Molly Jahn

What Next for Agriculture After Durban

Despite obstacles in the UN climate talks, modest progress and opportunities for scientific input on agriculture arose. Global agriculture must produce more food to feed a growing population. Yet scientific assessments point to climate change as a growing threat to agricultural yields and food security (1–4). Recent droughts and floods in the Horn of Africa, Russia, Pakistan, and Australia affected food production and prices. The Intergovernmental Panel on Climate Change predicts that the frequency of such extreme weather events will increase (5), which, when combined with poverty, weak governance, conflict, and poor market access, can result in hunger and famine. At the same time, agriculture exacerbates climate change when greenhouse gases (GHGs) are released by land clearing, inappropriate fertilizer use, and other practices (6).

Functional Dissection of Naturally Occurring Amino Acid Substitutions in eIF4E That Confers Recessive Potyvirus Resistance in Plants

Naturally existing variation in the eukaryotic translation initiation factor 4E (eIF4E) homolog encoded at the pvr1 locus in Capsicum results in recessively inherited resistance against several potyviruses. Previously reported data indicate that the physical interaction between Capsicum-eIF4E and the viral genome-linked protein (VPg) is required for the viral infection in the Capsicum-Tobacco etch virus (TEV) pathosystem. In this study, the potential structural role(s) of natural variation in the eIF4E protein encoded by recessive resistance alleles and their biological consequences have been assessed. Using high-resolution three-dimensional structural models based on the available crystallographic structures of eIF4E, we show that the amino acid substitution G107R, found in many recessive plant virus resistance genes encoding eIF4E, is predicted to result in a substantial modification in the protein binding pocket. The G107R change was shown to not only be responsible for the interruption of VPg binding in planta but also for the loss of cap binding ability in vitro, the principal function of eIF4E in the host. Overexpression of the Capsicum-eIF4E protein containing the G107R amino acid substitution in Solanum lycopersicum indicated that this polymorphism alone is sufficient for the acquisition of resistance against several TEV strains.

Molecular mapping of capsaicinoid biosynthesis genes and quantitative trait loci analysis for capsaicinoid content in Capsicum

Quantitative variation in the accumulation of two major capsaicinoids responsible for pungency in the fruit of chile peppers, capsaicin and dihydrocapsaicin, was analyzed in a cross between the non-pungent Capsicum annuum parent cv. Maor and a pungent Capsicum frutescens parent, accession BG 2816. In order to identify quantitative trait loci (QTLs) for capsaicinoid content, we employed the bulked segregant analysis method and screened bulked DNA from F2 individuals at the extremes of the distribution of capsaicinoid content with RAPD primers. Screening with 400 primers allowed the identification of three loci that were polymorphic between the bulks. These RAPD markers were converted to SCARs and subsequently mapped with additional RFLP markers to chromosome 7 of pepper. QTL interval analysis for individual and total capsaicinoid content identified a major QTL, termed cap, which explained 34–38% of the phenotypic variation for this trait in two growing environments. For all measurements, the allele of the pungent parent BG 2816 at cap contributed to the increased level of pungency. To determine whether known structural genes in the pathway could define a candidate for this QTL, 12 clones obtained from differentially expressed transcripts from placental tissue in pungent peppers were also mapped. None of them had a significant effect on this trait, nor did the allelic state at the locus C, the on/off switch for pungency in pepper, located on chromosome 2. The identity of cap and its effect on capsaicin content in other backgrounds will be addressed in future studies.

Genetic Mapping of the Tsw Locus for Resistance to the Tospovirus Tomato spotted wilt virus in Capsicum spp. and Its Relationship to the Sw-5 Gene for Resistance to the Same Pathogen in Tomato

The Tsw gene conferring dominant resistance to the Tospovirus Tomato spotted wilt virus (TSWV) in Capsicum spp. has been tagged with a random amplified polymorphic DNA marker and mapped to the distal portion of chromosome 10. No mapped homologues of Sw-5, a phenotypically similar dominant TSWV resistance gene in tomato, map to this region in C. annuum, although a number of Sw-5 homologues are found at corresponding positions in pepper and tomato. The relationship between Tsw and Sw-5 was also examined through genetic studies of TSWV. The capacity of TSWV-A to overcome the Tsw gene in pepper and the Sw-5 gene in tomato maps to different TSWV genome segments. Therefore, despite phenotypic and genetic similarities of resistance in tomato and pepper, we infer that distinct viral gene products control the outcome of infection in plants carrying Sw-5 and Tsw, and that these loci do not appear to share a recent common evolutionary ancestor.

A Dynamic Interface for Capsaicinoid Systems Biology

Capsaicinoids are the pungent alkaloids that give hot peppers (Capsicum spp.) their spiciness. While capsaicinoids are relatively simple molecules, much is unknown about their biosynthesis, which spans diverse metabolisms of essential amino acids, phenylpropanoids, benzenoids, and fatty acids. Pepper is not a model organism, but it has access to the resources developed in model plants through comparative approaches. To aid research in this system, we have implemented a comprehensive model of capsaicinoid biosynthesis and made it publicly available within the SolCyc database at the SOL Genomics Network (http://www.sgn.cornell.edu). As a preliminary test of this model, and to build its value as a resource, targeted transcripts were cloned as candidates for nearly all of the structural genes for capsaicinoid biosynthesis. In support of the role of these transcripts in capsaicinoid biosynthesis beyond correct spatial and temporal expression, their predicted subcellular localizations were compared against the biosynthetic model and experimentally determined compartmentalization in Arabidopsis (Arabidopsis thaliana). To enable their use in a positional candidate gene approach in the Solanaceae, these genes were genetically mapped in pepper. These data were integrated into the SOL Genomics Network, a clade-oriented database that incorporates community annotation of genes, enzymes, phenotypes, mutants, and genomic loci. Here, we describe the creation and integration of these resources as a holistic and dynamic model of the characteristic specialized metabolism of pepper.

QTL analysis for capsaicinoid content in Capsicum

Pungency or “heat” found in Capsicum fruit results from the biosynthesis and accumulation of alkaloid compounds known as capsaicinoids in the dissepiment, placental tissue adjacent to the seeds. Pepper cultivars differ with respect to their level of pungency because of quantitative and qualitative variation in capsaicinoid content. We analyzed the segregation of three capsaicinoids: capsaicin, dihydrocapsaicin and nordihydrocapsaicin in an inter-specific cross between a mildly pungent Capsicum annuum ‘NuMex RNaky’ and the wild, highly pungent C. frutescens accession BG2814-6. F3 families were analyzed in three trials in California and in Israel and a dense molecular map was constructed comprised mostly of loci defined by simple sequence repeat (SSR) markers. Six QTL controlling capsaicinoid content were detected on three chromosomes. One gene from the capsaicinoid biosynthetic pathway, BCAT, and one random fruit EST, 3A2, co-localized with QTL detected in this study on chromosomes 3 and 4. Because one confounding factor in quantitative determination of capsaicinoid is fruit size, fruit weight measurements were taken in two trials. Two QTL controlling fruit weight were detected, however, they did not co-localize with QTL detected for capsaicinoid content. The major contribution to the phenotypic variation of capsaicinoid content (24–42% of the total variation) was attributed to a digenic interaction between a main-effect QTL, cap7.1, and a marker located on chromosome 2 that did not have a main effect on the trait. A second QTL, cap7.2 is likely to correspond to the QTL, cap, identified in a previous study as having pronounced influence on capsaicinoid content.

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.

A GH3-like gene, CcGH3, isolated from Capsicum chinense L. fruit is regulated by auxin and ethylene*

Auxin, which has been implicated in multiple biochemical and physiological processes, elicits three classes of genes (Aux/IAAs, SAURs and GH3s) that have been characterized by their early or primary responses to the hormone. A new GH3-like gene was identified from a suppressive subtraction hybridization (SSH) library of pungent pepper (Capsicum chinense L.) cDNAs. This gene, CcGH3, possessed several auxin- and ethylene-inducible elements in the putative promoter region. Upon further investigation, CcGH3 was shown to be auxin-inducible in shoots, flower buds, sepals, petals and most notably ripening and mature pericarp and placenta. Paradoxically, this gene was expressed in fruit when auxin levels were decreasing, consistent with ethylene-inducibility. Further experiments demonstrated that CcGH3 was induced by endogenous ethylene, and that transcript accumulation was inhibited by 1-methylcyclopropene, an inhibitor of ethylene perception. When over-expressed in tomato, CcGH3 hastened ripening of ethylene-treated fruit. These results implicate CcGH3 as a factor in auxin and ethylene regulation of fruit ripening and suggest that it may be a point of intersection in the signaling by these two hormones.

Identification of quantitative trait loci associated with resistance to cucumber mosaic virus in Capsicum annuum

Abstract QTL analysis for resistance to cucumber mosaic virus (CMV) was performed in an intraspecific Capsicum annuum population. A total of 180 F3 families were derived from a cross between the susceptible bell-type cultivar Maor and the resistant small-fruited Indian line Perennial and inoculated with CMV in three experiments carried out in the USA and Israel using two virus isolates. Mostly RFLP and AFLP markers were used to construct the genetic map, and interval analysis was used for QTL detection. Four QTL were significantly associated with resistance to CMV. Two digenic interactions involving markers with and without an individual effect on CMV resistance were also detected. The QTL controlling the largest percentage (16–33%) of the observed phenotypic variation (cmv11.1) was detected in all three experiments and was also involved in one of the digenic interactions. This QTL is linked to the L locus that confers resistance to tobacco mosaic virus (TMV), confirming earlier anecdotal observations of an association between resistance to CMV and susceptibility to TMV in Perennial. An advanced backcross breeding line from an unrelated population, 3990, selected for resistance to CMV was analyzed for markers covering the genome, allowing the identification of genomic regions introgressed from Perennial. Four of these introgressions included regions associated with QTL for CMV resistance. Markers in two genomic regions that were identified as linked to QTL for CMV resistance were also linked to QTL for fruit weight, confirming additional breeding observations of an association between resistance to CMV originating from Perennial and small fruit weight.

The I Gene of Bean: A Dosage-Dependent Allele Conferring Extreme Resistance, Hypersensitive Resistance, or Spreading Vascular Necrosis in Response to the Potyvirus Bean common mosaic virus

The resistance to the potyvirus Bean common mosaic virus (BCMV) conferred by the I allele in cultivars of Phaseolus vulgaris has been characterized as dominant, and it has been associated with both immunity and a systemic vascular necrosis in infected bean plants under field, as well as controlled, conditions. In our attempts to understand more fully the nature of the interaction between bean with the I resistance allele and the pathogen BCMV, we carefully varied both I allele dosage and temperature and observed the resulting, varying resistance responses. We report here that the I allele in the bean cultivars we studied is not dominant, but rather incompletely dominant, and that the system can be manipulated to show in plants a continuum of response to BCMV that ranges from immunity or extreme resistance, to hypersensitive resistance, to systemic phloem necrosis (and subsequent plant death). We propose that the particular phenotypic outcome in bean results from a quantitative interaction between viral pathogen and plant host that can be altered to favor one or the other by manipulating I allele dosage, temperature, viral pathogen, or plant cultivar.