Gene expression in developing watermelon fruit

<p>Abstract</p> <p>Background</p> <p>Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [<it>Citrullus lanatus </it>(Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR.</p> <p>Results</p> <p>High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded, bright red flesh, dark green rind, etc., determined that ethylene levels were highest during the green fruit stage followed by a decrease during the white and pink fruit stages. Additionally, quantitative Real-Time PCR was used to validate modulation of 127 ESTs that were differentially expressed in developing and ripening fruits based on array analysis.</p> <p>Conclusion</p> <p>This study identified numerous ESTs with putative involvement in the watermelon fruit developmental and ripening process, in particular the involvement of the vascular system and ethylene. The production of ethylene during fruit development in watermelon gives further support to the role of ethylene in fruit development in non-climacteric fruits.</p

Identifying the genes involved in host root perception by root parasitic weeds: Genetic and transcriptomic analysis of Orobanche hybrids differing in signal response specificity

Seeds of the root parasitic plants of the genus Orobanchegerminate specifically in response to host-derived germination signals, which enables parasites to detect and attack preferred hosts. The best characterized class of germination stimulants is the strigolactones (SL), although some species respond to sesquiterpene lactones such as dehydrocostuslactone (DCL). Despite great progress in characterizing the SL signaling system in plants, the mechanism(s) by which parasite species detect specific compounds remains poorly understood. The goal of our project was to identify and characterize the genes responsible for stimulant specificity in O. cernuaand O. cumana. These two species are closely related, but differ in host range, with O. cernuaparasitizingSolanaceous crops such as tomato (and responding to SLs), and O. cumanaspecifically parasitizing sunflower (and responding to DCL). We used a genetic approach based on O. cernuax O. cumanahybrids to associate germination response with genes. We found that these parasite species each have multiple copies of KAI2d genes, which function in SL perception. In O. cernua, the OrceKAI2d2 responds to SL stimulants and is most consistently associated with hybrid lines that respond to SLs. For O. cumana, an apparently linked block of KAI2d genes was associated with response to DCL in hybrid lines, but we found no strong evidence that any of the OrcuKAI2d genes specifically recognize the DCL stimulant. Remarkably, one O. cumanagene, OrcuKAI2d5, responds to certain SLs in a genetic complementation assay, even though hybrid lines containing this gene show fidelity to DCL. In summary, we have identified the SL receptor in O. cernua, but the DCL receptor in O. cumanaremains unknown. Our data point to involvement of additional genes and yet greater levels of complexity regulating germination specificity in Orobanche. BARD Report - Project 4616 Page 2 of 8 </p

Genomic Approaches for Understanding Virulence and Resistance in the Sunflower-Orobanche Host-Parasite Interaction

Oroginal Objectives: (i) identify DNA markers linked to the avirulence (Avr) locus and locate the Avr locus through genetic mapping with an inter-race Orobanche cumana population; (ii) develop high-throughput fingerprint DNA markers for genotypingO. cumana races; (iii) identify nucleotide binding domain leucine rich repeat (NB-LRR) genes encoding R proteins conferring resistance to O. cumana in sunflower; (iv) increase the resolution of the chromosomal segment harboring Or₅ and related R genes through genetic and physical mapping in previously and newly developed mapping populations of sunflower; and (v) develop high-throughput DNA markers for rapidly and efficiently identifying and transferring sunflower R genes through marker-assisted selection. Revisions made during the course of project: Following changes in O. cumana race distribution in Israel, the newly arrived virulent race H was chosen for further analysis. HA412-HO, which was primarily chosen as a susceptible sunflower cultivar, was more resistant to the new parasite populations than var. Shemesh, thus we shifted sunflower research into analyzing the resistance of HA412-HO. We exceeded the deliverables for Objectives #3-5 by securing funding for complete physical and high-density genetic mapping of the sunflower genome, in addition to producing a complete draft sequence of the sunflower genome. We discovered limited diversity between the parents of the O. cumana population developed for the mapping study. Hence, the developed DNA marker resources were insufficient to support genetic map construction. This objective was beyond the scale and scope of the funding. This objective is challenging enough to be the entire focus of follow up studies. Background to the topic: O. cumana, an obligate parasitic weed, is one of the most economically important and damaging diseases of sunflower, causes significant yield losses in susceptible genotypes, and threatens production in Israel and many other countries. Breeding for resistance has been crucial for protecting sunflower from O. cumana, and problematic because new races of the pathogen continually emerge, necessitating discovery and deployment of new R genes. The process is challenging because of the uncertainty in identifying races in a genetically diverse parasite. Major conclusions, solutions, achievements: We developed a small collection of SSR markers for genetic mapping in O. cumana and completed a diversity study to lay the ground for objective #1. Because DNA sequencing and SNPgenotyping technology dramatically advanced during the course of the study, we recommend shifting future work to SNP discovery and mapping using array-based approaches, instead of SSR markers. We completed a pilot study using a 96-SNP array, but it was not large enough to support genetic mapping in O.cumana. The development of further SNPs was beyond the scope of the grant. However, the collection of SSR markers was ideal for genetic diversity analysis, which indicated that O. cumanapopulations in Israel considerably differ frompopulations in other Mediterranean countries. We supplied physical and genetic mapping resources for identifying R-genes in sunflower responsible for resistance to O. cumana. Several thousand mapped SNP markers and a complete draft of the sunflower genome sequence are powerful tools for identifying additional candidate genes and understanding the genomic architecture of O. cumana-resistanceanddisease-resistance genes. Implications: The OrobancheSSR markers have utility in sunflower breeding and genetics programs, as well as a tool for understanding the heterogeneity of races in the field and for geographically mapping of pathotypes.The segregating populations of both Orobanche and sunflower hybrids are now available for QTL analyses.</p

Discovery and Transfer of Genes from Wild Zea Germplasm to Improve Grain Oil and Protein Composition of Temperate Maize

Project Objectives 1. Develop and amplify two interspecific populations (annual and perennial teosintes x elite maize inbred) as the basis for genetic analysis of grain quality. 2. Identify quantitative trait loci (QTLs) from teosinte that improve oil, protein, and essential amino acid composition of maize grain. 3. Develop near isogenic lines (NILs) to quantify QTL contributions to grain quality and as a resource for future breeding and gene cloning efforts. 4. Analyze the contribution of these QTLs to hybrid performance in both the US and Israel. 5. Measure the yield potential of improved grain quality hybrids. (NOTE: Yield potential could not be evaluated due to environmentally-caused failure of the breeding nursery where seed was produced for this evaluation.) Background: Maize is a significant agricultural commodity worldwide. As an open pollinated crop, variation within the species is large and, in most cases, sufficient to supply the demand for modem varieties and for new environments. In recent years there is a growing demand for maize varieties with special quality attributes. While domesticated sources of genetic variation for high oil and protein content are limited, useful alleles for these traits may remain in maize's wild relative, teosinte. We utilized advanced backcross (AB) analysis to search for QTLs contributing to oil and protein content from two teosinte accessions: Zea mays ssp. mexicana Race Chalco, an annual teosinte (referred to as Chalco), and Z diploperennis Race San Miguel, a perennial teosinte (referred to as Diplo). Major Conclusions and Achievements Two NILs targeting a Diplo introgression in bin 1.04 showed a significant increase in oil content in homozygous sib-pollinated seed when compared to sibbed seed of their counterpart non-introgressed controls. These BC4S2 NILs, referred to as D-RD29 and D-RD30, carry the Diplo allele in bin 1.04 and the introgression extends partially into bins 1.03 and 1.05. These NILs remain heterozygous in bins 4.01 and 8.02, but otherwise are homozygous for the recurrent parent (RD6502) alleles. NILs were developed also for the Chalco introgression in bin 1.04 but these do not show any improvement in oil content, suggesting that the Chalco alleles differ from the Diplo alleles in this region. Testcross Fl seed and sibbed grain from these Fl plants did not show any effect on oil content from this introgression, suggesting that it would need to be present in both parents of a maize hybrid to have an effect on oil content. Implications, both Scientific and Agricultural The Diplo region identified increases oil content by 12.5% (from 4.8% to 5.4% oil in the seed). Although this absolute difference is not large in agronomic terms, this locus could provide additive increases to oil content in combination with other maize-derived loci for high oil. To our knowledge, this is the first confirmed report of a QTL from teosinte for improved grain oil content in maize. It suggests that further research on grain quality alleles from maize wild relatives would be of both scientific and agricultural interest.</p

Molecular Marker Mapping of Genes Enhancing Tocol and Carotenoid Composition of Maize Grain

The overall objective of this research was to identify chromosomal regions and candidate genes associated with control of concentration and forms of carotenoids (includes pro-Vitamin A) and tocopherols (Vitamin E), which are both antioxidants and are associated with health advantages. Vitamin A and E are included in animal feeding supplements and the eventual goal is to increase levels of these compounds in maize grain so that the cost of these supplements can be reduced or eliminated. Moreover, both compounds are antioxidants that protect unsaturated fatty acids from oxidation and thus maintaining maize oil quality for longer periods. We identified three SSR markers that are associated with 38% of the variation for total carotenoids and three SSR markers associated with 44% of the variation for total tocopherols in the cross W64a x A632. We identified two candidate genes associated with levels of carotenoids: phytoene synthase and zeta carotene desaturase. Evaluation of (Illinois High Oil x B73) B73 BC 1S1 population for tocopherols detected additional chromosomal regions influencing the level of total tocopherols, and detected a common region on chromosome 5 associated with ratio of the more desirable alpha from to the gamma form of tocopherol. The results suggest molecular marker assisted selection for higher levels of these antioxidants in corn grain should be feasible.</p

Discovery and Transfer of Genes from Wild Zea Germplasm to Improve Grain Oil and Protein Composition of Temperate Maize

Project Objectives 1. Develop and amplify two interspecific populations (annual and perennial teosintes x elite maize inbred) as the basis for genetic analysis of grain quality. 2. Identify quantitative trait loci (QTLs) from teosinte that improve oil, protein, and essential amino acid composition of maize grain. 3. Develop near isogenic lines (NILs) to quantify QTL contributions to grain quality and as a resource for future breeding and gene cloning efforts. 4. Analyze the contribution of these QTLs to hybrid performance in both the US and Israel. 5. Measure the yield potential of improved grain quality hybrids. (NOTE: Yield potential could not be evaluated due to environmentally-caused failure of the breeding nursery where seed was produced for this evaluation.) Background: Maize is a significant agricultural commodity worldwide. As an open pollinated crop, variation within the species is large and, in most cases, sufficient to supply the demand for modem varieties and for new environments. In recent years there is a growing demand for maize varieties with special quality attributes. While domesticated sources of genetic variation for high oil and protein content are limited, useful alleles for these traits may remain in maize's wild relative, teosinte. We utilized advanced backcross (AB) analysis to search for QTLs contributing to oil and protein content from two teosinte accessions: Zea mays ssp. mexicana Race Chalco, an annual teosinte (referred to as Chalco), and Z diploperennis Race San Miguel, a perennial teosinte (referred to as Diplo). Major Conclusions and Achievements Two NILs targeting a Diplo introgression in bin 1.04 showed a significant increase in oil content in homozygous sib-pollinated seed when compared to sibbed seed of their counterpart non-introgressed controls. These BC4S2 NILs, referred to as D-RD29 and D-RD30, carry the Diplo allele in bin 1.04 and the introgression extends partially into bins 1.03 and 1.05. These NILs remain heterozygous in bins 4.01 and 8.02, but otherwise are homozygous for the recurrent parent (RD6502) alleles. NILs were developed also for the Chalco introgression in bin 1.04 but these do not show any improvement in oil content, suggesting that the Chalco alleles differ from the Diplo alleles in this region. Testcross Fl seed and sibbed grain from these Fl plants did not show any effect on oil content from this introgression, suggesting that it would need to be present in both parents of a maize hybrid to have an effect on oil content. Implications, both Scientific and Agricultural The Diplo region identified increases oil content by 12.5% (from 4.8% to 5.4% oil in the seed). Although this absolute difference is not large in agronomic terms, this locus could provide additive increases to oil content in combination with other maize-derived loci for high oil. To our knowledge, this is the first confirmed report of a QTL from teosinte for improved grain oil content in maize. It suggests that further research on grain quality alleles from maize wild relatives would be of both scientific and agricultural interest.</p

three crops test for the ODO breeding method

Hybrid vigor is the leading concept that rules crops breeding for almost a century. Yet, the exact mechanism that underlies heterosis is not clear. Over dominance interaction between alleles is one of the possible explanations. Our preliminary results indicated that severe developmental mutations at the heterozygous state have significant potential to improve plant performance. This led us to propose the ‘ODO breeding method’ that is based replacing a parental line of a successful hybrid with its mutated from to improve hybrid performance. Our BARD research challenged this method in three crop systems: maize, tomato and melon. In maize we could not detect any effect of mutant heterozigosity on yield or yield components when hybrids were tested however when we analyzed the effect of heterozigosity in mutant genes at B73 genetic background we could detect ODO in yield components using certain mutants. Our results indicate that the potential of utilizing the ODO Breeding Method for maize remains unclear. In tomato we overcame technical problems we faced in creating an EMS mutation library in indeterminate glasshouse tomatoes and now we have in our hands advanced material to study the putative ODO hybrids. We transferred some of the promising ODO mutations from M82 to indeterminate glasshouse tomatoes and putative ODO hybrids are ready to be evaluated this winter. In addition, we tested the effect of In melon we compared putative ‘ODO hybrids’ with their isogenic hybrids lacking the mutant allele and our results indicated a potential for the ODO breeding method to improve yield, fruit number per plant, and carotenoids content. Additional experiments are required to estimate better the expected success percentage of the ODO breeding method in melon so that it will become a recommended practice for improving hybrid performance. Based on our results we can't yet recommend the 'ODO breeding method' as a general tool to improve hybrid performance and more efforts are necessary to evaluate the percent of success of this method. The increased carotenoid content we found in association with CRTISO heterozygosity is promising and additional experiments are currently being performed to characterize this finding.</p

System Approach to Understanding the Metabolic Diversity in Melon

Fruit quality is determined by numerous genetic factors that affect taste, aroma, ‎color, texture, nutritional value and shelf life. To unravel the genetic components ‎involved in the metabolic pathways behind these traits, the major goal of the project was to identify novel genes that are involved in, or that regulate, these pathways using correlation analysis between genotype, metabolite and gene expression data. The original and specific research objectives were: (1) Collection of replicated fruit from a population of 96 RI lines derived from parents distinguished by great diversity in fruit development and quality phenotypes, (2) Phenotypic and metabolic profiling of mature fruit from all 96 RI lines and their parents, (3) 454 pyrosequencing of cDNA representing mRNA of mature fruit from each line to facilitate gene expression analysis based on relative EST abundance, (4) Development of a database modeled after an existing database developed for tomato introgression lines (ILs) to facilitate online data analysis by members of this project and by researchers around the world. The main functions of the database will be to store and present metabolite and gene expression data so that correlations can be drawn between variation in target traits or metabolites across the RI population members and variation in gene expression to identify candidate genes which may impact phenotypic and chemical traits of interest, (5) Selection of RI lines for segregation and/or hybridization (crosses) analysis to ascertain whether or not genes associated with traits through gene expression/metabolite correlation analysis are indeed contributors to said traits. The overall research strategy was to utilize an available recombinant inbred population of melon (Cucumis melo L.) derived from phenotypically diverse parents and for which over 800 molecular markers have been mapped for the association of metabolic trait and gene expression QTLs. Transcriptomic data were obtained by high throughput sequencing using the Illumina platform instead of the originally planned 454 platform. The change was due to the fast advancement and proven advantages of the Illumina platform, as explained in the first annual scientific report. Metabolic data were collected using both targeted (sugars, organic acids, carotenoids) and non-targeted metabolomics analysis methodologies. Genes whose expression patterns were associated with variation of particular metabolites or fruit quality traits represent candidates for the molecular mechanisms that underlie them. Candidate genes that may encode enzymes catalyzingbiosynthetic steps in the production of volatile compounds of interest, downstream catabolic processes of aromatic amino acids and regulatory genes were selected and are in the process of functional analyses. Several of these are genes represent unanticipated effectors of compound accumulation that could not be identified using traditional approaches. According to the original plan, the Cucurbit Genomics Network (http://www.icugi.org/), developed through an earlier BARD project (IS-3333-02), was expanded to serve as a public portal for the extensive metabolomics and transcriptomic data resulting from the current project. Importantly, this database was also expanded to include genomic and metabolomic resources of all the cucurbit crops, including genomes of cucumber and watermelon, EST collections, genetic maps, metabolite data and additional information. In addition, the database provides tools enabling researchers to identify genes, the expression patterns of which correlate with traits of interest. The project has significantly expanded the existing EST resource for melon and provides new molecular tools for marker-assisted selection. This information will be opened to the public by the end of 2013, upon the first publication describing the transcriptomic and metabolomics resources developed through the project. In addition, well-characterized RI lines are available to enable targeted breeding for genes of interest. Segregation of the RI lines for specific metabolites of interest has been shown, demonstrating the utility in these lines and our new molecular and metabolic data as a basis for selection targeting specific flavor, quality, nutritional and/or defensive compounds. To summarize, all the specific goals of the project have been achieved and in many cases exceeded. Large scale trascriptomic and metabolomic resources have been developed for melon and will soon become available to the community. The usefulness of these has been validated. A number of novel genes involved in fruit ripening have been selected and are currently being functionally analyzed. We thus fully addressed our obligations to the project. In our view, however, the potential value of the project outcomes as ultimately manifested may be far greater than originally anticipated. The resources developed and expanded under this project, and the tools created for using them will enable us, and others, to continue to employ resulting data and discoveries in future studies with benefits both in basic and applied agricultural - scientific research.</p