Source: WEST VIRGINIA STATE UNIVERSITY submitted to NRP
STRENGTHENING VEGETABLE IMPROVEMENT CENTER WITH GENOMIC SELECTION TOOLS FOR NUTRACEUTICALS AND FRUIT QUALITY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1012058
Grant No.
2017-38821-26434
Cumulative Award Amt.
$597,406.00
Proposal No.
2016-06616
Multistate No.
(N/A)
Project Start Date
Apr 15, 2017
Project End Date
Apr 14, 2021
Grant Year
2017
Program Code
[EQ]- Research Project
Recipient Organization
WEST VIRGINIA STATE UNIVERSITY
PO BOX 1000
INSTITUTE,WV 25112
Performing Department
Biology
Non Technical Summary
Micronutrient malnutrition or "hidden hunger" is an alarming public health issue in most parts of the world including USA. More than half of the world's population is iron (Fe) deficient while around one-third is zinc (Zn) deficient. Besides this, about 40% of women and pre-school children in Asia and Africa have low hemoglobin levels mainly due to Fe deficiency. These deficiencies cause havoc to human through malnutrition including stunting, respiratory tract infections, malaria, diarrhea and others. The solution to these problems is possible through crops that naturally reduce anemia, cognitive impairment and other nutritionally related health problems in human and animals. For example, biofortified plants have the potential to nourish nutrient depleted soils, help increase crop productivity and provide nutritional benefits to plants, humans and livestock. Biofortification simply refers to producing staple foods whose edible portions are denser in bioavailable minerals and vitamins. Our students often have inadequate background knowledge to appreciate the impact of plant breeding programs in US agriculture. Furthermore, during the last 3 years, a plethora of genomic technologies have been launched, which has facilitated the use of genomic selection in various crop improvement programs via genome-wide association study (GWAS). Once our students have been exposed to modern metabolomics and GWAS techniques, they will be able to compete for jobs or pursue advanced or additional education in one of the fastest-growing areas of research and industry. We will generate unique opportunities for students to pursue biofortification research involving metabolomics and modern methods in plant breeding and will expose them to the concept of plant breeding for biofortification. In doing, so we will bring our students up to speed so that they can be part of the nation's strength in crop improvement. We thus here propose to address this important issue through the establishment of a multi institutional networking with the CGIAR (Consultative Group for International Agricultural Research) institutes across the world that played a major role in alleviating undernourishment across the world. We will create a plethora of common opportunities for students to pursue research in the area of genome analysis and modern methods in plant breeding for enhanced nutrition and in addition will expose them to the impact of genomics and metabolomics at a global scale. In doing so, we will bring our students up to speed so that they can be part of the nation's strength in crop genome analysis.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011429108150%
2021461108050%
Knowledge Area
202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1461 - Peppers; 1429 - Cucurbits, other;

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
A comparable range of fruit colors is available among watermelon cultivars. Pigment analyses have revealed striking similarities in carotenoid compositions between pepper and watermelon varieties of the equivalent flesh colors (Lewinsohn et al. 2005). Genetic basis for the control of flesh color in watermelon fruit is much less well understood. Henderson et al. (1998) described five fruit color watermelon phenotypes--Canary yellow, Salmon yellow, Orange, Red, and White--and defined the genetic relationships among these phenotypes on the basis of segregating populations. Thus, although peppers and watermelon are botanically distant, it seems that the tomato and pepper fruit colors have phenotypic equivalents in watermelon (Tadmor et al. 2005). Our proposal is to analyze natural variation for various metabolites that are important for carotenoid pathway and the other pathways involved in fruit aroma and perform high resolution QTL analysis using GWAS and composite interval mapping on diverse accessions and mapping populations. Most important objective of the current proposal is to expose our students to globally impacted biofortification programs conducted at Consultative Group on International Agricultural Research (CGIAR) institutes. Our goals will be accomplished through active cooperation among an amalgam of committed plant breeding specialists from the CGIAR institutions that made tremendous impact on food sustainability and nutrition at a global level. The resulting professional network will benefit the CGIAR and the US agriculture by advancing the exchange of ideas, by creating associations (for new collaborations and/or student career development), and by fostering technology transfers necessary for maintaining human health and well-being. Specifically, our intent is to: 1) analyze natural variation for various metabolites that are important for carotenoid pathway and the other pathways involved in fruit aroma and perform high resolution QTL analysis using GWAS and composite interval mapping on diverse accessions and mapping populations 2) strengthen the traditionally taught crop diversity course with the modern genomics and phenomic methods that can impact "Biofortification"; and 3) Implement summer internships and training programs in high throughput technologies and modern plant breeding that impacted biofortification research at various CGIAR institutes including ICRISAT (International Crop Research Institute for Semi-Arid Tropics), Hyderabad, India; CIMMYT (International Maize and Wheat Improvement Center) in Mexico and CIMMYT center located in Nairobi, Kenya, AVRDC (The world Vegetable Center) in Taiwan and International Rice Research Institute, Manila, Philippines.
Project Methods
The emergence of high-throughput SNP datasets has allowed for Genome-wide Association Studies (GWAS) of crop plants (Li et al. 2014; Pasam et al. 2012; Visioni et al. 2013). The team of researchers from WVSU has developed several resources including a 384 Veracode SNP chip (Nimmakayala et al. 2014), 14K SNPs using Genotyping By Sequencing for high resolution genetic map (Reddy et al. 2014), GWAS for soluble solid content (Nimmakayala et al. 2014) and RNAseq analysis of diploids and tetraploids (Saminathan et al. 2014). During this project, genomic selection will be conducted for fruit color and flavor using high density SNPs generated using reference genome sequencing. We will use 500 watermelon and hot pepper (Capsicum annuum and Capsicum chinense L.)-cultivated accessions collected across the world. The entire GWAS panel was already selfed to maintain their seed stocks. Large numbers of panel are generated for use in proposal. During the summers of 2017 and 2018, the entire GWAS panel will be grown at the Agricultural Experiment Station of WVSU for evaluation of fruit-related traits such as color and flavor. The experimental design will be a randomized block design (RBD) with three replications. Comparative fruit color and β-carotene analyses in watermelon and pepper will be performed by using fruit flesh color using the procedure narrated by Cuevas et al. (2009). Mesocarp and endocarp color will be categorized into RED (RED), Yellow (YELL), and white (WH) using the Royal Horticulture Society mini-color chart. QβC content of each variant will be determined by reverse phase high performance liquid chromatography (HPLC). After QβC content is determined for different carotenoids gene-variants, β-carotene can be estimated using the correlation of mean mesocarp color ratings and amount of QβC (e.g. YELL=15.62 µg g-1 β-carotene). Such identified natural variants will be evaluated in participatory selection approach for potential use as novel lines. Metabolomic profiles for flesh of ripe watermelons of the genetic populations will be carried out by the service center of University of Illinois. Polar compounds (such as the phenolic acids), and less polar compounds (such as anthocyanins) will be extracted from freeze dried flesh samples of the genetic populations using our established methods. Carotenoids and fat soluble vitamin, the least polar compounds, will be extracted using non polar solvents, such as ethanol, under protective lighting followed by hexane extraction (Davis et al., 2003). For GBS data, only the SNPs that are successfully mapped to the watermelon and pepper genome drafts will be considered, because knowing the chromosome location of SNPs helps prevent spurious results of LD and association mapping. Mapped SNPs will then be further filtered by successful call rate and Hardy-Weinberg equilibrium. Horticultural evaluation conducted during 2017 will be repeated during the summer of the year 2018 with the selected value added peppers and watermelons. Selection of the best parental lines will be based on the breeding performance estimated using the BLUPs (see the attached poster for the details) and the lines that are carriers of favorable QTLs. A course has been developed to sensitize and orient students in understanding the issues related to biofortification of food crops and to To enable the participants to acquire the knowledge on the latest techniques/strategies for enriching food crops through biofortification. The concepts, principles, and techniques of biofortification, crop improvement, evolution and domestication will be taught in the context of utilization of NGS methods. This integrative teaching style with hands on experiences makes students more responsible for their own learning and engages them more actively in the material, leading to better comprehension. The link between textbook sources and primary literature requires students to confront the reality of how scientific information is acquired and how small experimental findings can lead to general principles and models. In addition, we organize Summer internships in high throughput technologies at ICRISAT (Summer 2017), AVRDC (Summer 2018), International Rice Research Institute (Summer 2018) and CIMMYT (Summer 2019). Our goals will be accomplished through active cooperation among an amalgam of committed DNA technology specialists from the CGIAR institutions that made tremendous impact in food sustainability at global level. A summer travel program (stay of three weeks at any of the above mentioned CGIAR institute one every summer throughout the grant period of three years) is proposed to expose the students to the impact of genomic and genetic research on global agriculture and sustainability in developing countries. We will solicit applications from the undergraduate students of WVSU and the other 1890 land grant universities to select a final number of 5 students (3 students from WVSU and 2 from the other 1890 universities). Criteria for selection are based on their interest and involvement in various crop improvement programs, horticulture and biotechnology related projects. A committee will be made that also will consist a scientist from the collaborative CGIAR organizations and applications will be screened to select a final list of the students. Students will be provided travel, living expenses and bench fees that cover the expenses of training at respective CGIAR institutes. Main goal is to expose our students to the impact of various biofortified crops in alleviating poverty and hunger in Asia and Africa. High throughput genomics, germplasm conservation, biotechnology methods such as genetic engineering, marker assisted selection and the other practices that made tremendous impact on biofortification and food sustainability are some areas to expose and train the students.

Progress 04/15/17 to 04/14/21

Outputs
Target Audience:Pepper and watermelon growers, Geneticists and plant breeders, graduate and undergraduate students Changes/Problems:Travel funds could not be utilized as planned due to pandemic conditions. An equipment was purchased for teaching and research use, instead. What opportunities for training and professional development has the project provided?Six graduate students submitted their thesis research. Eight undergraduate students were trained in pepper metabolite analysis, DNA/RNA extractions and genotyping.Two postdoctoral associates and four technicians were also involved at various levels of the project. Student participation is restricted during the final year of the grant, due to the pandemic of 2019-2020. Bhagarathi Shahi.2020. Thesis Title: Genetic Analysis of fruit shape in Capsicum chinense. Thesis submitted to the Biotechnology Graduate Program at West Virginia State University. Tosin E. Akinsipe. 2019. Thesis Title: Comparative GWAS for Seedling Root Variation in Acidic Conditions across the Cultivated Pepper Species Complexes. Thesis submitted to the Biotechnology Graduate Program at West Virginia State University. Tolulope A. Akinmoju. 2019. Thesis Title: Metabolomic and transcriptomic insights for fruit cutin in habanero peppers. Thesis submitted to the Biotechnology Graduate Program at West Virginia State University. Marjan Nadimi, Spring 2018. Thesis title:Characterization of Phytochemicals in Peruvian Peppers. Thesis Submitted to the Faculty of the Biotechnology Graduate Program at West Virginia State University Brittany Davenport. 2017. Thesis Title: Molecular diversity analysis in conjunction with fruit color variation among C. annuum collections. Thesis submitted to the Biotechnology Graduate Program at West Virginia State University. Krittika Vilas Tonapi 2017. Thesis Title: Comparative fruit color analysis for Capsicum spp. using Genome-wide Microsatellites. Thesis submitted to the Biotechnology Graduate Program at West Virginia State University. How have the results been disseminated to communities of interest?We presented the results of the project in Regional, National and International conferences. Many seed companies showed keen interest in our nutraceutical research in peppers and associated SNPs to flavor compounds. Peer reviewed publications are generated in pepper and watermelon nutraceutical genomic research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Micronutrient malnutrition or "hidden hunger" is an alarming public health issue in most parts of the world including USA. Besides this, about 40% of women and pre-school children in Asia and Africa have low hemoglobin levels mainly due to Fe deficiency. These deficiencies cause havoc to human through malnutrition including stunting, respiratory tract infections, malaria, diarrhea and others. The solution to these problems is possible through crops that naturally reduce anemia, cognitive impairment and other nutritionally related health problems in human and animals. For example, nutraceuticals in plants have the potential to provide health benefits to humans and plants. Our students often have inadequate background knowledge to appreciate the impact of plant breeding programs in enriching nutraceuticals and natural variation of these compounds in germplasm. Furthermore, during the last 3 years, a plethora of genomic technologies have been launched, which has facilitated the use of genomic selection for nutraceuticals in various crop improvement programs via NGS technologies. Once our students have been exposed to modern metabolomics and genomics, they will be able to compete for jobs or pursue advanced or additional education in one of the fastest-growing areas of research and industry. We will generate unique opportunities for students to pursue nutraceutical research involving genomics and modern methods to exploit natural variation use in plant breeding. In doing, so we will bring our students up to speed so that they can be part of the nation's strength in crop improvement. The main goal of this proposal is to analyze natural variation for various nutraceuticals and metabolites that are important for human and plant health by performing functional genomics, metabolomics and high resolution QTL mapping using GWAS and composite interval mapping on diverse accessions and mapping populations in watermelon and peppers. In peppers, Capsaicin and dihydrocapsaicin make up approximately 90% (66% and 22%, respectively) of total capsaicinoids for which we explored natural variation in 1234 collections belonging to four cultivated species. In addition, anthocyanin and carotenoid pigments that determine fruit colors, cutin and other important vitamins were estimated in these collections. The most abundant carotenoids we quantified in green and ripe pepper fruits are β-carotene, α-carotene, lutein, capsanthin, zeaxanthin, lycopene, and capsorubin in 200 accessions of Capsicum annuum, 268 accessions of C. baccatum, 225 accessions of C. chinense and 175 accessions of C. frutescens for use in GWAS and genomic selection. In this process, we have standardized analytical methods for capsaicinoids, VOCs, capsanthin, zeaxanthin, lutein, β-cryptoxanthin, lycopene, α-carotene, and β-carotene. We genotyped all our pepper collections using GBS (genotyping by sequencing) technology and performed association mapping for the metabolites mentioned above.In addition, we also analyzed the transcriptome and metabolome pertaining to cutin in habanero peppers. These analyses improved our knowledge of the molecular mechanisms regulating the accumulation of cutin in habanero pepper fruits. In addition, we explored natural variation for volatile aroma compounds (VOCs) including 2-Hexenal, 2-Nonenal, 2-octonone, 5-Heptenal, 2,6-dimethyl-, and 3,6-Nonadien-1-ol, E,Z- content that are known for fruit aroma during ripening process. In summary, we have archived 20,000 SNPs that are common for all the pepper species and individual species groups have about ~50,000 SNPs each with 70% call rate and 0.05 minor allele frequencies. We resolved population structures and selective sweeps important for various fruit traits and nutraceutical traits in various cultivated species. All these resources will be made available for public use. Similarly, in watermelon, we identified single nucleotide polymorphism (SNP) markers in 1365 collections and used a subset of diverse accessions for resolving natural variation in flesh specific citrulline, lipid-derived VOCs including 2-Hexenal, 2-Nonenal, 2-octonone, 5-Heptenal, 2,6-dimethyl-, and 3,6-Nonadien-1-ol, E,Z- contents in fruit flesh, seed specific amino acids and total protein content in seeds. Further the underlying natural variation for these metabolites were subjected to genome-wide association study (GWAS) for identification of genes and BLUPs to identify potential breeding stocks for use in genomic selection. Various evolutionary tools were explored to understand the role of various metabolites in watermelon domestication. In addition to natural variation, we explored impact of grafting in watermelon on yield and fruit quality. Graft specific functional genomics was used to understand long distance signaling of various genes. Genetic, transcriptomic and epigenetic tools were employed to understand diploid and synthetic tetraploid watermelon that are progenitors for seedless cultivars. Fruit specific microbiome was studied in watermelon and identified its role in fruit ripening and storage. Key Out comes/impacts We generated a large number of genome anchored SNPs and linked markers for various fruit traits for 4 different pepper species and cultivated watermelon. In addition, this project impacted entire world pepper and watermelon research by providing several important genomic resources such as SNPs, miRNAs, RNAseq generated transcripts, GWAS pipelines and metabolome information for several important germplasm resources. Some of these resources are publicly available and rest will be made for public use after published. For the first time, the current research provided a comprehensive data on phytochemicals in the world collection of Capsicum spp. and watermelon genotypes that can serve as a key resource for functional food research. Six graduate students submitted their master's thesis on fruit colors and phytochemicals. We also published 4 peer reviewed journal articles from this research. Both Graduate and undergraduate students participated in local, regional and International conferences and presented the research results.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lopez-Ortiz C, Pe�a-Garcia Y, Bhandari M, Abburi VL, Natarajan P, Stommel J, Nimmakayala P, Reddy UK. Identification of miRNAs and Their Targets Involved in Flower and Fruit Development across Domesticated and Wild Capsicum Species. International Journal of Molecular Sciences. 2021; 22(9):4866. https://doi.org/10.3390/ijms22094866
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Natarajan P, Akinmoju TA, Nimmakayala P, Lopez-Ortiz C, Garcia-Lozano M, Thompson BJ, Stommel J, Reddy UK. Integrated Metabolomic and Transcriptomic Analysis to Characterize Cutin Biosynthesis between Low- and High-Cutin Genotypes of Capsicum chinense Jacq. International Journal of Molecular Sciences. 2020; 21(4):1397. https://doi.org/10.3390/ijms21041397
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Joshi, V., Shinde, S., Nimmakayala, P., Abburi, V. L., Alaparthi, S. B., Lopez-Ortiz, C., & Reddy, U. K. (2019). Haplotype Networking of GWAS Hits for Citrulline Variation Associated with the Domestication of Watermelon. International Journal of Molecular Sciences, 20(21), 5392.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Garcia-Lozano, M., Dutta, S., Natarajan, P., Tomason, Y., Lopez, C., Katam, R., Levi, A., Nimmakayala, P., & Reddy, U.K (2019). Transcriptome changes in reciprocal grafts involving watermelon and bottle gourd reveal molecular mechanisms involved in increase of the fruit size, rind toughness and soluble solids. Plant Molecular Biology. 1573-5028. doi10.1007/s11103-019-00942-7.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Natarajan P, Tolulope Abodunrin Akinmoju, Padma Nimmakayala, Carlos Lopez-Ortiz, Marleny Garcia-Lozano, Benjamin J. Thompson, John Stommel and Umesh K. Reddy. Integrated metabolomic and transcriptomic analysis to characterize cutin biosynthesis between low-and high-cutin genotypes of Capsicum chinense Jacq. International Plant & Animal Genome XXVIII Conference, San Diego, California, 2020
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Tosin Akinsipe, Lakshmi V. Abburi, Suhas Shinde, Thangasamy Saminathan, Padma Nimmakayala and Umesh K. Reddy. Acid Mitigating Mechanisms in Capsicum Spp. International Plant & Animal Genome XXVIII Conference, San Diego California, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Carlos Lopez-Ortiz, Yadira Pe�a-Garcia, Purushothaman Natarajan, Menuka Bhandari, Lakshmi V. Abburi, Lav Yadav, John Stommel, Padma Nimmakayala and Umesh K. Reddy. The Ankyrin Repeat Gene Family in Capsicum spp: Genome-Wide Survey, Characterization and Gene Expression Profile. International Plant & Animal Genome XXVIII Conference, San Diego California, 2020.


Progress 04/15/19 to 04/14/20

Outputs
Target Audience:Pepper and watermelon growers, Geneticists and plant breeders, graduate and undergraduate students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students submitted their thesis research. Three undergraduate students worked in the project. Two postdoctoral associates and four technicians were also involvedat various levels of the project. How have the results been disseminated to communities of interest?We presented the results of the project in Regional, National and International conferences. Many seed companies showed keen interest in our nutraceutical research in peppers and associated SNPs to flavor compounds. Peer reviewed publications are generated in peppers and watermelon nutraceutical genomic research. What do you plan to do during the next reporting period to accomplish the goals?We fine mapped genes for capsaicinoids in peppers, citrulline in watermelon. We are using parents with highest contents of nutraceutical compounds in our breeding program. This project enabled us thousands of mapped SNPs and Insertion Deletions for use in future nutraceutical research. Crosses involving various high nutraceutical lines are currently under genomic selection in advanced generations.Individual lines having high phytochemicals will be selected from these populations.

Impacts
What was accomplished under these goals? We have estimated Carotenoids (Carotene, Cryptoxanthin, Zeaxanthin, Violaxanthin, Capsanthin, Capsorubin, Neoxanthin and Cryptoxanthin), Flavonoids (Quercetin, Kaempferol, Myricetin, Quercetin and Luteolin), volatile compounds Hexanol; 2-hexanal; octanal; 2-Decenal, Z-; 5-Heptenal; 2,6-dimethyl-, 1-Heptanol; 6-methyl-; 5-Hepten-2-one; 6-methyl-; Nonanal; 4-Nonenal, E-; 3-Nonen-1-ol, Z-; 2-Nonenal, E-; 3,6-Nonadien-1-ol, E,Z- and 2,6-Nonadienal, E,Z) and cutin monomers for 185 watermelon accessions, 230 accessions of Capsicum annuum, 268 accessions of C. baccatum, 225 accessions of C. chinense and 175 accessions of C. frutescens for use in GWAS. The major carotenoids found in large amounts in peppers were the carotenes α-carotene, β-carotene, and lycopene; the xanthophylls β-cryptoxanthin, capsanthin, and lutein; and zeaxanthin. Lycopene was detected in orange-colored peppers. And the flavonoids detected in large amounts in peppers were the flavones apigenin, luteolin, myricetin, and quercetin and the flavonol kaempferol. We genotyped all these pepper germplasm lines and performed association mapping for the metabolites mentioned above. In addition, we also analyzed the transcriptome and metabolome pertaining to cutin in habanero peppers. These analyses improve our knowledge of the molecular mechanisms regulating the accumulation of cutin in habanero pepper fruits. In this study, we integrated metabolome and transcriptome profiling pertaining to cutin in two habanero genotypes: PI 222448 and PI 257145. The fruits were selected by the waxy or glossy phenotype on their surfaces. Metabolomics analysis with GC-MS showed a significant variation in cutin composition between the genotypes, with about 6-fold higher cutin content in PI 257145 than PI 222448. We predicted a putative cutin biosynthetic pathway for habanero peppers based on deep transcriptome analysis. This is the first study of the transcriptome and metabolome pertaining to cutin in habanero peppers. These resources can be built on for developing cultivars with high cutin content that show resistance to biotic and abiotic stresses with superior post-harvest appearance. Watermelon is a good source of citrulline, a non-protein amino acid. Citrulline has several therapeutic and clinical implications as it produces nitric oxide via arginine. In plants, citrulline plays pivotal role in nitrogen transport and osmoprotection. We identified single nucleotide polymorphism (SNP) markers associated with citrulline metabolism using a genome-wide association study (GWAS) and understand the role of citrulline in watermelon domestication. A watermelon collection consisting of 187 wild, landraces, and cultivated accessions were used to estimate citrulline content. An association analysis involved a total of 12,125 SNPs with a minor allele frequency (MAF) > 0.05 to understand the population structure and phylogenetic analysis in light of citrulline accumulation. Wild egusi types and landraces contained low to medium citrulline content, whereas cultivars had higher content, which suggests that obtaining higher content of citrulline is a domesticated trait. GWAS analysis identified candidate genes (Ferrochelatase and Acetolactate Synthase) showing a significant association of SNPs with citrulline content. Haplotype networking indicated positive selection from wild to domesticated watermelon. To our knowledge, this is the first study showing genetic regulation of citrulline variation in plants by using a GWAS strategy. These results provide new insights into the citrulline metabolism in plants and the possibility of incorporating high citrulline as a trait in watermelon breeding programs.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Natarajan, P., A. T. Akinmoju, P. Nimmakayala, C. Lopez-Ortiz, M. Garcia-Lozano, J. B. Thompson, J. Stommel and U. K. Reddy (2020). "Integrated Metabolomic and Transcriptomic Analysis to Characterize Cutin Biosynthesis between Low- and High-Cutin Genotypes of Capsicum chinense Jacq." International Journal of Molecular Sciences 21(4).
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Garcia-Lozano, M., Dutta, S., Natarajan, P., Tomason, Y., Lopez, C., Katam, R., Levi, A., Nimmakayala, P., & Reddy, U.K (2020). Transcriptome changes in reciprocal grafts involving watermelon and bottle gourd reveal molecular mechanisms involved in increase of the fruit size, rind toughness and soluble solids. Plant Molecular Biology. 1573-5028. doi10.1007/s11103-019-00942-7.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Natarajan P, Akinmoju T, Nimmakayala P, Ortiz C, Garcia-Lozano M, Thompson BJ, John Stommel and Umesh K. Reddy. Integrated metabolomic and transcriptomic analysis to characterize cutin biosynthesis between low-and high-cutin genotypes of Capsicum chinense Jacq. International Plant & Animal Genome XXVIII Conference, San Diego California, 2020.


Progress 04/15/18 to 04/14/19

Outputs
Target Audience:Pepper and watermelon growers, Geneticists and plant breeders, graduate and undergraduate students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student submitted his thesis research. Two undergraduate students worked in the project. We have trained two postdoctoral associates and four technicians to work at various levels of the project How have the results been disseminated to communities of interest?Emails and conference presentations. seed companies were interested in our nutraceutical research in peppers and associated SNPs to flavor compounds What do you plan to do during the next reporting period to accomplish the goals?We are currently performing carotenoid and flavor metabolite profiling on various watermelons that constitute GWAS panel. We plan to estimate various important volatiles and flavor and citrulline specific compounds. Comparable GWAS and gene annotation will be performed for carotenoid compound levels in watermelon and pepper. Crosses involving various pepper species are currently under evaluation.

Impacts
What was accomplished under these goals? We have estimated flavor compounds Hexanol; 2-hexanal; octanal; 2-Decenal, Z-; 5-Heptenal; 2,6-dimethyl-, 1-Heptanol; 6-methyl-; 5-Hepten-2-one; 6-methyl-; Nonanal; 4-Nonenal, E-; 3-Nonen-1-ol, Z-; 2-Nonenal, E-; 3,6-Nonadien-1-ol, E,Z- and 2,6-Nonadienal, E,Z) using standard protocols in entire watermelon and C. chinense GWAS panels consisting 185 and 250 accessions, respectively. We identified highly favorable genotypes for use in breeding program. We identified several associated SNPs for flavor compounds throughout pepper and watermelon using GWAS (EMMS model). Pepper genotypes exhibit a wide range of capsaicinoid accumulation as a result of environmental and genetic variability. Recently, our group determined that the major markers linked to capsaicinoid synthesis are ankyrin-like protein family and the ATP-binding cassette (ABC) transporter family, which suggests that their activity may be involved in the pungency variation in pepper cultivars. We performed a genome-wide identification and characterization of Ankyrin and ABC transporter genes in three Capsicum species (i.e., C. annuum, C. baccatum and C. chinense) including sequence alignment, phylogenetic analysis, chromosomal location and expression profile of C. annuum and C. chinense. We identified 572 putative ABC transporter proteins and 268 genes potentially encoding ANK proteins in the entire pepper genome sequences of three different Capsicum species. Based on our results, we suggest that ABC transporters may play an important role in the transport of secondary metabolites such as capsaicin and dihydrocapsaicin to the placenta vacuoles, effecting on their content in pepper fruits. As well, we suggest that the ABC transporter play a role in the H2O2 detoxification process to reduce capsaicin degradation, specifically in the C. chinense fruits. In another hand the Capsicum Ankyrin proteins may play a role in plant growth, stress responses and fruit development by interacting with other proteins and may alter the capsaicinoids content in pepper fruits. Our results provide a more comprehensive understanding of ABC transporter and Ankyrin gene family in different Capsicum species while allowing the identification of important candidate genes related to capsaicin content for subsequent functional validation. We genotyped 1,365 watermelon plant introduction (PI) lines maintained at the U.S. National Plant Germplasm System using genotyping-by-sequencing (GBS). These PI lines were collected throughout the world and belong to three Citrullus species, C. lanatus, C. mucosospermus and C. amarus. Approximately 25,000 high-quality single-nucleotide polymorphisms (SNPs) were derived from the GBS data using the 'Charleston Gray' genome as the reference. Population genomic analyses using these SNPs discovered a close relationship between C. lanatus and C. mucosospermus and identified four major groups in these two species correlated to their geographic locations. Citrullus amarus was found to have a distinct genetic makeup compared to C. lanatus and C. mucosospermus. The SNPs also enabled identification of genomic regions associated with important fruit quality and disease resistance traits through genome-wide association studies. The high-quality 'Charleston Gray' genome and the genotyping data of this large collection of watermelon accessions provide valuable resources for facilitating watermelon research, breeding and improvement.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Garcia M, Natarajan P, Dutta SK, Lopez C, Tomason Y, Saminathan T, Nimmakayala P and Reddy UK Genomewide Transcriptome Analysis across the Reciprocal Grafts Involving Watermelon and Bottle Gourd to Study Effects on Fruit Quality. International Plant & Animal Genome XXVII Conference, January 12-16, 2019, San Diego, CA.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lopez-Ortiz C, Dutta SK, Natarajan P, Pe�a-Garcia Y, Abburi V, et al. (2019) Genome-wide identification and gene expression pattern of ABC transporter gene family in Capsicum spp.. PLOS ONE 14(4): e0215901. https://doi.org/10.1371/journal.pone.0215901


Progress 04/15/17 to 04/14/18

Outputs
Target Audience:Graduate and undergraduate students, pepper and watermelon growers, Geneticists and Plant breeders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student submitted her thesis research. Three undergraduate students worked in the project. We have trained one postdoctoral associate and three technicians to work at various levels of the project. How have the results been disseminated to communities of interest?We made presentations in plant and animal genome conference and American society of Horticultural sciences. Two seed companies were interested in our nutraceutical research in peppers and associated SNPs to fruit colors. What do you plan to do during the next reporting period to accomplish the goals?We are currently performing carotenoid and flavor metabolite profiling on various watermelons that constitute GWAS panel. We plan to estimate various important volatiles and flavor and citrulline specific compounds. Comparable GWAS and gene annotation will be performed for carotenoid compound levels in watermelon and pepper. Crosses involving various species are currently under evaluation.

Impacts
What was accomplished under these goals? Our team has genetically characterized traits in watermelon, including increased carotenoid levels, increased ascorbic acid, elevated sugars, flavor traits, fruit size and shape characteristics. Currently, our group is introgressing genes for elevated levels of carotenoids, ascorbic acid, and total sugars. To date, we have collected 1600 different genotypes of Citrullus spp. from the USDA-ARS National Plant Germplasm System (NPGS) and seed companies. We have developed HPLC methods to quantify carotenoids in watermelon. Comparable range of fruit colors is available among various species of peppers and watermelon. Pigment analyses have revealed striking similarities in carotenoid compositions between pepper varieties of the equivalent flesh colors. Although pepper species are botanically distant, it seems that the pepper fruit colors have phenotypic equivalents in watermelon. Our proposal is to analyze natural variation for various metabolites that are important for carotenoid pathway and the other pathways involved in fruit color and perform high resolution QTL analysis using GWASon diverse accessions. During the first year of the project, we analyzed the HPLC method for capsanthin, zeaxanthin, lutein, β-cryptoxanthin, lycopene, α-carotene, and β-carotene in 100 accessions each of Capsicum baccatum, C. annuum and C. chinenese. We identified six types of carotenoids including capsanthin, zeaxanthin, lutein, β-cryptoxanthin, α-carotene, and β-carotene in red and six types of carotenoids such as zeaxanthin, lutein, β-cryptoxanthin, lycopene, α-carotene, and β-carotene in orange and yellow peppers. Carotenoid profiles in red peppers were simpler than those in yellow and orange peppers and carotenoid profiles in yellow matured peppers included very small amounts than red and orange peppers. Total carotenoid contents showed the greatest difference in orange paprika, followed by red and yellow paprika. The richest carotenoid in orange and red ripened fruits were β-carotene, which was over 40 and 65% in all cultivars respectively, and yellow ripened fruits are lutein, which was over 28% followed by lutein in all cultivars. Red peppers contain a high content of provitamin A, due to the high concentration of β-carotene and β-cryptoxanthin. In our study, capsanthin concentrations in red ripened fruits were in the range of 7.01 µg/g to 178.81 µg/gDW lower than the previously reported range of 46.74±9.44 mg/100g DW, 58.33±3.91mg/100g DW in red paprika var. Special. The total Carotenoids in the ripened fruits ranged from 61.20 to 802.61 µg g-1 DW. Among genotypes tested, the highest amount of carotenoids were found in a genotype, VI029124-A originated from Bolivia, which contained high levels of carotenoids are β-carotene (227.32µg/g DW) followed by lycopene (210.16 µg/g DW) and lutein (168.09 µg/g DW). Currently GWAS is in progress using genomewide SNPs and SSRs in both watermelon and pepper. We also analyzed the microbiome of ripe fruits of watermelon cultivars of yellow (PI227202, PI435990, and JBush) and red flesh (PI459074, Congo, and SDRose) by employing both 16S metagenomics and metatranscriptomics to understand and predict their role in ripe fruits. The plant microbiome is a key determinant of plant health and productivity, and changes in the plant microbiome can alter the tolerance to biotic and abiotic stresses and the quality of end produce. Metagenomics data showed that Proteobacteria were abundant in SDRose and PI227202, whereas Cyanobacteria were most abundant in Congo and PI4559074. In the case of metatranscriptome data, Proteobacteria was the most abundant in all cultivars. High expression of genes linked to infectious diseases and the expression of peptidoglycan hydrolases associated to pathogenicity of eukaryotic hosts was observed in SDRose, which could have resulted in low microbial diversity in this cultivar. The presence of GH28, associated with polygalacturonase activity in JBush and SDRose could be related to cell wall modifications including de-esterification and depolymerization, and consequent loss of galacturonic acid and neutral sugars. Moreover, based on the KEGG annotation of the expressed genes, nine α-galactosidase genes involved in key processes of galactosyl oligosaccharide metabolism, such as raffinose family were identified and galactose metabolism pathway was reconstructed. Results of this study underline the links between the host and fruit-associated microbiome in carbohydrate metabolism of the ripe fruits. The cultivar difference in watermelon reflects the quantum and diversity of the microbiome, which would benefit watermelon and other plant breeders aiming at the holobiont concept to incorporate associated microbiomes in breeding programs

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Reddy UK, Nimmakayala P, Abburi VL, Ortiz C, Akinmoju T, Akinspe T, Saminathan T, Alaparthi S, Davenport B, Nadimi M, Vajja G, Kim S, Choi D, Stommel J. 2018. Genomewide chromosomal rearrangements and impact of positional effect of associated SNPs for capsaicinoids and fruit weight in various Capsicum spp. International Plant & Animal Genome XX Conference, January 13-17, 2018, San Diego, CA.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Saminathan T, Garc�a M, Ghimire B, Lopez C, Bodunrin A, Nimmakayala P, Abburi V, Levi A, Balagurusamy N, and Reddy UK. Metagenomic and metatranscriptomic analyses of diverse watermelon cultivars reveal the role of fruit associated microbiome in carbohydrate metabolism and ripening of mature fruits. Frontiers in Plant Science 2018, 9, 4.