Source: WEST VIRGINIA STATE UNIVERSITY submitted to NRP
SPEED BREEDING FOR INTROGRESSION AND FINE MAPPING OF DIVERSE ALLELES OF PEPPER FRUIT SIZE, SHAPE, FLAVOR AND COLOR
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1018082
Grant No.
2019-38821-29064
Cumulative Award Amt.
$299,739.00
Proposal No.
2018-04969
Multistate No.
(N/A)
Project Start Date
Dec 15, 2018
Project End Date
Dec 14, 2022
Grant Year
2019
Program Code
[EQ]- Research Project
Recipient Organization
WEST VIRGINIA STATE UNIVERSITY
PO BOX 1000
INSTITUTE,WV 25112
Performing Department
BIOLOGY
Non Technical Summary
Combining genomic resources with speed breeding will help advance breeding cycles in peppers, thus further accelerating the introgression process. In a breeding context, rapid generation advance to homozygosity after crossing will facilitate genetic gains for key traits and allow for more rapid production of improved cultivars by breeding programs. Fruit weight, shape and color are major traits that determine the yield and quality of the pepper fruit. Fruit weight and shape are quantitatively inherited traits with large natural variation in Capsicum. However, most studies have used low-resolution mapping populations or lacked functional tests to prove the effect of potential candidate genes and therefore were not able to identify the underlying genes. By combing high-resolution mapping, enrichment of markers in the QTL regions using next-generation sequencing (NGS) technologies and the identification of differentially expressed genes by transcriptome analysis, we will identify a set of candidate genes for the fruit shape.
Animal Health Component
60%
Research Effort Categories
Basic
40%
Applied
60%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011461108050%
2021461108150%
Knowledge Area
202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1461 - Peppers;

Field Of Science
1081 - Breeding; 1080 - Genetics;
Goals / Objectives
Pepper (Capsicum spp.) exhibits wide variation in fruit morphological traits such as size, shape and color that are utilized for breeding diverse types. The genetic and molecular basis of the natural variation in pepper fruit size and shape has been studied mostly by QTL mapping. Previous quantitative trait loci (QTL) studies in our laboratory allowed for identifying several QTL related to various fruit traits. However, because of lack of sufficient mapping resolution and functional tests, a large number of genes underlying these QTL have not been validated. We propose to perform pepper introgression by using bridge accessions of diverse pepper species in fully enclosed, controlled-environment growth chambers that can accelerate plant growth, called "speed breeding". Speed breeding greatly shortens the generation time and accelerates advancement of breeding populations, allowing up to 6 generations per year. This rapid generation cycling by the single seed descent method has great potential for introgression of diverse alleles. This accelerated growth will aid in our fine-mapping endeavors, including phenotyping of weight, shape, flavor and color, involving introgression of value-added traits from hot peppers (Capsicum chinense) and Peruvian peppers (Capsicum baccatum) into American peppers (Capsicum annuum). This proposal, to build the capacity for use of controlled-environment growth chambers that can accelerate introgression and fine-mapping programs, will create a plethora of opportunities for students to pursue research in the area of genome analysis and modern methods in plant breeding for enhanced nutrition.The specific objectives of the current proposal areTo fine-map and identify candidate genes underlying the QTL explaining major fruit morphology traits;Use bridge accessions for introgression of value-added traits from Peruvian and hot peppers into American peppers;Improve and integrate speed breeding methods in a course taught at both undergraduate and graduate levels, "Introduction to Crop Improvement andBiodiversity ".
Project Methods
A Conviron BDW chamber (Conviron, Canada) will be programmed to run a 22-hr photoperiod, with temperature 22 °C during the photoperiod, and 17 °C during the 2-hr dark period. Light and temperature will be set to ramp up and down for 1 hr. 30 min to mimic natural dawn and dusk conditions. Humidity will be set to 70%. Lighting will be supplied by a mixture of white LED bars (Valoya; 6 units per 7.6 m2), far -red LED lamps (Valoya; 12 units per 7.6 m2) and ceramic metal hydrargyrum quartz iodide lamps (Valoya; 32 units per 7.6 m2). Light intensity will be adjusted to 360-380 µmol m−2 s−1 (highest value after ramping) at bench height, where the pot s will be kept, and 490-500 µmol m−2 s−1 (highest value after ramping) at adult plant height. We plan to advance eight generations (starting from summer 2018 at 6 generations per year) of single seed (using SSD) collected from each of selfed 600 F2 lines (Maor x Birds Eye Baby) in a speed chamber to generate Recombinant Inbred Lines (RILs). Speed breeding in fully enclosed, controlled-environment growth chambers can accelerate plant development. The use of supplemental lighting in a glasshouse environment allows for rapid generation cycling via SSD and potential for use in fine-mapping strategies. Recombinant inbred lines will be generated and grown under normal greenhouse conditions. Data on fruit traits will be recorded by using Tomato Analyzer (TA). Fruits scanned by using a scanner (Hewlett-Packard, Palo Alto, CA, USA) will be subjected to morphometric analysis with TA v3 to examine fruit shape traits: basic measurements, fruit shape index, blockiness, homogeneity, proximal fruit end shape, asymmetry, internal eccentricity, and slenderness. GBS will be performed on RIL population at WVSU. DNA samples will be used to make barcoded libraries and sequenced on the Illumina Nextseq500 platform, which is currently available and ready to use in the PI's' laboratory. The TASSEL-GBS will be used to process the GBS sequencing reads for SNP calling. During the summers of 2020 and 2021, the entire RIL population will be grown at the Agricultural Experiment Station of WVSU for evaluating fruit morphology and fruit weight traits. The experimental design will be a randomized block design with three replications.Metabolomic profiles for pepper fruits of the genetic populations will be carried out using our established methods. Both Polar compounds (such as the phenolic acids), and less polar compounds (such as carotenoids and capsaicinoids) will be extracted from freeze dried samples of the genetic populations. 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. All extracts will be analyzed by reverse-phase liquid chromatography (Waters Alliance HPLC 2695) coupled to a photodiode array detector and a quadrupole time of flight high-resolution MS (LC-PDA-QTOF-MS) system (Waters QTOF-Ultima). Data processing will involve the MetAlign software (www.?metalign.?nl) for baseline correction, noise estimation and ion-wise mass spectral alignment.

Progress 12/15/18 to 12/14/22

Outputs
Target Audience:Graduate and undergraduate students, plant breeders and seed companies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Three graduate students submitted their thesis research in pepper genomics and metabolomics . A workshop was conducted as part of Crop Diversity class for graduate and undergraduate students to understand fruit shape phenotyping and its implications for breeding. How have the results been disseminated to communities of interest?Through emails, conference presentations and peer reviewed publications What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We identified candidate genes forC. chinensefruit shape, explored their contribution to population structure, and characterized their potential function in pepper fruit shape. We conducted this study using a collection of 244 accessions ofC. chinensefrom Peru, Bolivia, Costa Rica, Venezuela, Mexico, Puerto Rico, Guatemala, Brazil, Panama, Caribbean Islands, Suriname, Brazil, Caribbean Islands, Jamaica, Suriname, Colombia, Ecuador, Cuba, Trinidad and Tobago, United States, Korea, Taiwan, India, China, Tanzania and Niger. Using genotyping by sequencing, we identified 43,081single nucleotide polymorphisms(SNPs) from diverse collections ofC. chinense. Principal component, neighbor-joining tree, and population structure analyses resolved 3 phylogenetically robust clusters associated with fruit shapes. Genome-wide association study (GWAS) was used to identify associated genomic regions with various fruit shape traits obtained from image analysis with Tomato Analyzer software. In our GWAS, we selected 12 SNPs associated with locule number trait and 8 SNP markers associated with other fruit shape traits such as perimeter, area, obovoid, ellipsoid and morphometrics (5y, 6y and 7y). The SNPs inCLAVATA1,WD-40,Auxin receptor,AAA typeATPasefamily protein, andRNA polymerase IIIgenes were the major markers identified for fruit locule number from our GWAS results. Furthermore, we found SNPs in tetratricopeptide-repeat thioredoxin-like 3(TTL3), enhancer ofABAco-receptor 1(EAR1), subunit ofexocystcomplex 8 (SEC8) and pleiotropic drug resistance proteins (PDR11) associated with various fruit shape traits.CLAVATA1,WD-40andAuxin receptorgenes are known genes that affect tomato fruit shape. In addition, we further validated the functions of genes identified in our GWAS as associated with fruit shape traits, includingPDR11,TTL3,SEC8andEAR1, by using Arabidopsis T-DNA insertion knockout mutants to determine their role in fruit development, because these genes had not previously been reported to be fruit-specific genes.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Dutta, S.K.; Nimmakayala, P.; Reddy, U.K. Genome-Wide Identification, Characterisation, and Expression of C3HC4-Type RING Finger Gene Family in Capsicum annuum L. J. Hortic. Sci. Biotechnol. 2022, 97, 603614.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Nimmakayala, P.; Lopez-Ortiz, C.; Shahi, B.; Abburi, V.L.; Natarajan, P.; Kshetry, A.O.; Shinde, S.; Davenport, B.; Stommel, J.; Reddy, U.K. Exploration into natural variation for genes associated with fruit shape and size among Capsicum chinense collections. Genomics 2021, 113, 30023014.


Progress 12/15/20 to 12/14/21

Outputs
Target Audience:Plant breeders, private seed companies, graduate and undergraduate students. Changes/Problems:Because of COVID pandemic we could not engage undergraduate students in pepper field and greenhouse studies. Growth chamber delivery and installation was delayed for almost a year. What opportunities for training and professional development has the project provided?One graduate student is working on GWAS analysis of cutin monomers using SNP data for 96 Capsicum annuum collections. Both graduate and undergraduate studentsparticipated in the research. One lab class was conducted as part of Crop Diversity class to understand fruit shape phenotyping and its implications for breeding. How have the results been disseminated to communities of interest?Through emails, classroom teaching, conference presentations and peer reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?We have already set up the speed breeding chamber and the F2 populations generated among three Capsicum species will be evaluated for fruit traits under field conditions in summer 2022, and candidate genes and QTLs will be identified for major fruit traits.

Impacts
What was accomplished under these goals? Bell peppers constantly face biotic and abiotic stresses such as pathogen/pest infections, extreme temperature, drought and UV radiation. In addition, the fruit cutin lipid composition plays an important role in post-harvest water loss rates, which in turn causes shriveling and reduced fruit quality and storage. During this funding cycle, we have generated raw cutin from 96 bell pepper genotypes and further depolymerized in 3N methanolic hydrochloride (Me-OH-HCl), and cutin composition was analyzed and quantified by using GC-MS. The cutin monomers from these genotypes mostly consisted of long-chain aliphatic ω-hydroxy acids, especially dihydroxy hexadecanoic acids, considered the most important component of most plant cutin materials, and showed 16-fold differences in cutin monomer amounts among the bell pepper accessions studied. The highest genotype had the most abundant cutin content, with about 1884 mg/g dry weight (DW), and the lowest had the lowest cutin content, 129.36 mg/g DW. Total cutin composition and relative proportion of individual monomers among the cultivars, with about 6-fold higher cutin content in bell pepper genotypes. Our data showed that 10,16-dihydroxy hexadecanoic acid was the dominant cutin monomer with portions from 35% to 96% total cutin in our collections. Of note, our study showed a similar pattern, with a genotype having the lowest amount of dihydroxy hexadecanoic acid, about 102 mg/g DW, and the highest genotype showing the highest amount, 1240 mg/g DW. Among the octadecanoic acids, 9,10,12,13,18-pentahydroxy octadecanoic acid was dominant, with the highest showing an amount, 47.6 mg/g DW. This compound was detected only in fruits, which suggests that they might play a major role in the cutin composition of plants. Levels of p-coumaric acid, a phenolic compound, also showed significant variations among the collection. These variations among the collections provided a good background to investigate the cutin biosynthesis mechanisms. We further performed GBS with 96 Bell pepper collection generated a total of approximately 350 million reads (350,358,722) of 75 bp in length. The average number of reads with tag per sample were 3.6 million. The GBS reads were aligned to the Capsicum annuum v1.6 reference genome sequence (http://peppergenome.snu.ac.kr/). Average of 2.8 million reads with tag per sample were successfully mapped to the reference genome which corresponds to the overall mapping percentage of 81 to the genome. Based on the alignments, we identified a total of 907, 384 SNPs with 96 selected accessions. The SNPs were retained based on (1) read depth, DP>2 (2) minor allele frequency, MAF>0.05 (3) call rate>0.7. After stringent SNP filtering, we obtained a total of 40, 067 SNPs. The SNPs were annotated for further analysis. Our GWAS identified several candidate SNPs located in the genes and transcription factors such as GDSL lipase, glycerol-3 phosphate acyltransferase 6, long-chain acyltransferase 2, cytochrome P450 86A/77A, SHN1, ANL2 and HDG1 highly contributed to the high cutin content. We performed BLUP analysis to identify genotypes with favorable alleles to be used in genomic selection for cutin content through speed breeding.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Nimmakayala, P., Lopez-Ortiz, C., Shahi, B., Abburi, V. L., Natarajan, P., Kshetry, A. O., Shinde, S., Davenport, B., Stommel, J., & Reddy, U. K. (2021). Exploration into natural variation for genes associated with fruit shape and size among Capsicum chinense collections. Genomics.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Lopez-Ortiz, C., Pe�a-Garcia, Y., Bhandari, M., Abburi, V. L., Natarajan, P., Stommel, J., Nimmakayala, P., & Reddy, U. K. (2021). Identification of miRNAs and Their Targets Involved in Flower and Fruit Development across Domesticated and Wild Capsicum Species. International journal of molecular sciences, 22(9), 4866.


Progress 12/15/19 to 12/14/20

Outputs
Target Audience:Graduate and undergraduate students, plant breeders and seed companies Changes/Problems:Due to COVID conditions, undergraduate students could not participate in field research What opportunities for training and professional development has the project provided?Two graduate students submitted their thesis research in pepper genomic and metabolomics research. How have the results been disseminated to communities of interest?Through emails, conference presentations and peer reviewed publications What do you plan to do during the next reporting period to accomplish the goals?Functional validation of various genes identified is in progress. The research team already set upspeed-breeding chamber to advance pepper selections having multiple favorable gene combinations.

Impacts
What was accomplished under these goals? More than 1000 Capsicum accessions belonging to C. annuum, C. pubescence, C. baccatum, C. chinense, C. frutescence and C. galapagoense are phenotyped at the breaker stage of development. The collected fruits were cut in horizontal and longitudinal sections, and cut fruits were scanned with a scanner. The saved scanned images were analyzed with tomato analyzer 3.0 freeware. The scanned images were loaded in Tomato Analyzer software. The dpi was set to 300 by selecting the Scanner DPI from setting the menu, and for measurement of data output, the unit was set in centimeter (cm). The attributes like basic measurements (perimeter, area, width mid-height, maximum width, height mid-width, maximum height, curved height), fruit shape index (fruit shape index external I, fruit shape index external II), blockiness (proximal fruit blockiness, distal fruit blockiness, fruit shape triangle), homogeneity (ellipsoid, circular, rectangular), proximal fruit end shape (shoulder height, proximal angle macro, proximal angle micro, proximal indentation area), distal fruit end shape (distal angle micro, distal angle macro, distal indentation area, distal end protrusion), asymmetry (obovoid, ovoid, v. asymmetry, h. asymmetry.ob, h. asymmetry.ov, width widest pos.), internal eccentricity (eccentricity, proximal eccentricity, distal eccentricity, fruit shape index internal, eccentricity area index), latitudinal section (lobedness degree, pericarp area, and pericarp thickness) and morphometrics were selected. Based on literature review we selected only 13 traits (perimeter, area, fruit shape triangle, ellipsoid, fruits shape index external II, ovoid, obovoid, V. Asymmetry, H. Asymmetry.obv, H. Asymmetry.ov, lobedness degree, pericarp area, pericarp thickness) Manual adjustments in selected images were done by adjusting the boundary and pericarp of fruits. After manual adjustments of an image, the phenotypic data were exported as a .csv file and stored in the desired file destination. It was not possible to count the locule number by using Tomato Analyzer, so we counted the locule number manually by taking 6 fruits from each accession grown in 3 replications. About 1000 accessions belonging to various species were genotyped using genotyping by sequencing to generate genomewide mapped SNPs. 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. Phenotypic diversity within cultivated Capsicum species is particularly evident for fruit shape and size; however, genetic analysis of fruit shape variation across the species is absent. To unravel the genetic mechanism underlying fruit shape variation, we used GWAS for diverse collections. In our GWAS, we selected 12 SNPs associated with locule number trait and 8 SNP markers associated with other fruit shape traits such as perimeter, area, obovoid, ellipsoid and morphometrics (5y, 6y and 7y). The SNPs in CLAVATA1, WD-40, auxin receptor, AAA type ATPase family protein, and RNA polymerase III genes were the major markers identified for fruit locule number from our GWAS results. Furthermore, we found SNPs in tetratricopeptide-repeat thioredoxin-like 3, enhancer of ABA co-receptor 1, subunit of exocyst complex 8 and pleiotropic drug resistance proteins associated with various fruit shape traits. Because CLAVATA1, WD-40 and auxin receptor genes are known genes that shape tomato fruit, in this study we focused on functional characterization of newly identified genes to understand their role in fruit shape by using Arabidopsis thaliana T-DNA insertion knockout mutants. Color perception is explained by color spaces; commonly used color spaces in plant studies are CIELAB and the Munsell color system. The individual contributions of the color components (L*, a*, b*, hue and chroma) of these color spaces contribute to the overall color variation seen in peppers and were analyzed in this study. Multiple color diversity analysis tests showed that there are three main clusters found in Capsicum spp. collections that was not based on geographical location and that lighter peppers clustered together for color component L*. The percentages of total variation contributed by color components L*, a*, b*, hue and chroma are as follows: 10.5%, 14.9%, 10.4%, 37.1% and 10.10%, respectively. A total of 30 linked markers were found using the association mapping approach for the 5 color components. HpmsE026 was found to be a significant marker for color component a* and may play a role in the carotenoid biosynthetic pathway. Marker HpmsE090 associated with hue was found to be linked to the intergenic space between homeodomain leucine zipper protein HD-Zip and Peptidyl-prolyl cis-trans isomerase which are involved in fruit senescence and maturity. Understanding the variation in the contribution of each color component as well as the markers linked to these components, will provide insight into pepper color variation. Breeding populations and genetic materials are being progressed for rapid multiplication.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Nimmakayala P, Bhagarathi Shahi, V. Abburi, C. Lopez-Ortiz, J. Stommel and U. K. Reddy Population structure and exploration of fruit shape and locule number diversity among Capsicum chinense collections. Genomics (in review)
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Lopez-Ortiz, C., Pe�a-Garcia, Y., Natarajan, P., Bhandari, M., Abburi, V., Dutta, S. K., ... & Reddy, U. K. (2020). The ankyrin repeat gene family in Capsicum spp: Genome-wide survey, characterization and gene expression profile. Scientific reports, 10(1), 1-16.


Progress 12/15/18 to 12/14/19

Outputs
Target Audience:Graduate and Undergraduate students, Plant breeders and seed companies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student submitted thesis on Transcriptomics and metabolomics analysis of cutinin habanero pepper fruits. Another graduate student thesis research on fruit color analysis is in progress. Two undergraduate students participated in the research. A workshop was conducted as part of Crop Diversity class to understand fruit shape phenotyping and its implications for breeding. How have the results been disseminated to communities of interest?Through emails, conference presentations and peer reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?We plan to set up a speed-breeding chamber to advance pepper selections having multiple favorable gene combinations. We have already placed an order for controlled growth chamber but the delivery was delayed for more than 6 months. Functional validation of various genes identified is in progress.

Impacts
What was accomplished under these goals? Using this research capacity-building grant, we phenotyped 258 accessions belong to various cultivated Capsicum spp. These accessions were grown for three times to observe fruit color, shape, size and flavor. Since color perception is explained by color spaces, we used CIELAB and the Munsell color system to understand contributions of the color components (L*, a*, b*, hue and chroma) for entire collection of peppers. Thirty linked markers were found using the association mapping approach for five color components. For flavor, we estimated Nonanal, 2,6-Nonadienal, E,Z, 3-Nonen-1-ol, Z, 3,6-Nonadien-1-ol, E,Z, 2-Nonenal, E and 2-Decenal, Z- for hot peppers. These compounds produce flavor and aroma to peppers and important for insect resistance. For fruit shape and size, we used Tomato Analyzer to understand fruit architectural traits. We identified tomato homologues including SUN, OVATE, LC and FAS in peppers. Currently breeding endeavor to produce bell peppers with important shape and size alleles is in progress.

Publications

  • Type: Conference Papers and Presentations 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. 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: Akinsipe T, Abburi VL, Shinde S, Saminathan T, Nimmakayala P and Reddy UK. Acid Mitigating Mechanisms in Capsicum Spp. International Plant & Animal Genome XXVIII Conference, San Diego California, 2020
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Ortiz C, Dutta SK, Natarajan P, Pena Garcia Y, Abburi V, Saminathan T Nimmakayala P and Reddy UK (2019). Genome-wide identification and gene expression pattern of ABC transporter gene family in Capsicum spp. PloS one 14, e0215901, https://doi.org/10.1371/journal.pone.0215901