Early flowering promotes early maturity, production, and the capacity to counteract biotic and abiotic stresses, making it an important agronomic trait in zucchini.  The present study demonstrated that the zucchini inbred line ‘19’ consistently flowered early, taking significantly fewer days to bloom the first female flower (DFF) than the inbred line ‘113’.  Genetic analysis revealed that DFF, an inheritable quantitative trait, is controlled by multiple genes.  Based on the strategy of quantitative trait locus (QTL) sequencing (QTL-seq) combined with linkage analysis, three QTLs for DFF were identified on chromosomes 4, 11, and 20.  This study used additional F₂ populations grown under different environmental conditions for QTL mapping analysis of DFF with insertion/deletion (InDel) markers to validate these results.  Using the composite interval mapping (CIM) method of R/qtl software, we only identified one major locus under all environmental conditions, located in a 117-kb candidate region on chromosome 20.  Based on gene annotation, gene sequence alignment, and qRT-PCR analysis, we found that the Cp4.1LG20g08050 gene encoding a RING finger protein may be a candidate gene for the opposite regulation of early flowering in zucchini.  In summary, these results lay a foundation for a better understanding of early flowering and improving early flowering-based breeding strategies in zucchini.

The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide (MDP)-mediated inflammatory response of bovine rumen epithelial cells (BRECs).  First, changes in the mRNA expression of pro-inflammatory factor genes in BRECs following 10 μg mL^–1 MDP treatments were examined.  RT-qPCR results showed that the expression of pro-inflammatory factor (IL-1β, IL-6, and TNF-α) mRNAs were significantly increased under MDP stimulation (P<0.001).  Moreover, SLC15A4-Knockout (SLC15A4-KO) cells were obtained through lentivirus packaging, transfection, screening, and cell monoclonal culture.  In order to gain further insight into the potential function of SLC15A4, we utilized transcriptome data, which revealed a change in the genes between WT-BRECs and SLC15A4-KO.  Five down-regulated pro-inflammatory genes and 13 down-regulated chemokine genes related to the inflammatory response were identified.  Meanwhile, the down-regulated genes were mostly enriched in the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways.  The results of RT-qPCR also verified these detected changes.  To further determine the mechanism of how WT and SLC15A4-KO BRECs are involved in inflammatory responses, we investigated the inflammatory responses of cells exposed to MDP.  WT-BRECs and SLC15A4-KO were treated with a culture medium containing 10 μg mL^–1 MDP, in comparison to a control without MDP.  Our results show that SLC15A4-KO BRECs had reduced the expression of genes (IL-6, TNF-α, CXCL2, CXCL3, CXCL9, and CCL2) and proteins (p-p65 and p-p44/42) from the MDP-mediated inflammatory response compared to WT-BRECs (P<0.05).  In this experiment, CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4, and its role was confirmed via the MDP-induced inflammatory response in BRECs.  This work will provide a theoretical basis for studying the pro-inflammatory mechanism of MDP and its application in the prevention and treatment of subacute rumen acidosis in dairy cows.