Search

The commercial and nutritional quality of chili peppers deteriorates rapidly after harvest. So far, little is known about the effect of temperature on postharvest chili pepper quality. This study elucidated the effects of two temperatures (20 °C and 30 °C) on chili peppers’ postharvest firmness, flavor, and nutritional attributes. We found that compared to 20 °C, 30 °C escalated the decline in fruit firmness, capsaicin content, and dihydrocapsaicin content, while enhancing the increment in water loss and electrical conductivity, as well as total carotenoids and ascorbic acid content. The contents of most amino acids (AAs) decreased significantly during postharvest storage compared to their initial values, whether stored at 20 °C or 30 °C; however, 30 °C had a more substantial impact than 20 °C. Meanwhile, as for soluble protein and amino acid compositions, the effect of storage temperature was genotype-dependent, as reflected by differential changes in total AA contents, single AA contents, essential AA ratio, delicious AA ratio, etc., under the 20 °C or 30 °C treatments. In conclusion, our findings reveal the influence of temperature on pepper quality, showing that the storage temperature of 20 °C was better for maintaining chili quality than 30 °C from the perspective of overall commercial attributes.

Superoxide dismutases (SODs), a family of antioxidant enzymes, are the first line of defense against oxidative damage and are ubiquitous in every cell of all plant types. The Cu/Zn SOD, one of three types of SODs present in plant species, is involved in many of the biological functions of plants in response to abiotic and biotic stresses. Here, we carried out a comprehensive analysis of the Cu/Zn SOD gene family in different plant species, ranging from lower plants to higher plants, and further investigated their organization, sequence features, and expression patterns in response to biotic and abiotic stresses. Our results show that plant Cu/Zn SODs can be divided into two subfamilies (group I and group II). Group II appeared to be conserved only as single- or low-copy genes in all lineages, whereas group I genes underwent at least two duplication events, resulting in multiple gene copies and forming three different subgroups (group Ia, group Ib, and group Ic). We also found that, among these genes, two important events—the loss of introns and the loss of and variation in signal peptides—occurred over the long course of their evolution, indicating that they were involved in shifts in subcellular localization from the chloroplast to cytosol or peroxisome and underwent functional divergence. In addition, expression patterns of Cu/Zn SOD genes from Arabidopsis thaliana and Solanum lycopersicum were tested in different tissues/organs and developmental stages and under different abiotic stresses. The results indicate that the Cu/Zn SOD gene family possesses potential functional divergence and may play vital roles in ROS scavenging in response to various stresses in plants. This study will help establish a foundation for further understanding these genes’ function during stress responses.

Fungal wilt and root rot diseases affecting tomato have become prevalent in China in recent years and have caused considerable damage. In 2016 to 2018, symptoms of putative wilt and root rot diseases were observed in several locations in tomato cultivars with resistance to Fusarium oxysporum f. sp. lycopersici races 1 and 2. The objective of this study was to identify the causative agents of wilt and root rot of tomato in China and provide a basis for disease prevention and resistance breeding programs. Based on DNA sequence analyses of the internal transcribed spacer (ITS) region, 91 isolates from the roots of tomato plants showing symptoms of wilt and root rot were identified, including F. oxysporum (64 isolates), Fusarium solani (11 isolates), Fusarium proliferatum (2 isolates), Fusarium graminearum (2 isolates), Fusarium equiseti (1 isolate), Pythium aphanidermatum (6 isolates), Ascomycota sp. (2 isolates), and Plectosphaerella cucumerina (3 isolates). F. oxysporum accounted for 70.33% of the isolates obtained. In this case, using PCR-based methods for differentiation of F. oxysporum, we identified several formae speciales and races of F. oxysporum: 7 isolates were identified as F. oxysporum f. sp. lycopersici race 1, 2 isolates as F. oxysporum f. sp. lycopersici race 2, 35 isolates as F. oxysporum f. sp. lycopersici race 3, and 13 isolates as F. oxysporum f. sp. radicis-lycopersici. Pathogenicity tests revealed 55 isolates of tomato wilt and root rot pathogens to be virulent. This study demonstrated that F. oxysporum f. sp. lycopersici race 3 was the most widespread and highly virulent race among these tomato pathogens in China, followed by F. oxysporum f. sp. radicis-lycopersici. Therefore, the development of resistant varieties of tomato against F. oxysporum f. sp. lycopersici race 3 and F. oxysporum f. sp. radicis-lycopersici would aid efforts to develop effective disease management strategies.

Invertases catalyze the irreversible hydrolysis of sucrose into glucose and fructose and thus play key roles in carbon metabolism and plant development. To gain insights into their evolutional and functional relationships, we conducted genome-wide analyses of invertase genes in tomato and other species, focusing on their evolution and expression dynamics. The analyses unexpectedly identified in the tomato genome 5 pseudo invertsase sequences and 5 non-functional cell wall invertases (CWINs) lacking the critical β-fructosidase motif or other amino acids required for hydrolyzing sucrose. Based on their phylogeny relationship and exon–intron structure, we speculated that the invertase gene family could arose from different ancestral genes. The acid invertase gene family, comprised of CWIN and vacuolar invertase (VIN), expanded through segmental and tandem duplication. Analysis of functional divergence suggests site-specific shifted evolutionary rate (Type-I) have played an important role in evolutionary novelties after acid invertase gene duplication in plants. Finally, paralogs within each of the CWIN, VIN and CIN subfamilies exhibited diverse expression responses to the same set of stress treatments including salt and temperature stresses, probably reflecting functional adaptability of the invertase genes during evolution.

Chinese wild pepper (Capsicum frutescens L.) acts as a source of genetic variability for cultivated pepper breeding. High-resolution quantitative train locus (QTL) mapping is essential for identifying genetic regions related to important phenotype variation. In this study, a set of 120 F2 populations derived from a cross between cultivar Capsicum annuum ‘007EA’ and the wild relative Capsicum frutescens ‘P1512’ (Chinese wild pepper) were generated, and genotyping was performed with genotyping-by-sequencing. Next, an interspecific high-density genetic map containing 3708 bins was constructed using a sliding window approach. The genetic distance of the total map was 5601.79 cM, and the average interval between bins was 1.51 cM. Based on the genetic map, a QTL analysis of the pepper horticultural traits was performed. A total of 25 significant QTLs controlling nine horticultural traits were detected on 12 linkage groups (LGs), and contributed values of phenotypic variance explained (PVE) ranging from 1.90% to 59.03%, with LOD scores ranging from 4.2 to 24.6. In addition, the QTLs for the main stem length, flowering date and numbers of leaves were observed at the same position. Next, we mapped a set of significant QTLs for yield-related traits and other horticultural traits in pepper, based on a high-density genetic linkage map, using GBS. These genetic map and markers are valuable genomic resources for marker-assisted selection (MAS) of excellent agronomic traits and will provide a basis for further fine mapping and comparative genomics research involving pepper.

Sugar transporter proteins (STPs) play an important role in plant growth and development and stress resistance. Pepper (Capsicum annuum L.) is a significant economic crop and is widely cultivated worldwide. However, the evolution and the roles of STP genes in the growth and development and in coping with abiotic stresses in pepper are poorly known. Here, we characterized STP gene family in pepper through integration of chromosomal location, gene structure, conserved motif, phylogeny, expression patterns in different tissues and abiotic stresses. Using bioinformatics-based methods, we identified 17 putative STP genes in pepper. Chromosome mapping indicated that they are unevenly distributed on 9 chromosomes and there are tandem duplication events. Gene structure analysis revealed that intron numbers among these CaSTP genes ranged from 2 to 5, except that CaSTP12 and CaSTP13 genes contained no introns. The phylogenetic tree of STP genes from pepper and other plant species revealed that STP genes were grouped into 4 subfamilies, suggesting that these gene subfamilies may have diverged from a single common ancestor prior to the mono-dicot split. Gene expression analysis based on RNA-seq data showed that three genes were expressed constitutively in all the tissues analyzed, implying that these genes might have specific function in pepper. Stress data analysis showed that the expression profiles of some members of the CaSTP gene family were altered under cold, heat, salt and hormone stress conditions. The present results will provide valuable information into the evolutionary relationships and functional divergence of the STP family in the future.

Supplemental material, Table_S2_xyz33056ee822a92 for Genomic Organization and Comparative Phylogenic Analysis of NBS-LRR Resistance Gene Family in Solanum pimpinellifolium and Arabidopsis thaliana by Huawei Wei, Jia Liu, Qinwei Guo, Luzhao Pan, Songlin Chai, Yuan Cheng, Meiying Ruan, Qingjing Ye, Rongqing Wang, Zhuping Yao, Guozhi Zhou and Hongjian Wan in Evolutionary Bioinformatics

The invertase gene family in plants is composed of two subfamilies of enzymes, namely, acid- and neutral/alkaline invertases (cytosolic invertase, CIN). Both can irreversibly cleave sucrose into fructose and glucose, which are thought to play key roles in carbon metabolism and plant growth. CINs are widely found in plants, but little is reported about this family. In this paper, a comparative genomic approach was used to analyze the CIN gene family in Solanum, including Solanum tuberosum, Solanum lycopersicum, Solanum pennellii, Solanum pimpinellifolium, and Solanum melongena. A total of 40 CINs were identified in five Solanum plants, and sequence features, phylogenetic relationships, motif compositions, gene structure, collinear relationship, and expression profile were further analyzed. Sequence analysis revealed a remarkable conservation of CINs in sequence length, gene number, and molecular weight. The previously verified four amino acid residues (D188, E414, Arg430, and Ser547) were also observed in 39 out of 40 CINs in our study, showing to be deeply conserved. The CIN gene family could be distinguished into groups &alpha, and &beta, and &alpha, is further subdivided into subgroups &alpha, 1 and &alpha, 2 in our phylogenetic tree. More remarkably, each species has an average of four CINs in the &alpha, groups. Marked interspecies conservation and collinearity of CINs were also further revealed by chromosome mapping. Exon&ndash, intron configuration and conserved motifs were consistent in each of these &alpha, groups on the basis of in silico analysis. Expression analysis indicated that CINs were constitutively expressed and share similar expression profiles in all tested samples from S. tuberosum and S. lycopersicum. In addition, in CIN genes of the tomato and potato in response to abiotic and biotic stresses, phytohormones also performed. Overall, CINs in Solanum were encoded by a small and highly conserved gene family, possibly reflecting structural and functional conservation in Solanum. These results lay the foundation for further expounding the functional characterization of CIN genes and are also significant for understanding the evolutionary profiling of the CIN gene family in Solanum.

As a conserved protein family, WRKY has been shown to be involved in multiple biological processes in plants. However, the mechanism of functional diversity for WRKYs in pepper has not been well elucidated. Here, a total of 223 WRKY members from solanaceae crops including pepper, tomato and potato, were analyzed using comparative genomics. A tremendous genetic variation among WRKY members of different solanaceous plants or groups was demonstrated by the comparison of some WRKY features, including number/size, group constitution, gene structure, and domain composition. The phylogenetic analysis showed that except for the known WRKY groups (I, IIa/b/c/d/e and III), two extra WRKY subgroups specifically existed in solanaceous plants, which were named group IIf and group IIg in this study, and their genetic variations were also revealed by the characteristics of some group IIf and IIg WRKYs. Except for the extensive genetic variations, certain degrees of conservatism for solanaceae WRKYs were also revealed. Moreover, the variant zinc-finger structure (CX4,7CX22-24HXC) in group III of solanaceae WRKYs was identified. Expression profiles of CaWRKY genes suggested their potential roles in pepper development and stress responses, and demonstrated a functional division pattern for pepper CaWRKYs. Furthermore, functional analysis using virus induced gene silencing (VIGS) revealed critical roles of two CaWRKYs (CaWRKY45 and CaWRKY58) in plant responses to disease and drought, respectively. This study provides a solid foundation for further dissection of the evolutionary and functional diversity of solanaceae WRKYs in crop plants.

NBS-LRR (nucleotide-binding site and leucine-rich repeat) is one of the largest resistance gene families in plants. The completion of the genome sequencing of wild tomato Solanum pimpinellifolium provided an opportunity to conduct a comprehensive analysis of the NBS-LRR gene superfamily at the genome-wide level. In this study, gene identification, chromosome mapping, and phylogenetic analysis of the NBS-LRR gene family were analyzed using the bioinformatics methods. The results revealed 245 NBS-LRRs in total, similar to that in the cultivated tomato. These genes are unevenly distributed on 12 chromosomes, and ~59.6% of them form gene clusters, most of which are tandem duplications. Phylogenetic analysis divided the NBS-LRRs into 2 subfamilies (CNL-coiled-coil NBS-LRR and TNL-TIR NBS-LRR), and the expansion of the CNL subfamily was more extensive than the TNL subfamily. Novel conserved structures were identified through conserved motif analysis between the CNL and TNL subfamilies. Compared with the NBS-LRR sequences from the model plant Arabidopsis thaliana, wide genetic variation occurred after the divergence of S. pimpinellifolium and A thaliana. Species-specific expansion was also found in the CNL subfamily in S. pimpinellifolium. The results of this study provide the basis for the deeper analysis of NBS-LRR resistance genes and contribute to mapping and isolation of candidate resistance genes in S. pimpinellifolium.

Carotenoid cleavage dioxygenases (CCDs) in plant species is one of the most important enzymes in the carotenoid metabolism. In this study, we performed a comprehensive analysis for the CCDs family in Solanum lycopersicum based on the whole tomato genome sequences and explored their expression pattern. At least seven CCD genes were discovered in the tomato genome sequence. Two pairs of them were arranged in tandem. The tandem duplication events could be dating to approximately 14 and 21 Mya, and the tandem duplication genes experienced a purifying selection during the course of evolution after diversification. Additionally, subcellular localization revealed that four members were predicted to be cytoplasm-localized and the three remaining members plastids-localized. Subsequently, a number of cis-regulatory elements, which were involved in light responsiveness, hormone regulation, and abiotic and biotic stresses, were identified in the promoter sequences of SlCCD genes. Phylogenetic tree revealed that the CCDs from Solanaceae crops have a closer genetic relationship. The difference in abundance and distinct expression patterns during the vegetative and reproductive development suggests different functions for these seven SlCCDs. Our findings suggest that SlCCDs family play important roles throughout the whole life course and will lay the foundation for further elaborating the regulatory mechanism of each member in tomato.

Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR) is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs). Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified thirty-eight most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red) using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170), SlFRG12 (Solyc04g009770), SlFRG16 (Solyc10g081190), SlFRG27 (Solyc06g007510), and SlFRG37 (Solyc11g005330)were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070) and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species.

Due to its high sensitivity and reproducibility, quantitative real-time PCR (qPCR) is practiced as a useful research tool for targeted gene expression analysis. For qPCR operations, the normalization with suitable reference genes (RGs) is a crucial step that eventually determines the reliability of the obtained results. Although pepper is considered an ideal model plant for the study of non-climacteric fruit development, at present no specific RG have been developed or validated for the qPCR analyses of pepper fruit. Therefore, this study aimed to identify stably expressed genes for their potential use as RGs in pepper fruit studies. Initially, a total of 35 putative RGs were selected by mining the pepper transcriptome data sets derived from the PGP (Pepper Genome Platform) and PGD (Pepper Genome Database). Their expression stabilities were further measured in a set of pepper (Capsicum annuum L. var. 007e) fruit samples, which represented four different fruit developmental stages (IM: Immature; MG: Mature green; B: Break; MR: Mature red) using the qPCR analysis. Then, based on the qPCR results, three different statistical algorithms, namely geNorm, Normfinder, and boxplot, were chosen to evaluate the expression stabilities of these putative RGs. It should be noted that nine genes were proven to be qualified as RGs during pepper fruit development, namely CaREV05 (CA00g79660); CaREV08 (CA06g02180); CaREV09 (CA06g05650); CaREV16 (Capana12g002666); CaREV21 (Capana10g001439); CaREV23 (Capana05g000680); CaREV26 (Capana01g002973); CaREV27 (Capana11g000123); CaREV31 (Capana04g002411); and CaREV33 (Capana08g001826). Further analysis based on geNorm suggested that the application of the two most stably expressed genes (CaREV05 and CaREV08) would provide optimal transcript normalization in the qPCR experiments. Therefore, a new and comprehensive strategy for the identification of optimal RGs was developed. This strategy allowed for the effective normalization of the qPCR analysis of the pepper fruit development at the whole pepper genome level. This study not only explored the optimal RGs specific for studying pepper fruit development, but also introduced a referable strategy of RG mining which could potentially be implicated in other plant species.

WRKY transcription factors play important roles in plant development and stress responses. Here, global expression patterns of pepper CaWRKYs in various tissues as well as response to environmental stresses and plant hormones were systematically analyzed, with an emphasis on fruit ripening. The results showed that most CaWRKYs were expressed in at least two of the tissues tested. Group I, a subfamily of the entire CaWRKY gene family, had a higher expression level in vegetative tissues, whereas groups IIa and III showed relatively lower expression levels. Comparative analysis showed that the constitutively highly expressed WRKY genes were conserved in tomato and pepper, suggesting potential functional similarities. Among the identified 61 CaWRKYs, almost 60% were expressed during pepper fruit maturation, and the group I genes were in higher proportion during the ripening process, indicating an as-yet unknown function of group I in the fruit maturation process. Further analysis suggested that many CaWRKYs expressed during fruit ripening were also regulated by abiotic stresses or plant hormones, indicating that these CaWRKYs play roles in the stress-related signaling pathways during fruit ripening. This study provides new insights to the current research on CaWRKY and contributes to our knowledge about the global regulatory network in pepper fruit ripening.

The Pto gene from the wild tomato (Solanum pimpinellifolium Mill.) encodes a serine/threonine kinase that plays an important role in bacterial speck resistance in the cultivated tomato (Solanum lycopersicum Mill.). In this paper, 10 classes of Pto-like genes are identified using degenerate polymerase chain reaction (PCR) primers and database mining in pepper. Sequences alignment reveals that many features of the gene family, such as subdomains, autophosphorylation sites, and important amino acid residues for tomato Pto, are well conserved in pepper. A phylogenetic tree of pepper Pto-like genes along with those of other plant species, including tomato Pto genes, suggests that these genes share a common evolutionary origin, and they may have evolved prior to the divergence of monocotyledonous and dicotyledonous plants. Expression analysis has revealed that nine selected Pto-like genes can be detected in at least one of the tissues grown under normal growth conditions; however, these genes are differentially expressed. In addition, some of these genes are regulated by at least one of the subjected treatments, including hormones, abiotic stress, and pathogen infection. These findings will contribute to expanding our knowledge of the roles of Pto-like genes in growth, development, and stress tolerance in pepper.

WRKY transcription factors play important roles in plant development and stress responses. Here, global expression patterns of pepper CaWRKYs in various tissues as well as response to environmental stresses and plant hormones were systematically analyzed, with an emphasis on fruit ripening. The results showed that most CaWRKYs were expressed in at least two of the tissues tested. Group I, a subfamily of the entire CaWRKY gene family, had a higher expression level in vegetative tissues, whereas groups IIa and III showed relatively lower expression levels. Comparative analysis showed that the constitutively highly expressed WRKY genes were conserved in tomato and pepper, suggesting potential functional similarities. Among the identified 61 CaWRKYs, almost 60% were expressed during pepper fruit maturation, and the group I genes were in higher proportion during the ripening process, indicating an as-yet unknown function of group I in the fruit maturation process. Further analysis suggested that many CaWRKYs expressed during fruit ripening were also regulated by abiotic stresses or plant hormones, indicating that these CaWRKYs play roles in the stress-related signaling pathways during fruit ripening. This study provides new insights to the current research on CaWRKY and contributes to our knowledge about the global regulatory network in pepper fruit ripening.

Superoxide dismutases (SODs) are critical antioxidant enzymes that protect organisms from reactive oxygen species (ROS) caused by adverse conditions, and have been widely found in the cytoplasm, chloroplasts, and mitochondria of eukaryotic and prokaryotic cells. Tomato (Solanum lycopersicum L.) is an important economic crop and is cultivated worldwide. However, abiotic and biotic stresses severely hinder growth and development of the plant, which affects the production and quality of the crop. To reveal the potential roles of SOD genes under various stresses, we performed a systematic analysis of the tomato SOD gene family and analyzed the expression patterns of SlSOD genes in response to abiotic stresses at the whole-genome level. The characteristics of the SlSOD gene family were determined by analyzing gene structure, conserved motifs, chromosomal distribution, phylogenetic relationships, and expression patterns. We determined that there are at least nine SOD genes in tomato, including four Cu/ZnSODs, three FeSODs, and one MnSOD, and they are unevenly distributed on 12 chromosomes. Phylogenetic analyses of SOD genes from tomato and other plant species were separated into two groups with a high bootstrap value, indicating that these SOD genes were present before the monocot-dicot split. Additionally, many cis-elements that respond to different stresses were found in the promoters of nine SlSOD genes. Gene expression analysis based on RNA-seq data showed that most genes were expressed in all tested tissues, with the exception of SlSOD6 and SlSOD8, which were only expressed in young fruits. Microarray data analysis showed that most members of the SlSOD gene family were altered under salt- and drought-stress conditions. This genome-wide analysis of SlSOD genes helps to clarify the function of SlSOD genes under different stress conditions and provides information to aid in further understanding the evolutionary relationships of SOD genes in plants.

The Hsp20 genes are involved in the response of plants to environment stresses including heat shock and also play a vital role in plant growth and development. They represent the most abundant small heat shock proteins (sHsps) in plants, but little is known about this family in tomato (Solanum lycopersicum), an important vegetable crop in the world. Here, we characterized heat shock protein 20 (SlHsp20) gene family in tomato through integration of gene structure, chromosome location, phylogenetic relationship, and expression profile. Using bioinformatics-based methods, we identified at least 42 putative SlHsp20 genes in tomato. Sequence analysis revealed that most of SlHsp20 genes possessed no intron or a relatively short intron in length. Chromosome mapping indicated that inter-arm and intra-chromosome duplication events contributed remarkably to the expansion of SlHsp20 genes. Phylogentic tree of Hsp20 genes from tomato and other plant species revealed that SlHsp20 genes were grouped into 13 subfamilies, indicating that these genes may have a common ancestor that generated diverse subfamilies prior to the mono-dicot split. In addition, expression analysis using RNA-seq in various tissues and developmental stages of cultivated tomato and the wild relative Solanum pimpinellifolium revealed that most of these genes (83%) were expressed in at least one stage from at least one genotype. Out of 42 genes, 4 genes were expressed constitutively in almost all the tissues analyzed, implying that these genes might have specific housekeeping function in tomato cell under normal growth conditions. Two SlHsp20 genes displayed differential expression levels between cultivated tomato and S. pimpinellifolium in vegetative (leaf and root) and reproductive organs (floral bud and flower), suggesting inter-species diversification for functional specialization during the process of domestication. Based on genome-wide microarray analysis, we showed that the transcript levels of SlHsp20 genes could be induced profusely by abiotic and biotic stresses such as heat, drought, salt, Botrytis cinerea, and Tomato Spotted Wilt Virus (TSWV), indicating their potential roles in mediating the response of tomato plants to environment stresses. In conclusion, these results provide valuable information for elucidating the evolutionary relationship of Hsp20 gene family and functional characterization of the SlHsp20 gene family in the future.

Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technology for gene expression and transcriptome analysis. Normalization is a process that is necessary to accurately analyze qRT-PCR data. Stability of reference gene expression is required for this process. Due to the large variation in expression levels of reference genes obtained from different experimental conditions, gene expression stabilities must be evaluated and identified in all experimental systems. In the present paper, the stability of the expression levels of seven potential reference genes in pepper are assessed using qRT-PCR analysis to determine optimal reference genes. These reference genes are evaluated in different pepper tissues, abiotic stress, and hormonal treatment samples. Three common statistical algorithms, geNorm, NormFinder, and BestKeeper, are used to identify expression stability and provide an accurate selection of reference genes. Two reference genes, beta tubulin and ubiquitin-conjugating protein (UBI-3), showed high stability in sample pools with abiotic stress and hormonal treatments. Among the sample pools tested, UBI-3 and glyceraldehyde-3-phosphate dehydrogenase expression levels were the most stable in different tissues. Therefore, these reference genes are selected for qRT-PCR analysis under the experimental conditions tested in pepper. In contrast, ubiquitin-conjugating enzyme and actin genes are identified as the least stable reference genes in all the groups tested, confirming that they are not suitable for normalization. Validation of these candidate genes could provide useful guidelines for reference gene selection in qRT-PCR studies in pepper.

Heat shock proteins 90 (Hsp90) are a kind of specific proteins in plant which were produced under environmental stresses. By referring to the tomato genome database, we identified and analyzed Hsp90 gene family members using bioinformatics methods. Results indicated that the tomato genome contained at least 7 Hsp90 genes, which were distributed unevenly on 6 chromosomes. Amino acid sequence length of these proteins ranged from 267 to 794aa. Numbers of intron ranged from 2 to 19. Microsynteny analysis showed that two pairs of Hsp90 genes (Hsp90-1and Hsp90-3, Hsp90-5 and Hsp90-7) were identified by segment duplication. In addition, multiple conservation motifs were found in Hsp90 proteins. Phylogenetic analysis revealed that Hsp90 genes from tomato, rice and Arabidopsis can be divided into 5 groups. Three pair of orthologous genes and four pairs of homologous genes were found. Expression analysis based on RNA-seq showed that the expression of three genes (Hsp90-5, Hsp90-6 and Hsp90-7) was high in vegetable and reproductive organs, while the expression of other four genes (Hsp90-1, Hsp90-2, Hsp90-3 and Hsp90-4) was relatively low except for its expression at the breaking stage of fruit. Analysis of promoter regions of Hsp90 genes showed that multiple cis-elements were involved in plant responses to biotic and abiotic stresses. The expression of 7 genes under heat stress was also detected by qRT-PCR. Expression of all Hsp90 genes in tomato leaf was enhanced. The results indicated that these genes could be participated in tomato leaf response to heat stresses. Together, these results will lay a foundation for analyzing Hsp90 gene function and molecular evolution in the future.

Background Pepper (Capsicum annuum L.) is one of the most important vegetable crops worldwide. However, its yield and fruit quality can be severely threatened by several pathogens. The plant nucleotide-binding site (NBS)-leucine-rich repeat (LRR) gene family is the largest class of known disease resistance genes (R genes) effective against such pathogens. Therefore, the isolation and identification of such R gene homologues from pepper will provide a critical foundation for improving disease resistance breeding programs. Results A total of 78 R gene analogues (CaRGAs) were identified in pepper by degenerate PCR amplification and database mining. Phylogenetic tree analysis of the deduced amino acid sequences for 51 of these CaRGAs with typically conserved motifs ( P-loop, kinase-2 and GLPL) along with some known R genes from Arabidopsis and tomato grouped these CaRGAs into the non-Toll interleukin-1 receptor (TIR)-NBS-LRR (CaRGAs I to IV) and TIR-NBS-LRR (CaRGAs V to VII) subfamilies. The presence of consensus motifs (i.e. P-loop, kinase-2 and hydrophobic domain) is typical of the non-TIR- and TIR-NBS-LRR gene subfamilies. This finding further supports the view that both subfamilies are widely distributed in dicot species. Functional divergence analysis provided strong statistical evidence of altered selective constraints during protein evolution between the two subfamilies. Thirteen critical amino acid sites involved in this divergence were also identified using DIVERGE version 2 software. Analyses of non-synonymous and synonymous substitutions per site showed that purifying selection can play a critical role in the evolutionary processes of non-TIR- and TIR-NBS-LRR RGAs in pepper. In addition, four specificity-determining positions were predicted to be responsible for functional specificity. qRT-PCR analysis showed that both salicylic and abscisic acids induce the expression of CaRGA genes, suggesting that they may primarily be involved in defence responses by activating signaling pathways. Conclusion The identified CaRGAs are a valuable resource for discovering R genes and developing RGA molecular markers for genetic map construction. They will also be useful for improving disease resistance in pepper. The findings of this study provide a better understanding of the evolutionary mechanisms that drive the functional diversification of non-TIR- and TIR-NBS-LRR R genes in pepper.