2024 Volume 93 Issue 2 Pages 143-152
Capsaicinoids are compounds that generate the characteristic pungent taste of chili peppers, the presence or absence of which determines the utilization of the chili peppers as spices or vegetables. Loss of pungency is a qualitative trait resulting from dysfunction in any of four capsaicinoid biosynthesis genes (Pun1, pAMT, CaKR1, and CaMYB31). However, the lack of pungency in sweet peppers cannot be explained by known mutation alleles in these four genes. Herein, we report a novel dysfunctional allele of Pun1, which encodes acyltransferase 3 (capsaicin synthase), in a Japanese sweet pepper, ‘Sampo Oamanaga’. Firstly, PCR genotyping of ‘Sampo Oamanaga’ Pun1 showed that it was not a known mutant allele. We also performed whole-genome resequencing and found a large genomic deletion around the ‘Sampo Oamanaga’ Pun1 (XM_016704778.1). We then examined the precise size and breakpoint of the Pun1-deletion region via de novo assembly and Sanger sequencing analysis. We found an 18.5-kbp deletion, including the Pun1, on chromosome 2, and we designated this novel allele pun15. The genotypic effects of pun15 were investigated using F2 progeny derived from a ‘Sampo Oamanaga’ (pun15/pun15) × pungent cultivar ‘Takanotsume’ (Pun1/Pun1) cross. Only pun15-homozygous F2 plants showed non-pungency; co-segregation between Pun1 genotypes and pungency traits was observed. These results demonstrated that the deficiency of pungency in ‘Sampo Oamanaga’ is associated with the pun15 allele. The present study is the first to discover a large genomic deletion, including a gene among dysfunctional pun1 alleles, and provides new insights into the regulation mechanism of pungency in chili peppers.
Chili peppers (Capsicum spp.) are members of the Solanaceae family, characterized by their pungency traits. The sensory effects of pungency are derived from capsaicin and its analogues, known as capsaicinoids. The presence and contents of capsaicinoids determine the use of chili peppers as spices or vegetables (Matsushima, 2020). Capsaicinoids are also known to exert various effects on the body, such as pain relief, antioxidant effects, arthritis relief, and fat reduction (Luo et al., 2011; Materska and Perucka, 2005). Regarding biosynthesis, capsaicinoids are synthesized and accumulated in epidermal cells of the placental tissue, and their synthesis is activated at 20–40 days after flowering (DAF) (Iwai et al., 1979; Suzuki et al., 1980; Zhang et al., 2016). The capsaicinoid biosynthesis pathway consists of two sub-pathways: the phenylpropanoid pathway, which biosynthesizes vanillylamine from phenylamine, and the branched-chain fatty acid pathway, which synthesizes branched-chain fatty acids from valine or leucine (Aza-Gonzalez et al., 2011). Capsaicinoids are ultimately produced by condensing vanillylamine and a branched-chain fatty acid as the final products. Previous studies have indicated that numerous genes are involved in these metabolic processes, and the underlying genotypes and transcriptional levels affect the capsaicinoid content (i.e., pungency level) (Ben Chaim et al., 2006; Han et al., 2018; Park et al., 2019). In contrast, deficiency in pungency is a qualitative trait controlled by a single recessive gene, which can lead to dysfunction in any of the capsaicinoid biosynthesis genes (Arce-Rodríguez and Ochoa-Alejo, 2019).
Previous studies identified four genes that are responsible for the loss of pungency in chili peppers. The Pun1 gene plays a key role in determining the presence of pungency, found from C. annuum. This gene is located on chromosome 2 and encodes acyltransferase 3 (capsaicin synthase) that is responsible for the final reaction of capsaicinoid biosynthesis. Stewart et al. (2005) initially revealed that a dysfunctional pun1 allele (pun11) leads to pungency deficiency; three other alleles (pun12–pun14) were subsequently reported (Kirii et al., 2017; Stellari et al., 2010; Stewart et al., 2007). Of these, pun11 and pun14 are dysfunctional Pun1 alleles found in C. annuum. Putative aminotransferase (pAMT), located on chromosome 3, is also a critical gene for pungency (Lang et al., 2009). This gene is involved in the biosynthetic conversion of vanillin to vanillylamine in the phenylpropanoid pathway. Compared with Pun1, more information is available regarding mutations in pAMT. Previous studies identified 10 different pAMT alleles (pamt1–pamt10) (Koeda et al., 2014; Lang et al., 2009; Park et al., 2015; Tanaka et al., 2010a, b, 2015, 2017, 2018; Tsurumaki and Sasamuma, 2019). Additionally, putative ketoacyl-ACP reductase (CaKR1) was identified by Koeda et al. (2019) as a novel gene controlling the presence of pungency. This gene is located on chromosome 6 and mediates the elongation of branched-chain fatty acids used in capsaicinoid biosynthesis. Furthermore, Han et al. (2019) identified a dysfunctional allele of CaMYB31 (Pun3), located on chromosome 7, which causes a pungency deficiency. CaMYB31 encodes the R2R3-MYB transcription factor, which is thought to markedly affect the expression of capsaicinoid biosynthesis genes (Arce-Rodríguez and Ochoa-Alejo 2017). The above noted genes and their mutant alleles are responsible for the presence of pungency in chili peppers. However, some non-pungent cultivars are available for which the pungency deficiency cannot be explained by either known pungency-related genes or their known mutant alleles. Investigating non-pungency traits in such varieties may therefore provide novel insights regarding the genetic regulation of pungency.
In this study, we attempted to elucidate the loss-of-pungency mechanism in ‘Sampo Oamanaga’ (C. annuum), a Japanese landrace sweet pepper. This cultivar, cultivated only in Tottori Prefecture, Japan, produces large, elongated fruits (approximately 20 cm). Most sweet pepper cultivars have bell-type fruits, and elongate-type fruits are rare in Japan. This implies that a unique mechanism controls pungency deficiency in this cultivar. Herein, we report a novel dysfunctional pun1 allele, designated pun15, in ‘Sampo Oamanaga’. The discovery of pun15 provides new insights regarding the mechanism of pungency deficiency in chili peppers.
In the present study, we used C. annuum cultivar ‘Sampo Oamanaga’ (JP accession: 32567, Gene Bank, National Agriculture and Food Research Organization [NARO]) and two non-pungent cultivars, ‘California Wonder’ and ‘Nara Murasaki’, which lose their pungency due to dysfunctional pun1 alleles: pun11 and pun14, respectively (Kirii et al., 2017; Stewart et al., 2005). We also used the pungent C. annuum variety ‘Takanotsume’. To investigate the heredity of non-pungency in ‘Sampo Oamanaga’, we prepared two kinds of F1 progeny, one derived from ‘Sampo Oamanaga’ × ‘California Wonder’ and the other from ‘Sampo Oamanaga’ × ‘Takanotsume’. Then, as in the latter F1 progeny, we obtained the F2 progeny by self-pollination. These plants were grown in 2020 at a farm at Shinshu University (Nagano, Japan). For the gene expression analyses, we cultivated ‘Sampo Oamanaga’, ‘California Wonder’, and ‘Takanotsume’ at the greenhouse of Shinshu University in 2020.
Amplification of pun1 locus using known primer setIn ‘Sampo Oamanaga’, ‘Takanotsume’, ‘California Wonder’, and ‘Nara Murasaki’, gDNA was extracted from young leaves using a QuickGene DNA tissue kit S (DT-S; Kurabo Industries Ltd., Osaka, Japan) and QuickGene-810 (QG-810; Kurabo Industries Ltd.), based on the manufacturer’s instructions. We then prepared polymerase chain reaction (PCR) mixtures (18 μL each) consisting of 0.1 μL of Ex Taq DNA polymerase (Takara Bio. Inc., Shiga, Japan), 2.0 μL of 10× Ex Taq buffer, 1.6 μL of 2.5 mM dNTP mixture, 1.2 μL each of forward and reverse primers (50/3 μM), and 2.0 μL of template DNA (10 ng·μL−1). We used a primer set developed by Lee et al. (2005), and the sequences are shown in Table 1. PCR was conducted using the following thermal conditions: 1 cycle at 94°C for 2 min, followed by 35 cycles of 98°C for 10 s, 60°C for 30 s, 72°C for 4 min, and a final extension at 72°C for 4 min. The PCR products were electrophoresed on a 1.5% agarose gel, stained with ethidium bromide, and visualized using a UV transilluminator.
Sequences of primers used in this study.
Whole-genome resequencing of ‘Sampo Oamanaga’ was performed using a sequencing service (Chemical-Dojin Co., Ltd., Tokyo, Japan). First, gDNA of ‘Sampo Oamanaga’ was fragmented to an average size of 350 bp and subjected to library construction using Illumina paired-end protocols. On average, 150-bp DNA fragments were generated, and paired-end sequencing was conducted using a NovaSeq 6000 system (Illumina Inc., San Diego, CA, USA). Quality filtering was then conducted using Fastp, and unusable reads were removed using Fastp v0.20.0 (Chen et al., 2018). Sequence reads with quality values of < Q5 and uncertain nucleotide rates > 10% were removed. Thereafter, trimmed reads were mapped to the C. annuum genome ‘Zunla 1’ (Ref_v1.0; Qin et al., 2014) using Burrows-Wheeler Alignment (v0.7.8; Li and Durbin, 2009). After mapping, structure variant (SV) analysis was performed using Breakdancer v1.4.4 (Chen et al., 2009), and deletions, insertions, inversions, and duplications were detected using the bam format file. The parameters were as follows: -q 20 and -c 4. Mapping data of ‘Sampo Oamanaga’ were visualized using Integrative Genomics Viewer (IGV v2.9.4; Thorvaldsdótti et al., 2013); the BAM format file was loaded and the genomic region around the Pun1 (XM_016704778.1) in ‘Sampo Oamanaga’ was compared with the reference genome ‘Zunla1’.
Exploration of the breakpoint of Pun1-deletion region in ‘Sampo Oamanaga’In the SV analysis, ‘Sampo Oamanaga’ was estimated to have a large deletion that included the Pun1; therefore, de novo assembly was performed to identify the genomic breakpoints of the Pun1-deletion region. ABySS 2.3.4 (Jackman et al., 2017) was used for this analysis with the following parameters: k = 84, j = 64, B = 100 G, H = 3, kc = 2, q = 3, v = −v, s = 1,000, n = 10, S = 10,000, and N = 10. We identified a contig that included genomic breakpoints of the Pun1-deletion region in ‘Sampo Oamanaga’ by comparing the results with the ‘Zunla 1’ genome. We then verified the accuracy of the de novo assembly by Sanger sequencing. Using Primer3Plus (Untergasser et al., 2007), we designed a primer set (SP-F2/SP-R1_2) that amplified most of the above contig. This primer set was also used as a sequence tag specific (STS) marker (Fig. 5a) to determine Pun1 genotypes in the F2 population, as described below. The same PCR mixture (16 μL) described above was used with 4.0 μL of template DNA (10 ng·μL−1). The thermal conditions were as follows: 1 cycle at 94°C for 2 min, followed by 35 cycles of 98°C for 10 s, 66°C for 30 s, 72°C for 40 s, with a final extension at 72°C for 40 s. The PCR products were electrophoresed on a 1.0% agarose gel and purified using a QIAquick Gel Extraction kit (QIAGEN) according to the manufacturer’s instructions. Sanger sequencing was then performed by the Eurofins DNA sequencing service (Eurofins Genomics Inc., Tokyo, Japan). The entire nucleotide sequences of the PCR fragments were determined using MEGA X (Kumar et al., 2018), and the overall sequence was compared with the contig sequence from the ‘Sampo Oamanaga’ and ‘Zunla 1’ genomes using ClustalW.
Heredity analysis of the pun1 allele of ‘Sampo Oamanaga’We first investigated the pungency trait of the F1 progeny between ‘Sampo Oamanaga’ and the pun11-dependent non-pungent cultivar ‘California Wonder’ to elucidate the details of genetic complementation. We also investigated those in the F1 and F2 progeny (‘Sampo Oamanaga’ × pungent cultivar ‘Takanotsume’) to understand the heredity of non-pungency in ‘Sampo Oamanaga’. For phenotyping, we implemented an organoleptic test to determine the presence of pungency in nine of the F1 plants and 67 of the F2 plants; three researchers randomly collected fruits 35 DAF from each plant and licked the placental septa and the plants were regarded as pungent if even one researcher sensed pungency.
In addition to the organoleptic test, we also quantified the capsaicinoid content of the progeny, as described by Kondo et al. (2023). Briefly, we lyophilized bulk placental septum tissues collected from fruits 35 DAF (5–10 fruits from each plant). We then extracted and quantified the capsaicinoids (total amount of capsaicin and dihydrocapsaicin) using high-performance liquid chromatography. Furthermore, to elucidate the genotypic effects of Pun1 alleles in the F1 and F2 progeny (‘Sampo Oamanaga’ × ‘Takanotsume’), we determined the respective Pun1 genotypes and investigated the relationship with pungency traits. We first extracted gDNA from young leaves in each progeny, as described above, and then performed Pun1 genotyping of the progeny using co-dominant STS markers. The primer sequences and amplified genomic region are shown in Table 1 and Figure 5a. Regarding PCR and gel electrophoresis, the methods as described for Sanger sequence analysis were used.
Analysis of Pun1 expressionQuantitative reverse-transcription PCR (qRT-PCR) was conducted to investigate expression levels of capsaicinoid biosynthesis genes. For this analysis, we targeted four capsaicinoid biosynthesis genes: Pun1, pAMT, CaMYB31, and WRKY9, which are known to have a significant effect on capsaicinoid biosynthesis (Han et al., 2018; Lang et al., 2009; Stewart et al., 2005; Zhu et al., 2019). Actin was examined as a reference gene. The sequences of primers used for qRT-PCR are listed in Table 1. Total RNA was extracted from placental septum tissues of 20- and 35-DAF fruits and used to synthesize cDNAs. qRT-PCR was then performed as described by Kondo et al. (2021a). Relative expression levels for each sample were calculated based on the comparative CT method.
We first attempted to amplify the genomic region around the Pun1 locus in ‘Sampo Oamanaga’ and several pungent/non-pungent cultivars via genomic PCR using a known primer set (BF6/CSR4) (Lee et al., 2005, Fig. 1). No amplicon was detected in ‘Sampo Oamanaga’, but as expected, a 3.6-kbp amplicon was obtained from the pungent cultivar ‘Takanotsume’, and 1.0-kbp and 3.6-kbp amplicons were obtained from the non-pungent cultivars ‘California Wonder’ (pun11/pun11), and ‘Nara Murasaki’ (pun14/pun14), respectively. These results suggest that ‘Sampo Oamanaga’ has a unique pun1 allele not reported in previous studies.
Amplification of the genomic region around the Pun1 locus. (a) Structures of the functional Pun1 allele and known dysfunctional alleles (pun11, pun14). Black arrows with characters represent positions of known primer set (BF6/CSR4, Lee et al., 2005) amplification of the genomic region (3,626 bp) around the Pun1 locus. (b) Gel showing products of amplification of the Pun1 locus with the above primer set in several cultivars: M: DNA ladder marker, TK: ‘Takanotsume’ (Pun1/Pun1, pungent), SP: ‘Sampo Oamanaga’, CW: ‘California Wonder’ (pun11/pun11, non-pungent), NM: ‘Nara Murasaki’ (pun14/pun14, non-pungent). In the case of functional Pun1, a 3,626-bp amplicon was obtained. In contrast, the amplicon of pun11 was 1,045 bp due to the 2.5-kbp deletion, and pun14 produced a 3,627-bp amplicon because of a 1-bp insertion.
Whole-genome resequencing and SV analyses were performed to explore genomic variations in ‘Sampo Oamanaga’. In whole-genome resequencing, we obtained a total of 528,849,258 raw reads (approximately 95.7 Gb), equivalent to 30 times the ‘Zunla 1’ genome size (2.93 Gb). Also, the reference genome mapping rate and average depth were 98.5% and 29.14×, respectively. In the SV analysis, two large deletions were detected around the Pun1 locus; one deletion of 18,625 bp and the other of 18,532 bp (Table 2). These genomic regions included 1,897 bp of the Pun1 (XM_016704778.1) on chromosome 2 (NC_029978.1). To verify these results, we visualized the read mapping data in comparison with the reference genome using IGV software. Few reads were mapped in the 19-kbp region, as expected from the SV analysis results (Fig. 2), indicating that ‘Sampo Oamanaga’ has a large deletion around the Pun1.
Structure variants detected around the Pun1 in ‘Sampo Oamanaga’.
Visualization of mapped reads around the Pun1-deletion region in ‘Sampo Oamanaga’ (143,420–143,440 kbp on chromosome 2 in the ‘Zunla 1’ genome [Ref_v1.0]), as determined using IGV software. The black symbols at the bottom of the diagram indicate the position of Pun1 (XM_016704778.1).
To identify the precise variant size and breakpoint of the Pun1-deletion region in ‘Sampo Oamanaga’, we performed de novo assembly and obtained a total of 2,948,021 contigs (Table 3), but only one of these contigs included the breakpoint of the deletion. We then designed a primer set to amplify most parts of the above contig (SP-F2/SP-R1_2) and performed PCR using gDNA from ‘Sampo Oamanaga’. The amplicon size corresponded to the expected size, and the sequence perfectly matched the contig (Fig. 3), indicating the present de novo assembly was precisely implemented. By alignment between the obtained sequence and the ‘Zunla 1’ genome, we identified an 18,588-bp deletion in chromosome 2 of ‘Sampo Oamanaga’, which included the Pun1 (Fig. 4). In the present study, we named this novel allele pun15. Based on the ‘Zunla 1’ genome, no genes other than Pun1 were identified on this deleted genomic region. Two candidate breakpoints could then be estimated: BP1 (143,416,825–143,435,412 bp) and BP2 (143,416,828–143,435,415 bp), as shown in Figures 3 and 4. This was because the same 3-bp sequence (-ACA-) was present in both the 5'- and 3'-terminal regions of the Pun1-deletion region of the ‘Zunla 1’ genome (Fig. 4), and we could thus not determine the single breakpoint.
Statistics regarding contigs/scaffolds built using de novo assembly (k = 84).
Sanger sequencing verification of the accuracy of contig sequences obtained by de novo assembly, which included the break point of the Pun1-deletion region in ‘Sampo Oamanaga’. Sampo-contig: part of contig including break point obtained using de novo assembly; Sampo-amp: sequence of DNA fragment amplified with SP-F2/SP-R1_2 markers. BP1 and BP2 denote putative breakpoints 1 and 2 respectively, as shown in Figure 3. *Indicates the consensus of each nucleotide between the two sequences.
Structure of 18.5-kbp Pun1-deletion region in ‘Sampo Oamanaga’, compared with chromosome 2 of the ‘Zunla 1’ genome (Ref_v1.0). The precise deletion size and breakpoints in ‘Sampo Oamanaga’ were identified via de novo assembly and Sanger sequencing. Gray box shows the Pun1-deletion region (18,588 bp), and black symbols above the illustration indicate the position of Pun1 (XM_016704778.1). BP1 and BP2 denote two putative breakpoints: putative breakpoint 1 (143,416,825–143,435,412 bp) and putative breakpoint 2 (143,416,828–143,435,415 bp), respectively.
We first compared the pungency traits of the F1 progeny between ‘Sampo Oamanaga’ (pun15/pun15) and ‘California Wonder’ (pun11/pun11) by organoleptic testing and capsaicinoid quantification. The F1 progeny showed non-pungency, and no capsaicinoids were detected, as with the parents (Table 4) (i.e., genetic complementation was not observed). This indicated that the deficiency of pungency in ‘Sampo Oamanaga’ depends on pun15.
Relationship between Pun1 genotypes and pungency traits in parents and crossed progeny.
We then investigated pungency traits in the F1 and F2 progeny derived from ‘Sampo Oamanaga’ (pun15/pun15) × ‘Takanotsume’ (Pun1/Pun1). All F1 plants showed pungency, and the mean capsaicinoids content was 15,842 μg·g−1DW, similar to the pungent parent ‘Takanotsume’ (capsaicinoids content: 19,144 μg·g−1DW). For the 67 F2 progeny, 49 exhibited pungency, and their capsaicinoid content was within the range: 253–36,972 μg·g−1DW, whereas the other 18 showed non-pungency (no capsaicinoids were detected). We also conducted genotyping of Pun1 alleles in the F1 and F2 progeny using the designed STS markers (Fig. 5a; Table 1). In the genomic PCR and gel electrophoresis analysis using the designed STS markers, 457-bp and 527-bp fragments were detected in the case of functional Pun1 and pun15 alleles, respectively (Fig. 5b). The F2 progeny were therefore classified as 13 Pun1-homozygous plants, 36 heterozygous plants, and 18 pun15-homozygous plants (Table 4), and the segregation ratio of Pun1 alleles was compatible with the expected ratio of 1:2:1 (X2 = 1.1194, p = 0.5714). Focusing on the relationship with phenotypes, Pun1-homozygous and heterozygous plants exhibited pungency, whereas pun15-homozygous plants showed non-pungency, and co-segregation between the Pun1 genotype and pungency traits was observed (Fig. 5b). Collectively, these results revealed that non-pungency in ‘Sampo Oamanaga’ is a recessive and qualitative trait controlled by pun15.
Pun1 genotyping in the F1 and F2 progeny derived from ‘Sampo Oamanaga’ × ‘Takanotsume’. (a) Schematic diagram of targeted genomic regions for STS markers for Pun1 genotyping. Light gray boxes show functional Pun1, and dark gray box below indicates the Pun1-deletion region. We designed STS markers to distinguish functional Pun1 and pun15 based on differences in their sequences; primer sequences are listed in Table 1. (b) Co-segregation of the pun15 genotype in the F2 population derived from ‘Sampo Oamanaga’ × ‘Takanotsume’. The bar chart and band image show the capsaicinoid content and Pun1 genotypes determined using the above markers in each plant, respectively: M: DNA ladder, SP: ‘Sampo Oamanaga’ (pun15/pun15), TK: ‘Takanotsume’ (Pun1/Pun1), F1: F1 progeny, 1–9: F2 progeny. The size of the Pun1 amplicon was 457 bp, whereas that of pun15 was 527 bp.
qRT-PCR analyses of Pun1 and other capsaicinoid genes (pAMT, CaMYB31, and WRKY9) were conducted using the placental septum of 20- and 35-DAF fruits of ‘Sampo Oamanaga’, ‘Takanotsume’, and ‘California Wonder’ plants. Neither Pun1 nor the other three genes were expressed in ‘Sampo Oamanaga’ at any developmental stage, although conversely, ‘Takanotsume’ showed high expression of these genes (Fig. 6). Similar expression patterns were also observed in ‘California Wonder’.
Expression levels of capsaicinoid biosynthesis genes (Pun1, pAMT, CaMYB31, and WRKY9) in the placental septum of 20- and 35-DAF fruits: TK: ‘Takanotsume’, SP: ‘Sampo Oamanaga’, CW: ‘California Wonder’. Error bars indicate standard errors (n = 3), and nd indicates not detected.
The use of chili peppers depends on the presence and level of pungency. In particular, the non-pungency traits of vegetable cultivars stand out among chili peppers, so much so that the genetic mechanism of non-pungency has been studied by many researchers. The presence of pungency in chili peppers was known as a qualitative trait controlled by a single gene until previous studies identified four genes (Pun1, pAMT, CaKR1, and CaMYB31) responsible for the trait (Han et al., 2019; Koeda et al., 2019; Lang et al., 2009; Stewart et al., 2005). However, there are several non-pungent cultivars for which loss of pungency cannot be explained by the known dysfunctional alleles of these genes. The Japanese landrace sweet cultivar ‘Sampo Oamanaga’ (C. annuum) is one such example, and so we focused on its non-pungency trait. Regarding this cultivar, we initially tried to amplify the genomic region around the Pun1, but no amplicon was obtained (Fig. 1), implying the presence of a novel gene mutation. Therefore, we performed whole-genome resequencing and SV analysis of ‘Sampo Oamanaga’ plants to explore the genomic variation. An approximately 19-kb deletion around the Pun1 was identified in ‘Sampo Oamanaga’ (Fig. 2). To determine the precise deletion size and breakpoints, we also performed de novo assembly and Sanger sequencing. These analyses revealed that ‘Sampo Oamanaga’ has an 18,588-bp deletion (143,416,825–143,435,412 bp or 143,416,828–143,435,415 bp) on chromosome 2 compared with the ‘Zunla 1’ genome, and this deletion includes Pun1 (Fig. 4). As this mutation has not been previously reported, we designated it pun15, as the fifth pun1 allele.
Heredity analysis using several kinds of crossed progeny demonstrated that the non-pungency trait in ‘Sampo Oamanaga’ is controlled by pun15 (Fig. 5b; Table 4). Previous studies have reported four dysfunctional pun1 alleles (pun11–pun14) (Table 5). The pun11 allele has a deletion of 2.5 kb, spanning from the promoter region to most of the first exon, and this deletion is widely distributed among sweet C. annuum varieties (Stewart et al., 2005). The second allele, pun12, was discovered in C. chinense and has a 4-bp deletion in the first exon region (Stewart et al., 2007). The third allele, pun13, which was identified in C. frutescens, is truncated at the end of the second exon (Stellari et al., 2010). Finally, the pun14 allele was identified in the C. annuum cultivar ‘Nara Murasaki’, a Japanese sweet pepper cultivar. The pun14 allele has a 1-nucleotide insertion in the second exon (Kirii et al., 2017). Collectively, these data indicate that the known pun1 allele mutants contain deletions and insertions in the promoter or exon regions. On the other hand, regarding pun15, Pun1 is absolutely deficient, and the mutation type varies markedly from known mutations in terms of scale.
Dysfunctional pun1 alleles reported in previous studies and this study.
We also found that Pun1 was never expressed in ‘Sampo Oamanaga’, similar to the non-pungent cultivar ‘California Wonder’ (Fig. 6). Considering that Pun1 mediates the final reaction in capsaicinoid biosynthesis, the lack of Pun1 expression due to the genomic mutation may contribute to the pungency deficiency in ‘Sampo Oamanaga’. Interestingly, we also observed that other capsaicinoid biosynthesis genes (pAMT, CaMYB31, and WRKY9) are not expressed in ‘Sampo Oamanaga’. Previous studies reported similar insights to those of this study. For example, Stewart et al. (2005, 2007) observed that the other capsaicinoid biosynthesis genes are expressed in low levels in non-pungent cultivars due to the dysfunctional pun1 allele. Arce-Rodríguez and Ochoa-Alejo (2015) reported that virus-induced gene silencing of Pun1 suppressed the expression of pAMT and several genes responsible for the branched-chain fatty acid pathway. Collectively, these results suggest that a transcriptional deficiency of Pun1 due to the mutation affects the expression of capsaicinoid biosynthesis genes.
Although plants have DNA repair mechanisms that overcome damage in nucleotide sequences, genetic information can be lost by incomplete repairs due to mutation size or chromosomal position during repair. Such repair errors can negatively affect plant growth (Waterworth et al., 2007). However, we did not observe any effect of large genomic deletions, including the Pun1, on survival in ‘Sampo Oamanaga’; there were no abnormities in pollen fertility, fruiting, or growth of ‘Sampo Oamanaga’ plants, so we could easily generate F1 and F2 progeny (data not shown). These results suggest that chili peppers remain viable even if Pun1 and its peripheral region are completely lost. It has not been previously mentioned in studies on pun11–pun14 alleles that mutations in Pun1 affect their fertility or growth (Kirii et al., 2017; Stellari et al., 2010; Stewart et al., 2005, 2007). Notably, except for Pun1, the other genes were not located within the deleted 18.5-kbp region; thus, ‘Sampo Oamanaga’ retained normal biological activity.
Finally, we focused on the derivation of ‘Sampo Oamanaga’. It is said that the seeds were brought from Southeast Asia to Japan in the late 1920s, and cultivation was started by a local farmer in Tottori Prefecture (Teragishi, 2022). It is unclear whether ‘Sampo Oamanaga’ already exhibited non-pungency traits before the seeds were brought from Southeast Asia. However, non-pungent cultivars have few uses in Southeast Asia, except for sweet bell peppers bred in Europe and the United States, and pun15 has not been observed in other chili pepper cultivars. Considering these data, it may be inferred that the pun15 mutation occurred in Japan and the non-pungency trait was selected by local farmers or breeders. However, further phylogenetic analysis of the Pun1 locus in Southeast Asian peppers and ‘Sampo Oamanaga’ is needed to reach a conclusion.
In conclusion, we discovered a novel pun1 allele, pun15, which results in non-pungency in the Japanese sweet pepper ‘Sampo Oamanaga’. The pun15 allele consists of an 18.5-kb deletion that includes the Pun1, and is a rare case of genomic variation observed in chili peppers. These results provide novel insights regarding pungency regulation in chili peppers. Additionally, ‘Sampo Oamanaga’ and the pun15-specific markers developed in the present study will be useful for breeding sweet pepper varieties in the future.
We thank the Gene Bank of the National Agriculture and Food Research Organization for providing plant material: ‘Sampo Oamanaga’ (JP:32567). Computations were partially performed on the NIG supercomputer at the ROIS National Institute of Genetics.
