**5. Conclusions**

Improving NUE is urgently needed for the development of sustainable pepper production. However, present knowledge about the molecular basis governing the genetic variation of N-use efficiency (NUE) among pepper cultivars remains unclear. In this study, transcriptome of two pepper genotypes with contrary low-N tolerance (750-1, tolerant; ZCFB, sensitive) were compared using RNA-Seq and various bioinformatics methods after N starvation for 28 days. The results showed that the transcriptomic responses to low-N stress differed considerably between 750-1 and ZCFB, especially in genes that were not directly involved in N metabolism. The unique low-N-responsive genes between the two genotypes provide new insights for a comprehensive understanding of genotypic variation in NUE.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/horticulturae7050110/s1, Figure S1: DEGs involved in nitrogen metabolism. Table S1: Primers for qRT-PCR. Table S2: Relative values of each character. Table S3: DEGs of TCL1vs.TTL3. Table S4: DEGs of TCR2 vs. TTR4. Table S5: DEGs of SCL5 vs. STL7. Table S6: DEGs of SCR vs. STR8.

**Author Contributions:** Data curation, C.W., Y.L. and W.B.; formal analysis, C.W., K.L., R.H., Q.H. and Q.L.; investigation, C.W., S.Z., W.B., X.Y., H.W., Y.L.; methodology, C.W., Q.H. and Q.L.; project administration, R.H., S.Z., Q.H. and Q.L.; resources, S.Z., X.Y., R.H. and Q.H.; software, C.W. and W.B.; writing—original draft, C.W. and Y.L.; writing—review & editing, C.W., Y.L., K.L., R.H., Q.H. and Q.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from the China Agriculture Research System (CARS-24-A-04) and the Chongqing Science and Technology Bureau of China (cstc2019jscx-gksbX0123; cstc2020jscx-msxmX0054).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data supporting the findings of this study are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare no competing interest.

## **References**

