Pungent traits caused by capsaicinoids are characteristic of chili peppers (Capsicum spp.), and the pungent-variable sweet chili pepper ‘Shishito’ (Capsicum annuum) is unique in being known for the pungency in fruits with few seeds. In the present study, we tried to clarify the relationship between the number of seeds and pungency in ‘Shishito’. First, we investigated the pungency of ‘Shishito’ by simple sensory evaluations and quantifications of capsaicinoids by high-performance liquid chromatography. As a result, few-seeded fruits had a larger fluctuation of capsaicinoid content than many-seeded ones. This indicates that the number of seeds, in particular a decrease of the seeds, has some sort of connection with the pungency of ‘Shishito’. Then, we analyzed the relationship between pungency and gene expression involving capsaicinoid biosynthesis at the individual fruit level. We vertically separated the placental septum in which capsaicinoids are synthesized and performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for 18 genes involved in capsaicinoid biosynthesis. We also used the placental septum for capsaicinoid quantification so that the gene expression levels and capsaicinoid contents in the same fruits were obtained, and their correlations were analyzed using 20 biological replicates. Among the 18 genes, expression levels of 11 genes (WRKY9, CaMYB31, AT3, BCAT, BCKDH, KAS I, KAS III, ACL, CaKR1, FAT, and pAMT) had a significant positive correlation with the capsaicinoid concentration, and they were considered to upregulate capsaicinoid biosynthesis. These results provide new insights regarding the environmental variation of the pungency traits in chili peppers and the relationship between pungency, the number of seeds, and gene expression involved in capsaicinoid biosynthesis.

由辣椒素类引起的刺激性状是辣椒（Capsicum spp。）和刺激性可变甜辣椒“ Shishito” （Capsicum annuum）的特征。）独特之处在于，它以很少种子的水果中的辛辣味闻名。在本研究中，我们试图阐明“ Shitoto”中种子数量与刺激性之间的关系。首先，我们通过简单的感官评估和通过高效液相色谱对辣椒素类化合物的定量研究了“ Shishito”的刺激性。结果，很少播种的果实的辣椒素含量波动要比许多播种的果实大。这表明种子的数量，特别是种子的减少，与“ Shitoto”的刺激性有某种联系。然后，我们在单个果实水平上分析了涉及辣椒素类生物合成的刺激性与基因表达之间的关系。我们垂直分离了其中合成了类辣椒素的胎盘隔，并对参与类辣椒素生物合成的18个基因进行了定量逆转录聚合酶链反应（qRT-PCR）。我们还使用胎盘隔进行了辣椒素定量分析，从而获得了相同果实中的基因表达水平和辣椒素含量，并使用20个生物学重复进行了相关性分析。在18个基因中，有11个基因的表达水平（WRKY9，CaMYB31，AT3，BCAT，BCKDH，KAS I，KAS III，ACL，CaKR1，FAT和pAMT）与类辣椒素浓度呈显着正相关，被认为上调了类辣椒素的生物合成。这些结果为辣椒中的辛辣性状的环境变化以及辛辣性，种子数量和参与辣椒素类生物合成的基因表达之间的关系提供了新的见解。