Low chlorophyll content in the exocarp of 17C827 affects light-green immature-fruit formation
We examined the cross-section of 17C658 (green immature fruits) and 17C827 (light-green immature fruits) immature fruits. As shown in Figure 1a, both 17C827 and 17C658 immature fruits formed a notable division of green coloration at the boundary between the exocarp and mesocarp; the green portion was located on the exocarp side, and the light-green portion was located on the mesocarp side. The degree of green in the 17C658 and 17C827 immature fruits is, therefore, mainly influenced by the exocarp.
Next, the pigment contents in the exocarp and endocarp (including the mesocarp) of the two pepper accessions were compared. We found that chlorophyll content (chlorophyll a [Figure 1b] and chlorophyll b [Figure 1c]) in the exocarp was significantly different between 17C827 and 17C658 fruits, and the content in 17C827 fruits was lower than in 17C658 fruits. However, there was no significant difference in chlorophyll content in the endocarp (including the mesocarp) between 17C827 and 17C658 fruits during the various periods of fruit development (Figure 1b, c). These results showed that low chlorophyll content in the 17C827 fruit exocarp was the main factor influencing the formation of its light-green immature fruits. Furthermore, chlorophyll content (chlorophyll a [Figure 1b] and chlorophyll b [Figure 1c]) in the exocarp and endocarp (including the mesocarp) of the two pepper accessions decreased from day 5 to day 9, increased from day 9 to day 12, and gradually decreased and eventually stabilized from day 12 to the M stage. The change in carotenoid content was similar to that in chlorophyll content (Supplementary Figure 1). The results showed that the degree of green in the immature fruits of 17C658 and 17C827 began to stabilize after day 12. Meanwhile, total chlorophyll content (0.048–0.382 mg/g·FW) in both 17C827 and 17C658 fruits was markedly higher than their carotenoid content (0.003–0.047 mg/g·FW) during the various periods of fruit development (Supplementary Figure 1), which indicates that the formation of green-colored immature pepper fruits is mainly affected by chlorophyll.
Abnormal chloroplast thylakoids in 17C827 exocarp affects light-green immature-fruit formation
To explore whether light-green immature-fruit formation of 17C827 plants was affected by chloroplasts, we first examined the number of chloroplasts in a single cell and the number of chloroplast grana in a single chloroplast. Regardless of the evaluation day—day 9 or day 20—there were no significant difference in the numbers of chloroplasts and chloroplast grana between 17C827 and 17C658 fruits (Supplementary Figure 2a). We then measured the perimeter and area of the chloroplast cross section in the exocarp and found that there were significant differences between 17C827 and 17C658 fruits (Supplementary Figure 2b, c). The chloroplast cross-section perimeter and area values of 17C827 fruits were higher than those of 17C658 fruits. Meanwhile, we calculated the increments of the chloroplast section perimeter and area from day 9 to day 20, and we found that the increments of these values in 17C827 were also higher than those in 17C658 (Supplementary Figure 2b, c). At day 9, dividing chloroplasts were found in both 17C827 and 17C658 fruits, indicating that both accessions were able to proliferate chloroplasts (Supplementary Figure 3a). At day 20, we also found that the chloroplast grana of the mature chloroplasts in the light-green immature fruits of 17C827 were thinner and more scattered than those of 17C658, and there were fewer stroma lamella in 17C827 than in 17C658 (Supplementary Figure 3b). The above results indicate that the abnormal chloroplast thylakoids in the exocarp of 17C827 affected the formation of light-green immature fruits.
Light-green color of 17C827 immature fruits is controlled by a single dominant locus
In the present study, F1 and F2 were constructed from a cross between 17C827 (light-green immature fruits) and 17C658 (green immature fruits). All F1 individuals showed a light-green immature-fruit color, which was the same as that of the female parent 17C827 (Figure 2). In contrast, the immature-fruit colors of the 941 F2 individuals were segregated into light green (726 individuals) and green (215 individuals), with a ratio of 3:1 (χ2 = 2.32 < 6.64, df = 1, p = 0.01), which was similar to the separation ratio of red (701 individuals) and yellow (240 individuals) mature fruits. These results indicate that the formation of light-green immature-fruit color could be attributed to the genetics of a single dominant locus compared with the formation of green immature-fruit color. Finally, we found that F2 could be segregated according to fruit color, light-green immature-fruit and red mature-fruit (534 individuals), light-green immature-fruit and yellow mature-fruit (174 individuals), green immature-fruit and red mature-fruit (167 individuals), and green immature-fruit and yellow mature-fruit (66 individuals) color, which followed the ratio of 9:3:3:1 (χ2 = 1.45 < 11.35, df = 3, p = 0.01). This result suggest that the genetic mechanisms of immature-fruit color (light green and green) and mature fruit color (red and yellow) are relatively independent from each other.
LG locus position revealed by GWAS and BSA
To efficiently map the LG locus, a GWAS on immature-fruit color, comprising yellowish white, milky yellow, yellowish green, light green, green, deep green, purple, and purplish black, in 287 pepper accessions was conducted (Supplementary Table 1). As shown in Figure 3a, there was a candidate region spanning from 157,194,038 bp to 190,675,776 bp on chromosome 10 that was significantly associated with immature-fruit color. Additionally, a GWAS of immature-fruit colors according to lightness (L*), red and green coordinates (a*), and yellow and blue coordinates (b*) of 287 pepper accessions was conducted, and a candidate region was found on chromosome 10. The candidate region significantly associated with L* spanned from 176,498,851 bp to 176,669,957 bp on chromosome 10, while that associated with a* spanned from 55,687,612 bp to 62,184,580 bp, and that associated with b* spanned from 175,987,065 bp to 186,812,911 bp. Therefore, we assumed that the LG locus controlling color in light-green immature fruits was likely to be located in the region spanning from 157,194,038 bp to 190,675,776 bp on chromosome 10. In addition, the BSA of the extreme mixed pool based on F2 showed that there was a candidate region from 156,650,000 bp to 178,330,000 bp on chromosome 10 that was significantly associated with the light-green color of immature pepper fruits (Figure 3b). Finally, we selected the intersection of the GWAS candidate region and the BSA candidate region to determine that the LG locus was located in the region spanning from 157,494,038 bp to 178,330,000 bp (size: 20.8 Mbp) on chromosome 10. Seven primer pairs from the above region were used for linkage analysis of the LG locus. The results showed that the LG locus was located between the InDel-106 and InDel-109 primers, with a physical distance of 35.07 kbp (from 174,787,932 bp to 174,823,007 bp) (Supplementary Figure 4a). The QTL analysis for the colorimetry indices (L*, a*, b*, chroma [SD], and shade [SG]) of the immature-fruit surfaces of 315 F2 individuals showed similar results (Supplementary Figure 4b).
CaPP2C35 is the candidate gene of the LG locus
Using the JBrowser tool of the Sol Genomics Network (https://solgenomics.net/), we found that there was only one gene, Capana10g001710, located between 174,787,932 bp and 174,823,007 bp on chromosome 10. Sequence analysis revealed a G-A substitution at the 313th base of the Capana10g001710 coding sequence (CDS) in 17C827 (Supplementary Figure 5). Based on this base substitution, a derived cleaved amplified polymorphic sequence (dCAPS) molecular marker, dCAPS10-1, was designed. As shown in Figure 4a, b, dCAPS10-1 had a stable polymorphism between the two parents and was closely linked to LG (0.2 cM). Meanwhile, the QTL analysis of the colorimetry indices (L*, a*, b*, SD, and SG) of the immature-fruit surfaces of 315 F2 individuals showed that the QTLs of L*, a *, b *, SD, and SG were all located near dCAPS10-1 (Figure 4c). Additionally, the percentage of variation explained (PVE%) of dCAPS10-1 for L*, a *, b *, SD, and SG was 27.35%, 21.43%, 20.42%, 17.76%, and 18.14%, respectively. Furthermore, the expression of Capana10g001710 in 17C827 with light-green pools was significantly higher than that of 17C658 with green pools (Figure 4d, e). Finally, we analyzed the structure of the protein encoded by Capana10g001710 and found that a V-I substitution occurred at the 105th amino acid, which was also the last amino acid in the PP2C35 protein domain. This substitution caused the α-helix of the PP2C35 protein to turn into a β-fold in 17C827 (Supplementary Figure 6). Hence, Capana10g001710 was named CaPP2C35. Evolutionary analysis showed that the PP2C35 proteins belonged to the C subgroup of the PP2C protein family (Supplementary Figure 7).
Silencing of CaPP2C35 leads to green stripes on the surface of 17C827 immature fruits
To further explore the role of CaPP2C35, we silenced this gene in 17C827 (light-green immature fruits) via tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS). The immature fruits of 17C827 plants subjected to TRV2-CaPP2C35 silencing continued to have green stripes on their surfaces that were unable to transition into the light-green color of the surface of the fruits (Figure 5). Additionally, chlorophyll contents in the green and light-green exocarps were determined for fruits treated with TRV2-CaPP2C35. The results showed that chlorophyll content in the green exocarp was 0.248 mg/g·FW and that in the light-green exocarp was 0.095 mg/g·FW; thus, there was a significant difference between them. In addition, the green and light-green parts of the exocarp of CaPP2C35-silenced fruits were sampled for CaPP2C35 expression analysis. The exocarp of plants subjected to no treatment or TRV2 treatment was used as negative controls, and the exocarp of plants treated with TRV2-PDS was used as a positive control. The results revealed that the expression of CaPP2C35 in the light-green exocarp of fruits treated with TRV2-CaPP2C35 was not significantly different from that of the control treatments (Figure 6a). However, the expression of CaPP2C35 in the green exocarp of fruits treated with TRV2-CaPP2C35 was significantly lower than that in the light-green exocarp of the same fruits. In addition, the tissue-specific expression pattern of CaPP2C35 shows that it was specifically and highly expressed in the fruit tissues of 17C827 (Supplementary Figure 8). These results demonstrate that CaPP2C35 plays a role in the formation of light-green immature pepper fruits.
CaPP2C35 forms light-green immature fruits in 17C827 by affecting the accumulation of chlorophyll in the exocarp
To investigate the effect of CaPP2C35 down-regulation on the expression of genes involved in chlorophyll metabolism, we analyzed the expression of key chlorophyll metabolism-related genes in the green and light-green portions of the same fruits treated with TRV2-CaPP2C35. As shown in Figure 6b, the expression of the chlorophyll biosynthesis metabolism-related genes—GSA, HEMA1, CHLD, and CAO—in the green portion of the exocarp was significantly higher than that in the light-green portion. However, there was no significant difference in the expression of genes involved in chlorophyll degradation metabolism (Figure 6c). In addition, we analyzed the expression of chlorophyll metabolism-related genes between 17C827 and 17C658 (Supplementary Figure 9). The results showed that there was a significant difference in the expression of chlorophyll biosynthesis metabolism-related genes between 17C827 and 17C658 at day 9 or day 12, including the expression of GSA, HEMA1, CHLD, CHLH, CAO, CHLG, and CRD1, which were expressed at lower levels in 17C827 than in 17C658. Moreover, there was a significant difference in the expression of the chlorophyll degradation metabolism-related genes—SGR1, PAO, RCCR, and SGR2—between 17C827 and 17C658 at day 9 or day 12. In addition, the expression of some of them (RCCR and SGR2) was higher in 17C827 than in 17C658 at day 12. Finally, the co-expression analysis of CaPP2C35 and the key chlorophyll metabolism-related genes showed that the expression levels of the genes (HEMA1, CHLH, CAO, CRD1, and CHLD) involved in the chlorophyll biosynthetic pathway were negatively associated with that of CaPP2C35. In contrast, the expression level of SGR1, involved in chlorophyll degradation, was negatively correlated to that of CaPP2C35, whereas SGR2 was positively correlated to that of CaPP2C35 (Figure 7 and Supplementary Figure 10). These results indicate that CaPP2C35 may participate in the formation of light-green immature fruits in 17C827 by affecting the accumulation of chlorophyll in the exocarp.