Rating System for Phytophthora Root Rot Influences Quantitative Trait Locus Detection and Reveals Incomplete Dominance and Duplicative Recessive Epistatic Gene Action in Capsicum annuum.

Phytophthora capsici is one of the most devastating pathogens facing pepper (Capsicum annuum) producers worldwide. Numerous factors, such as the race of the pathogen, the growing environment, and the source of resistance, have resulted in an overall lack of widely applicable molecular markers associated with resistance. Our objective was to determine the effect of the rating system on quantitative trait locus (QTL) detection and understand inheritance patterns of host resistance that can influence selection and molecular marker accuracy. We evaluated an F2:11 recombinant inbred line population screened against the highly virulent strain (Pc134) and scored using two widely used methods, developed by Bosland and Lindsey and by Black. The rating system developed by Bosland and Lindsey resulted in slightly higher logarithm of odds for the QTL on chromosome 5, and we detected a QTL on chromosome 12 uniquely using this rating system. A QTL on chromosome 10 was detected using both rating systems, but Black resulted in considerably higher logarithm of odds for this QTL compared with the Bosland and Lindsey system. Molecular markers developed were nominally better at accurately predicting the phenotype than previously published molecular markers but did not completely explain resistance in our validation populations. The inheritance pattern of resistance in one of our F2 populations did not significantly deviate from a 7:9 segregation ratio, indicating duplicative recessive epistasis. However, these results could be confounded by the presence of incomplete gene action, which was found through the improved selection accuracy when the phenotypes of heterozygous individuals were grouped with those with susceptible alleles.

Single nucleotide polymorphisms reveal genetic diversity in New Mexican chile peppers (Capsicum spp.).

BACKGROUND: Chile peppers (Capsicum spp.) are among the most important horticultural crops in the world due to their number of uses. They are considered a major cultural and economic crop in the state of New Mexico in the United States. Evaluating genetic diversity in current New Mexican germplasm would facilitate genetic improvement for different traits. This study assessed genetic diversity, population structure, and linkage disequilibrium (LD) among 165 chile pepper genotypes using single nucleotide polymorphism (SNP) markers derived from genotyping-by-sequencing (GBS). RESULTS: A GBS approach identified 66,750 high-quality SNP markers with known map positions distributed across the 12 chromosomes of Capsicum. Principal components analysis revealed four distinct clusters based on species. Neighbor-joining phylogenetic analysis among New Mexico State University (NMSU) chile pepper cultivars showed two main clusters, where the C. annuum genotypes grouped together based on fruit or pod type. A Bayesian clustering approach for the Capsicum population inferred K = 2 as the optimal number of clusters, where the C. chinense and C. frutescens grouped in a single cluster. Analysis of molecular variance revealed majority of variation to be between the Capsicum species (76.08 %). Extensive LD decay (~ 5.59 Mb) across the whole Capsicum population was observed, demonstrating that a lower number of markers would be required for implementing genome wide association studies for different traits in New Mexican type chile peppers. Tajima's D values demonstrated positive selection, population bottleneck, and balancing selection for the New Mexico Capsicum population. Genetic diversity for the New Mexican chile peppers was relatively low, indicating the need to introduce new alleles in the breeding program to broaden the genetic base of current germplasm. CONCLUSIONS: Genetic diversity among New Mexican chile peppers was evaluated using GBS-derived SNP markers and genetic relatedness on the species level was observed. Introducing novel alleles from other breeding programs or from wild species could help increase diversity in current germplasm. We present valuable information for future association mapping and genomic selection for different traits for New Mexican chile peppers for genetic improvement through marker-assisted breeding.

Genomic regions and candidate genes linked with Phytophthora capsici root rot resistance in chile pepper (Capsicum annuum L.).

BACKGROUND: Phytophthora root rot, caused by Phytophthora capsici, is a major disease affecting Capsicum production worldwide. A recombinant inbred line (RIL) population derived from the hybridization between 'Criollo de Morellos-334' (CM-334), a resistant landrace from Mexico, and 'Early Jalapeno', a susceptible cultivar was genotyped using genotyping-by-sequencing (GBS)-derived single nucleotide polymorphism (SNP) markers. A GBS-SNP based genetic linkage map for the RIL population was constructed. Quantitative trait loci (QTL) mapping dissected the genetic architecture of P. capsici resistance and candidate genes linked to resistance for this important disease were identified. RESULTS: Development of a genetic linkage map using 1,973 GBS-derived polymorphic SNP markers identified 12 linkage groups corresponding to the 12 chromosomes of chile pepper, with a total length of 1,277.7 cM and a marker density of 1.5 SNP/cM. The maximum gaps between consecutive SNP markers ranged between 1.9 (LG7) and 13.5 cM (LG5). Collinearity between genetic and physical positions of markers reached a maximum of 0.92 for LG8. QTL mapping identified genomic regions associated with P. capsici resistance in chromosomes P5, P8, and P9 that explained between 19.7 and 30.4% of phenotypic variation for resistance. Additive interactions between QTL in chromosomes P5 and P8 were observed. The role of chromosome P5 as major genomic region containing P. capsici resistance QTL was established. Through candidate gene analysis, biological functions associated with response to pathogen infections, regulation of cyclin-dependent protein serine/threonine kinase activity, and epigenetic mechanisms such as DNA methylation were identified. CONCLUSIONS: Results support the genetic complexity of the P. capsici-Capsicum pathosystem and the possible role of epigenetics in conferring resistance to Phytophthora root rot. Significant genomic regions and candidate genes associated with disease response and gene regulatory activity were identified which allows for a deeper understanding of the genomic landscape of Phytophthora root rot resistance in chile pepper.

Comparative transcriptional analysis of Capsicum flower buds between a sterile flower pool and a restorer flower pool provides insight into the regulation of fertility restoration.

BACKGROUND: Cytoplasmic male sterility (CMS) and its restoration of fertility (Rf) system is an important mechanism to produce F1 hybrid seeds. Understanding the interaction that controls restoration at a molecular level will benefit plant breeders. The CMS is caused by the interaction between mitochondrial and nuclear genes, with the CMS phenotype failing to produce functional anthers, pollen, or male gametes. Thus, understanding the complex processes of anther and pollen development is a prerequisite for understanding the CMS system. Currently it is accepted that the Rf gene in the nucleus restores the fertility of CMS, however the Rf gene has not been cloned. In this study, CMS line 8A and the Rf line R1, as well as a sterile pool (SP) of accessions and a restorer pool (RP) of accessions analyzed the differentially expressed genes (DEGs) between CMS and its fertility restorer using the conjunction of RNA sequencing and bulk segregation analysis. RESULTS: A total of 2274 genes were up-regulated in R1 as compared to 8A, and 1490 genes were up-regulated in RP as compared to SP. There were 891 genes up-regulated in both restorer accessions, R1 and RP, as compared to both sterile accessions, 8A and SP. Through annotation and expression analysis of co-up-regulated expressed genes, eight genes related to fertility restoration were selected. These genes encode putative fructokinase, phosphatidylinositol 4-phosphate 5-kinase, pectate lyase, exopolygalacturonase, pectinesterase, cellulose synthase, fasciclin-like arabinogalactan protein and phosphoinositide phospholipase C. In addition, a phosphatidylinositol signaling system and an inositol phosphate metabolism related to the fertility restorer of CMS were ranked as the most likely pathway for affecting the restoration of fertility in pepper. CONCLUSIONS: Our study revealed that eight genes were related to the restoration of fertility, which provides new insight into understanding the molecular mechanism of fertility restoration of CMS in Capsicum.

Race Characterization of Phytophthora Root Rot on Capsicum in Taiwan as a Basis for Anticipatory Resistance Breeding.

Peppers (Capsicum sp.) are an increasingly important crop because of their use as a vegetable, spice, and food colorant. The oomycete Phytophthora capsici is one of the most devastating pathogens to pepper production worldwide, causing more than $100 million in losses annually. Developing cultivars resistant to P. capsici is challenging because of the many physiological races that exist and new races that are continuously evolving. This problem is confounded by the lack of a universal system of race characterization. As a basis to develop a global anticipatory breeding program, New Mexico recombinant inbred lines (NMRILs) functioned as a host differential for Phytophthora root rot to characterize the race structure of P. capsici populations in Taiwan. Using the NMRILs, 24 new races were identified, illustrating the utility and usefulness of the NMRILs for anticipatory breeding. Virulence of P. capsici was observed to be geographically specific and in two virulence clusters. Interestingly, all but two isolates collected in 2016 were the A2 mating type, which is a shift from the predominantly A1 mating type isolates collected prior to 2008. The NMRILs host differential provides an approach for scientists to work together on a global scale when breeding for resistance as well as on a local level for regional gene deployment. Additionally, we propose that the current race numbering system, which has no biological meaning, be supplemented with the virulence phenotype, based on the susceptible NMRILs to a given isolate. This work provides insights into the population dynamics of P. capsici and interactions within the highly complex Capsicum-Phytophthora pathosystem, and offers a basis for similar research in other crops.

Sensory properties of chile pepper heat - and its importance to food quality and cultural preference.

Chile peppers are one of the most important vegetable and spice crops in the world. They contain capsaicinoids that are responsible for the characteristic burning (pungency) sensation. Currently, there are 22 known naturally occurring capsaicinoids that can cause the heat sensation when consumed. Each produces a different heat sensation effect in the mouth. A need exists for a standard and new terminology to describe the complex heat sensation one feels when eating a chile pepper. A comprehensive set of descriptors to describe the sensory characteristics of chile pepper heat was developed. It was validated with trained panelists tasting samples representing the five domesticated species and 14 pod-types within these species. Five key attributes that define and reference a lexicon for describing the heat sensation of chile peppers were determined to describe the heat sensation in any product, and importantly, can be used in the food industry worldwide.

A novel Capsicum gene inhibits host-specific disease resistance to Phytophthora capsici.

A novel disease resistance inhibitor gene (inhibitor of P. capsici resistance [Ipcr]), found in the chile pepper (Capsicum annuum) variety 'New Mexico Capsicum Accession 10399' (NMCA10399), inhibits resistance to Phytophthora capsici but not to other species of Phytophthora. When a highly P. capsici-resistant variety was hybridized with NMCA10399, the resultant F1 populations, when screened, were completely susceptible to P. capsici for root rot and foliar blight disease syndromes, despite the dominance inheritance of P. capsici resistance in chile pepper. The F2 population displayed a 3:13 resistant-to-susceptible (R:S) ratio. The testcross population displayed a 1:1 R:S ratio, and a backcross population to NMCA10399 displayed complete susceptibility. These results demonstrate the presence of a single dominant inhibitor gene affecting P. capsici resistance in chile pepper. Moreover, when lines carrying the Ipcr gene were challenged against six Phytophthora spp., the nonhost resistance was not overcome. Therefore, the Ipcr gene is interfering with host-specific resistance but not the pathogen- or microbe-associated molecular pattern nonhost responses.

Chile Pepper (Capsicum) Breeding and Improvement in the "Multi-Omics" Era.

Chile pepper (Capsicum spp.) is a major culinary, medicinal, and economic crop in most areas of the world. For more than hundreds of years, chile peppers have "defined" the state of New Mexico, USA. The official state question, "Red or Green?" refers to the preference for either red or the green stage of chile pepper, respectively, reflects the value of these important commodities. The presence of major diseases, low yields, decreased acreages, and costs associated with manual labor limit production in all growing regions of the world. The New Mexico State University (NMSU) Chile Pepper Breeding Program continues to serve as a key player in the development of improved chile pepper varieties for growers and in discoveries that assist plant breeders worldwide. Among the traits of interest for genetic improvement include yield, disease resistance, flavor, and mechanical harvestability. While progress has been made, the use of conventional breeding approaches has yet to fully address producer and consumer demand for these traits in available cultivars. Recent developments in "multi-omics," that is, the simultaneous application of multiple omics approaches to study biological systems, have allowed the genetic dissection of important phenotypes. Given the current needs and production constraints, and the availability of multi-omics tools, it would be relevant to examine the application of these approaches in chile pepper breeding and improvement. In this review, we summarize the major developments in chile pepper breeding and present novel tools that can be implemented to facilitate genetic improvement. In the future, chile pepper improvement is anticipated to be more data and multi-omics driven as more advanced genetics, breeding, and phenotyping tools are developed.

A predicted NEDD8 conjugating enzyme gene identified as a Capsicum candidate Rf gene using bulk segregant RNA sequencing.

Cytoplasmic male sterility (CMS) is an important tool for producing F1 hybrids, which can exhibit heterosis. The companion system, restorer-of-fertility (Rf), is poorly understood at the molecular level and would be valuable in producing restorer lines for hybrid seed production. The identity of the Rf gene in Capsicum (pepper) is currently unclear. In this study, using bulked segregant RNA sequencing (BSR-seq), a strong candidate Rf gene, Capana06g002866, which is annotated as a NEDD8 conjugating enzyme E2, was identified. Capana06g002866 has an ORF of 555 bp in length encoding 184 amino acids; it can be cloned from F1 plants from the hybridization of the CMS line 8A and restorer line R1 but is not found in CMS line 8A. With qRT-PCR validation, Capana06g002866 was found to be upregulated in restorer accessions compared to sterile accessions. The relative expression in flower buds increased with the developmental stage in F1 plants, while the expression was very low in all flower bud stages of the CMS lines. These results provide new insights into the Rf gene in pepper and will be useful for other crops utilizing the CMS system.

Challenges and Strategies for Breeding Resistance in Capsicum annuum to the Multifarious Pathogen, Phytophthora capsici.

Phytophthora capsici is the most devastating pathogen for chile pepper production worldwide and current management strategies are not effective. The population structure of the pathogen is highly variable and few sources of widely applicable host resistance have been identified. Recent genomic advancements in the host and the pathogen provide important insights into the difficulties reported by epidemiological and physiological studies published over the past century. This review highlights important challenges unique to this complex pathosystem and suggests strategies for resistance breeding to help limit losses associated with P. capsici.

Enzymatic changes in phenylalanine ammonia-lyase, cinnamic-4-hydroxylase, capsaicin synthase, and peroxidase activities in capsicum under drought stress.

Penylalanine ammonia-lyase (PAL), cinnamic-4-hydroxylase (C4H), capsaicin synthase (CS), and peroxidase (POD) are involved in the capsaicinoid biosynthesis pathway and may be altered in cultivars with different pungency levels. This study clarified the action of these enzymes under drought stress for hot Capsicum cultivars with low, medium,and high pungency levels. At the flowering stage, control plants were watered at field capacity, whereas drought-induced plants were subjected to gradual drought stress. Under drought stress, PAL, C4H, CS, and POD enzyme activities increased as compared to the non-drought-stressed plants. A novel discovery was that PAL was the critical enzyme in capsaicinoid biosynthesis under drought stress because its activities and capsaicinoid increased across the different pungency levels of hot pepper cultivars examined.

Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species.

Hot pepper (Capsicum annuum), one of the oldest domesticated crops in the Americas, is the most widely grown spice crop in the world. We report whole-genome sequencing and assembly of the hot pepper (Mexican landrace of Capsicum annuum cv. CM334) at 186.6× coverage. We also report resequencing of two cultivated peppers and de novo sequencing of the wild species Capsicum chinense. The genome size of the hot pepper was approximately fourfold larger than that of its close relative tomato, and the genome showed an accumulation of Gypsy and Caulimoviridae family elements. Integrative genomic and transcriptomic analyses suggested that change in gene expression and neofunctionalization of capsaicin synthase have shaped capsaicinoid biosynthesis. We found differential molecular patterns of ripening regulators and ethylene synthesis in hot pepper and tomato. The reference genome will serve as a platform for improving the nutritional and medicinal values of Capsicum species.

Variability of Carotenoid Biosynthesis in Orange Colored Capsicum spp.

Pepper, Capsicum spp., is a worldwide crop valued for heat, nutrition, and rich pigment content. Carotenoids, the largest group of plant pigments, function as antioxidants and as vitamin A precursors. The most abundant carotenoids in ripe pepper fruits are ß-carotene, capsanthin, and capsorubin. In this study, the carotenoid composition of orange fruited Capsicum lines was defined along with the allelic variability of the biosynthetic enzymes. The carotenoid chemical profiles present in seven orange pepper varieties were determined using a novel UPLC method. The orange appearance of the fruit was due either to the accumulation of ß-carotene, or in two cases, due to only the accumulation of red and yellow carotenoids. Four carotenoid biosynthetic genes, Psy, Lcyb, CrtZ-2, and Ccs were cloned and sequenced from these cultivars. This data tested the hypothesis that different alleles for specific carotenoid biosynthetic enzymes are associated with specific carotenoid profiles in orange peppers. While the coding regions within Psy and CrtZ-2 did not change in any of the lines, the genomic sequence contained introns not previously reported. Lcyb and Ccs contained no introns but did exhibit polymorphisms resulting in amino acid changes; a new Ccs variant was found. When selectively breeding for high provitamin A levels, phenotypic recurrent selection based on fruit color is not sufficient, carotenoid chemical composition should also be conducted. Based on these results, specific alleles are candidate molecular markers for selection of orange pepper lines with high ß-carotene and therefore high pro-vitamin A levels.