Recurrent excision of a hAT-like transposable element in CmAPRR2 leads to the "shooting star" melon phenotype.

The external appearance of fruit commodities is an essential trait that has profound effects on consumer preferences. A natural melon variety, characterized by an uneven and patchy arrangement of dark green streaks and spots on the white-skinned rind, resembles shooting stars streaking across the sky; thus, this variety is called "Shooting Star" (SS). To investigate the mechanism underlying the SS melon rind pattern, we initially discovered that the variegated dark green color results from chlorophyll accumulation on the white skin. We then constructed a segregation population by crossing a SS inbred line with a white rind (WR) inbred line and used bulk segregant analysis (BSA) revealed that the SS phenotype is controlled by a single dominant gene, CmAPRR2, which has been previously confirmed to determine dark green coloration. Further genomic analysis revealed a hAT-like transposable element (TE) inserted in CmAPRR2. This TE in CmAPRR2 is recurrently excised from rind tissues, activating the expression of CmAPRR2. This activation promotes the accumulation of chlorophyll, leading to the variegated dark green color on the rind, and ultimately resulting in the SS rind phenotype. Therefore, we propose that the SS phenotype results from the recurrent excision of the hAT-like TE in CmAPRR2.

CaAOS as a hub gene based on physiological and transcriptomic analyses of cold-resistant and cold-sensitive pepper cultivars.

The yield and quality of pepper are considerably influenced by the cold conditions. Herein, we performed morphological, physiological and transcriptomic analyses by using two pepper seedlings, '2379' (cold-resistant) and '2380' (cold-sensitive). Briefly, 60 samples from each cultivar were analyzed at four distinct time points (0, 6, 24 and 48 h) at 5 °C in darkness. The physiological indices and activities of enzymes exhibited marked differences between the two cultivars. Transcriptomic analysis indicated that, compared to the control group, 11,415 DEGs were identified in '2379' and '2380' at 24 h. In the early stage, the number of DEGs in '2379' was 5.68 times higher than that in '2380', potentially explaining the observed differences in tolerance to colds. Processes such as protein targeting to membranes, jasmonic acid (JA)-mediated signalling, cold response and abscisic acid-activated signalling were involved. Subsequently, we identified a hub gene, CaAOS, that is involved in JA biosynthesis, positively influences cold tolerance and is a target of CaMYC2. Variations in the GC-motif of the CaAOS's promoter may influence the expression levels of CaAOS under cold treatment. The result of this study may lead to the development of more effective strategies for enhancing cold tolerance, potentially benefitting pepper breeding in cold regions.

Development of a 45K pepper GBTS liquid-phase gene chip and its application in genome-wide association studies.

Introduction: Pepper (Capsicum spp.) is a vegetable that is cultivated globally and has undergone extensive domestication, leading to a significant diversification in its agronomic traits. With the advancement of genomics in pepper and the reduction in sequencing costs, the high-throughput detection of single nucleotide polymorphisms (SNPs) and small insertions-deletions (indels) has become increasingly critical for analyzing pepper germplasms and improving breeding programs. As a result, there is a pressing need for a cost-effective, high-throughput, and versatile technique suitable for both foreground and background selection in pepper breeding. Methods: In the present study, Python-based web scraping scripts were utilized to systematically extract data from published literatures and relevant sequence databases focusing on pepper genomes. Subsequent to data extraction, SNPs and indels were meticulously identified and filtered. This process culminated in the delineation of core polymorphic sites, which were instrumental in the development of specific probes. Following this, comprehensive phenotypic and genotypic analyses were conducted on a diverse collection of 420 pepper germplasms. Concurrently, a genome-wide association study (GWAS) was conducted to elucidate the genetic determinants of helical fruit shape in peppers. Results: In this study, a 45K pepper Genotyping-By-Target-Sequencing (GBTS) liquid-phase gene chip was developed on the GenoBaits platform. This chip is composed of 45,389 probes, of which 42,535 are derived from core polymorphic sites (CPS) in the background genetic landscape, while 2,854 are associated with foreground agronomic traits, spanning across 43 traits. The CPS probes are spaced at an average interval of 68 Kb. We have assessed the performance of this chip on 420 pepper germplasms, with successful capture of target DNA fragments by 45,387 probes. Furthermore, the probe capture ratio surpassed 70% in 410 of the 420 germplasms tested. Using this chip, we have efficiently genotyped 273 germplasms for spiciness levels and elucidated the genetic relationships among 410 pepper germplasms. Our results allowed for precise clustering of sister lines and C. chinense germplasms. In addition, through a GWAS for helical fruit shape, we identified three quantitative trait loci (QTLs): heli2.1, heli11.1, and heli11.2. Within the heli11.1 QTL, a gene encoding the tubulin alpha chain was identified, suggesting its potential role in the helical growth pattern of pepper fruits. Discussion: In summary, the 45K pepper GBTS liquid-phase gene chip offers robust detection of polymorphic sites and is a promising tool for advancing research into pepper germplasm and the breeding of new pepper varieties.

Quantitative Trait Loci Mapping and Comparative Transcriptome Analysis of Fruit Weight (FW) in Watermelon (Citrullus lanatus L.).

The watermelon (Citrullus lanatus L.) holds substantial economic value as a globally cultivated horticultural crop. However, the genetic architecture of watermelon fruit weight (FW) remains poorly understood. In this study, we used sh14-11 with small fruit and N14 with big fruit to construct 100 recombinant inbred lines (RILs). Based on whole-genome resequencing (WGR), 218,127 single nucleotide polymorphisms (SNPs) were detected to construct a high-quality genetic map. After quantitative trait loci (QTL) mapping, a candidate interval of 31-38 Mb on chromosome 2 was identified for FW. Simultaneously, the bulked segregant analysis (BSA) in the F2 population corroborated the identification of the same interval, encompassing the homologous gene linked to the known FW-related gene fas. Additionally, RNA-seq was carried out across 11 tissues from sh14-11 and N14, revealing expression profiles that identified 1695 new genes and corrected the annotation of 2941 genes. Subsequent differential expression analysis unveiled 8969 differentially expressed genes (DEGs), with 354 of these genes exhibiting significant differences across four key developmental stages. The integration of QTL mapping and differential expression analysis facilitated the identification of 14 FW-related genes, including annotated TGA and NAC transcription factors implicated in fruit development. This combined approach offers valuable insights into the genetic basis of FW, providing crucial resources for enhancing watermelon cultivation.

A 14-3-3 Protein Ca16R Acts Positively in Pepper Immunity against Ralstonia solanacearum by Interacting with CaASR1.

Although 14-3-3 proteins have been implicated in plant growth, development, and stress response, their roles in pepper immunity against R. solanacearum remain poorly understood. In this study, a 14-3-3-encoding gene in pepper, Ca16R, was found to be upregulated by R. solanacearum inoculation (RSI), its silencing significantly reduced the resistance of pepper plants to RSI, and its overexpression significantly enhanced the resistance of Nicotiana benthamiana to RSI. Consistently, its transient overexpression in pepper leaves triggered HR cell death, indicating that it acts positively in pepper immunity against RSI, and it was further found to act positively in pepper immunity against RSI by promoting SA but repressing JA signaling. Ca16R was also found to interact with CaASR1, originally using pull-down combined with a spectrum assay, and then confirmed using bimolecular fluorescence complementation (BiFC) and a pull-down assay. Furthermore, we found that CaASR1 transient overexpression induced HR cell death and SA-dependent immunity while repressing JA signaling, although this induction and repression was blocked by Ca16R silencing. All these data indicate that Ca16R acts positively in pepper immunity against RSI by interacting with CaASR1, thereby promoting SA-mediated immunity while repressing JA signaling. These results provide new insight into mechanisms underlying pepper immunity against RSI.

Characterization of the Molecular Events Underlying the Establishment of Axillary Meristem Region in Pepper.

Plant architecture is a major motif of plant diversity, and shoot branching patterns primarily determine the aerial architecture of plants. In this study, we identified an inbred pepper line with fewer lateral branches, 20C1734, which was free of lateral branches at the middle and upper nodes of the main stem with smooth and flat leaf axils. Successive leaf axil sections confirmed that in normal pepper plants, for either node n, Pn (Primordium n) < 1 cm and Pn+1 < 1 cm were the critical periods between the identification of axillary meristems and the establishment of the region, whereas Pn+3 < 1 cm was fully developed and formed a completely new organ. In 20C1734, the normal axillary meristematic tissue region establishment and meristematic cell identity confirmation could not be performed on the axils without axillary buds. Comparative transcriptome analysis revealed that "auxin-activated signaling pathway", "response to auxin", "response to abscisic acid", "auxin biosynthetic process", and the biosynthesis of the terms/pathways, such as "secondary metabolites", were differentially enriched in different types of leaf axils at critical periods of axillary meristem development. The accuracy of RNA-seq was verified using RT-PCR for some genes in the pathway. Several differentially expressed genes (DEGs) related to endogenous phytohormones were targeted, including several genes of the PINs family. The endogenous hormone assay showed extremely high levels of IAA and ABA in leaf axils without axillary buds. ABA content in particular was unusually high. At the same time, there is no regular change in IAA level in this type of leaf axils (normal leaf axils will be accompanied by AM formation and IAA content will be low). Based on this, we speculated that the contents of endogenous hormones IAA and ABA in 20C1734 plant increased sharply, which led to the abnormal expression of genes in related pathways, which affected the formation of Ams in leaf axils in the middle and late vegetative growth period, and finally, nodes without axillary buds and side branches appeared.

Transcriptomic and Proteomic Analyses of Celery Cytoplasmic Male Sterile Line and Its Maintainer Line.

Male sterility is a common phenomenon in the plant kingdom and based on the organelles harboring the male-sterility genes, it can be classified into the genic male sterility (GMS) and the cytoplasmic male sterility (CMS). In every generation, CMS can generate 100% male-sterile population, which is very important for the breeders to take advantage of the heterosis and for the seed producers to guarantee the seed purity. Celery is a cross-pollinated plant with the compound umbel type of inflorescence which carries hundreds of small flowers. These characteristics make CMS the only option to produce the commercial hybrid celery seeds. In this study, transcriptomic and proteomic analyses were performed to identify genes and proteins that are associated with celery CMS. A total of 1255 differentially expressed genes (DEGs) and 89 differentially expressed proteins (DEPs) were identified between the CMS and its maintainer line, then 25 genes were found to differentially expressed at both the transcript and protein levels. Ten DEGs involved in the fleece layer and outer pollen wall development were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, most of which were down-regulated in the sterile line W99A. These DEGs and DEPs were mainly enriched in the pathways of "phenylpropanoid/sporopollenin synthesis/metabolism", "energy metabolism", "redox enzyme activity" and "redox processes". Results obtained in this study laid a foundation for the future investigation of mechanisms of pollen development as well as the reasons for the CMS in celery.

Mapping of the AgWp1 gene for the white petiole in celery (Apium graveolens L.).

Celery (Apium graveolens L.) is one of the most popular leafy vegetables worldwide. The main edible parts of celery are the leaf blade and especially the petiole, which typically has a white, green and red color. To date, there are very few reports about the inheritance and gene cloning of celery petiole color. In this study, bulked segregant analysis-sequencing (BSA-Seq) and fine mapping were conducted to delimit the white petiole (wp1) loci into a 668.5-kb region on Chr04. In this region, AgWp1 is a homolog of a DAG protein in Antirrhinum majus and a MORF9 protein in Arabidopsis, and both proteins are involved in chloroplast development. Sequencing alignment shows that there is a 27-bp insertion in the 3'-utr region in AgWp1 in the white petiole. Gene expression analysis indicated that the expression level of AgWp1 in the green petiole was much higher than that in the white petiole. Further cosegregation revealed that the 27-bp insertion was completely cosegregated with the petiole color in 45 observed celery varieties. Therefore, AgWp1 was considered to be the candidate gene controlling the white petiole in celery. Our results could not only improve the efficiency and accuracy of celery breeding but also help in understanding the mechanism of chlorophyll synthesis and chloroplast development in celery.

Host-induced gene silencing of PcCesA3 and PcOSBP1 confers resistance to Phytophthora capsici in Nicotiana benthamiana through NbDCL3 and NbDCL4 processed small interfering RNAs.

Host-induced gene silencing (HIGS) is a RNA-based system depend on the biological macromolecules generated in plants to control diseases. However, the effector proteins active in the HIGS are uncertain, which impedes its further application, especially for oomycete that lack efficient HIGS targets. Phytophthora capsici is an important oomycete causes blight in over 70 crops. Here, we comprehensively screened efficient HIGS vectors targeting PcCesA3 or PcOSBP1 in P. capsici to better control it and explore the characteristics of efficient HIGS vectors. Among the 26 vectors with different lengths and structures, we found that hairpin vectors with a 70 nt loop and ~ 500 bp stem showed the highest control efficacy, with the expressing of the screened vectors, the infection and fertility of P. capsici were greatly inhibited in transgenic Nicotiana benthamiana. Based on these efficient vectors, we demonstrated that the amount of HIGS vector generated small interfering RNAs (siRNAs) was positively related to gene silencing efficiency and resistance, and that NbDCL3 and NbDCL4 were the key effectors producing siRNAs. This work discovers the principles for efficient HIGS vectors design, and elucidates the molecular mechanism of HIGS, which could benefit the control of many other plant diseases based on HIGS.

Loci underlying leaf agronomic traits identified by re-sequencing celery accessions based on an assembled genome.

Celery is one of the most popular vegetables in the world. The main edible parts of celery are the leaf blade and petiole. The celery petiole is usually green, red, or white, with a hollow or solid pith. However, the loci/genes controlling these petiole-related traits have not been reported. In this study, we present a chromosome-level celery genome assembly with a total size of 3.339 Gb. Simultaneous bursts of long-terminal repeats (78.43%) contributed greatly to the large genome size. Re-sequencing and population structure analysis of 79 celery accessions revealed that they could be divided into Chinese celery and Western celery. By combining genome-wide association studies (GWAS) and mapping data, we located the hollow petiole (hp) loci in an 807.6-kb region on chromosome 11. This study provides valuable resources for genetic research on celery and is also helpful for the identification and cloning of genes controlling leaf agronomic traits in celery.

Identification of the Regulatory Genes of UV-B-Induced Anthocyanin Biosynthesis in Pepper Fruit.

Fruit peels of certain pepper (Capsicum annum L.) varieties accumulate a large amount of anthocyanins and exhibit purple color under medium-wave ultraviolet (UV-B) conditions, which severely impacts the commodity value of peppers. However, the regulatory mechanism of the above process has not been well studied so far. To explore which key genes are involved in this regulatory mechanism, pepper variety 19Q6100, the fruit peels of which turn purple under UV-B conditions, was investigated in this study. Transcription factors with expression levels significantly impacted by UV-B were identified by RNA-seq. Those genes may be involved in the regulation of UV-B-induced anthocyanin biosynthesis. Yeast one-hybrid results revealed that seven transcription factors, CabHLH143, CaMYB113, CabHLH137, CaMYBG, CaWRKY41, CaWRKY44 and CaWRKY53 directly bound to the putative promotor regions of the structural genes in the anthocyanin biosynthesis pathway. CaMYB113 was found to interact with CabHLH143 and CaHY5 by yeast two-hybrid assay, and those three genes may participate collaboratively in UV-B-induced anthocyanin biosynthesis in pepper fruit. Virus-induced gene silencing (VIGS) indicated that fruit peels of CaMYB113-silenced plants were unable to turn purple under UV-B conditions. These findings could deepen our understanding of UV-B-induced anthocyanin biosynthesis in pepper.

Fruit shape loci sun, ovate, fs8.1 and their interactions affect seed size and shape in tomato.

Seed size and shape are not only critical for plant reproduction and dispersal, but also important agronomic traits. Tomato fruit shape loci sun, ovate and fs8.1 regulate the morphology of fruit, flower, leaf and stem, and recently their functions in seed morphogenesis have also been noticed. However, mechanism underlying seed morphology variation has not been systematically investigated yet. Thus, using the near isogenic lines (NILs) harboring one, two or three of the fruit shape loci, histological, physiological and transcriptional bases of seed morphology change have been studied. sun and ovate showed potential abilities in decreasing seed size, whereas, fs8.1 had a potential ability in increasing this parameter. Interactions between two loci and the interaction among three loci all led to significant decrease of seed size. All the loci significantly down-regulated seed shape index (SSI), except for sun/fs8.1 double NIL, which resulted in the reductions in both seed length and width and finally led to a decreased trend of SSI. Histologically, seed morphological changes were mainly attributed to the cell number variations. Transcriptional and physiological analyses discovered that phytohormone-, cytoskeleton- as well as sugar transportation- and degradation-related genes were involved in the regulation of seed morphology by the fruit shape loci.

Mapping of CaPP2C35 involved in the formation of light-green immature pepper (Capsicum annuum L.) fruits via GWAS and BSA.

KEY MESSAGE: Genome-wide association study, bulked segregant analysis, and genetic analysis delimited the LG locus controlling light-green immature pepper fruits into a 35.07 kbp region on chromosome 10. A strong candidate gene, CaPP2C35, was identified in this region. In pepper (Capsicum annuum L.), the common colors of immature fruits are yellowish white, milky yellow, green, purple, and purplish black. Genes related to dark green, white, and purple immature fruits have been cloned; however, only a few studies have investigated light-green immature fruits. Here, we performed a genetic study using light-green (17C827) and green (17C658) immature fruits. The light-green color of immature fruits was controlled by a single locus-dominant genetic trait compared with the green color of immature fruits. We also performed a genome-wide association study and bulked segregant analysis of immature-fruit color and mapped the LG locus to a 35.07 kbp region on chromosome 10. Only one gene, Capana10g001710, was found in this region. A G-A substitution occurred at the 313th base of the Capana10g001710 coding sequence in 17C827, resulting in the conversion of the α-helix of its encoded PP2C35 protein into a ß-fold. The expression of Capana10g001710 (termed CaPP2C35) in 17C827 was significantly higher than in 17C658. Silencing CaPP2C35 in 17C827 resulted in an increase in chlorophyll content in the exocarp and the appearance of green stripes on the surface of the fruit. These results indicate that CaPP2C35 may be involved in the formation of light-green immature fruits by regulating the accumulation of chlorophyll content in the exocarp. Thus, these findings lay the foundation for further studies and genetic improvement of immature-fruit color in pepper.

Complete Mitochondrial Genome Sequence and Identification of a Candidate Gene Responsible for Cytoplasmic Male Sterility in Celery (Apium graveolens L.).

Celery (Apium graveolens L.) is an important leafy vegetable worldwide. The development of F1 hybrids in celery is highly dependent on cytoplasmic male sterility (CMS) because emasculation is difficult. In this study, we first report a celery CMS, which was found in a high-generation inbred line population of the Chinese celery "tanzhixiangqin". Comparative analysis, following sequencing and assembly of the complete mitochondrial genome sequences for this celery CMS line and its maintainer line, revealed that there are 21 unique regions in the celery CMS line and these unique regions contain 15 ORFs. Among these ORFs, only orf768a is a chimeric gene, consisting of 1497 bp sequences of the cox1 gene and 810 bp unidentified sequences located in the unique region, and the predicted protein product of orf768a possesses 11 transmembrane domains. In summary, the results of this study indicate that orf768a is likely to be a strong candidate gene for CMS induction in celery. In addition, orf768a can be a co-segregate marker, which can be used to screen CMS in celery.

Hairiness Gene Regulated Multicellular, Non-Glandular Trichome Formation in Pepper Species.

Trichomes are unicellular or multicellular epidermal structures that play a defensive role against environmental stresses. Although unicellular trichomes have been extensively studied as a mechanistic model, the genes involved in multicellular trichome formation are not well understood. In this study, we first classified the trichome morphology structures in Capsicum species using 280 diverse peppers. We cloned a key gene (Hairiness) on chromosome 10, which mainly controlled the formation of multicellular non-glandular trichomes (types II, III, and V). Hairiness encodes a Cys2-His2 zinc-finger protein, and virus-induced gene silencing of the gene resulted in a hairless phenotype. Differential expression of Hairiness between the hairiness and hairless lines was due to variations in promoter sequences. Transgenic experiments verified the hypothesis that the promoter of Hairiness in the hairless line had extremely low activity causing a hairless phenotype. Hair controlled the formation of type I glandular trichomes in tomatoes, which was due to nucleotide differences. Taken together, our findings suggest that the regulation of multicellular trichome formation might have similar pathways, but the gene could perform slightly different functions in crops.

Fine mapping of the Ca3GT gene controlling anthocyanin biosynthesis in mature unripe fruit of Capsicum annuum L.

KEY MESSAGE: The anthocyanin biosynthesis gene Ca3GT was fine-mapped in a 110.5-kb region through a map-based cloning strategy. Gene expression and promoter analyses confirmed the strong candidate gene Capana10g001978. Pepper (Capsicum annum L.) fruit can be dark green, green, light green, purple, yellow, or ivory at the juvenile stage. Anthocyanins are responsible for fruit color formation in mature unripe pepper fruit, and transient accumulation of anthocyanins is the main problem in breeding pepper plants with mature purple fruit. Only a few genes controlling this trait have been cloned. The present study aimed to map and identify an anthocyanin biosynthesis gene from pepper using an F2 population derived from a cross between line '17C3808' (purple mature unripe fruit) and line '17C3807' (green mature unripe fruit). The trait was mapped on a 110.5-kb interval between markers SSR18213 and SSR18228 on chromosome 10. There were three open reading frames in this region; Capana10g001978 was predicted in this region as markers CAPS-78-708 and InDel146 co-segregated with it. Capana10g001978 is a structural gene encoding the GTB transcription factor involved in the biosynthesis of anthocyanins. Comparing parental sequences, two base mutations were identified in the exon of Capana10g001978, at positions + 528 bp and + 708 bp, which resulted in changes in the 176th and 236th amino acid residues, from glutamine (CAA) to histidine (CAC), causing a nonsense mutation (from CAG to CAA). Additionally, Capana10g001978 was highly expressed in the pericarp of mature, unripe pepper fruit. There were four single nucleotide polymorphisms, three sequence deletions, and one sequence insertion in the promoter region of purple, mature, and unripe pepper fruit, leading to the formation of a W-box and a GT1-motif. Thus, Capana10g001978 is a strong candidate gene of Ca3GT involved in anthocyanin biosynthesis in mature unripe pepper fruit. These results provide important information regarding the isolation and characterization of Ca3GT, and they are the starting point for studying the regulatory pathway responsible for anthocyanin biosynthesis in pepper.

Transcription of lncRNA ACoS-AS1 is essential to trans-splicing between SlPsy1 and ACoS-AS1 that causes yellow fruit in tomato.

Phytoene synthase (PSY) has been considered as an important regulatory enzyme in carotenoids biosynthesis pathway. Previous study finds that the yellow fruit in Solanum lycopersicum var. cerasiforme accession PI 114490 is caused by loss-of-function of SlPSY1 due to trans-splicing between SlPsy1 and an unknown gene transcribed from neighbour opposite strand DNA of SlPsy1. The genomic DNA sequences of SlPsy1 between red and yellow-fruited tomato lines have one single-nucleotide polymorphism (SNP) in the fourth intron and one SSR in the intergenic region. In the current study, the cause of trans-splicing event was further investigated. The data showed that the previously defined unknown gene was a putative long non-coding RNA ACoS-AS1 with three variants in many yellow-fruited tomato lines. The intronic SNP and intergenic SSR were tightly associated with trans-splicing event SlPsy1-ACoS-AS1. However, transgenic tomato lines carrying the genomic DNA of SlPsy1 from PI 114490 did not generate transcripts of ACoS-AS1and SlPsy1-ACoS-AS1 suggesting that only the intronic SNP could not cause the trans-splicing event. Over-expression of SlPsy1-ACoS-AS1 in red-fruited tomato line M82 did not have any phenotype change while over-expression of wild type SlPsy1 resulted in altered leaf colour. Sub-cellular localization analysis showed that SlPSY1-ACoS-AS1 could not enter plastids where SlPSY1 has its enzyme activity. Mutation of ACoS-AS1 in PI 114490 generated by CRISPR/Cas9 techniques resulted in red fruits implying that ACoS-AS1 was essential to trans-splicing event SlPsy1-ACoS-AS1. The results obtained here will extend knowledge to understand the mechanism of trans-splicing event SlPsy1-ACoS-AS1 and provide additional information for the regulation of carotenoids biosynthesis.

DNA methylation is involved in the regulation of pepper fruit ripening and interacts with phytohormones.

There is growing evidence to suggest that epigenetic tags, especially DNA methylation, are critical regulators of fruit ripening. To examine whether this is the case in sweet pepper (Capsicum annuum) we conducted experiments at the transcriptional, epigenetic, and physiological levels. McrBC PCR, bisulfite sequencing, and real-time PCR demonstrated that DNA hypomethylation occurred in the upstream region of the transcription start site of some genes related to pepper ripening at the turning stage, which may be attributed to up-regulation of CaDML2-like and down-regulation of CaMET1-like1, CaMET1-like2, CaCMT2-like, and CaCMT4-like. Silencing of CaMET1-like1 by virus-induced gene silencing led to DNA hypomethylation, increased content of soluble solids, and accumulation of carotenoids in the fruit, which was accompanied by changes in expression of genes involved in capsanthin/capsorubin biosynthesis, cell wall degradation, and phytohormone metabolism and signaling. Endogenous ABA increased during fruit ripening, whereas endogenous IAA showed an opposite trend. No ethylene signal was detected during ripening. DNA hypomethylation repressed the expression of auxin and gibberellin biosynthesis genes as well as cytokinin degradation genes, but induced the expression of ABA biosynthesis genes. In mature-green pericarp, exogenous ABA induced expression of CaDML2-like but repressed that of CaCMT4-like. IAA treatment promoted the transcription of CaMET1-like1 and CaCMT3-like. Ethephon significantly up-regulated the expression of CaDML2-like. Treatment with GA3 and 6-BA showed indistinct effects on DNA methylation at the transcriptional level. On the basis of the results, a model is proposed that suggests a high likelihood of a role for DNA methylation in the regulation of ripening in the non-climacteric pepper fruit.

Phenotypic, genetic, and molecular function of msc-2, a genic male sterile mutant in pepper (Capsicum annuum L.).

KEY MESSAGE: Bulked segregant analysis and fine mapping delimited the pepper genic male sterile (msc-2) locus into a 336 kb region on chromosome 5. A strong candidate gene, Capana05g000766, a homolog of AtMS1, was indentified in this region. Genic male sterility (msc-2) is used to produce hybrid seeds in Northern China. However, no co-segregated markers have been reported or candidate genes controlling this trait have been cloned. Here, bulked segregant analysis and genotyping of an F2 population and a 18Q5431AB line were employed to fine map msc-2, which was delimited to a 336 kb region. In this region, Capana05g000766 was a homolog of AtMS1, which encodes a plant homeodomain finger involved in tapetum development. A "T" deletion in the Capana05g000766 locus leads to a premature stop codon, which may cause a loss-of-function mutation. Real-time PCR analysis revealed that Capana05g000766 was an anther-specific gene and down-regulation of the gene resulted in male sterility. Therefore, Capana05g000766 was identified as the strongest candidate gene for the msc-2 locus. Allelism tests showed that msc-1 and msc-2 were nonallelic, and bimolecular fluorescence complementation analysis indicated that the two genes did not interact directly with each other at the protein level. As msc-1 and msc-2 are homologs of AtDYT1 and AtMS1 in Arabidopsis, they may play similar roles in tapetum development in genic male sterile peppers, and Msc-1 might be up stream of Msc-2 in the regulation of other genes involved in tapetum development.