Functional identification of DNA demethylase gene CaROS1 in pepper (Capsicum annuum L.) involved in salt stress.

Pepper, which is a widely cultivated important vegetable, is sensitive to salt stress, and the continuous intensification of soil salinization has affected pepper production worldwide. However, genes confer to salt tolerance are rarely been cloned in pepper. Since the REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylase that plays a crucial regulatory role in plants in response to various abiotic stresses, including salt stress. We cloned a ROS1 gene in pepper, named CaROS1 (LOC107843637). Bioinformatic analysis showed that the CaROS1 protein contains the HhH-GPD glycosylase and RRM_DME domains. qRT-PCR analyses showed that the CaROS1 was highly expressed in young and mature fruits of pepper and rapidly induced by salt stress. Functional characterization of the CaROS1 was performed by gene silencing in pepper and overexpressing in tobacco, revealed that the CaROS1 positively regulates salt tolerance ability. More detailly, CaROS1-silenced pepper were more sensitive to salt stress, and their ROS levels, relative conductivity, and malondialdehyde content were significantly higher in leaves than those of the control plants. Besides, CaROS1-overexpressing tobacco plants were more tolerant to salt stress, with a higher relative water content, total chlorophyll content, and antioxidant enzyme activity in leaves compared to those of WT plants during salt stress. These results revealed the CaROS1 dose play a role in salt stress response, providing the theoretical basis for salt tolerance genetic engineering breeding in pepper.

A point mutation in MC06g1112 encoding FLOWERING LOCUS T decreases the first flower node in bitter gourd (Momordica charantia L.).

In Cucurbitaceae crops, the first flower node (FFN) is an important agronomic trait which can impact the onset of maturity, the production of female flowers, and yield. However, the gene responsible for regulating FFN in bitter gourd is unknown. Here, we used a gynoecious line (S156G) with low FFN as the female parent and a monoecious line (K8-201) with high FFN as the male parent to obtain F1 and F2 generations. Genetic analysis indicated that the low FFN trait was incompletely dominant over the high FFN trait. A major quantitative trait locus (QTL)-Mcffn and four minor effect QTLs-Mcffn1.1, Mcffn1.2, Mcffn1.3, and Mcffn1.4 were detected by whole-genome re-sequencing-based QTL mapping in the S156G×K8-201 F2 population (n=234) cultivated in autumn 2019. The Mcffn locus was further supported by molecular marker-based QTL mapping in three S156G×K8-201 F2 populations planted in autumn 2019 (n=234), autumn 2020 (n=192), and spring 2022 (n=205). Then, the Mcffn locus was fine-mapped into a 77.98-kb physical region on pseudochromosome MC06 using a large S156G×K8-201 F2 population (n=2,402). MC06g1112, which is a homolog of FLOWERING LOCUS T (FT), was considered as the most likely Mcffn candidate gene according to both expression and sequence variation analyses between parental lines. A point mutation (C277T) in MC06g1112, which results in a P93S amino acid mutation between parental lines, may be responsible for decreasing FFN in bitter gourd. Our findings provide a helpful resource for the molecular marker-assisted selective breeding of bitter gourd.

Fine mapping and gene silencing pinpoint Capana10g002229 as a strong candidate gene regulating the deciduous character of ripe pepper fruit (Capsicum spp.).

KEY MESSAGE: The pepper S locus, which controls the deciduous character of ripe fruit, was first fine mapped into an interval with a physical length of ~ 38.03 kb on chromosome P10. Capana10g002229, encoding a polygalacturonase, was proposed as a strong candidate gene based on sequence comparison, expression pattern analysis and virus-induced gene silencing (VIGS). The deciduous character of ripe fruit, which is controlled by the dominant S locus, is a domesticated trait with potential value in the pepper processing industry (Capsicum spp.). However, the gene associated with the S locus has not been identified. Here, one major QTL designated S10.1 was detected by using the F2 population (n = 155) derived from BA3 (Capsicum annuum) × YNXML (Capsicum frutescens) and was further verified in an intraspecific backcross population (n = 254) derived from the cross between BB3 (C. annuum) and its wild relative Chiltepin (C. annuum var. glabriusculum) with BB3 as the recurrent parent. Then, a large BC1F2 population derived from the self-pollination of BB3 × (BB3 × Chiltepin) individuals and comprising 4217 individuals was used to screen the recombinants, and the S locus was ultimately delimited into a 38.03-kb region on chromosome P10 harbouring four annotated genes. Capana10g002229, encoding a polygalacturonase (PG), was proposed as the best candidate gene for S based on sequence comparison and expression pattern analyses. Downregulation of Capana10g002229 in fruits through VIGS significantly delayed fruit softening and abscission from the fruit-receptacle junction. Taken together, the results show that Capana10g002229 could be regarded as a strong candidate gene associated with the S locus in pepper. These findings not only lay a foundation for deciphering the molecular mechanisms underlying pepper domestication but also provide a strategy for genetic improvement of the deciduous character of ripe fruit using a marker-assisted selection approach.

Fine-mapping and candidate gene analysis of the Mcgy1 locus responsible for gynoecy in bitter gourd (Momordica spp.).

KEY MESSAGE: The Mcgy1 locus responsible for gynoecy was fine-mapped into a 296.94-kb region, in which four single-nucleotide variations and six genes adjacent to them might be associate with sex differentiation in bitter gourd. Gynoecy plays an important role in high-efficiency hybrid seed production, and gynoecious plants are excellent materials for dissecting sex differentiation in Cucurbitaceae crop species, including bitter gourd. However, the gene responsible for gynoecy in bitter gourd is unknown. Here, we first identified a gynoecy locus designated Mcgy1 using the F2 population (n = 291) crossed from the gynoecious line S156G and the monoecious line K8-201 via bulked segregant analysis with whole-genome resequencing (BSA-seq) and molecular marker linkage analysis. Then, a large S156G × K8-201 F2 population (n = 5,656) was used for fine-mapping to delimit the Mcgy1 locus into a 296.94-kb physical region on pseudochromosome MC01, where included 33 annotated genes different from any homologous gynoecy genes previously reported in Cucurbitaceae species. Within this region, four underlying single-nucleotide variations (SNVs) that might cause gynoecy were identified by multiple genomic sequence variation analysis, and their six neighbouring genes were considered as potential candidate genes for Mcgy1. Of these, only MC01g1681 showed a significant differential expression at two-leaf developmental stage between S156G and its monoecious near-isogenic line S156 based on RNA sequencing (RNA-seq) and qRT-PCR analyses. In addition, transcriptome analysis revealed 21 key differentially expressed genes (DEGs) and possible regulatory pathways of the formation of gynoecy in bitter gourd. Our findings provide a new clue for researching on gynoecious plants in Cucurbitaceae species and a theoretical basis for breeding gynoecious bitter gourd lines by the use of molecular markers-assisted selection.

A 163-bp insertion in the Capana10g000198 encoding a MYB transcription factor causes male sterility in pepper (Capsicum annuum L.).

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.

MC03g0810, an Important Candidate Gene Controlling Black Seed Coat Color in Bitter Gourd (Momordica spp.).

Seed coat color is one of the most intuitive phenotypes in bitter gourd (Momordica spp.). Although the inheritance of the seed coat color has been reported, the gene responsible for it is still unknown. This study used two sets of parents, representing, respectively, the intersubspecific and intraspecific materials of bitter gourd, and their respective F1 and F2 progenies for genetic analysis and primary mapping of the seed coat color. A large F2:3 population comprising 2,975 seedlings from intraspecific hybridization was used to fine-map the seed coat color gene. The results inferred that a single gene, named McSC1, controlled the seed coat color and that the black color was dominant over the yellow color. The McSC1 locus was mapped to a region with a physical length of ∼7.8 Mb and 42.7 kb on pseudochromosome 3 via bulked segregant analysis with whole-genome resequencing (BSA-seq) and linkage analysis, respectively. Subsequently, the McSC1 locus was further fine-mapped to a 13.2-kb region containing only one candidate gene, MC03g0810, encoding a polyphenol oxidase (PPO). Additionally, the variations of MC03g0810 in the 89 bitter gourd germplasms showed a complete correlation with the seed coat color. Expression and PPO activity analyses showed a positive correlation between the expression level of MC03g0810 and its product PPO and the seed coat color. Therefore, MC03g0810 was proposed as the causal gene of McSC1. Our results provide an important reference for molecular marker-assisted breeding based on the seed coat color and uncover molecular mechanisms of the seed coat color formation in bitter gourd.

Fine mapping and candidate gene analysis of the up locus determining fruit orientation in pepper (Capsicum spp.).

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.

Fine mapping of restorer-of-fertility gene based on high-density genetic mapping and collinearity analysis in pepper (Capsicum annuum L.).

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.