*3.2. RNA-Seq Analysis of Two Pepper Cultivars with Contrasting Low-N Tolerances*

To better understand the molecular mechanism governing the genetic variation of N-use efficiency (NUE) in pepper cultivars, the global gene expression profile of two pepper genotypes (750-1 and ZCFB) with contrasting low-N tolerance was next analyzed using RNA-Seq. Samples were taken 28 days after low-N treatment, and the following samples were used for sequencing: TCL1 (750-1-leaf control), TCR2 (750-1-root control), TTL3 (750-1-leaf low-N treatment), TTR4 (750-1-root low-N treatment), SCL5 (ZCFB-leaf control), SCR6 (ZCFB-root control), STL7 (ZCFB-leaf low-N treatment), and STR8 (ZCFBroot low-N treatment). Sequence data are available from the NCBI SRA database with accession number PRJNA700470. Over 65 million clean reads were generated from each library. A total of 91.75–93.55% of these clean reads could be well aligned to the pepper reference genome, at least 69% could be uniquely mapped to the reference genome (71.88% on average). The number of genes with at least one mapped transcript were 29,167, 29,817, 28,970, 29,981, 29,330, 29,814, 29,078, and 30,335 for libraries TCL1, TCR2, TTL3, TTR4, SCL5, SCR6, STL7, and STR8, respectively (Table 3).


**Table 3.** Summary of genome mapping.

*3.3. Identification of DEGs and Validation of RNA-Seq by qRT-PCR*

To identify low-N-responsive genes, comparisons were made between the low-N treated samples and controls (Figure 2; Tables S3–S6). We found that 548/2073 differentially expressed genes (DEGs) (TCL1 vs. TTL3) were up/down-regulated in leaves, while 1385/1085 DEGs (TCR2 vs. TTR4) were up/down-regulated in the roots of 750-1. Additionally, 1239/2697 DEGs (TCL5 vs. TTL7) were up/down-regulated in leaves, and 2956/1262 DEGs (TCR6 vs. TTR8) were up/down-regulated in the roots of ZCFB.

**Figure 2.** Volcano plot of DEGs. The X-axis represents the value of the difference multiple after log2 conversion, and the Y-axis indicates the significance value after log10 conversation. The three colors red, blue, and gray, represent up-regulated DEGs, down-regulated DEGs, and unchanged genes, respectively.

To validate our RNA-Seq data, the expression levels of eight genes (*LOC107841845*, *LOC107855963*, *LOC107858815*, *LOC107867964*, *LOC107870519*, *LOC107875600*, *LOC107875602*, and *LOC107875603*) in the leaves and roots of 750-1 and ZCFB were next confirmed by qRT-PCR. The expression trends of the eight genes were consistent with our results from RNA-Seq in the eight libraries (Figure 3).

**Figure 3.** RNA-Seq results confirmed by qRT-PCR. A total of eight genes (*LOC107841845*, *LOC107855963*, *LOC107858815*, *LOC107867964*, *LOC107870519*, *LOC107875600*, *LOC107875602*, and *LOC107875603*) were examined by qRT-PCR. The pepper actin gene (AY572427) was used as an internal control.
