Genetic analysis of broad spectrum resistance to potyviruses using doubled haploid lines of pepper (Capsicum annuum L.)

Summary Genetic analysis of resistance to PVY in androgenetic doubled haploid lines, F1, F2 and backcross progenies of the Mexican pepper line, CM 334 (Capsicum annuum L.), was performed. Three reaction types were observed when seedlings were inoculated with several PVY strains of different pathotypes and with an American PeMV strain. Resistant genotypes never showed systemic symptoms although some individuals sporadically developed necrotic local lesions on inoculated cotyledons. Susceptible genotypes exhibited either a typical systemic mosaic or a systemic necrosis that caused the death of the inoculated seedlings. Segregation analyses indicated that resistance to pepper potyviruses in CM 334 is conferred by two genes. The first one, tentatively named Pr4, is dominant and confers the resistance to all now known pathotypes of PVY and to PeMV. The second one, tentatively named pr5, is recessive; it confers only the resistance to common strains of PVY. The systemic necrotic response is conferred by an independent dominant gene, tentatively named Pn1.     

Early selection for extreme resistance to potato virus Y and tobacco etch virus in potato using a β-glucuronidase-tagged virus

The Ry_sto gene from Solanum stoloniferum introduced into potato cultivars (Solanum tuberosum L. ssp. tuberosum) confers resistance to potato virus A, potato virus V and potato virus Y (PVY). In addition to PVY, tobacco etch virus (TEV) and a TEV construct that encodes β‐glucuronidase (TEV‐GUS) were inoculated to determine the inheritance of resistance to these viruses in progenies obtained from potato cultivars containing the Ry_sto gene. While cultivars ‘Karlena’ and ‘Delikat’ were susceptible, ‘Bettina’ and clone 927eY were resistant to PVY, TEV and TEV‐GUS, as determined by enzyme linked immunosorbent assay, biotest and GUS assay, respectively. The segregation ratios obtained from the progenies of ‘Bettina’בDelikat’ and 816eY בKarlena’ indicate that resistances to PVY and TEV are governed by one dominant gene or two genes tightly linked in coupling phase. Evidently, Ry_sto confers broad spectrum resistance to potyviruses. TEV resistance could be reliably detected 4 days after inoculation with the TEV‐GUS construct by GUS assay. Therefore, the GUS‐tagged TEV construct can be used for early selection for resistances based on the gene Ry_sto or closely linked genes.     

Elucidation of virus-host interactions to enhance resistance breeding for control of virus diseases in potato

Potato virus Y (PVY) and Potato mop-top virus (PMTV) are viruses whose geographical distribution is expanding and economic losses are increasing, in contrast to most of other viruses infecting potato crops. Most potato cultivars lack broad-spectrum resistance to the new, genetically complex strains of PVY, and no efficient resistance to PMTV is known in potato. Control of the vectors of these viruses is not an efficient or possible strategy to prevent infections. Studies on molecular virus-host interactions can discover plant genes that are important to viral infection or antiviral defence. Both types of genes may be utilized in resistance breeding, which is discussed in this paper. The advanced gene technologies provide means to fortify potato cultivars with effective virus resistance genes or mutated, non-functional host factors that interfere with virus infection.     

Combining resistance to potato leafroll virus (PLRV) with immunity to potato viruses X and Y (PVX and PVY)

Summary Degeneration of seed tubers caused by viruses is a major constraint to potato cultivation in many warm tropical areas. Cultivars resistant to the most important viruses present a promising solution to such a problem. In a breeding study conducted at the International Potato Center (CIP), ten cultivars resistant to PLRV were crossed to five clones immune to PVX and PVY, as well as to a susceptible control. The resulting progenies were screened for PVX and PVY immunity. Afterwards, the resistant genotypes were fieldexposed to a PLRV-viruliferous aphid population. One tuber was harvested from each plant and used to assess, using ELISA serology, the presence of leafroll virus. The cultivars Serrana and Pentland Crown gave the best progenies in terms of percentage of healthy plants, with values of 47.4 and 33.3 respectively. The presence of joint infection of PLRV and PVY or PVX in the progenies of the susceptible control resulted in an increased susceptibility to leafroll.     

Individual reaction of <Emphasis Type="Italic">Capsicum</Emphasis> F<Subscript>2</Subscript> hybrid genotypes in anther cultures

The present study evaluated the individual plants reaction of F₂ hybrid generation of C. annuum: ATZ1 × PO and ATZ1 × CDT as well as two interspecific hybrids: C. frutescens × C. annuum ATM1 and C. frutescens × C. chinense on androgenesis conditions in in vitro anther cultures. The experiment was carried out following a modified method of Dumas de Vaulx et al. (Agronomie 1:859–864, 1981). There were demonstrated clear differences in the effectiveness of androgenesis both between the pepper hybrid forms as well as among individual plants of all the genotypes tested. The highest effectiveness of androgenic embryos development was observed for the cultivated form of C. annuum: (ATZ1 × PO)F₂. Anthers of most of the plants of this hybrid produced embryos at the level higher than 5%, while in anther cultures of the second C. annuum hybrid (ATZ1 × CDT)F₂ almost 3-fold fewer embryos and plants were produced. Anthers isolated from flower buds of interspecific hybrids formed much lower number of embryos. A positive reaction was recorded for five hybrid plants of (C. frutescens × C. annuum ATM1)F₂, while in case of (C. frutescens × C. chinense)F₂ androgenic embryos were obtained from anthers of two plants. Only in the case of a one of these plants did the effectiveness of androgenesis exceed 5%. The ploidy level of the regenerants was determined by flow cytometry. Among the regenerants there were observed both haploid forms and the plants with the diploid number of chromosomes.     

Screening wild plants of Capsicum annuum for resistance to pepper huasteco virus (PHV): Presence of viral DNA and differentiation among populations

Plants collected in thirteen wild populations of Capsicum annuum from Northwest Mexico were tested for resistance to the pepper huasteco begomovirus (formerly subgroup III) (PHV) that is transmitted by the white fly Bemisia tabaci Genadius. Plants were inoculated using both grafting and biolistic methods. Presence of viral DNA was detected by dot blot hybridization and densitometry. Populations varied in their resistance to PHV. Plants of only two of the populations either did not develop disease symptoms or showed very light symptoms after inoculation. In some cases, symptoms appeared several days after inoculation. In plants of these populations viral DNA was detected by dot-blot hybridization assays but they appear to be a good source of resistance (symptomless) for use in breeding programmes. This revised version was published online in August 2006 with corrections to the Cover Date.     

New inheritance studies related to Potato Virus Y (PVY) resistance in Capsicum annuum L. ‘Serrano Criollo de Morelos-334’

Summary C. annuum L. ‘Serrano Criollo de Morelos-334’ (SCM-334) is a well-known source of resistance against pepper diseases, including Potato Virus Y (PVY). In this work, inheritance studies involving ‘SCM-334’ and related to PVY resistance were described. For this purpose, ‘SCM-334’ derived lines were used, and plant material from the cross between ‘SCM-334’ and ‘Yolo Wonder’ (YW) was employed as well. Five PVY isolates were used for the inoculations. The first resistance described is a monogenic and recessive gene, controlling a specific-isolate resistance, and provisionally named as pvr1 ⁴. The second one is related to the inheritance of systemic necrotic symptoms after PVY inoculation. In this case a codominant gene is proposed and expressed only when the Pvr4 gene is not present.     

Screening of pepper accessions for resistance against two thrips species (<Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> and <Emphasis Type="Italic">Thrips parvispinus</Emphasis>)

Thrips are damaging pests in pepper worldwide. They can cause damage directly by feeding on leaves, fruits or flowers, and also indirectly by transferring viruses, especially tomato spotted wilt virus (TSWV). Although thrips are among the most damaging pests in pepper, until now there is no commercial variety with a useful level of resistance to thrips. This is at least partly due to the lack of knowledge on resistance levels in pepper germplasm of QTLs and/or genes for resistance, and of information about resistance mechanisms to thrips in pepper. This paper describes our research aimed at developing practical and reliable screening methods for thrips resistance in pepper and at identifying pepper accessions showing a strong resistance to thrips. Thirty-two pepper accessions from four species of pepper (Capsicum annuum, C. baccatum, C. chinense and C. frutescens) and two species of thrips (Frankliniella occidentalis and Thrips parvispinus) were used in this study. Our results indicate that the laboratory based leaf disc test and the detached leaf test can be used as reliable screening methods for thrips resistance in pepper. We observed a large variation for resistance to thrips in Capsicum that can be exploited in breeding programs.     

Inheritance of adult-plant resistance to Phytophthora capsici in pepper

Summary Inheritance studies were conducted to determine the genetic basis of adult-plant resistance in pepper (Capsicum annuum L.) to Phytophthora capsici. F₁, backcrosses and F₂ populations were developed using the resistant parent Criollo de Morellos 334 and susceptible parents Agronômico 10-G and Yolo Wonder. Pepper plants, at 36 days post-emergence, were inoculated near the base of the stem with an inoculum suspension of 5×10⁴ zoospores/ml. Segregation ratios in the F₂ generation of 13 resistant to 3 susceptible plants fit a 2-gene model for resistance with dominant and recessive epistasis.     

Characterization of graft-induced change in capsaicin content of Capsicum annuum L.

Summary Capsaicin contents were measured in the progenies from the test crosses between the strain, G₅S₁₇ derived from fruit with the graft-induced change in fruit shape and the original two cultivars, Yatsubusa (Capsicum annuum L. var fasciculatum Ilish) and Spanish Paprika (C. annuum L. var. grossum Sendt) used for grafting.The strain, G₅S₁₇ contained a lower amount of capsaicin than the original pungent cultivar Yatsubusa used as scion. The characteristic of the lowered capsaicin content in the strain has been stably inherited through seed propagation, and has also been transmitted to the progenies from the crosses with the original two cultivars used for scion or stock. These features in the capsaicin content agreed with those in fruit shape in the graft-induced change.     

Relationship between tolerance and resistance to pepper mottle virus in a cross between Capsicum annuum L. × Capsicum chinense Jacq.

Summary Progenies of a cross between pepper cultivar Delray Bell (Capsicum annuum L.) tolerant to pepper mottle virus (PeMoV) and P.I. 159236 (Capsicum chinense Jacq.), resistant to PeMoV were evaluated in greenhouse experiments. The F₁ generation was susceptible to PeMoV, whereas the backeross generations to both parents and the F₂ segregated in a ratio of 1:1. This indicates that PeMoV resistance is controlled at the same locus in both parents or at two closely linked loci. Each parent is homozygous for one locus and the homozygous condition of either allele results in failure to support virus; in heterozygous condition as observed in the F₁ generation, the effect of a single allele is insufficient even when one allele of each type is present, resulting in systemic infection.The abstract of this paper was presented at the IVth Eucarpia Capsicum meetings held in October at Wageningen, Netherlands.Florida Agricultural Experiment Stations Journal Series Paper No. 3175.     

Three AFLP markers tightly linked to the genic male sterility <Emphasis Type="Italic">ms</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript> gene in chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) and conversion to a CAPS marker

Genic male sterility (GMS) has long been used as a tool for hybrid seed production in chili pepper (Capsicum annuum L.). We developed DNA markers linked to the GMS ms ₃ gene in a segregating population using bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) techniques. The segregating population was subjected to BSA-AFLP with 512 primer combinations. Three AFLP markers (Eagg/Mccc₂₇₆, Eagc/Mctt₁₇₈, and Ecag/Mtgc₂₀₄) were identified as tightly linked to the ms ₃ locus. Among them, we converted the AFLP marker Ecag/Mtgc₂₀₄ to the cleavage amplified polymorphic sequence (CAPS) marker, named GMS3-CAPS, based on sequencing analysis of internal and flanking regions for the markers between male-fertile and sterile plants. This marker will be useful for pepper breeding using the GMS system.     

Breeding transgressive lines of pepper for resistance to Phytophthora capsici in a recurrent selection system

Summary In order to increase the resistance level of pepper (Capsicum annuum) to Phytophthora capsici the main sources of genetic resistance were intercrossed. The parents included 7 varieties from different origins of partial resistance to P. capsici and to other soilborne pathogens. Two cycles of recurrent selection were performed by 7 plant breeders who screened the plants at two developmental stages with various isolates and at two temperatures. Analysis of data revealed a strong influence of the testing conditions on the evaluation of the plants. The level of resistance of the progenies further depended on the pathogen isolate used to screen the plants. Improvement of the whole population was more significant during the first cycle and lines with enhanced level of resistance as compared to the parents i.e. transgressive lines were fixed from the two cycles. These lines were resistant to much higher inoculum concentrations than the original parents and the expression of resistance was stable at high temperature (32°C). These new sources of polygenic resistance were included into a new breeding program to improve the agronomic characters.     

Recurrent selection for resistance to Verticillium dahliae in pepper

Summary Two cycles of recurrent selection were performed in a population including 7 small fruited varieties of pepper (Capsicum annuum) with low resistance level to Verticillium and resistance to other soilborne pathogens. The collaboration of 7 plant breeders in this work and the use of 2 artificial methods of inoculation allowed screening of numerous plants for resistance to V. dahliae under different conditions. The population was simultaneously improved for resistance to V. dahliae and for resistance to Phytophthora capsici, and at each cycle, intercrosses were performed between all these plants. The general level of resistance to V. dahliae increased continuously during the 2 cycles of seles of selection. Selfed progenies of plants from the 2 cycles displayed a higher resistance than the original parents, whatever the method of inoculation used. This gave evidence of transgressions for resistance. Crosses involving parents that did not seem resistant by themselves also produced transgressive lines, indicating that resistance genes were present but not active in these varieties. The transgressive lines with polygenic resistance to V. dahliae were included into a further breeding program to introduce genes of agronomic value.     

Estimating agronomic traits of transgenic tobacco lines

Tobacco transgenic lines belonging to four cultivars modified with three genetic constructions providing the resistance to Potato virus Y (PVY) had been described as regards their agronomic and biological traits. The constructions included viral genes: the coat protein gene of Lettuce mosaic virus (LMV) and the replicase gene of PVY used in sense and antisense orientation. Evaluation of the chosen farm traits of the transgenic tobacco lines was performed by comparing them with their nontransgenic equivalents. The traits such as growth and development rate, height of stalks, leaf area, morphology of plants, course of meiosis and pollen viability were described. The cured leaves were analysed as regards to their physical traits and chemical composition. The individual transgenic lines differed from control cultivars to a little extent. Only insignificant changes, mainly in the quality of raw material were observed. Genetic modification did not cause any changes in the course of meiosis and pollen viability.     

Evaluation and inheritance of the Lycopersicon hirsutum resistance against potato virus Y

Summary Potato virus Y (PVY) infects most Solanaceous crops grown in Mediterranean countries in open fields and in greenhouses. Necrogenic strains, which have been isolated from diseased tomatoes in France since the 1980's, seriously cause yield and quality loss of tomato fruits. Lycopersicon hirsutum PI 247087 was found to be resistant to PVY. Virus could not be detected in inoculated leaves by ELISA and/or by back-inoculation on susceptible plants. This resistance was efficient against the 16 tested isolates or strains. Temperature and inoculum concentration did not affect its expression. All the F₁ plants of (Momor × PI 247087), (PI 134417 × PI 247087) and (PI 247087 × PI 134417) had symptom scores and ELISA values similar to those of the susceptible parents. The mechanism of resistance could be immunity-like or inhibition of virus migration from cell to cell. The resistance of L. hirsutum PI 247087 appeared to be governed by two independent recessive genes. In a few F₂ plants of (PI 134417 × PI 247087) and F₂ (Momor × PI 247087), virus was able to multiply in the inoculated leaves but could not establish a systemic infection. This finding may suggest a mechanism which interfers with the long distance migration of the virus in the plant.     

Inheritance of closed flower in pepper

Summary Progenies of pepper (Capsicum annuum L.) crosses between the closed flower pepper line UFBG 8209-1 and cultivars Permagreen and Early Calwonder representing the normal, open flower type, were evaluated in a field experiment. The F₁ generation was open flowered. Backcrosses and F₂ generations indicated that the closed flower trait was controlled by a single recessive gene.Florida Agricultural Experiment Stations Journal Series No. 4918.     

Heavy-ion-induced mutants in sweet pepper isolated by M1 plant selection

We examined the effects of heavy-ion bombardment on mutagenesis in sweet pepper (Capsicum annuum L.). Dose–response studies indicated that 10 Gy irradiation of ¹²C or ²⁰Ne ions on dry seeds is suitable for inducing mutations in plants. From ²⁰Ne-irradiated M₁ plants, putative mutants included two dwarf plants and one plant whose pericarp was yellow were isolated. Phenotypes of their M₂ progeny were similar to those of the M₁ plants and did not segregate. F₁ plants resulting from reciprocal crosses between the mutants and wild-type plants showed the wild-type phenotype, but phenotypes of F₂ and BC₁F₁ segregated at 1:3 (mutant:wild) and 1:1, respectively. These crossing experiments indicate that the three mutants have monogenic recessive mutations in nuclear genes. In light of these data, we discuss the effectiveness of using heavy-ion bombardment to mutate sweet peppers.     

Conversion of the genic male sterility (GMS) system of bell pepper (Capsicum annuum L.) to cytoplasmic male sterility (CMS)

Non‐pungent bell pepper (Capsicum annuum L.) lacks the cytoplasmic male sterility (CMS) nuclear restorer allele, Rf, and CMS cannot be employed in its F₁ hybrid seed production. To demonstrate that the genic male sterility (GMS) system in non‐pungent bell pepper can be converted to the CMS male sterility system, the conversion of GMS to CMS for non‐pungent bell pepper line GC3 was conducted by introgression of S‐type cytoplasm and the Rf allele from tropical pungent donors. After morphological traits were evaluated, two lines from BC₁F₁ containing S‐type cytoplasm and four lines from BC₂F₂ containing Rf allele, phenotypically similar to GC3, were obtained and could be employed as CMS male sterile lines and restorer lines for non‐pungent bell pepper. Four molecular markers potentially linked to traits of interest were also evaluated in BC₁F₁ and BC₁F₂ populations. This is the first time that GMS has been successfully converted to CMS in bell pepper, a significant contribution for bell pepper hybrid seed production.     

Cytogenetics of interspecific hybrids in the genus Capsicum L.

Summary Crosses between a wild species C. chacoense and three cultivated species of chili pepper viz. C. annuum, C. frutescens and C. chinense yielded hybrids when C. chacoense was the seed parent but the reciprocal crosses were unsuccessful. C. chacoense × C. annuum F₁ hybrids were partly fertile and therefore an F₂ population could be raised; the other two F₁ hybrids were totally sterile. Chromosome pairing in the F₁ plants resulted largely in bivalents and a few multivalents and univalents. The genomes of the four species share large homologies and the role of chromosome structural changes in genome differentiation is suggested. Hybrid sterility is the major reproductive isolation mechanism.