*3.1. Screening for Pepper Cultivars with Contrasting Low-N Tolerance*

To identify pepper cultivars with contrasting low-N tolerance, 100 pepper cultivars were screened based on various metrics. To avoid the effects of natural variation in individual embryos and/or endosperm size on initial growth from different genotypes, the relative value (low-N treated sample/control) of each character was used in the following analyses. Our results showed that peppers from these 100 cultivars had a wide range of relative values of leaf areas, shoots, roots, and plant dry weights, and the coefficient of variation (CV) of all of the relative values was above 15%, demonstrating the high variation in tolerance to low-N among cultivars (Table 1 and Table S2).

**Table 1.** Range of relative values of growth characteristics in pepper seedlings.


Cluster analysis was then carried out based on the relative values of leaf areas, shoot dry weights, root dry weights, and plant dry weights. The 100 pepper cultivars could be grouped into three clusters, consisting of five (Group I), six (Group II), and 89 (Group III) cultivars (Figure 1). There were significant differences in each characteristic between the three groups (Table 2). The mean relative values of each characteristic in Group I were significantly higher than those of Group II, and the mean relative values of each characteristic in Group III were between those of Groups I and II, indicating that Group I was a low-N-tolerant group, while Group II was a low-N-sensitive group.


**Table 2.** The low-nitrogen tolerance of each cluster group.

Note: Values followed by different letters within the same column are significantly different at the 0.05 level.

750-1 had the highest values for the relative leaf area as well as shoot and plant dry weights (Table S2) and was classified into Group I. Thus, it was considered low-N tolerant. ZCFB had the lowest relative values of shoot, root and plant dry weights (Table S2) and was classified into Group II. This cultivar was therefore considered low-N sensitive.

**Figure 1.** Cluster analysis of the low-nitrogen tolerance from 100 pepper cultivars.Cluster analysis was carried out based on the relative values of leaf area and shoot, root, and plant dry weights. Agglomerative hierarchical clustering was performed using the hclust algorithm in R, with dst = dist (dat1, method = "euclidean") and hclust (dst, method = "average"). Group I, low-N-tolerant group; Group II, low-N-sensitive group; the low-nitrogen tolerance of Group III was between that of Groups I and II.
