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SUMMARY: Male sterility provides an efficient approach for commercial exploitation of heterosis. Despite more than 20 genic male sterile (GMS) mutants documented in pepper (Capsicum annuum L.), only two causal genes have been successfully identified. Here, a novel spontaneous recessive GMS mutant, designated msc‐3, is identified and characterized at both phenotypic and histological levels. Pollen abortion of msc‐3 mutant may be due to the delayed tapetum degradation, leading to the non‐degeneration of tetrads callosic wall. Then, a modified MutMap method and molecular marker linkage analysis were employed to fine mapping the msc‐3 locus, which was delimited to the ~139.91‐kb region harboring 10 annotated genes. Gene expression and structure variation analyses indicate the Capana10g000198, encoding a R2R3‐MYB transcription factor, is the best candidate gene for the msc‐3 locus. Expression profiling analysis shows the Capana10g000198 is an anther‐specific gene, and a 163‐bp insertion in the Capana10g000198 is highly correlated with the male sterile (MS) phenotype. Additionally, downregulation of Capana10g000198 in male fertile plants through virus‐induced gene silencing resulted in male sterility. Finally, possible regulatory relationships of the msc‐3 gene with the other two reported pepper GMS genes, msc‐1 and msc‐2, have been studied, and comparative transcriptome analysis reveals the expression of 16 GMS homologs are significantly downregulated in the MS anthers. Overall, our results reveal that Capana10g000198 is the causal gene underlying the msc‐3 locus, providing important theoretical clues and basis for further in‐depth study on the regulatory mechanisms of pollen development in pepper. Significance Statement: In this study, a novel pepper genic male sterile mutant and the causal gene were identified, and the regulatory mechanism for complex fertility development was preliminarily studied. Our findings provide important theoretical clues and basis for further in‐depth study on the regulatory mechanisms of pollen development in pepper. [ABSTRACT FROM AUTHOR]

Fruit colour is one of the most intuitive economic traits attracting consumers' attention for horticultural crops. Many genes associated with mature fruit colour have been identified in pepper, but genes influencing immature fruit colour are mainly unknown. In the present study, pepper inbred lines with light‐yellow (CSJ009) and green (CSJ010) immature fruit colour were used as female and male parents, respectively, to generate an F2 segregation population. Genetic analysis indicated that the light‐yellow immature fruit colour in CSJ009 is controlled by a single recessive gene, tentatively named as ly. The single‐nucleotide polymorphism (SNP)‐index analysis and genetic linkage analysis delimited the ly locus into a 6.51 Mbp region on chromosome 9, and four candidate genes Capana09g000680, Capana09g000671, Capana09g000715 and Capana09g000692 were identified in the linkage region. Nonsense mutation of APRR2 may be responsible for the low carotenoid content in immature CSJ009 fruits. These results lay a foundation for future immature fruit colour improvement via molecular genetics approaches and further identification of new immature fruit colour‐related genes in pepper. [ABSTRACT FROM AUTHOR]

Pungency is a unique characteristic of chili peppers (Capsicum spp.) caused by capsaicinoids. The evolutionary emergence of pungency is thought to be a derived trait within the genus Capsicum. However, it is not well-known how pungency has varied during Capsicum domestication and specialization. In this study, we applied a comparative metabolomics along with transcriptomics analysis to assess various changes between two peppers (a mildly pungent cultivated pepper BB3 and its hot progenitor chiltepin) at four stages of fruit development, focusing on pungency variation. A total of 558 metabolites were detected in two peppers. In comparison with chiltepin, capsaicinoid accumulation in BB3 was almost negligible at the early stage. Next, 412 DEGs associated with the capsaicinoid accumulation pathway were identified through coexpression analysis, of which 18 genes (14 TFs, 3 CBGs, and 1 UGT) were deemed key regulators due to their high coefficients. Based on these data, we speculated that downregulation of these hub genes during the early fruit developmental stage leads to a loss in pungency during Capsicum domestication (from chiltepin to BB3). Of note, a putative UDP-glycosyltransferase, GT86A1, is thought to affect the stabilization of capsaicinoids. Our results lay the foundation for further research on the genetic diversity of pungency traits during Capsicum domestication and specialization. [ABSTRACT FROM AUTHOR]

Key message: The up locus determining fruit orientation was fine-mapped into a region with a physical length of ~169.51 kb on chromosome P12 in pepper. Capana12g000958, encoding a developmentally regulated G protein 2, was proposed as the strongest candidate via sequence comparison and expression analysis. Fruit orientation is an important horticultural and domesticated trait, which is controlled by a single semi-dominant gene (up) in pepper. However, the gene underlying up locus has not yet been identified. In this study, the previously detected major QTL UP12.1 was firstly verified using a backcross population (n = 225) stem from the cross of BB3 (C. annuum) and its wild relative Chiltepin (C. annuum var. glabriusculum) using BB3 as the recurrent parent. Then, a large BC1F2 population (n = 1827) was used for recombinant screening to delimit the up locus into an interval with ~ 169.51 kb in length. Sequence comparison and expression analysis suggested that Capana12g000958, encoding a developmentally regulated G protein 2, was the most likely candidate gene for the up locus. There is no difference within the coding sequences of Capana12g000958 between BB3 and Chiltepin, while a SNP in the upstream of Capana12g000958 showed a complete correlation with the fruit orientation among a panel of 40 diverse pepper inbred lines. These findings will form a basis for gene isolation and reveal of genetic mechanism underlying the fruit orientation domestication in pepper. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas9 system is an efficient genome editing tool that possesses the outstanding advantages of simplicity and high efficiency. Genome-wide identification and specificity analysis of editing sites is an effective approach for mitigating the risk of off-target effects of CRISPR/Cas9 and has been applied in several plant species but has not yet been reported in pepper. In present study, we first identified genome-wide CRISPR/Cas9 editing sites based on the 'Zunla-1' reference genome and then evaluated the specificity of CRISPR/Cas9 editing sites through whole-genome alignment. Results showed that a total of 603,202,314 CRISPR/Cas9 editing sites, including 229,909,837 (~38.11%) NGG-PAM sites and 373,292,477 (~61.89%) NAG-PAM sites, were detectable in the pepper genome, and the systematic characterization of their composition and distribution was performed. Furthermore, 29,623,855 highly specific NGG-PAM sites were identified through whole-genome alignment analysis. There were 26,699,38 (~90.13%) highly specific NGG-PAM sites located in intergenic regions, which was 9.13 times of the number in genic regions, but the average density in genic regions was higher than that in intergenic regions. More importantly, 34,251 (~96.93%) out of 35,336 annotated genes exhibited at least one highly specific NGG-PAM site in their exons, and 90.50% of the annotated genes exhibited at least 4 highly specific NGG- PAM sites, indicating that the set of highly specific CRISPR/Cas9 editing sites identified in this study was widely applicable and conducive to the minimization of the off-target effects of CRISPR/Cas9 in pepper. [ABSTRACT FROM AUTHOR]

Key message: The pepper restorer-of-fertility (CaRf) gene was fine mapped based on conjoint analysis of recombinants and collinearity between the two pepper reference genomes. Capana06g003028, encoding an Rf-like PPR protein, was proposed as the strongest candidate for pepper CaRf based on sequence comparison and expression analysis. The cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) system not only provides an excellent model to dissect genetic interactions between the mitochondria and nucleus but also plays a vital role in high-efficiency hybrid seed production in crops including pepper (Capsicum spp.). Although two important CMS candidate genes, orf507 and Ψatp6-2, have previously been suggested, the pepper Rf gene (CaRf) has not yet been isolated. In this study, a high-density genetic map comprising 7566 SNP markers in 1944 bins was first constructed with the array genotyping results from 317 F2 individuals. Based on this map, the CaRf gene was preliminarily mapped to a region of 1.15 Mb in length at the end of chromosome P6. Then, by means of a conjoint analysis of recombinants and collinearity between the two pepper reference genomes, an important candidate interval with 270.10 kb in length was delimited for CaRf. Finally, Capana06g003028, which encodes an Rf-like PPR protein, was proposed as the strongest candidate for CaRf based on sequence analysis and expression characteristics in sterile and fertile plants. The high-density genetic map and fine mapping results provided here lay a foundation for the application of molecular breeding, as well as cloning and functional analysis of CaRf, in pepper. [ABSTRACT FROM AUTHOR]