Pepper Genome Research Articles

Genome-wide identification, phylogenetic relationships, and expression analysis of the carotenoid cleavage oxygenase gene family in pepper.

Carotenoid cleavage oxygenases (CCOs) are a family of dioxygenases, which specifically catalyze the cleavage of conjugated double bonds in carotenoids and apocarotenoids in plants. In this study, genome-wide analysis of CCO genes in pepper plants was performed using bioinformatic methods. At least 11 members of the CCO gene family were identified in the pepper genome. Phylogenetic analysis showed that pepper and tomato CCO genes could be divided into two groups (CCDs and NCEDs). The CCD group included five sub-groups (CCD1, CCD4, CCD7, CCD8, and CCD-like). These results indicate that there is a close genetic relationship between the two species. Sequence analysis using the online tool, Multiple Expectation Maximization for Motif Elicitation (MEME), showed that the CCO proteins comprise multiple conserved motifs, with 20 to 41 amino acids. In addition, multiple cis-acting elements in the promoter of CCO genes were identified using the online tool PlantCARE, and were found to be involved in light responsiveness, plant hormone regulation, and biotic and abiotic stresses, suggesting potential roles of these proteins under different conditions. RNA-seq analysis revealed that the CCO genes exhibit distinct patterns of expression in the roots, stems, leaves, and fruit. These findings suggest that the CCO genes have important roles in the vegetative and reproductive development of pepper plants.

In silico identification and characterization of the WRKY gene superfamily in pepper (Capsicum annuum L.).

The WRKY family is one of the most important transcription factor families in plants, involved in the regulation of a broad range of biological roles. The recent releases of whole-genome sequences of pepper (Capsicum annuum L.) allow us to perform a genome-wide identification and characterization of the WRKY family. In this study, 61 CaWRKY proteins were identified in the pepper genome. Based on protein structural and phylogenetic analyses, these proteins were classified into four main groups (I, II, III, and NG), and Group II was further divided into five subgroups (IIa to IIe). Chromosome mapping analysis indicated that CaWRKY genes are distributed across all 12 chromosomes, although the location of four CaWRKYs (CaWRKY58-CaWRKY61) could not be identified. Two pairs of CaWRKYs located on chromosome 01 appear to be tandem duplications. Furthermore, the phylogenetic tree showed a close evolutionary relationship of WRKYs in three species from Solanaceae. In conclusion, this comprehensive analysis of CaWRKYs will provide rich resources for further functional studies in pepper.

Comparative transcriptome analysis reveals differential transcription in heat-susceptible and heat-tolerant pepper (Capsicum annum L.) cultivars under heat stress

Heat stress is an important agricultural problem around the world. In pepper (Capsicum annum L.), heat stress seriously affects pollination and yield. However, to date, the molecular basis of heat stress has not been extensively studied. Using the HiSeq™ 2000 sequencing platform, the seedling transcriptome of heat-susceptible C.annuum ‘S590’(CaS) and heat-tolerant ‘R597’ (CaR) under the heat stress was examined. Over five million clean reads were generated from each library, each corresponding to a coverage of >250,000 nt. About 73% of the reads were mapped to the pepper genome, and 3,799 and 4,010 differentially expressed genes (DEGs) were identified in ‘R597’ (CaR) and ‘S590’(CaS), respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses determined that the identified DEGs were involved in heat shock protein, heat shock transcription factors, hormone, as well as calcium and kinase signaling. Further validation identified 35 genes that were involved in stress response, and that most of the heat shock proteins were upregulated in two genotypes, and highly expressed in susceptible S590 than in tolerant cultivar R597; the transcription factors and hormone signaling genes showed higher levels of expression in the heat-tolerant cultivar R597 than that observed in the heat-susceptible S590. These findings facilitate in better understanding of the molecular mechanism underlying heat stress in different pepper genotypes.

Marker-assisted backcross breeding for development of pepper varieties (Capsicum annuum) containing capsinoids

Capsinoids have similar biological effects as capsaicinoids, including anticancer and anti-obesity properties. The Capsicum chinense ‘SNU11-001’ variety of pepper was previously reported to contain high levels of capsinoids due to a mutation in the putative aminotransferase (pAMT) gene, which leads to production of the capsinoid precursor vanillyl alcohol. Here, to develop Capsicum annuum pepper varieties with high capsinoid contents, marker-assisted backcrossing was performed during backcross breeding. For foreground selection, plants carrying the pAMT/pamt genotype were selected from BC1F1 and BC2F1 populations using SCAR markers derived from the unique pamt mutation of ‘SNU11-001.’ To obtain background selection markers, 412 single nucleotide polymorphism (SNP) markers were screened to identify SNP markers polymorphic between the ‘Shinhong’ paternal lines and ‘SNU11-001.’ Of the 412 SNP markers, 204 polymorphic SNP markers evenly distributed in pepper genome were selected. BC1F1 and BC2F1 plants carrying the pAMT/pamt genotype were subjected to background selection using these SNP markers. Multiple genotypes were analyzed using a high-throughput genotyping system. As a result, one BC1F1 plant 84 % similar to the recurrent parent and seven BC2F1 plants showing more than 96 % recovery of the recurrent parent genotype were selected. Genetic backgrounds of the selected BC2F1 plants were evaluated using the genotype-by-sequencing (GBS) method to confirm the background selection results from the SNP marker set. GBS results showed that the recovery rate and positions of introgressed segments were well matched between two methods, demonstrating MABC can be successfully performed with only ~200 SNP markers.

Ultra-High Density, Transcript-Based Genetic Maps of Pepper Define Recombination in the Genome and Synteny Among Related Species.

Our ability to assemble complex genomes and construct ultradense genetic maps now allows the determination of recombination rates, translocations, and the extent of genomic collinearity between populations, species, and genera. We developed two ultradense genetic linkage maps for pepper from single-position polymorphisms (SPPs) identified de novo with a 30,173 unigene pepper genotyping array. The Capsicum frutescens × C. annuum interspecific and the C. annuum intraspecific genetic maps were constructed comprising 16,167 and 3,878 unigene markers in 2108 and 783 genetic bins, respectively. Accuracies of marker groupings and orders are validated by the high degree of collinearity between the two maps. Marker density was sufficient to locate the chromosomal breakpoint resulting in the P1/P8 translocation between C. frutescens and C. annuum to a single bin. The two maps aligned to the pepper genome showed varying marker density along the chromosomes. There were extensive chromosomal regions with suppressed recombination and reduced intraspecific marker density. These regions corresponded to the pronounced nonrecombining pericentromeric regions in tomato, a related Solanaceous species. Similar to tomato, the extent of reduced recombination appears to be more pronounced in pepper than in other plant species. Alignment of maps with the tomato and potato genomes shows the presence of previously known translocations and a translocation event that was not observed in previous genetic maps of pepper.

High genetic abundance of Rpi-blb2/Mi-1.2/Cami gene family in Solanaceae.

BackgroundThree NBS-LRR genes, Rpi-blb2, Mi-1.2, and Cami, constitute a very special plant resistance gene family. These genes confer resistance against 4 distantly related pathogen species in 3 different Solanaceae hosts. To characterize this noted resistance, we conducted a series of studies on this gene family.ResultsFirst, homologs of this gene family were identified in the pepper, tomato and potato genomes. This revealed a large variation in copy number within this gene family among species and a great divergence was found both between and within species. To gain more information pertaining to gene resistance within this family, 121 LRR regions were cloned in 16 different wild/cultivated potato accessions. Again, frequent copy number variations and a high level of divergence between homolog were observed common among accessions. The divergence within species was so high that it reaches the level of divergence between species. Also, frequent frameshift mutations and abundant gene conversion events were identified in these LRR regions.ConclusionsOur findings suggest that this family harbors an unusually high level of genetic abundance, making it of particular interest. Together with other reported examples, our study also provides evidence that multi-resistance is a common trait in R gene families like this.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0493-z) contains supplementary material, which is available to authorized users.

Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum.

The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper.

The complete chloroplast genome of Capsicum annuum var. glabriusculum using Illumina sequencing.

Chloroplast (cp) genome sequences provide a valuable source for DNA barcoding. Molecular phylogenetic studies have concentrated on DNA sequencing of conserved gene loci. However, this approach is time consuming and more difficult to implement when gene organization differs among species. Here we report the complete re-sequencing of the cp genome of Capsicum pepper (Capsicum annuum var. glabriusculum) using the Illumina platform. The total length of the cp genome is 156,817 bp with a 37.7% overall GC content. A pair of inverted repeats (IRs) of 50,284 bp were separated by a small single copy (SSC; 18,948 bp) and a large single copy (LSC; 87,446 bp). The number of cp genes in C. annuum var. glabriusculum is the same as that in other Capsicum species. Variations in the lengths of LSC; SSC and IR regions were the main contributors to the size variation in the cp genome of this species. A total of 125 simple sequence repeat (SSR) and 48 insertions or deletions variants were found by sequence alignment of Capsicum cp genome. These findings provide a foundation for further investigation of cp genome evolution in Capsicum and other higher plants.

Genome-wide analysis, expression profile of heat shock factor gene family (CaHsfs) and characterisation of CaHsfA2 in pepper (Capsicum annuum L.).

BackgroundHeat shock factors (Hsfs) play crucial roles in plant developmental and defence processes. The production and quality of pepper (Capsicum annuum L.), an economically important vegetable crop, are severely reduced by adverse environmental stress conditions, such as heat, salt and osmotic stress. Although the pepper genome has been fully sequenced, the characterization of the Hsf gene family under abiotic stress conditions remains incomplete.ResultsA total of 25 CaHsf members were identified in the pepper genome by bioinformatics analysis and PCR assays. They were grouped into three classes, CaHsfA, B and C, based on highly conserved Hsf domains, were distributed over 11 of 12 chromosomes, with none found on chromosome 11, and all of them, except CaHsfA5, formed a protein–protein interaction network. According to the RNA-seq data of pepper cultivar CM334, most CaHsf members were expressed in at least one tissue among root, stem, leaf, pericarp and placenta. Quantitative real-time PCR assays showed that all of the CaHsfs responded to heat stress (40 °C for 2 h), except CaHsfC1 in thermotolerant line R9 leaves, and that the expression patterns were different from those in thermosensitive line B6. Many CaHsfs were also regulated by salt and osmotic stresses, as well as exogenous Ca2+, putrescine, abscisic acid and methyl jasmonate. Additionally, CaHsfA2 was located in the nucleus and had transcriptional activity, consistent with the typical features of Hsfs. Time-course expression profiling of CaHsfA2 in response to heat stress revealed differences in its expression level and pattern between the pepper thermosensitive line B6 and thermotolerant line R9.ConclusionsTwenty-five Hsf genes were identified in the pepper genome and most of them responded to heat, salt, osmotic stress, and exogenous substances, which provided potential clues for further analyses of CaHsfs functions in various kinds of abiotic stresses and of corresponding signal transduction pathways in pepper.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0512-7) contains supplementary material, which is available to authorized users.

Rapid identification of transgenic black pepper using loop-mediated isothermal amplification (LAMP) and real-time LAMP assays

A loop-mediated isothermal amplification (LAMP) and real-time LAMP based assays were developed for quick and sensitive detection of transgenic black pepper plants. Primers (six each) were designed based on the nucleotide sequence of two target regions [kanamycin and Cauliflower mosaic virus (CaMV) 35S promoter] integrated into the genome of transgenic black pepper. Both assays successfully detected the transgenic plants and no cross-reaction was recorded with non-transgenic plants. The sensitivity of LAMP was up to 104 times that of conventional PCR while real-time LAMP was up to 103 times that of LAMP and 107 times to that of PCR. The addition of 6 mM magnesium sulphate and 0.4 M betaine with 1 h reaction time proved optimal for amplification through LAMP assay. The assays were validated by testing putative transformants of black pepper. The present study clearly established that LAMP and real-time LAMP assays can provide a rapid and simple approach for screening transgenic black pepper and other plants transformed by using the above target gene sequences.

Construction of an interspecific genetic map based on InDel and SSR for mapping the QTLs affecting the initiation of flower primordia in pepper (Capsicum spp.).

Re-sequencing permits the mining of genome-wide variations on a large scale and provides excellent resources for the research community. To accelerate the development and application of molecular markers and identify the QTLs affecting the flowering time-related trait in pepper, a total of 1,038 pairs of InDel and 674 SSR primers from different sources were used for genetic mapping using the F2 population (n = 154) derived from a cross between BA3 (C. annuum) and YNXML (C. frutescens). Of these, a total of 224 simple PCR-based markers, including 129 InDels and 95 SSRs, were validated and integrated into a map, which was designated as the BY map. The BY map consisted of 13 linkage groups (LGs) and spanned a total genetic distance of 1,249.77 cM with an average marker distance of 5.60 cM. Comparative analysis of the genetic and physical map based on the anchored markers showed that the BY map covered nearly the whole pepper genome. Based on the BY map, one major and five minor QTLs affecting the number of leaves on the primary axis (Nle) were detected on chromosomes P2, P7, P10 and P11 in 2012. The major QTL on P2 was confirmed based on another subset of the same F2 population (n = 147) in 2014 with selective genotyping of markers from the BY map. With the accomplishment of pepper whole genome sequencing and annotations (release 2.0), 153 candidate genes were predicted to embed in the Nle2.2 region, of which 12 important flowering related genes were obtained. The InDel/SSR-based interspecific genetic map, QTLs and candidate genes obtained by the present study will be useful for the downstream isolation of flowering time-related gene and other genetic applications for pepper.

An InDel-based linkage map of hot pepper (Capsicum annuum).

Two independent pepper (Capsicum annuum) genomes were published recently, opening a new era of molecular genetics research on pepper. However, pepper molecular marker technologies are still mainly focusing on the simple sequence repeats derived from public database or genomic library. The development and application of the third generation marker system such as single nucleotide polymorphisms, structure variations as well as insertion/deletion polymorphisms (InDels) is still in its infancy. In the present study, we developed InDel markers for pepper genetic mapping with the convenience of two whole-genome re-sequenced inbred lines BA3 (C. annuum) and B702 (C. annuum). A total of 154,519 and 149,755 InDel (1–5 bp) sites were identified for BA3 and B702, respectively, by the alignment of re-sequencing reads to Zunla-1 reference genome. Then, 14,498 InDel sites (only 4 and 5 bp) that are different between BA3 and B702 were predicted. Finally, within a random set of 1,000 primer pairs, 251 InDel markers were validated and mapped onto a linkage map using F2 population derived from the intraspecific cross BA3 × B702. The first InDel-based map, named as BB-InDel map, consisted of 12 linkage groups, covered a genetic distance of 1,178.01 cM and the average distance between bin markers was 5.01 cM. Compared to the Zunla-1 reference physical map, high consistency was observed on all 12 chromosomes, and the total length of scaffold anchored and physical distance covered by this map was 299.66 and 2,558.68 Mb, respectively, which accounted for 8.95 and 76.38 % of the Zunla-1 reference genome (3.35 Gb), respectively. Furthermore, 37 scaffolds (total length of 36.21 Mb) from the pseudo-chromosome (P0) of the current genome assembly were newly assigned to the corresponding chromosomes by 40 InDel markers. Thus, this map provided good genome coverage and would be useful for basic and applied research in pepper.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-015-0219-3) contains supplementary material, which is available to authorized users.

An EST-based linkage map reveals chromosomal translocation in Capsicum

To facilitate marker-assisted breeding and analysis of the structure and/or organization of Capsicum (pepper) genomes, this study utilized expressed sequence tags (ESTs) to develop single-nucleotide polymorphism (SNP) markers. Three different types of PCR-based markers derived from pepper ESTs were developed: intron-based polymorphic markers (IBPs), conserved ortholog sets (COSIIs), and eSNPs (EST–SNPs). For scanning and detection of SNPs, high-resolution melting analysis was performed and the resultant markers were used for linkage analysis. A total of 512 markers, comprising 214 IBP, 143 COSII, 48 eSNP, and 107 previously reported markers, were mapped on 12 linkage groups (LGs) of the “AC99” F2 population. This newly constructed interspecific map (AC2) covered 2,335.6 cM with an average marker interval distance of 4.5 cM and was aligned directly with another interspecific map (AF) for validation. Most LGs showed collinear relationships, except for the alignment of chromosomes 1 and 8 of the AC2 map to LG P1 of the AF map. Using our newly developed SNP markers, we generated chromosome-specific markers, and the previously predicted reciprocal translocation event between chromosomes 1 and 8 was revealed between wild and cultivated Capsicum by fluorescent in situ hybridization analysis. The results from this study will promote subsequent evolutionary studies of Capsicum species.

Evolution of a hot genome

The chile pepper has a long and important human history. Chiles, members of the plant genus Capsicum in the Solanaceae family, evolved in the New World and were domesticated there several millennia ago (1). Although it is hard to imagine a Mexican, Szechuan, or Indian dinner without chiles, these characteristic cuisine elements are a relatively recent development, having only achieved their enormous Old World culinary impact in the centuries after Columbus. Indeed, several other Solanaceae crops are of entirely New World origin, with tomato ( Solanum lycopersicum ) and potato ( Solanum tuberosum ) of particular importance, but also pepino ( Solanum muricatum ) and tomatillo ( Physalis philadelphica ) (Fig. 1 A ). In testimony to the importance of this plant group, whole-genome sequences of tomato (2) and potato (3) were among the first 50 plant genomes available for study. Now, we can add chile pepper to the mix, as published in PNAS by Qin et al. (4) and independently by Kim et al. (5). Although both papers provide excellent resources to the plant science community and share several similar findings (e.g., on the enormous expansion of the chile pepper genome by proliferation of mobile elements and on the biochemical genetics of the fruit’s characteristic pungency), the work of Qin et al. (4), which includes considerable genomic resequencing of multiple cultivated varieties and a wild pepper, the chiltepin, provides important added data for evaluating the (in part artificially derived) adaptive landscape of cultivated peppers. Fig. 1. The genome of chile pepper in an evolutionary context. ( A ) A phylogeny of lamiid angiosperms, the family Solanaceae in yellow and chile pepper in red. ( B ) Syntenic dotplot of tomato chromosome 2 (vertical) versus chiltepin pepper chromosomes 2 ( Upper ) and 4 ( Lower ), with syntenic gene duplicates colored according to their Ks values (synonymous substitutions per synonymous site), as plotted in the x axis of C … [↵][1]1To whom correspondence should be addressed. E-mail: vaalbert{at}buffalo.edu. [1]: #xref-corresp-1-1

Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization

As an economic crop, pepper satisfies people's spicy taste and has medicinal uses worldwide. To gain a better understanding of Capsicum evolution, domestication, and specialization, we present here the genome sequence of the cultivated pepper Zunla-1 (C. annuum L.) and its wild progenitor Chiltepin (C. annuum var. glabriusculum). We estimate that the pepper genome expanded ∼0.3 Mya (with respect to the genome of other Solanaceae) by a rapid amplification of retrotransposons elements, resulting in a genome comprised of ∼81% repetitive sequences. Approximately 79% of 3.48-Gb scaffolds containing 34,476 protein-coding genes were anchored to chromosomes by a high-density genetic map. Comparison of cultivated and wild pepper genomes with 20 resequencing accessions revealed molecular footprints of artificial selection, providing us with a list of candidate domestication genes. We also found that dosage compensation effect of tandem duplication genes probably contributed to the pungent diversification in pepper. The Capsicum reference genome provides crucial information for the study of not only the evolution of the pepper genome but also, the Solanaceae family, and it will facilitate the establishment of more effective pepper breeding programs.

QTL hotspots in eggplant (Solanum melongena) detected with a high resolution map and CIM analysis

Fifty-eight F2 individuals derived from an interspecific cross between cultivated eggplant, Solanum melongena, and its wild relative, S. linnaeanum, were phenotyped for 42 plant, leaf, flower, and fruit traits. Composite interval mapping analysis using genotypic data from 736 molecular markers revealed the positions of 71 statistically significant (P ≤ 0.05) quantitative trait loci (QTL) influencing 32 of the morphological traits. Although most QTL were location-specific, QTL governing three traits (leaf lobing, leaf prickles and prickle anthocyanin) were detected in both experimental locations. Analysis of three additional traits (stem prickles, fruit calyx prickles and fruit length) in both locations yielded QTL in similar but non-overlapping map positions. The majority (69 %) of the QTL corresponded closely with those detected in previous analyses of this data set. However the increased resolution of the linkage map combined with advances in QTL mapping permitted more precise localization, such that the average interval length of these QTL was reduced by 93 %. Thirty-one percent of the QTL were novel, suggesting that simple linear regression with a low density linkage map (the method used in previous studies of this population) missed a substantial portion of significant QTL. Hotspots of QTL affecting plant hairiness, prickliness, and pigmentation were identified on chromosomes 3, 6, and 10, respectively, and may reflect the pleiotropic activity of single structural or regulatory genes at these positions. Based on synteny between the eggplant, tomato, potato and pepper genomes, putative orthologs were identified for 35 % of the QTL suggesting strong conservation of gene function within the Solanaceae. These results should make it easier to target particular loci for map-based cloning and marker-assisted selection studies.

A pepper (Capsicum annuum L.) metacaspase 9 (Camc9) plays a role in pathogen‐induced cell death in plants

Metacaspases, which belong to the cysteine-type C14 protease family, are most structurally similar to mammalian caspases than any other caspase-like protease in plants. Atmc9 (Arabidopsis thaliana metacaspase 9) has a unique domain structure, and distinct biochemical characteristics, such as Ca²⁺ binding, pH, redox status, S-nitrosylation and specific protease inhibitors. However, the biological roles of Atmc9 in plant-pathogen interactions remain largely unknown. In this study, a metacaspase gene present as a single copy in the pepper genome, and sharing 54% amino acid sequence identity with Atmc9, was isolated and named Capsicum annuum metacaspase 9 (Camc9). Camc9 encodes a 318-amino-acid polypeptide with an estimated molecular weight of 34.6 kDa, and shares approximately 40% amino acid sequence identity with known type II metacaspases in plants. Quantitative reverse transcription-polymerase chain reaction analyses revealed that the expression of Camc9 was induced by infections of Xanthomonas campestris pv. vesicatoria race 1 and race 3 and treatment with methyl jasmonate. Suppression of Camc9 expression using virus-induced gene silencing enhanced disease resistance and suppressed cell death symptom development following infection with virulent bacterial pathogens. By contrast, overexpression of Camc9 by transient or stable transformation enhanced disease susceptibility and pathogen-induced cell death by regulation of reactive oxygen species production and defence-related gene expression. These results suggest that Camc9 is a possible member of the metacaspase gene family and plays a role as a positive regulator of pathogen-induced cell death in the plant kingdom.

A molecular framework for risk assessment of a virus-tolerant transgenic pepper line

The Cucumber mosaic virus coat protein (CMV-CP) gene-transgenic pepper lines exhibit high tolerance to Cucumber mosaic virus (CMV) strains. In this study, E7, one of the CMVP0-CP transgenic chili pepper events selected by screening was further characterized. Southern blotting and inverse PCR analysis revealed that the E7 event contains a single copy of the inserted gene cassette whose flanking sequences appear to be noncoding and intergenic. We searched for pepper-specific DNA sequence candidates as an endogenous reference gene for GM-pepper detection. We found that only one copy of CaSIG4 and lipocalin genes are present in the pepper genome and their sequences were determined to be pepper-specific. The characterization of the genomic sequences flanking the transgene, as well as the availability of the pepper-specific single copy CaSIG4 and lipocalin genes as endogenous reference genes, enabled the design of E7-event-specific PCR-based quantitative detection methods. The CMV-CP protein levels in the CMV-inoculated wild-type pepper tissues were approximately 60 times higher than those in the uninoculated and CMV-inoculated E7 pepper tissues. These results suggested that the amount of CMV-CP expressed in transgenic pepper tissue was negligible relative to the amount of CMV-CP in the virus-infected wild-type pepper consumed by human beings. This work may prove useful for risk assessment studies of transgenic pepper lines. Furthermore, the characterized single copy genes, lipocalin and CaSIG4, may be used to develop a method to detect gene copy number variations in the pepper genome.

Identification of BABY BOOM and LEAFY COTYLEDON genes in sweet pepper (Capsicum annuum L.) genome by their partial gene sequences

Haploid plant production via anther culture is a valuable tool to rapidly create pure lines for plant breeding programs. Some sweet pepper (Capsicum annuum L.) varieties produce embryogenic anther culture readily, while others do not respond at all. The switching of microspore developmental program from gametophyte- to sporophyte-type is regulated by transcription factors like BABY BOOM (BBM) and LEAFY COTYLEDON (LEC). Genes encoding these transcription factors have not been found in sweet pepper yet. The sequences of BBM and LEC from other plants, annotated in National Center for Biotechnology Information (NCBI), were used to design degenerative primers for conservative regions in these genes and to amplify BBM- and LEC-like sequences from genomic DNA of sweet pepper by PCR. The PCR products were cloned and sequenced. Sequence comparison revealed high similarity between the isolated sequences and those annotated in NCBI for BBM and LEC1. The obtained sequences were subjected to bioinformatics analyses to determine the exon–intron structure. Exons were translated in protein products in silico. The protein blast of the pepper BBM in the NCBI database revealed presence of an AP2-type functional domain, which is typical for the BBM family. The pepper LEC sequence was found to carry CBFD-NFYB-HMF motif, typical for the LEAFY COTYLEDON gene product family. RT-PCR confirmed high levels of expression of the BBM and LEC genes at the early stages of direct embryos development in anther culture. Expression of the two genes was not detected either in mature pepper plants or in non-embryogenic anthers. The two genes were detected in genomes of 14 pepper varieties, some of which do not respond with formation of direct embryos. We assume that differences in responses between varieties should be attributed not to presence/absence of two genes, but to their interaction with other genes’ products and different factor like micro-RNAs involved in the regulation of their expression.

De novo assembly of the pepper transcriptome (Capsicum annuum): a benchmark for in silico discovery of SNPs, SSRs and candidate genes

BackgroundMolecular breeding of pepper (Capsicum spp.) can be accelerated by developing DNA markers associated with transcriptomes in breeding germplasm. Before the advent of next generation sequencing (NGS) technologies, the majority of sequencing data were generated by the Sanger sequencing method. By leveraging Sanger EST data, we have generated a wealth of genetic information for pepper including thousands of SNPs and Single Position Polymorphic (SPP) markers. To complement and enhance these resources, we applied NGS to three pepper genotypes: Maor, Early Jalapeño and Criollo de Morelos-334 (CM334) to identify SNPs and SSRs in the assembly of these three genotypes.ResultsTwo pepper transcriptome assemblies were developed with different purposes. The first reference sequence, assembled by CAP3 software, comprises 31,196 contigs from >125,000 Sanger-EST sequences that were mainly derived from a Korean F1-hybrid line, Bukang. Overlapping probes were designed for 30,815 unigenes to construct a pepper Affymetrix GeneChip® microarray for whole genome analyses. In addition, custom Python scripts were used to identify 4,236 SNPs in contigs of the assembly. A total of 2,489 simple sequence repeats (SSRs) were identified from the assembly, and primers were designed for the SSRs. Annotation of contigs using Blast2GO software resulted in information for 60% of the unigenes in the assembly. The second transcriptome assembly was constructed from more than 200 million Illumina Genome Analyzer II reads (80–120 nt) using a combination of Velvet, CLC workbench and CAP3 software packages. BWA, SAMtools and in-house Perl scripts were used to identify SNPs among three pepper genotypes. The SNPs were filtered to be at least 50 bp from any intron-exon junctions as well as flanking SNPs. More than 22,000 high-quality putative SNPs were identified. Using the MISA software, 10,398 SSR markers were also identified within the Illumina transcriptome assembly and primers were designed for the identified markers. The assembly was annotated by Blast2GO and 14,740 (12%) of annotated contigs were associated with functional proteins.ConclusionsBefore availability of pepper genome sequence, assembling transcriptomes of this economically important crop was required to generate thousands of high-quality molecular markers that could be used in breeding programs. In order to have a better understanding of the assembled sequences and to identify candidate genes underlying QTLs, we annotated the contigs of Sanger-EST and Illumina transcriptome assemblies. These and other information have been curated in a database that we have dedicated for pepper project.