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Pepper (Capsicum spp.) has a long domestication history and has accumulated diverse fruit shape variations. The illustration of the mechanisms underlying different fruit shape is not only important for clarifying the regulation of pepper fruit development but also critical for fully understanding the plant organ morphogenesis. Thus, in this study, morphological, histological and transcriptional investigations have been performed on pepper accessions bearing fruits with five types of shapes. From the results it can be presumed that pepper fruit shape was determined during the developmental processes before and after anthesis, and the anthesis was a critical developmental stage for fruit shape determination. Ovary shape index variations of the studied accessions were mainly due to cell number alterations, while, fruit shape index variations were mainly attributed to the cell division and cell expansion variations. As to the ovary wall thickness and pericarp thickness, they were regulated by both cell division in the abaxial-adaxial direction and cell expansion in the proximal-distal and medio-lateral directions. Transcriptional analysis discovered that the OFP-TRM and IQD-CaM pathways may be involved in the regulation of the slender fruit shape and the largest ovary wall cell number in the blocky-shaped accession can be attributed to the higher expression of CYP735A1, which may lead to an increased cytokinin level. Genes related to development, cell proliferation/division, cytoskeleton, and cell wall may also contribute to the regulation of helical growth in pepper. The insights gained from this study are valuable for further investigations into pepper fruit shape development.

IntroductionPepper (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.MethodsIn 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.ResultsIn 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.DiscussionIn 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.

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.

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.

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.

Summary: 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.

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.

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.

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.

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.

Pepper flowers are hermaphroditic; the plant's male sterility trait is characterized by its inability to produce pollen grains. In the ABC model of flower development, B-function genes play roles in petal and stamen development in the angiosperm. In this study, a B-class gene designated as PAP3 (GenBank accession no. HM104635) was isolated in pepper. The gene encoded 226 amino acids and shared high similarity with the MADS-box protein family, with a conservative MADS domain and semiconservative K domain. Furthermore, the expression of PAP3 was abundant only in petals and anthers but not in leaves. A functional study employing virus-induced gene silencing (VIGS) showed that knockdown of PAP3 led to the shriveling of pollen grains and male sterility; however, it did not affect petal development. These results suggest an essential role for PAP3 in the development of the pepper stamen and in contributing to the variation of floral traits.

Elite parental lines are more likely to breed fine varieties, but knowledge about elite parents and their genetic backgrounds is limited. In this paper, we investigated the pedigree relationships of potato varieties bred worldwide and in China. Several elite parents were identified, and these parents were more frequently used as parents in breeding programs across different time periods and countries. We next used 2b-RAD, a reduced-representation sequencing method, to genotype the elite parent Mira and 24 of its offspring. These cultivars span 5 generations, making this lineage the longest continuous pedigree among Chinese bred potatoes. A total of 47,314 tetraploid single nucleotide polymorphisms (SNPs) identified by FreeBayes were used to trace the conserved segments of the Mira genome. The conserved segments had identical or similar allele-specific SNPs across the analyzed genotypes. In Mira, 3,788 segments comprising over 10,000 bp, or 20.8% of the genome, were defined as conserved segments. These segments contain genes involved in crucial biological processes that are of special interest to breeders. These regions, which have been conserved across generations of highly selective breeding, may be helpful for further breeding and performing genome-wide breeding by design.

Phytophthora capsici (Leonian), classified as an oomycete, seriously threatens the production of pepper (Capsicum annuum). Current understanding of the defense responses in pepper to P. capsici is limited. In this study, RNA-sequencing analysis was utilized to identify differentially expressed genes in the resistant line “PI 201234”, with 1220 differentially expressed genes detected. Of those genes, 480 were up-regulated and 740 were down-regulated, with 211 candidate genes found to be involved in defense responses based on the gene annotations. Furthermore, the expression patterns of 12 candidate genes were further validated via quantitative real-time PCR (qPCR). These genes were found to be significantly up-regulated at different time points post-inoculation (6 hpi, 24 hpi, and 5 dpi) in the resistant line “PI 201234” and susceptible line “Qiemen”. Seven genes were found to be involved in cell wall modification, phytoalexin biosynthesis, symptom development, and phytohormone signaling pathways, thus possibly playing important roles in combating exogenous pathogens. The genes identified herein will provide a basis for further gene cloning and functional verification studies and will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.

Tomato (Solanum lycopersicum) fruit weight (FW), soluble solid content (SSC), fruit shape and fruit color are crucial for yield, quality and consumer acceptability. In this study, a 192 accessions tomato association panel comprising a mixture of wild species, cherry tomato, landraces, and modern varieties collected worldwide was genotyped with 547 InDel markers evenly distributed on 12 chromosomes and scored for FW, SSC, fruit shape index (FSI), and color parameters over 2 years with three replications each year. The association panel was sorted into two subpopulations. Linkage disequilibrium ranged from 3.0 to 47.2 Mb across 12 chromosomes. A set of 102 markers significantly (p < 1.19–1.30 × 10−4) associated with SSC, FW, fruit shape, and fruit color was identified on 11 of the 12 chromosomes using a mixed linear model. The associations were compared with the known gene/QTLs for the same traits. Genetic analysis using F2 populations detected 14 and 4 markers significantly (p < 0.05) associated with SSC and FW, respectively. Some loci were commonly detected by both association and linkage analysis. Particularly, one novel locus for FW on chromosome 4 detected by association analysis was also identified in F2 populations. The results demonstrated that association mapping using limited number of InDel markers and a relatively small population could not only complement and enhance previous QTL information, but also identify novel loci for marker-assisted selection of fruit traits in tomato.

Pepper (Capsicum annuum L.) is a globally important horticultural crop. Use of the genic male-sterile (GMS) line enables efficient commercial hybrid pepper seed production. However, the mechanisms of pepper GMS functioning remain unclear. In this study, we used proteomic and transcriptomic analysis to identify proteins and genes related to genic male sterility. A total of 764 differentially expressed proteins (DEPs) and 1069 differentially expressed genes (DEGs) were identified in the proteomic and transcriptomic level respectively, and 52 genes (hereafter “cor-DEGs-DEPs„ genes) were detected at both levels. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 13 DEPs and 14 DEGs involved in tapetum and pollen development. Among the 13 DEPs identified, eight were involved in pollen exine formation, and they were all up-regulated in the fertile line 16C1369B. For the 14 DEGs identified, ABORTED MICROSPORES (AMS) and DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1) were involved in tapetum development, and both are possibly regulated by Msc-1. All of these genes were detected and confirmed by qRT-PCR. The presence of these genes suggests their possible role in tapetum and pollen exine formation in GMS pepper. Most key genes and transcription factors involved in these processes were down-regulated in the sterile line 16C1369A. This study provides a better understanding of GMS (msc-1) molecular functioning in pepper.

Cytoplasmic male sterility (CMS), which is controlled by mitochondrial genes, is an important trait for commercial hybrid seed production. So far, genes controlling this trait are still not clear in pepper. In this study, complete mitochondrial genomes were sequenced and assembled for the CMS line 138A and its maintainer line 138B. The genome size of 138A is 504,210 bp, which is 8618 bp shorter than that of 138B. Meanwhile, more than 214 and 215 open reading frames longer than 100 amino acids (aas) were identified in 138A and 138B, respectively. Mitochondrial genome structure of 138A was quite different from that of 138B, indicating the existence of recombination and rearrangement events. Based on the mitochondrial genome sequence and structure variations, mitochondrion of 138A and FS4401, a Korean origin CMS line, may have inherited from a common female ancestor, but their CMS traits did originate separately. Candidate gene selection was performed according to the published characteristics of the CMS genes, including the presence SNPs and InDels, located in unique regions, their chimeric structure, co-transcription, and transmembrane domain. A total of 35 ORFs were considered as potential candidate genes and 14 of these were selected, with orf300a and 0rf314a as strong candidates. A new marker, orf300a, was developed which did co-segregate with the CMS trait.

Class B gene APETALA3 (AP3) plays a key role in the development of petals and stamens. Here, we investigated the expression pattern of PAP3 gene (genbank accession number: HM104635) in the buds of cytoplasmic male sterility line 121A and its near-isogenic restorer line 121C at four developmental stages and analyzed the possible association between Class B genes and cytoplasmic male sterility of pepper. Semi-quantitative PCR and quantitative real-time RT-PCR (qRT-PCR) as well as RNA in situ hybridization showed increased expression of PAP3 at late phase of anther development and its higher expression in restorer line compared with sterility line indicating PAP3’s role at late developmental stage of anther and suppressed expression in sterility line. RNA in situ hybridization showed Class B gene features: high abundance in stamen and petal; lower expression in pistil; no expression in sepal. Results of transient expression in onion epidermal cells also showed PAP3 localized in the nucleus, which is consistent with the expression pattern of transcription factors of MADS-box gene family.

Pepper (Capsicum annuum L.) is an economically important vegetable crop worldwide. Although many genes associated with anther and pollen development have been identified, little is known about the mechanism of pollen abortion in pepper. Here, we identified and isolated two putative aborted microspore (AMS) isoforms from pepper flowers: CaAMS1 and CaAMS2. Sequence analysis showed that CaAMS2 was generated by retention of the fourth intron in CaAMS1 pre-mRNA. CaAMS1 encodes a putative protein with a basic helix-loop-helix (bHLH) domain belonging to the MYC subfamily of bHLH transcription factors, and it is localized to the nucleus. Truncated CaAMS2-1 and CaAMS2-2 are produced by alternative splicing. Quantitative real-time PCR analysis showed that CaAMS (referred to CaAMS1 and CaAMS2-2) was preferentially expressed in stamens and its expression level gradually decreases with flower development. RNA in situ hybridization analysis showed that CaAMS is strongly expressed in the tapetum at the tetrad and uninucleate stages. Downregulation of CaAMS led to partial shortened filaments, shriveled, indehiscent stamens and abortive pollens in pepper flowers. Several genes involved in pollen exine formation were downregulated in defective CaAMS-silenced anthers. Thus, CaAMS seems to play an important role in pepper tapetum and pollen development by regulating a complex genetic network.

low level of genetic variation has limited the application of molecular markers for characterizing important traits in cultivated tomato. To detect polymorphisms in tomato conserved ortholog sets (COS), expressed sequence tags (ESTs) were searched against tomato and Arabidopsis genomic sequences to define the positions of introns. Introns were amplified from 12 different accessions of tomato by polymerase chain reaction and nucleotide sequences were determined by sequencing. Results indicated that there was a possibility of 71% to amplify introns from tomato genomic DNA through this approach. A total of 201 introns were sequenced from 86 COS unigenes. The intron positions and numbers were conserved between tomato and Arabidopsis, but average intron length was three times longer in tomato than in Arabidopsis. A total of 307 single nucleotide polymorphisms (SNPs) and 75 indels were detected in introns of 57 COS unigenes among 12 tomato lines. Within cultivated tomato germplasm 172 SNPs and 47 indels were detected in introns of 33 COS unigenes. In addition, 41 SNPs were identified in the exons of 27 COS unigenes. The frequency of SNPs was 2.4 times higher in introns than in exons in the 22 COS unigenes having both intronic and exonic polymorphisms. These results indicate that intronic regions may contain sufficient variation to develop sufficient marker resources for genome-wide analysis in cultivated tomato. [ABSTRACT FROM AUTHOR]

Head splitting resistance (HSR) in cabbage is an important trait closely related to appearance, yield, storability, and mechanical harvestability. In this study, a doubled haploid (DH) population derived from a cross between head splitting-susceptible inbred cabbage line 79-156 and resistant line 96-100 was used to analyze inheritance and detect quantitative trait loci (QTLs) for HSR during 2011-12 in Beijing, China. The analysis was performed using a mixed major gene/polygene inheritance method and QTL mapping. This approach, which uncovered no cytoplasmic effect, indicated that HSR can be attributed to additive-epistatic effects of three major gene pairs combined with those of polygenes. Major gene and polygene heritabilities were estimated to be 88.03% to 88.22% and 5.65% to 7.60%, respectively. Using the DH population, a genetic map was constructed with simple sequence repeat (SSR) markers anchored on nine linkage groups spanning 906.62 cM. Eight QTLs for HSR were located on chromosomes C4, C5, C7, and C9 based on 2 years of phenotypic data using both multiple-QTL mapping and inclusive composite interval mapping. The identified QTLs collectively explained 37.6% to 46.7% of phenotypic variation. Three or four major QTLs (Hsr 4.2,7.2,9.3, and/or 9.1) showing a relatively larger effect were robustly detected in different years or with different mapping methods. The HSR trait was shown to have a complex genetic basis. Results from QTL mapping and classical genetic analysis were consistent. Our results provide a foundation for further research on HSR genetic regulation and molecular marker-assisted selection (MAS) for HSR in cabbage. [ABSTRACT FROM AUTHOR]