Resistance to three thrips species in Capsicum spp. depends on site conditions and geographic regions

Capsicum species are commercially grown for pepper production. This crop suffers severely from thrips damage and the identification of natural sources of thrips resistance is essential for the development of resistant cultivars. It is unclear whether resistance to Frankliniella occidentalis as assessed in a specific environment holds under different conditions. Additionally, other thrips species may respond differently to the plant genotypes. Screening for robust and general resistance to thrips encompasses testing different Capsicum accessions under various conditions and with different thrips species. We screened 11 Capsicum accessions (C. annuum and C. chinense) for resistance to F. occidentalis at three different locations in the Netherlands. Next, the same 11 accessions were screened for resistance to Thrips palmi and Scirtothrips dorsalis at two locations in Asia. This resulted in a unique analysis of thrips resistance in Capsicum at five different locations around the world. Finally, all accessions were also screened for resistance to F. occidentalis in the Netherlands using a leaf disc choice assay, allowing direct comparison of whole plant and leaf disc assays. Resistance to F. occidentalis was only partially consistent among the three sites in the Netherlands. The most susceptible accessions were consistently susceptible, but which accession was the most resistant differed among sites. In Asia, one C. chinense accession was particularly resistant to S. dorsalis and T. palmi, but this was not the most resistant accession to F. occidentalis. Overall, resistance to F. occidentalis correlated with S. dorsalis but not with T. palmi resistance in the C. annuum accessions. Damage inflicted on leaf discs reflected damage on the whole plant level. Our study showed that identifying broad spectrum resistance to thrips in Capsicum may prove to be challenging. Breeding programmes should focus on developing cultivars suitable for growing in defined geographic regions with specific thrips species and abiotic conditions.     

Combined use of bulked segregant analysis and microarrays reveals SNP markers pinpointing a major QTL for resistance to Phytophthora capsici in pepper

Key message

Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed candidate genes underlying the major QTL for Phytophthora capsici resistance in Capsicum . Using the candidate genes, reliable markers for Phytophthora resistance were developed and validated.

Abstract

Phytophthora capsici L. is one of the most destructive pathogens of pepper (Capsicum spp.). Resistance of pepper against P. capsici is controlled by quantitative trait loci (QTL), including a major QTL on chromosome 5 that is the predominant contributor to resistance. Here, to maximize the effect of this QTL and study its underlying genes, an F₂ population and recombinant inbred lines were inoculated with P. capsici strain JHAI1-7 zoospores at a low concentration (3 × 10³/mL). Resistance phenotype segregation ratios for the populations fit a 3:1 and 1:1 (resistant:susceptible) segregation model, respectively, consistent with a single dominant gene model. Bulked segregant analysis (BSA) using Affymetrix GeneChips revealed a single position polymorphism (SPP) marker mapping to the major QTL. When this SPP marker (Phyto5SAR) together with other SNP markers located on chromosome 5 was used to confirm the position of the major QTL, Phyto5SAR showed the highest LOD value at the QTL. A scaffold sequence (scaffold194) containing Phyto5SAR was identified from the C. annuum genome database. The scaffold contained two putative NBS-LRR genes and one SAR 8.2A gene as candidates for contributing to P. capsici resistance. Markers linked to these genes were developed and validated by testing 100 F₁ commercial cultivars. Among the markers, Phyto5NBS1 showed about 90 % accuracy in predicting resistance phenotypes to a low-virulence P. capsici isolate. These results suggest that Phyto5NBS1 is a reliable marker for P. capsici resistance and can be used for identification of a gene(s) underlying the major QTL on chromosome 5.     

Genetic diversity in Capsicum germplasm based on microsatellite and random amplified microsatellite polymorphism markers

A sound knowledge of the genetic diversity among germplasm is vital for strategic germplasm collection, maintenance, conservation and utilisation. Genomic simple sequence repeats (SSRs) and random amplified microsatellite polymorphism (RAMPO) markers were used to analyse diversity and relationships among 48 pepper (Capsicum spp.) genotypes originating from nine countries. These genotypes covered 4 species including 13 germplasm accessions, 30 improved lines of 4 domesticated species and 5 landraces derived from natural interspecific crosses. Out of 106 SSR markers, 25 polymorphic SSR markers (24 %) detected a total of 76 alleles (average, 3.04; range, 2–5). The average polymorphic information content (PIC) was 0.69 (range, 0.29–0.92). Seventeen RAMPO markers produced 87 polymorphic fragments with average PIC of 0.63 (range, 0.44–0.81). Dendrograms based on SSRs and RAMPOs generated two clusters. All 38 Capsicum annuum genotypes and an interspecific landrace clustered together, whereas nine non-annuum (three Capsicum frutescens, one Capsicum chinense, one Capsicum baccatum and four interspecific landraces) genotypes clustered separately. Genetic variation within non-annuum genotypes was greater than the C. annuum genotypes. Distinctness of interspecific derivative landraces grown in northeast India was validated; natural crossing between sympatric Capsicum species has been proposed as the mechanism of their origin.     

Resistance of pepper germplasm to Meloidogyne incognita

In this work, we have evaluated the host suitability of 29 pepper genotypes of Capsicum annuum and 9 of the related cultivated species C. chinense (4), C. frutescens (4) and C. pubescens (1) to Meloidogyne incognita in field conditions. The presence/absence of resistance genes in the pepper germplasm were also assessed using PCR‐specific markers linked to the N, Me1‐Mech2, Me3‐Me4 and Me7‐Mech1 genes. Intraspecific variability for M. incognita resistance was found. According to gall index (GI) and reproduction index (RI) the most resistant genotypes, which may contribute to nematode management, include three of C. frutescens (Fru‐2, Fru‐3 and Fru‐4) and seven of C. annuum (Ca‐3, Ca‐4, Ca‐5, Ca‐11, Ca‐15, Ca‐24 and Ca‐25). No egg masses or eggs were found in Fru‐3 and Fru‐4 genotypes as occurred in the resistant controls ‘SCM’, ‘CH’ and ‘Charlot’. The amplification of markers linked to resistance genes in genotypes with a suitable degree of resistance, together with the differences found between genotypes with regard to the gene and/or number of amplified markers, make this germplasm a valuable tool for further characterisation and pepper breeding.     

QTL mapping of anthracnose (<Emphasis Type="Italic">Colletotrichum</Emphasis> spp.) resistance in a cross between <Emphasis Type="Italic">Capsicum annuum</Emphasis> and <Emphasis Type="Italic">C. chinense</Emphasis>

Anthracnose fruit rot is an economically important disease that affects pepper production in Indonesia. Strong resistance to two causal pathogens, Colletotrichum gloeosporioides and C. capsici, was found in an accession of Capsicum chinense. The inheritance of this resistance was studied in an F₂ population derived from a cross of this accession with an Indonesian hot pepper variety (Capsicum annuum) using a quantitative trait locus (QTL) mapping approach. In laboratory tests where ripe fruits were artificially inoculated with either C. gloeosporioides or C. capsici, three resistance-related traits were scored: the infection frequency, the true lesion diameter (averaged over all lesions that actually developed), and the overall lesion diameter (averaged over all inoculation points, including those that did not develop lesions). One main QTL was identified with highly significant and large effects on all three traits after inoculation with C. gloeosporioides and on true lesion diameter after inoculation with C. capsici. Three other QTL with smaller effects were found for overall lesion diameter and true lesion diameter after inoculation with C. gloeosporioides, two of which also had an effect on infection frequency. Interestingly, the resistant parent carried a susceptible allele for a QTL for all three traits that was closely linked to the main QTL. The results with C. capsici were based on less observations and therefore less informative. Although the main QTL was shown to have an effect on true lesion diameter after inoculation with C. capsici, no significant QTL were identified for overall lesion diameter or infection frequency.     

Isolation and viral infection of Capsicum leaf protoplasts

Summary A protocol for protoplast isolation was developed and tested with five Capsicum genotypes representing two cultivated species, C. annuum and C. chinense. Key variables included growth conditions for source plants and the concentration of mannitol used as osmoticum. Protoplasts isolated from each of the genotypes became infected when inoculated via electroporation with viral RNA from either pepper mottle potyvirus, tobacco etch potyvirus or cucumber mosaic cucumovirus.     

Conservation of floral,fruit and chromosomal diversity: a review on diploid and polyploid Capsicum annuum complex in India

Capsicum as a spice crop, has wild and cultivated forms admired globally, including Indian subcontinent with vast climatic ranges. Systematic representation of the Indian Capsicum is required to address species relationships and sustainable agriculture, in face of unpredictable climatic conditions. We have updated the catalogue of Indian ‘C. annuum complex’ with 28 landraces and populations from different agro-climatic regions. The agro-climatic influence on the origin of stable chili landraces in India is remarkable, especially in the North East. The floral and fruit morphotype standards and chromosomal attributes have been considered for four distinct ‘C. annuum complex’ members under three species. The highlights of study are: (1) comparative profiling of Indian Capsicum species revealing less infraspecific variation within C. frutescens and C. chinense than C. annuum, at par with cultivation status, (2) karyotype analysis of some unique diploid landraces of C. annuum, (3) karyotypic confirmation of the polyploid Dalle Khursani landraces exclusive to India. To obtain more information, we attempted to correlate diversity of fruit and floral morphotype with chromosomal diversity. Existence of elite and rare germplasm found in the regional pockets offer great scope for enriching the agricultural tradition. The present dataset may serve as a template to be continuously upgraded by taxonomists, genomicists and breeders.

     

Molecular Profiling for Genetic Variability in <Emphasis Type="Italic">Capsicum</Emphasis> Species Based on ISSR and RAPD Markers

The taxonomic identity of Capsicum species is found to be difficult as it displays variations at morpho-chemical characters. Twenty-two accessions of six Capsicum species, namely, C. annuum, C. baccatum, C. chinense, C. eximium, C. frutescens, and C. luteum were investigated for phenotypic diversity based on flower color and for genetic differences by molecular makers. The genetic cluster analyses of 27 RAPD and eight ISSR primers, respectively, revealed genetic similarities in the ranges of 23–88% and 11–96%. Principal component analysis of the pooled RAPD and ISSR data further supports the genetic similarity and groupings. Different species showed variations in relation to corolla shade of flower. C. annuum accessions formed a single cluster in the molecular analysis as maintaining their flower characteristic. C. chinense accession shared flower features with the accessions of C. frutescens and were found to be closer at genotypic level. C. luteum was found to be rather closer to C. baccatum complex, both phenotypically and genetically. The only accession of C. eximium presenting purple flowers falls apart from the groupings. The floral characteristics and the molecular markers are found to be useful toward the delineation of the species specificity in Capsicum collection and identification of genetic stock.     

Single-base cytosine methylation analysis in fruits of three Capsicum species

Single-base cytosine methylation analysis across fruits of Capsicum annuum, C. chinense and C. frutescens showed global average methylation ranging from 82.8–89.1%, 77.6–83.9%, and 22.4–25% at CG, CHG and CHH contexts, respectively. High gene-body methylation at CG and CHG was observed across Capsicum species. The C. annuum showed the highest proportion (>80%) of mCs at different genomic regions compared to C. chinense and C. frutescens. Cytosine methylation for transposable-elements were lower in C. frutescens compared to C. annuum and C. chinense. A total of 510,165 CG, 583112 CHG and 277,897 CHH DMRs were identified across three Capsicum species. The differentially methylated regions (DMRs) distribution analysis revealed C. frutescens as more hypo-methylated compared to C. annuum and C. chinense, and also the presence of more intergenic DMRs in Capsicum genome. At CG and CHG context, gene expression and promoter methylation showed inverse correlations. Furthermore, the observed correlation between methylation and expression of genes suggested the potential role of methylation in Capsicum fruit development/ripening.     

Patterns of genetic variation of the genusCapsicum (Solanaceae) in Mexico

The evolutionary relationships of 186 accessions ofCapsicum from Mexico were studied through enzyme electrophoresis. A total of 76 alleles representing 20 genetic loci coding for nine enzyme systems were observed and the allelic variations of enzymes were studied for geographical distribution. Allele frequencies were used to estimate the apportionment of gene diversity within and between populations and to construct a dendrogram based on a similarity matrix containingNei genetic distances. — The gene diversity estimates suggest that the structure ofCapsicum populations in Mexico consists of predominantly homozygous genotypes presumably due to a self-pollinated breeding system and population bottlenecks. Significant genetic differentiation was found mainly between populations of differing geographical regions.—Based on the results of this study, three species of domesticatedCapsicum can be identified in Mexico,C. annuum var.annuum, C. chinense, andC. pubescens. Semidomesticated and wild forms include two species,C. frutescens andC. annuum var.glabriusculum. A sharp geographical division results between the latter species;C. frutescens was collected exclusively in the southeastern states of Oaxaca, Chiapas, and Tabasco; whereas wild and semidomesticated forms from the rest of the country areC. annuum. Based upon the similarity of enzyme genotypes of semidomesticated and wild forms, the primary center of domestication of cultivatedC. annuum was estimated to be the region comprising the states of Tamaulipas, Nuevo Leon, San Luis Potosi, Veracruz, and Hidalgo in eastern Mexico. A possible second center of domestication is suggested to be localized in the state of Nayarit, western Mexico.     

Genetic analysis of stunted growth by nuclear-cytoplasmic interaction in interspecific hybrids of Capsicum by using RAPD markers

When eight cultivars of Capsicum annuum were used as female parents in interspecific crosses with two accessions of C. chinense, dwarfism occurred in hybrids originating from 10 out of 16 combinations, while hybrids of the remaining 6 combinations grew normally. In contrast, when C. chinense was used as female parent, all of the hybrids showed severely stunted growth as if affected by a virus. These results suggested that the stunted growth expressed in the cross of C. chinense x C. annuum is caused by an interaction between nuclear gene(s) from C. annuum and the cytoplasm of C. chinense. To examine the number of nuclear gene(s) which cause(s) the stunted growth, we backcrossed F₁ hybrids of C. annuum x C. chinense to C. chinense. About one-quarter of the progeny in the backcrossed hybrids of C. chinense x (C. annuum x C. chinense) showed the same stunted growth shown by the f₁ hybrids of C. chinense x C. annuum, suggesting that two complementary genes of C. annuum cause the stunted growth. However, the higher abortion rates of ovules and lower germination percentage of seeds in C. chinense x C. annuum than in the selfed C. chinense implied that the genetic ratio of the stunted type would have been higher than that observed in the C. chinense x (C. annuum x C. chinense) progeny. We then attempted a linkage analysis between the stunted growth and randomly amplified polymorphic DNA (RAPD) of C. chinense x (C. annuum x C. chinense) progeny. A RAPD marker that associated with 94% of the stunted plants but not with 94% of the normal one was identified. This confirmed that a single nuclear gene of C. annuum which is linked to the RAPD marker with a recombination value of 6% causes the stunted growth in an interaction with the cytoplasm of C. chinense.     

Identification,validation and survey of a single nucleotide polymorphism (SNP) associated with pungency in <Emphasis Type="Italic">Capsicum</Emphasis> spp.

A single nucleotide polymorphism (SNP) associated with pungency was detected within an expressed sequence tag (EST) of 307 bp. This fragment was identified after expression analysis of the EST clone SB2-66 in placenta tissue of Capsicum fruits. Sequence alignments corresponding to this new fragment allowed us to identify an SNP between pungent and non-pungent accessions. Two methods were chosen for the development of the SNP marker linked to pungency: tetra-primer amplification refractory mutation system-PCR (tetra-primer ARMS-PCR) and cleaved amplified polymorphic sequence. Results showed that both methods were successful in distinguishing genotypes. Nevertheless, tetra-primer ARMS-PCR was chosen for SNP genotyping because it was more rapid, reliable and less cost-effective. The utility of this SNP marker for pungency was demonstrated by the ability to distinguish between 29 pungent and non-pungent cultivars of Capsicum annuum. In addition, the SNP was also associated with phenotypic pungent character in the tested genotypes of C. chinense, C. baccatum, C. frutescens, C. galapagoense, C. eximium, C. tovarii and C. cardenasi. This SNP marker is a faster, cheaper and more reproducible method for identifying pungent peppers than other techniques such as panel tasting, and allows rapid screening of the trait in early growth stages.     

Optimization of Culturing Conditions of a Strain of Phytophthora capsici Pathogenic to Habanero Pepper (Capsicum chinense)

Phytophthora capsici is an oomycete known as the causal agent of wilting disease in Capsicum spp., which causes rotting of roots, crowns, stems, leaves and fruits. To date, little is known about the production of phytotoxic metabolites by P. capsici or their role in the infection process. As part of a project directed towards the isolation and identification of phytotoxins produced by a strain of P. capsici pathogenic to habanero pepper (Capsicum chinense), we have evaluated the effect of factors such as aeration, light and culture medium on the production of mycelium and phytotoxic metabolites by P. capsici. The results showed that culturing P. capsici in potato dextrose broth (PDB) containing habanero pepper leaf infusion, in the dark and under still conditions, results in a high production of mycelium and a high phytotoxicity of the culture filtrate, in the shortest period of time.     

Polymorphic Microsatellite Markers Transferable Across <Emphasis Type="Italic">Capsicum</Emphasis> Species

Pepper (Capsicum annuum L.) is one of the most important crops in the family Solanaceae. However, the number of polymorphic molecular loci detected in this important crop is far behind that of other cultivated plant species. In the present study, a total of 45 microsatellite primer pairs were developed using Capsicum expressed sequence tags databases. Microsatellite primer pairs were tested using several species of Capsicum and several genera in the family Solanaceae including tomato, potato, eggplant, and tobacco. Results indicated that microsatellite primer pairs amplified genomic targets of C. annuum L., Capsicum baccatum L., Capsicum chacoense L., Capsicum chinense L., Capsicum frutescens L., and Capsicum pubescens Ruiz et Pavon, indicating species transferability within Capsicum. Further analyses revealed that amplicons of these primer pairs segregated 1:2:1 or 3:1 Mendelian fashions in 38 F₂ individuals of pepper. It was also noted that markers derived from sequences containing dinucleotide repeats were generally more polymorphic at the intraspecific level than sequences containing trinucleotide repeats. All the microsatellite primer pairs developed in this study will be useful for marker-assisted selection and mapping studies in pepper.     

Comparison of resistance to potyviruses within Solanaceae: infection of potatoes with tobacco etch potyvirus and peppers with potato A and Y potyviruses

The reaction of several cultivated potato varieties (Solarium tuberosum L.) to three strains of tobacco etch potyvirus (TEV-F, TEV-Mex21 and TEV-ATCC) and the reaction of several pepper lines (Capsicum annuum L. and C. chinense L.) to two strains of potato Y potyvirus (PVY^O and PVY^N) and one strain of potato A potyvirus (PVA-M) was tested. The potato varieties included in this study carried resistance genes against PVY, PVA and potato V potyvirus, but all were susceptible to TEV and developed mottle and mosaic symptoms. TEV was readily transmitted by mechanical inoculation from tobacco and potato to potato, whereas transmission from pepper to potato occurred infrequently. TEV was transmitted through potato tubers, and from pepper to potato plants by aphids. Lack of detectable systemic infection following graft-inoculation indicated extreme resistance to PVY^O and PVA in several pepper lines. No pepper line was systemically infected with PVY^N following mechanical inoculation (graft-inoculation was not carried out with PVY^N). The development of necrotic lesions following mechanical and graft-inoculation indicated hypersensitive response to PVY^O in several pepper lines which resembled the resistance responses to these potyvirus strains in potato. Results of this study together with previous work indicate that C. annuum cv. Avelar is resistant to four potyviruses [PVY, PVA, pepper mottle potyvirus (PepMoV) and some isolates of TEV]; C. annuum cv. Criollo de Morelos and C. chinense PI 152225 and PI 159236 are resistant to three potyviruses (PVY, PepMoV and PVA; and PVY, PepMoV and TEV, respectively); C. annuum 9093–1 and 92016–1 are resistant to PVY and PepMoV; and C. annuum cv. Jupiter and C. annuum cv. RNaky are resistant to PVY^N and PVA.     

Morphological, Pathological and Molecular Variability in Colletotrichum capsici, the Cause of Fruit Rot of Chillies in the Subtropical Region of North-western India

Fruit rot of chillies (Capsicum annuum L.), caused by Colletotrichum capsici under tropical and subtropical conditions, results in qualitative and quantitative yield losses. Based on variation in cultural and morphological traits of C. capsici populations, 37 isolates were categorized into five groups designated, respectively, as Cc‐I, Cc‐II, Cc‐III, Cc‐IV and Cc‐V. In culture, most of the isolates produced cottony, fluffy or suppressed colonies. However, no significant differences were noticed in shape and size of conidia. The reaction of the 37 isolates on an indigenously developed differential set of Capsicum cultivars indicated the existence of different virulences in Himachal Pradesh (HP) chilli populations. Fifteen pathotypes of the pathogen were characterized from various chilli‐growing regions of HP. Pathotype CCP‐1 was most virulent and attacked all the differential cultivars. The genetic relationship between five morphological groups recognized within C. capsici was investigated using random amplified polymorphic DNA (RAPD) analysis. Molecular polymorphism generated by RAPD confirmed the variation in virulences of C. capsici and different isolates were grouped into five clusters. However, four isolates (Cc‐5, Cc‐33, Cc‐29 and Cc‐37) exhibited identical RAPD haplotypes. The pathological and RAPD grouping of isolates suggested no correlation among the test isolates.     

New Insights into Capsicum spp Relatedness and the Diversification Process of Capsicum annuum in Spain

The successful exploitation of germplasm banks, harbouring plant genetic resources indispensable for plant breeding, will depend on our ability to characterize their genetic diversity. The Vegetable Germplasm Bank of Zaragoza (BGHZ) (Spain) holds an important Capsicum annuum collection, where most of the Spanish pepper variability is represented, as well as several accessions of other domesticated and non-domesticated Capsicum spp from all over the five continents. In the present work, a total of 51 C. annuum landraces (mainly from Spain) and 51 accessions from nine Capsicum species maintained at the BGHZ were evaluated using 39 microsatellite (SSR) markers spanning the whole genome. The 39 polymorphic markers allowed the detection of 381 alleles, with an average of 9.8 alleles per locus. A sizeable proportion of alleles (41.2%) were recorded as specific alleles and the majority of these were present at very low frequencies (rare alleles). Multivariate and model-based analyses partitioned the collection in seven clusters comprising the ten different Capsicum spp analysed: C. annuum, C. chinense, C. frutescens, C. pubescens, C. bacatum, C. chacoense and C. eximium. The data clearly showed the close relationships between C. chinense and C. frutescens. C. cardenasii and C. eximium were indistinguishable as a single, morphologically variable species. Moreover, C. chacoense was placed between C. baccatum and C. pubescens complexes. The C. annuum group was structured into three main clusters, mostly according to the pepper fruit shape, size and potential pungency. Results suggest that the diversification of C. annuum in Spain may occur from a rather limited gene pool, still represented by few landraces with ancestral traits. This ancient population would suffer from local selection at the distinct geographical regions of Spain, giving way to pungent and elongated fruited peppers in the South and Center, while sweet blocky and triangular types in Northern Spain.     

A key QTL cluster is conserved among accessions and exhibits broad-spectrum resistance to Phytophthora capsici: a valuable locus for pepper breeding

Developing cultivars carrying effective resistance against destructive pathogens has become a priority for breeders. While little is currently known about the genetic basis of durable resistance, it is generally associated with polygenic and broad-spectrum resistance. In this study, we assessed the spectrum of resistance to Phytophthora capsici conferred by a major effect quantitative trait locus (QTL) that has been detected in all of the resistant pepper accessions studied to date. After adding new markers derived from tomato sequences and those from pepper reported in the literature to three maps of pepper chromosome P5, we detected a QTL cluster involved in P. capsici resistance. By means of meta-analyses, we determined the occurrence of these QTLs in different genetic backgrounds and with different P. capsici isolates. Comparative mapping with tomato and potato highlighted a complex mosaic of Phytophthora resistance loci on colinear chromosome segments. We tested different lines with and without one of these QTLs, Pc5.1, with four isolates that we determined to be genetically distinct. Our data demonstrate that Pc5.1 is active against 12 isolates from different geographical origins and that it is conserved in all of the resistant accessions tested. We propose that this QTL is a key element responsible for the broad-spectrum resistance to P. capsici and, therefore, is a valuable locus for improving the effective resistance of pepper to P. capsici.