Root-knot nematode (Meloidogyne spp.) Me resistance genes in pepper (Capsicum annuum L.) are clustered on the P9 chromosome

The root-knot nematode (Meloidogyne spp.) is a major plant pathogen, affecting several solanaceous crops worldwide. In Capsicum annuum, resistance to this pathogen is controlled by several independent dominant genes—the Me genes. Six Me genes have previously been shown to be stable at high temperature in three highly resistant and genetically distant accessions: PI 322719, PI 201234, and CM334 (Criollo de Morelos 334). Some genes (Me4, Mech1, and Mech2) are specific to certain Meloidogyne species or populations, whereas others (Me1, Me3, and Me7) are effective against a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita, the most common species in Mediterranean and tropical areas. These genes direct different response patterns in root cells depending on the pepper line and nematode species. Allelism tests and fine mapping using the BSA-AFLP approach showed these genes to be different but linked, with a recombination frequency of 0.02–0.18. Three of the PCR-based markers identified in several genetic backgrounds were common to the six Me genes. Comparative mapping with CarthaGene software indicated that these six genes clustered in a single genomic region within a 28 cM interval. Four markers were used to anchor this cluster on the P9 chromosome on an intraspecific reference map for peppers. Other disease resistance factors have earlier been mapped in the vicinity of this cluster. This genomic area is colinear to chromosome T12 of tomato and chromosome XII of potato. Four other nematode resistance genes have earlier been identified in this area, suggesting that these nematode resistance genes are located in orthologous genomic regions in Solanaceae.     

The inheritance of chilling tolerance in tomato (Lycopersicon spp.)

During the past 25 years, chilling tolerance of the cultivated (chilling-sensitive) tomato Lycopersicon esculentum and its wild, chilling-tolerant relatives L. peruvianum and L. hirsutum (and, less intensively studied, L. chilense) has been the object of several investigations. The final aim of these studies can be seen in the increase in chilling tolerance of the cultivated genotypes. In this review, we will focus on low-temperature effects on photosynthesis and the inheritance of these traits to the offspring of various breeding attempts. While crossing L. peruvianum (male symbol) to L. esculentum (female symbol) so far has brought the most detailed insight with respect to physiological questions, for practical purposes, e.g., the readily cross ability, crossing programmes with L. hirsutum as pollen donor at present seem to be a promising way to achieve higher chilling-tolerant genotypes of the cultivated tomato. This perspective is due to the progress that has been made with respect to the genetic basis of chilling tolerance of Lycopersicon spp. over the past five years.     

An integrated genetic linkage map of pepper (Capsicum spp.)

An integrated genetic map of pepper including 6 distinct progenies and consisting of 2262 markers covering 1832 cM was constructed using pooled data from six individual maps by the Keygene proprietary software package INTMAP. The map included: 1528 AFLP, 440 RFLP, 288 RAPD and several known gene sequences, isozymes and morphological markers. In total, 320 anchor markers (common markers in at least two individual maps) were used for map integration. Most anchor markers (265) were common to two maps, while 27, 26 and 5 markers were common to three, four and five maps, respectively. Map integration improved the average marker density in the genome to 1 marker per 0.8 cM compared to 1 marker per 2.1 cM in the most dense individual map. In addition, the number of gaps of at least 10 cM between adjacent markers was reduced in the integrated map. Although marker density and genome coverage were improved in the integrated map, several small linkage groups remained, indicating that further marker saturation will be needed in order to obtain a full coverage of the pepper genome. The integrated map can be used as a reference for future mapping studies in Capsicum and to improve the utilization of molecular markers for pepper breeding.These authors contributed equally to the work described in this paper(e-mail:     

Molecular biology of capsaicinoid biosynthesis in chili pepper (Capsicum spp.)

Capsicum species produce fruits that synthesize and accumulate unique hot compounds known as capsaicinoids in placental tissues. The capsaicinoid biosynthetic pathway has been established, but the enzymes and genes participating in this process have not been extensively studied or characterized. Capsaicinoids are synthesized through the convergence of two biosynthetic pathways: the phenylpropanoid and the branched-chain fatty acid pathways, which provide the precursors phenylalanine, and valine or leucine, respectively. Capsaicinoid biosynthesis and accumulation is a genetically determined trait in chili pepper fruits as different cultivars or genotypes exhibit differences in pungency; furthermore, this characteristic is also developmentally and environmentally regulated. The establishment of cDNA libraries and comparative gene expression studies in pungent and non-pungent chili pepper fruits has identified candidate genes possibly involved in capsaicinoid biosynthesis. Genetic and molecular approaches have also contributed to the knowledge of this biosynthetic pathway; however, more studies are necessary for a better understanding of the regulatory process that accounts for different accumulation levels of capsaicinoids in chili pepper fruits.     

EMS-induced lincomycin resistance in red pepper (Capsicum annuum L.)

Summary Ethyl methane sulphonate (EMS) is a potential mutagen to induce lincomycin resistance in Capsicum annuum. Mutagenized cotyledons were cultured on shoot regenerating medium containing lincomycin (100 mgl⁻¹). Approximately 14% of regenerated shoots were chlorophyll deficient and about 4% of regenerated shoots were green from mutaganized cotyledons. The regenerated green plants were resistant to lincomycin but sensitive to chloramphenicol, kanamycin, spectinomycin, and streptomycin. Reciprocal crosses were made between resistant and sensitive plants. Inheritance of lincomycin resistance was transmitted as a non-Mendelian trait. Lincomycin resistance is a first selectable and maternally inherited organelle encoded genetic marker described in chili pepper. Such mutants should be useful in designing biochemical selection schemes to recover somatic hybrids and cybrids.     

Molecular mapping of the chlorophyll retainer (cl) mutation in pepper (Capsicum spp.) and screening for candidate genes using tomato ESTs homologous to structural genes of the chlorophyll catabolism pathway.

The chlorophyll retainer (cl) mutation causes inhibition of chlorophyll degradation during pepper fruit ripening and is controlled by a single recessive gene. The retention of chlorophyll in mature red or yellow fruits produces brown- or green-colored ripe fruits, respectively. We mapped CL on chromosome 1 of pepper corresponding to chromosome 8 in tomato in which a homologous mutation, green flesh, was previously assigned. To test whether known structural genes from the chlorophyll catabolism pathway could correspond to CL, we mapped tomato expressed sequence tag clones corresponding to three loci of CHLOROPHYLLASE and one locus of PHEOPHORBIDE A OXYGENASE in the tomato introgression lines population. The three CHLOROPHYLLASE loci mapped to chromosomes 6, 9, and 12, while PHEOPHORBIDE A OXYGENASE mapped to chromosome 11, indicating that CL may correspond to an as yet unavailable gene from the chlorophyll catabolism pathway or to a regulator of the pathway.     

High-resolution genetic mapping of the pepper (Capsicum annuum L.) resistance loci Me3 and Me4 conferring heat-stable resistance to root-knot nematodes (Meloidogyne spp.)

The PM687 line of Capsicum annuum L. has a single dominant gene, Me ₃ , that confers heat-stable resistance to root-knot nematodes (RKN). Me ₃ was mapped using doubled-haploid (DH) lines and F₂ progeny from a cross between the susceptible cultivar ’Yolo Wonder’ (’YW’) and the highly resistant line ’PM687’. Bulked-segregant analysis with DNA pools, from susceptible or resistant DH lines, was performed to identify RAPD and AFLP markers linked to Me ₃ . There was no polymorphism between bulks of ten DH lines using over 800 RADP primers (4,000 amplified fragments analysed). Using 512 AFLP primers (74,000 amplified fragments analysed), and bulked DNA templates from 20 resistant and 20 susceptible plants, we identified eight repulsion-phase and four coupling-phase markers linked to Me _3. Analysed in 103 DH progeny, they defined a 56.1-cM interval containing the target gene. The nearest were located 0.5, 1.0, 1.5 and 3.0 centimorgans (cM) on both sides of the gene. Analysis of the F₂ progeny (162 plants) with the nearest coupling-phase marker confirmed its close position. Another resistance gene to RKN, present in ’PM687’ (Me ₄ ), was shown to be linked to Me ₃ , 10 cM from it. In order to localize Me ₃ and Me ₄ on our reference intraspecific pepper linkage map, two AFLP markers were mapped. The Me ₃ nearest marker was 10.1cM from a RAPD marker named Q04_0.3 and 2.7cM from a RFLP marker named CT135. We investigated map-position orthologies between Me ₃ and two other nematode resistance genes, the tomato Mi-3 and the potato Gpa ₂ genes, which mapped in the telomeric region of the short arm of the tomato and potato chromosome 12 (or XII for potato). Received: 23 March 2000 / Accepted: 2 January 2001     

Growth substances in roots of tomato (Lycopersicon esculentum Mill.) infected with root-knot nematodes (Meloidogyne spp.)

Roots of tomato plants with galls caused by larvae of Meloidogyne spp. contained a similar concentration of auxin as uninfected roots, but a larger total amount because the roots of infected plants were heavier. The body contents and saliva or excretions of M. incognita larvae contained too little auxin to account for the increased amounts in infected roots. Roots with galls contained more bound auxin, released by alkaline hydrolysis or incubation after maceration, and more tryptophan and other amino acids, than uninfected roots. The larvae may hydrolyse the plant proteins to yield tryptophan, which may then react with the endogenous phenolic acids to produce auxin.     

Knock-down of both eIF4E1 and eIF4E2 genes confers broad-spectrum resistance against potyviruses in tomato

Background

The eukaryotic translation initiation factor eIF4E plays a key role in plant-potyvirus interactions. eIF4E belongs to a small multigenic family and three genes, eIF4E1, eIF4E2 and eIF(iso)4E, have been identified in tomato. It has been demonstrated that eIF4E-mediated natural recessive resistances against potyviruses result from non-synonymous mutations in an eIF4E protein, which impair its direct interaction with the potyviral protein VPg. In tomato, the role of eIF4E proteins in potyvirus resistance is still unclear because natural or induced mutations in eIF4E1 confer only a narrow resistance spectrum against potyviruses. This contrasts with the broad spectrum resistance identified in the natural diversity of tomato. These results suggest that more than one eIF4E protein form is involved in the observed broad spectrum resistance.

Methodology/Principal Findings

To gain insight into the respective contribution of each eIF4E protein in tomato-potyvirus interactions, two tomato lines silenced for both eIF4E1 and eIF4E2 (RNAi-4E) and two lines silenced for eIF(iso)4E (RNAi-iso4E) were obtained and characterized. RNAi-4E lines are slightly impaired in their growth and fertility, whereas no obvious growth defects were observed in RNAi-iso4E lines. The F1 hybrid between RNAi-4E and RNAi-iso4E lines presented a pronounced semi-dwarf phenotype. Interestingly, the RNAi-4E lines silenced for both eIF4E1 and eIF4E2 showed broad spectrum resistance to potyviruses while the RNAi-iso4E lines were fully susceptible to potyviruses. Yeast two-hybrid interaction assays between the three eIF4E proteins and a set of viral VPgs identified two types of VPgs: those that interacted only with eIF4E1 and those that interacted with either eIF4E1 or with eIF4E2.

Conclusion/Significance

These experiments provide evidence for the involvement of both eIF4E1 and eIF4E2 in broad spectrum resistance of tomato against potyviruses and suggest a role for eIF4E2 in tomato-potyvirus interactions.     

Spectrum of resistance to root-knot nematodes and inheritance of heat-stable resistance in in pepper (Capsicum annuum L.)

Capsicum annuum L. has resistance to root-knot nematodes (RKN) (Meloidogyne spp.), severe polyphagous pests that occur world-wide. Several single dominant genes confer this resistance. Some are highly specific, whereas others are effective against a wide range of species. The spectrum of resistance to eight clonal RKN populations of the major Meloidogyne species, M. arenaria (2 populations), M. incognita (2 populations), M. javanica (1 population), and M. hapla (3 populations) was studied using eight lines of Capsicum annuum. Host susceptibility was determined by counting the egg masses (EM) on the roots. Plants were classified into resistant (R; EM ≤ 5) or susceptible (H; EM >5) classes. The french cultivar Doux Long des Landes was susceptible to all nematodes tested. The other seven pepper lines were highly resistant to M. arenaria, M. javanica and one population of M. hapla. Variability in resistance was observed for the other two populations of M. hapla. Only lines PM687, PM217, Criollo de Morelos 334 and Yolo NR were resistant to M. incognita. To investigate the genetic basis of resistance in the highly resistant line PM687, the resistance of two progenies was tested with the two populations of M. incognita: 118 doubled-haploid (DH) lines obtained by androgenesis from F₁ hybrids of the cross between PM687 and the susceptible cultivar Yolo Wonder, and 163 F₂ progenies. For both nematodes populations, the segregation patterns 69 R / 49 S for DH lines and 163 R / 45 S for F₂ progenies were obtained at 22°C and at high temperatures (32°C and 42°C). The presence of a single dominant gene that totally prevented multiplication of M. incognita was thus confirmed and its stability at high temperature was demonstrated. This study confirmed the value of C. annuum as a source of complete spectrum resistance to the major RKN. Received: 2 July 1998 / Accepted: 11 March 1999     

Both common and specific genetic factors are involved in polygenic resistance of pepper to several potyviruses

Absolute resistance to potato virus Y pathotype 0 (PVY 0), potyvirus E and chili veinal mottle virus (CVMV) and a partial resistance to potato virus Y pathotype 1,2 (PVY 1,2) were found in an Indian pepper line, Perennial. In the doubled haploid (DH) progeny from the F₁ of a cross Perennial by Yolo Wonder, resistance to CVMV was confered by two independent genes, one with a clear dominant effect. Resistance to PVY and potyvirus E was quantitatively expressed and controlled by several recessive genetic factors. Genetic analysis showed that fewer resistance factors were necessary to explain resistance to PVY (0) and potyvirus E than resistance to PVY(1,2). Genetic correlations between resistances to the different potyviruses in the DH progeny showed that most of genetic factors involved in PVY(0) resistance appear to be also involved in potyvirus E resistance, and some of these polyvalent factors may be also involved in PVY(1,2) resistance but, in this case, additional specific genes were necessary. One of the two CVMV resistance genes seems to be implicated in potyvirus E resistance. Thus, the polygenic resistance of Perennial to these potyviruses was due both to polyvalent genetic factors, i.e. factors that apparently interact with several viruses, and strain-specific genetic factors.     

Single‐molecule real‐time sequencing reveals diverse allelic variations in carotenoid biosynthetic genes in pepper (Capsicum spp.)

The diverse colours of mature pepper (Capsicum spp.) fruit result from the accumulation of different carotenoids. The carotenoid biosynthetic pathway has been well elucidated in Solanaceous plants, and analysis of candidate genes involved in this process has revealed variations in carotenoid biosynthetic genes in Capsicum spp. However, the allelic variations revealed by previous studies could not fully explain the variation in fruit colour in Capsicum spp. due to technical difficulties in detecting allelic variation in multiple candidate genes in numerous samples. In this study, we uncovered allelic variations in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene β‐cyclase, β‐carotene hydroxylase, zeaxanthin epoxidase and capsanthin‐capsorubin synthase (CCS) genes, in 94 pepper accessions by single‐molecule real‐time (SMRT) sequencing. To investigate the relationship between allelic variations in the candidate genes and differences in fruit colour, we performed ultra‐performance liquid chromatography analysis using 43 accessions representing each allelic variation. Different combinations of dysfunctional mutations in PSY1 and CCS could explain variation in the compositions and levels of carotenoids in the accessions examined in this study. Our results demonstrate that SMRT sequencing technology can be used to rapidly identify allelic variation in target genes in various germplasms. The newly identified allelic variants will be useful for pepper breeding and for further analysis of carotenoid biosynthesis pathways.     

Development and application of a suite of non-pungency markers for the Pun1 gene in pepper (Capsicum spp.)

Pungency in peppers is due to the presence of capsaicinoid molecules, which are only produced in Capsicum species. The major gene Pun1 is required for the production of capsaicinoids. Three distinct mutant alleles of Pun1 have been found in three cultivated Capsicum species, one of which has been widely utilized by breeders. Although these mutations have been previously identified, a robust collection of molecular markers for the set of alleles is not available. This has been hindered by the existence of at least one paralogous locus that tends to amplify with Pun1. We present a suite of markers that can differentiate the four Pun1 alleles and test them on a diverse panel of pepper lines and in an F2 population segregating for pungency. These markers will be useful for pepper breeding, germplasm characterization, and seed purity testing.     

Relationship between epistasis and aggressiveness in resistance of pepper (Capsicum annuum L.) to Phytophthora nicotianae

This study evaluated the types of gene action governing the inheritance of resistance to Phytophthora nicotianae necrosis in populations derived from two crosses involving two susceptible (Beldi and Nabeul II) and one resistant (CM334) cultivars of pepper (Capsicum annuum L.). Populations, composed of Pr, Ps, F(1) , F (2) , BC (1) Pr, and BC (1) Ps generations, were inoculated with six P. nicotianae isolates. Generation means analysis indicated that an additive-dominance model was appropriate for P. nicotianae isolates Pn (Ko1) , Pn (Ko2) and Pn (Kr1) , which showed low aggressiveness in the two crosses. For the more aggressive isolates Pn (Bz1) , Pn (Bz2) and Pn (Kr2) , epistasis was an integral component of resistance in the two crosses. The presence of epistasis in the resistance of pepper to P. nicotianae was dependent on the level of aggressiveness of the isolates. Selection in pepper with less aggressive isolates was efficient, but not with more aggressive isolates; on the other hand, selection with more aggressive isolates was more stable. The minimum number of genes controlling resistance was estimated at up to 2.71. In the majority of cases, the additive variance was significant and greater than the environmental and dominance variance.     

Molecular characteristics and expression of cytoplasmic genes in cytoplasmic male sterility of pepper (Capsicum annuum L.)

This study compared the sequence variations and expressions of 12 chloroplastic and 8 mitochondrial genes in three pepper (Capsicum annuum L.) cytoplasmic male sterile (CMS) lines, their maintainers and two control cultivars. The results showed that the three CMS lines were highly similar in chloroplastic and mitochondrial fragment sequences, with average similarities of 96.81 and 98.73?%, respectively, and their chloroplastic trnH?CpsbA intergenic spacer, photosystem II 47?kDa protein (psbB) genes, mitochondrial apocytochrome b (cob) and RNAD fragments have 1, 9, 8 and 7 distinct sites from the maintainer lines, respectively, and could be used as informative sites to distinguish CMS lines from the maintainer lines. Meanwhile, the expressions of mitochondrial cob, RNAD and pvs in the reproductive organs (flowers) of CMS lines are different from those of the maintainer lines, but their expressions in the vegetative organs (roots and leaves) are similar. The results indicate that cytoplasmic DNA polymorphisms are rare in CMS lines, and mitochondrial cob, RNAD and pvs genes are closely related to pollen abortion.     

Antimutagenic effect of one variety of green pepper (Capsicum spp.) and its possible interference with the nitrosation process

It is known that the poblano green pepper, a significant component in the Mexican diet, contains certain natural compounds such as chlorophyll, beta-carotene, and vitamins, which have antimutagenic and/or anticarcinogenic properties. Using the somatic mutation and recombination test in wing cells of Drosophila melanogaster, an extract of the poblano pepper (Capsicum spp.) was evaluated to determine its antimutagenic effect against the nitrosation process, simulating the process occurring in the human stomach caused by known food additives. Larvae of 72h old D. melanogaster of standard (ST) and high bioactivation (HB) crosses were exposed in a simultaneous, chronic treatment with the juice expressed from the crushed, whole, fresh pepper fruit, plus the mixture of 20mM methyl urea (MU) and sodium nitrite (SN), mixed with the animals' food. Three doses of pepper juice (12.5, 25, and 50%) were used. The background mutation rate given as spots per wing was 0.36 and 0.48 for ST and HB, respectively. Mutation frequencies produced by the MU and SN mixture was 1.73 (ST) and 26.46 (HB) mutations per wing. The poblano juice decreased the above rates between 40 and 80%, respectively. The experiments suggest that some compounds present in the green pepper may cause this antimutagenic effect by interfering with the nitrosation process. The role of the extract and one of its components, such as vitamin C, in the nitrosation process will be discussed.     

Management of root-knot nematode (Meloidogyne spp.) on sweet pepper (Capsicum annuum L.) with moringa (Moringa oleifera Lam.) leaf powder

A major constraint facing sweet pepper production is infestation by nematodes leading to reduced yields. Field studies were conducted during the 2012 cropping season at the Experimental Farms of the University for Development Studies, Nyankpala, Northern region, Ghana, to determine efficacy of various levels of moringa leaf powder for the control of root-knot nematodes in sweet pepper (Capsicum annuum L.) in the savanna ecology of Ghana. Treatments consisted of three levels of moringa leaf powder (40, 60 and 80?g/L) per plot and 0?g/L (control). The experiment was laid out in a randomised complete block design with each treatment replicated four times. The infestations of root-knot nematodes were significantly lower in the moringa leaf powder-treated plots than the control. Although significant differences were not observed in all the parameters evaluated among the moringa leaf powder treatments, sweet pepper plants treated with 80?g/L of moringa leaf powder per plot recorded the highest mean value of plant height, number of leaves, number of fruits per plant, fruit weight per plant total yield per plot and the thickest plant girth. Similarly, the sweet pepper plants treated with 80?g/L of moringa leaf powder had the lowest infection index (root gall) and nematode population. Application of moringa leaf powder at 40, 60 and 80?g/L increased sweet pepper yield and decreased nematode population confirming their potential in management of root-knot nematodes.