leaf mutants, rl-1 and rl-2, were identified from EMS-induced mutagenesis. High throughput sequencing and map-based cloning suggested CsPID encoding a Ser/Thr protein kinase as the most possible candidate for rl-1. Rl-2 was allelic to Rl-1. Abstract Leaf shape is an important plant architecture trait that is affected by plant hormones, especially auxin. In Arabidopsis, PINOID (PID), a regulator for the auxin polar transporter PIN (PIN-FORMED) affects leaf shape formation, but this function of PID in crop plants has not been well studied. From an EMS mutagenesis population, we identified two round-leaf (rl) mutants, C356 and C949. Segregation analysis suggested that both mutations were controlled by single recessive genes, rl-1 and rl-2, respectively. With map-based cloning, we show that CsPID as the candidate gene of rl-1; a non-synonymous SNP in the second exon of CsPID resulted in an amino acid substitution and the round leaf phenotype. As compared in the wild type plant, CsPID had significantly lower expression in the root, leaf and female flowers in C356, which may result in the less developed roots, round leaves and abnormal female flowers, respectively in the rl-1 mutant. Among the three copies of PID genes, CsPID, CsPID2 and CSPID2L (CsPID2-like) in the cucumber genome, CsPID was the only one with significantly differential expression in adult leaves between WT and C356 suggesting CsPID plays a main role in leaf shape formation. The rl-2 mutation in C949 was also cloned, which was due to another SNP in a nearby location of rl-1 in the same CsPID gene. The two round leaf mutants and the work presented herein provide a good foundation for understanding the molecular mechanisms of CsPID in cucumber leaf development.