Source: OHIO STATE UNIVERSITY submitted to NRP
AN APPLE A DAY: INTERROGATING APPLE FOR CROP NUTRITIONAL QUALITY IMPROVEMENT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1030267
Grant No.
2023-67013-40267
Cumulative Award Amt.
$647,000.00
Proposal No.
2022-08458
Multistate No.
(N/A)
Project Start Date
Jun 1, 2023
Project End Date
May 31, 2027
Grant Year
2023
Program Code
[A1103]- Foundational Knowledge of Plant Products
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
(N/A)
Non Technical Summary
Apples are the most consumed fruit in America, and "an apple a day keeps the doctor away" has both anecdotal and scientific backing. The commercial importance of apples has led to varietal improvement; however, progress is limited by a long juvenile period, delaying fruit evaluation for traits of interest. As a result, apple breeders have implemented genomics approaches to enhance selection for some traits, but strategies for nutritionally relevant phytochemicals are lacking. We have developed a platform for genome-metabolome integration to better understand marker-metabolite relationships in apple fruit and used it to discover a strong association between chlorogenic acid content in fruit and a region on chromosome 17. We aim to conduct the foundational plant science needed to create well characterized and divergent apple germplasm for future nutritional testing in animal studies and human clinical trials. In Objective 1, we will screen diverse apple germplasm to understand variation in chlorogenic acid and other phenolics across cultivated and wild apples. In Objective 2, we will refine our knowledge on genetic control of chlorogenic acid and phenolics in apple fruit by combining whole genome sequencing, transcriptomics, and phytochemical quantitative analysis. In Objective 3, we will develop an understanding of the inheritance and genetic components involved in chlorogenic acid and phenolic content in apple fruits using transgenic apple germplasm with reduced juvenility. Our interdisciplinary team with expertise in apple diversity and germplasm evaluation, fruit genetics and genomics, phytochemical analysis, bioinformatics, and human nutrition has the unique expertise to complete the project we describe.
Animal Health Component
10%
Research Effort Categories
Basic
50%
Applied
10%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011110108040%
2011110108120%
2011110100040%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1110 - Apple;

Field Of Science
1081 - Breeding; 1000 - Biochemistry and biophysics; 1080 - Genetics;
Goals / Objectives
Our long-term goal is to produce unique apples that have enhanced health benefits, worthy of government-sanctioned health claims, that provide value to farmers, processors, and consumers. We propose the following specific objectives.Objective 1: Screen diverse apple germplasm to understand variation in chlorogenic acid and other phenolics across cultivated and wild apples.Apples will be sampled across an array including the most common commercially grown varieties, up-and-coming selections, progeny populations and their associated pedigree, and wild material. Environmental replication will be included for a subset of apples.A quantitative liquid-liquid extraction and liquid chromatography, tandem mass spectrometry (LC-MS/MS) analysis method will be developed and applied to fruits for chlorogenic acid and other common phenolics (e.g., flavanols, flavan-3-ols, anthocyanins, procyanidins, dihydrochalcones, and phenolic acids) in apples for which authentic standards are available. This approach will allow both a focused screening of chlorogenic acid and a broader understanding of phenolic distribution and profile among the selected germplasm.The outcomes of this objective will enable selection of the germplasm to be screened genomically and transcriptomically (see Objective 2 for details).Objective 2: Refine knowledge of genetic control of chlorogenic acid and phenolics in apple fruit by combining whole genome sequencing, transcriptomics, and phytochemical quantitative analysis.Germplasm across the chlorogenic acid and phenolic content range will be subjected to 1) long-read PacBio sequencing (n=10 most divergent genotypes), 2) gene expression analysis via Illumina RNA sequencing (top 5 five cultivars replicated over at least three environments), and 3) quantitative metabolite analysis using LC-MS/MS.This information will be used in order to refine genomic polymorphism-phenolics associations we have demonstrated previouslyon chromosomes 17 (and possibly 3). This will focus our understanding of the genetic control of chlorogenic acid and phenolics content in apple fruit at the transcript level.The outcome of this objective will be a set of haplotypes associated with chlorogenic acid/phenolic compounds across a diversity of germplasm that includes commercial cultivars, selections, and segregating populations.Objective 3: Understanding inheritance and genetic components involved in chlorogenic acid and phenolic content in apple fruits.We will evaluate germplasm generated through rapid cycle, fast breeding, and accelerated introgression with reduced juvenility. The material under current development has potential horticultural value (i.e., genetic background from commercially proven cultivars) with ~1800 seedlings already produced.Based on the findings in objectives 1 and 2, we will select existing seedlings, in addition to creating new reduced juvenility material. This will enable the production of fruit in less than two years (as opposed to the usual 6-12 years) in several small segregating families.Development of divergent apple germplasm for chlorogenic acid and phenolics is in progress, and additional progeny will be produced to contrast gene expression in bulk-segregantand inheritance analysesto identify genetic components (i.e., alleles) governing phytochemical production.To advance in allele identification and transcript association we will use RNA-sequencing using SMRT sequencing through PacBio Iso-seq of the most divergent segregating germplasm.The outcome of this objective will allow development of DNA tests that will enable the selection of desirable germplasm for future basic studies, clinical studies with animal models and human trials, as well as for future breeding endeavors.
Project Methods
Our methods are as follows for our 3 objectives:Objective 1: Screen diverse apple germplasm to understand variation in chlorogenic acid and other phenolics across cultivated and wild apples.Apples will be sampled across an array including the most common commercially grown varieties, up-and-coming selections, progeny populations and their associated pedigree, and wild material. Environmental replication will be included for a subset of apples.A quantitative liquid-liquid extraction and liquid chromatography, tandem mass spectrometry (LC-MS/MS) analysis method will be developed and applied to fruits for chlorogenic acid and other common phenolics (e.g., flavanols, flavan-3-ols, anthocyanins, procyanidins, dihydrochalcones, and phenolic acids) in apples for which authentic standards are available. This approach will allow both a focused screening of chlorogenic acid and a broader understanding of phenolic distribution and profile among the selected germplasm.Objective 2: Refine knowledge of genetic control of chlorogenic acid and phenolics in apple fruit by combining whole genome sequencing, transcriptomics, and phytochemical quantitative analysis.Germplasm across the chlorogenic acid and phenolic content range will be subjected to 1) long-read PacBio sequencing (n=10 most divergent genotypes), 2) gene expression analysis via Illumina RNA sequencing (top 5 five cultivars replicated over at least three environments), and 3) quantitative metabolite analysis using LC-MS/MS.This information will be used in order to refine genomic polymorphism-phenolics associations we have demonstrated previouslyon chromosomes 17 (and possibly 3). This will focus our understanding of the genetic control of chlorogenic acid and phenolics content in apple fruit at the transcript level.Objective 3: Understanding inheritance and genetic components involved in chlorogenic acid and phenolic content in apple fruits.We will evaluate germplasm generated through rapid cycle, fast breeding, and accelerated introgression with reduced juvenility. The material under current development has potential horticultural value (i.e., genetic background from commercially proven cultivars) with ~1800 seedlings already produced.Based on the findings in objectives 1 and 2, we will select existing seedlings, in addition to creating new reduced juvenility material. This will enable the production of fruit in less than two years (as opposed to the usual 6-12 years) in several small segregating families.Development of divergent apple germplasm for chlorogenic acid and phenolics is in progress, and additional progeny will be produced to contrast gene expression in bulk-segregantand inheritance analyses to identify genetic components (i.e., alleles) governing phytochemical production.To advance in allele identification and transcript association we will use RNA-sequencing using SMRT sequencing through PacBio Iso-seq of the most divergent segregating germplasm.

Progress 06/01/24 to 05/31/25

Outputs
Target Audience:Our targeted audience in 2024-2025 is other scientists, especially apple/phenolics researchers and orchardists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1.5 graduate students and one technician have been supported on this project, where they have developed molecular biology, chemistry, and plant biology skills. The graduate student on this project has presented his work at the annual meetings of the American Society for Horticultural Science and the Ohio Mass Spectrometry Symposium. Data has also been presented to apple growers atOhio Produce Growers and Marketers Association's Ohio Produce Network. How have the results been disseminated to communities of interest?Research has been disseminated at conferences (American Society for Horticultural Science, Ohio Mass Spectrometry Symposium), and at grower meetings. Research is slated to additionally be presented at the American Society for Mass Spectrometry. Manuscripts are in preparation. What do you plan to do during the next reporting period to accomplish the goals?We plan to select apples for sequencing in objective 2, conduct the sequencing, and analyze resultant data. Trangenic crosses and analysis will continue in objective 3.

Impacts
What was accomplished under these goals? Objective 1: We have developed an extraction and LC-MS/MS analysis method enabling the quantitative analysis of 12 phenolics (including chlorogenic acid and its isomers) in apple fruits. We have collected > 350 diverse apple fruits selected across apple species, disease resistance, fruit and flesh color, and previously reported phenolic compound content. We have also collected the top 5 apple varieties (Honeycrisp, Red Delicious, Golden Delicious, Fuji, Gala) in triplicate directly from 7 orchards (OH, NY, PA, MI, MN, NC, WA) in two years, and the top 10 apple varieties (Honeycrisp, Red Delicious, Golden Delicious, Fuji, Gala, Granny Smith, Pink Lady, McIntosh, Ambrosia, Cosmic Crisp) from grocery stores in 5 locations (OH, FL, TX, CA, NY) across the United States. These apples have all been extracted and run via LC-MS/MS for their analysis and the highest and lowest apples for chlorogenic acid content (and other fruits of interest based on their profile) will be selected for DNA and RNA sequencing in objective 2. Objective 3: We have made significant efforts to develop horticultural growth methods for the transgenic 19-1G08, which is heterozygous for BpMADS4 (early flowering). We have made crosses between 10 cultivars of interest and our transgenic parent to induce early flowering, have generated progeny which have been analyzed for phenolics via LC-MS/MS.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2025 Citation: Balogh L, Miller DD, Cooperstone JL. A new way to think about apple quality. Ohio Produce Growers and Marketers Associations Ohio Produce Network, January 20, 2025.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Balogh L, Quiroz Moreno CD, Miller DD, Simons C, Fresnedo Ram�rez J, Cooperstone JL. Linking apple fruit phenolics to human taste perception. Ohio Mass Spectrometry Symposium (OMSS), Columbus, OH, October 15, 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Quiroz Moreno CD, Cooperstone JL. Quantitative analysis of plant phenolics by LC-MS/MS, and PhenolicsDB: a publicly available high-resolution MS/MS spectral library. Ohio Mass Spectrometry Symposium (OMSS), Columbus, OH, October 15, 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Balogh L, Quiroz Moreno CD, Miller DD, Simons C, Fresnedo Ram�rez J, Cooperstone JL. Characterizing unique apple selections for U-pick growers in the Midwest using descriptive analysis, consumer liking tests, and phytochemical analysis. American Society for Horticultural Science Annual Meeting, Oral Presentation, Honolulu, HI, September 23-27, 2024.


Progress 06/01/23 to 05/31/24

Outputs
Target Audience:Our targeted audience in 2023-2024 is other apple researchers and orchardists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student and one technician have been supported on this project, where they have developed molecular biology, chemistry, and plant biology skills. The graduate student on this project has presented his work at the annual meetings of the American Society for Mass Spectrometry and the Metabolomics Society, and locally at the Ohio Mass Spectrometry Symposium. How have the results been disseminated to communities of interest?Research has been disseminated at conferences (American Society for Mass Spectrometry, the Metabolomics Society), and at grower meetings. Research is slated to additionally be presented at the American Society for Horticultural Science. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to complete analysis of fruits from Objective 1, select apples for sequencing in Objective 2, and begin sequencing and analysis.

Impacts
What was accomplished under these goals? Objective 1: We have developed an extraction and LC-MS/MS analysis method enabling the quantitative analysis of 12 phenolics (including chlorogenic acid and its isomers) in apple fruits. We have collected > 200 diverse apple fruits selected across apple species, disease resistance, fruit and flesh color, and previously reported phenolic compound content. We have also collected the top 5 apple varieties (Honeycrisp, Red Delicious, Golden Delicious, Fuji, Gala) in triplicate directly from 7 orchards (OH, NY, PA, MI, MN, NC, WA), and the top 10 apple varieties (Honeycrisp, Red Delicious, Golden Delicious, Fuji, Gala, Granny Smith, Pink Lady, McIntosh, Ambrosia, Cosmic Crisp) from grocery stores in 5 locations (OH, FL, TX, CA, NY) across the United States. These apples are all in the process of being analyzed for phenolics. Objective 3: We have made significant efforts to develop horticultural growth methods for the transgenic 19-1G08, which is heterozygous for BpMADS4 (early flowering). We have made crosses between 10 cultivars of interest and our transgenic parent to induce early flowering.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Quiroz Moreno CD, Cooperstone JL. Quantitative analysis of plant phenolics by LC-MS/MS, and PhenolicsDB: a publicly available high-resolution MS/MS spectral library. American Society for Mass Spectrometry, Anaheim, CA, June 2-6, 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Quiroz Moreno CD, Cooperstone JL. MS2extract: an R package for a scalable LC-MS/MS compound library creation. Ohio Mass Spectrometry Symposium (Oral Presentation), Columbus, OH, October 18-19, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Quiroz Moreno CD, Cooperstone JL. MS2extract: an R package for a scalable LC-MS/MS compound library creation. Metabolomics Society, Niagara Falls, Canada, June 18-22, 2023.