Source: CORNELL UNIVERSITY submitted to
BREEDING METHODS AND GERMPLASM FOR IMPROVED NUTRITIONAL QUALITY OF SWEET CORN
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1003971
Grant No.
(N/A)
Project No.
NYC-149429
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2014
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Project Director
Gore, MI, AL.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Plant Breeding
Non Technical Summary
As the 4th highest producer of fresh-market sweet corn in the nation, New York's sweet corn crop had a value of $68.4 million in 2012. Even though, sweet corn is the third most commonly consumed vegetable in the United States, it still does not provide adequate daily levels of provitamin A (carotenoids) and vitamin E (tocochromanols). While the vitamin A requirement in the U.S. is largely met through fortification, surprisingly large proportions of the U.S. population still do not obtain the daily recommended dietary amount of vitamin E and the two non-provitamin A carotenoids, lutein and zeaxanthin, that delay onset of age-related macular degeneration -- a leading cause of irreversible blindness in elderly populations of Western societies. Improving the nutritional quality of crops through plant breeding, termed biofortification, is a cost-effective and sustainable approach to potentially help address such nutritional deficiencies in the human population. In that light, this project will conduct an association mapping study to identify genes and favorable alleles that are responsible for quantitative variation in grain provitamin A and vitamin E levels in a sweet corn. The findings from the association mapping study will be used to develop and validate marker-based prediction models to rapidly and cost-effectively convert New York adapted sweet corn germplasm to dark orange grain with high provitamin A and vitamin E. The genetic marker assays, validated marker-based prediction models, and developed early generation sweet corn germplasm with elevated levels of provitamin A carotenoids and vitamin E tocochromanols will be available for use by stakeholders. The proposed research will benefit seed companies, growers, processors, consumers, and rural communities in New York State. Ultimately, this work is intended to serve, promote, and benefit the health and nutrition of New York State residents and beyond.
Animal Health Component
0%
Research Effort Categories
Basic
70%
Applied
(N/A)
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011480108150%
7011480108150%
Goals / Objectives
Breeders, growers, processors, and consumers of sweet corn in NY and throughout the U.S. will benefit from locally adapted, superior-performing orange-grain sweet corn germplasm with high provitamin A and vitamin E content. The two major objectives of this research are to (i) conduct a genome-wide association study (GWAS) to identify genes and favorable alleles responsible for quantitative variation of grain provitamin A and vitamin E levels in a sweet corn diversity panel (years 1 and 2); and (ii) development and validation of marker-based prediction models to convert locally adapted sweet corn germplasm to dark orange grain with high provitamin A and vitamin E content (years 1-3).
Project Methods
An association panel of ~500 diverse sweet corn inbred lines that captures a significant proportion of the genetic diversity existing in public U.S. sweet corn breeding programs will be evaluated at field locations in Wisconsin, Indiana, and New York in years 1 and 2, followed by the use of reverse-phase high-performance liquid chromatography (HPLC) to quantify provitamin A (carotenoids) and vitamin E (tocochromanols) in self-pollinated grain samples harvested from lines at the immature (21 days after pollination) and physiological mature stages of development. This effort will allow the extent of natural variation and heritability of carotenoids and tocochromanols in sweet corn at two developmental time points to be comprehensively evaluated (#1 change in knowledge). In addition, the sweet corn diversity panel will be genotyped with high-density single-nucleotide polymorphism (SNP) markers, allowing the patterns and levels of nucleotide diversity to be examined at a genome-wide level (#2 change in knowledge). Through the integration of SNP genotype and HPLC phenotype data sets, association mapping will be used to identify genes and favorable alleles that control levels of provitamin A (carotenoids) and vitamin E (tocochromanols) in the grain of sweet corn at two developmental time points (#3 change in knowledge). The findings from the GWAS effort will be used to develop and validate marker-based prediction models to convert locally adapted sweet corn germplasm to dark orange grain with high provitamin A and vitamin E content (#4 change in knowledge) in years 1-3. In addition, an analysis of variance will be conducted in effort to determine the importance of genotype-by-environment interactions (#5 change in knowledge).The outcomes of this proposed research will be delivered as science-based knowledge to people through the following efforts (#6 change in knowledge): 1) presentations of data at scientific conferences and invited seminars, symposiums, and workshops; 2) the goals and results of the project taught via class room examples to students in a short course at the Tucson Plant Breeding Institute (http://www.plantbreedinginstitute.bio5.org/), and two fall semester courses (PLBR 4070 - Nutritional Quality Improvement of Food Crops and PLBR 4060 - Methods of Plant Breeding Laboratory) at Cornell University that will include the participation of students in the field evaluation and selection of orange sweet corn with high provitamin A and vitamin E; 3) training of undergraduate and graduate students through their involvement with research activities proposed in the project; 4) findings conveyed and newly developed germplasm showcased to stakeholders at the Vegetable Breeding Institute's annual field day outreach event; and 5) information about the research project shared with stakeholders at Cornell Cooperative Extension events.The following project evaluation plan will be used to monitor and measure project performance through the completion of eight outcomes (milestones):1) Years 1-2: Revealed the extent of phenotypic variation and heritability for provitamin A and vitamin E levels in grain from a sweet corn diversity panel.2) Year 1: Characterized the patterns and levels of nucleotide diversity in a sweet corn diversity panel.3) Years 1-2: Discovered genes and favorable alleles that control provitamin A and vitamin E levels in grain of sweet corn.4) Year 2: Submit manuscript on GWAS results to a peer-reviewed journal.5) Years 1-3: Identified and validated optimal marker-based prediction models for the development of sweet corn germplasm with increased provitamin A and vitamin E content.6) Year 3: Quantified genotype-by-environment (G×E) interactions for levels of provitamin A and vitamin E in grain of sweet corn.7) Year 3: Submit manuscript on validated optimal marker-based prediction models and G×E interactions to a peer-reviewed journal.8) Years 1-3: Enhanced stakeholder awareness of sweet corn nutritional content and its importance to human health and nutrition.Monitoring and measurement of project performance will also be through the following indicators of success: 1) the number of peer-reviewed publications from the project; 2) the number of visits to the project website, number of downloads for peer-reviewed publications and annual reports; 3) the number of citations for peer-reviewed publications; 4) increased utilization of germplasm with high nutritional content in private and public sweet corn breeding programs (#1 change in action); 5) the number of direct requests by private and public breeders for validated genetic marker assays, marker-based prediction breeding methods, and seed of nutrient-dense sweet corn germplasm (#1 and #2 change in action); and 6) number of undergraduate and graduate students trained on the research project.

Progress 10/01/14 to 09/30/17

Outputs
Target Audience:PI Gore was an instructor at the Tucson Plant Breeding Institute (Tucson, Arizona) for the two modules: Introduction to Plant Quantitative Genetics and Advanced Statistical Plant Breeding. Classroom teaching examples included goals, background information, and preliminary results from this Hatch project and the 50 students in attendance were representing public institutions, private companies, and non-profit organizations from NY, the US and several other countries. While teaching at Cornell in 2016/17, Gore presented the project's sweet corn biofortification research efforts in several lectures to undergraduate and graduate students that included those from local farming communities and the New York metropolitan area over the semester in Nutritional Quality Improvement of Food Crops (PLBR/BIOPL/HORT 4070). It was the first introduction for many of them to biofortification and its role in addressing human nutritional deficiencies in New York State and beyond. In addition, PI Gore presented preliminary results from this Hatch project at several scientific conferences and seminars at the national and international levels. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The Hatch project has been the cornerstone to training the next-generation of plant breeders in the Gore lab and helped to address the paucity of women and racial and ethnic minorities in agriculture. PI Gore (field, lab, and computational), Technician III Nicholas Kaczmar (field and lab), and Research Support Specialist III James Clohessy (computational) provided training in field (pollinations and phenotyping), lab (ear phenotyping and post-processing of fresh harvested kernels), and computational (mobile applications and programming) research to Brazilian (1 male), Chinese (1 female), and American (1 female) graduate students. In the past year, the Hatch project involved four undergraduate students that participated in field and lab research and of which two were women. How have the results been disseminated to communities of interest?As an instructor at the Tucson Plant Breeding Institute (two modules: Introduction to Plant Quantitative Genetics and Advanced Statistical Plant Breeding) over the past year in Arizona (January), PI Gore included classroom teaching examples on the Hatch project's sweetcorn biofortification research efforts to the more than 50 students in attendance from public institutions, private companies, and non-profit organizations. Furthermore, collectively, these students represented several countries. While teaching at Cornell in 2016/17, PI Gore presented the project's sweet corn biofortification research efforts in several lectures to undergraduate and graduate students over the semester in Nutritional Quality Improvement of Food Crops (PLBR/BIOPL/HORT 4070), as well as a lecture on biofortification to undergraduate and graduate students in Methods of Plant Breeding Laboratory (PLBR 4060). In terms of outreach activities, PI Gore discussed the biofortification research with SeedWorld (video and print) and Ezra Magazine (video and print). The Ph.D. student (Matt Baseggio) conducting research on the project presented Hatch project results at two scientific conferences: Gordon Research Conference in Galveston, TX, and International Sweet Corn Development Association in Chicago, IL. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A panel of ~400 sweet corn inbred lines was grown in replicated field trials at University of Wisconsin's West Madison Research Station in Verona, WI (collaborator William Tracy) and Cornell University's Musgrave Research Farm in Aurora, NY (PI Gore and Co-PI Smith) during summers of 2014 and 2015. The lines from public breeding programs capture major levels of genetic diversity found in the U.S. sweet corn germplasm pool. Each of the genotypes selected for this study was homozygous for at least one of the different maize endosperm carbohydrate mutations. The experiment had 880 experimental units in 2014 and 968 in 2015. In each year, all experimental units were arranged as an incomplete block design with two replicates. The diversity panel was scored for several agronomic traits, including emergence, plant height, ear height, lodging, flowering time, and tiller number. A single replicate of the complete experiment from each year was used for tocochromanol and carotenoid quantification. Two self-pollinated ears were harvested from each plot at 400 growing-degree days [~21 days after pollination (DAP)] to represent the immature (fresh) stage of kernel development. Harvested ears from each plot were shelled and bulked to form a representative, composite kernel sample. Samples were stored at -80ºC until they were ground into a fine powder using a commercial Tube Mill control by IKA with the addition of liquid nitrogen during the process to prevent thawing. Frozen samples from 859 plots were sent to a collaborator (Dr. Dean DellaPenna) at Michigan State University for tocochromanol and carotenoid quantification using reverse-phase high-performance liquid chromatography (HPLC). Six tocochromanol and nine carotenoid compounds were measured and a series of 25 sums, ratios, and proportions were calculated and analyzed. For each trait, a best linear unbiased predictor (BLUP) for each inbred line was calculated after controlling for field and lab effects. Genotyping-by-sequencing (GBS) data from all the inbred lines were received in the first quarter of 2017 and single-nucleotide polymorphism (SNP) markers were called using the Tassel 5 production pipeline. Missing SNP genotypes were then imputed using Fast Inbred Line Library ImputatioN (FILLIN) procedure. These SNPs were filtered using several criteria, resulting in about 175,000 high quality markers, which were then used for genome-wide association studies (GWAS) and whole-genome prediction (WGP). For GWAS of tocochromanols, a total of 364 unique SNPs were significant for at least one and up to eight traits at a genome-wide false discovery rate (FDR) of 5%. Two SNPs located within an intron of the gene encoding gamma-tocopherol methyltransferase (ZmVTE4) were associated with the alpha-tocopherol trait. ZmVTE4 is a gene from the core tocochromanol pathway and catalyzes the conversion of gamma- to alpha- tocopherol. Each of these SNPs accounted for 7.6% of the total phenotypic variation for alpha-tocopherol. For traits gamma-tocotrienol and total tocotrienols, we identified one significant SNP on chromosome 9 that was close to a gene committed to tocotrienol biosynthesis. This SNP was located 138 Kb upstream of the start codon of a gene that encodes a homogentisate geranylgeranyl transferase (ZmHGGT1), an enzyme that catalyzes the condensation of homogentisate and geranylgeranyl diphosphate and yields the committed precursor to tocotrienols. We also found 24 significant SNPs within +/- 200 Kb of the gene encoding tocopherol cyclase (ZmVTE1), and these SNPs were associated with at least one of three tocotrienol-related traits. Most of the other SNPs were from regions on chromosomes 2, 3, and 4 in the vicinity of the endosperm kernel types sugary-enhancer (se1), shrunken2 (sh2), and sugary1 (su1), respectively. For GWAS of carotenoids, 17 SNPs located in the proximity of the gene that encodes beta-carotene hydroxylase (crtRB1) were associated with beta-carotene and two ratios. This enzyme converts beta-carotene into beta-cryptoxanthin and further to zeaxanthin, which have no provitamin A activity. Additionally, for the ratio beta-xanthophylls/alpha-xanthophylls, which are classes of oxygen-containing carotenoids, we identified an association with a SNP located in the proximity of lycopene epsilon-cyclase (LYCE), which encodes an enzyme that combined with lycopene beta-cyclase is responsible for converting lycopene to alpha-carotene. Similar to tocochromanols, SNPs in the proximity of sugary1 gene were associated with lutein. Three SNPs on chromosome 1, and 1 SNP on chromosome 3 were also associated with different carotenoid traits, but were not in proximity of any candidate gene. We calculated genomic estimated breeding values (GEBVs) of the inbred lines for all tocochromanol and carotenoid traits using ridge regression best linear unbiased prediction (RR-BLUP). The genomic relationship matrix was calculated with 175K high-quality SNPs, which is the same subset filtered for GWAS, and accuracies were estimated using a 5-fold cross validation. Overall, prediction for all the traits in the two classes of compounds presented moderate mean accuracies, ranging from 0.38 (alpha-tocopherol) to 0.65 (delta-tocotrienol) for tocochromanols, and from 0.15 (neoxanthin) to 0.66 (lutein) for carotenoids. In order to select the lines for the establishment of a breeding program for nutritional quality in sweet corn, inbred lines were ranked for each trait based on their GEBV. Top-ranked lines for alpha-tocopherol (greatest vitamin E activity), tocotrienols (increased antioxidant capacity), lutein and zeaxanthin (decreased risk of age-related macular degeneration), beta- and alpha-carotenoid (provitamin A) as well as total tocochromanols and carotenoids were selected for crossing. In total, 46 best performing sweet corn lines with different kernel types were chosen. Within each kernel endosperm type group, crosses were made between lines with complementary haplotypes for key regions identified by GWAS and further haplotype block analysis. Each line was used for crosses from two to eight times, depending on their importance ('sh2' > 'su1') and number of favorable haplotypes. During the summers of 2016 and 2017, more than one hundred and fifty crosses were made. In November of each year, a sample from each cross was sent to a winter nursery in Chile, where they were planted and self-pollinated in order to advance a generation. The resulting seed will then be planted again in 2018 in New York to be evaluated and compose a breeding population for nutritional quality in sweet corn. In addition to the fresh harvest for the nutritional analysis, in 2014 and 2015, two ears from self-pollinated plants from each plot were harvested at physiological maturity (~55 DAP) to be stored for seed. Also, two ears from open-pollinated plants were harvested for ear phenotyping, including traits such as kernel row number, kernel type, ear and cob length and width, and 20-kernel dry weight. Kernel color, measured both during the fresh and mature stages from self-pollinated ears using a Chroma Meter CR-400, showed this panel does capture a high range of endosperm color, and both a* and b* (positive values of a* and b* indicate red and yellow in the Hunter L*a*b* color space, respectively) showed high correlation (r2 = 0.79 and r2=0.71, respectively) to total carotenoids at fresh stage. These results suggest that selection of visibly darker orange grain for increased synthesis and retention of total carotenoids could be combined with marker-assisted selection for favorable alleles for specific carotenoids.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Baseggio, M., Murray, M., Kaczmar, N., Magallanes-Lundback, M., Buckler, E. S., DellaPenna, D., Tracy, W., Smith, M. E., and Gore, M. A. The genetic dissection and selection of natural variation for provitamin A and vitamin E levels in sweet corn kernels. Poster presented at the Quantitative Genetics and Genomics Gordon Research Conference in Galveston, TX. February 26 - March 3, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Baseggio, M., Murray, M., Kaczmar, N., Magallanes-Lundback, M., Buckler, E. S., DellaPenna, D., Tracy, W., Smith, M. E., and Gore, M. A. Genetic analysis of kernel nutritional quality and agronomic traits in sweet corn. Poster presented at the International Sweet Corn Development Association (ISCDA) Meeting in Chicago, IL. December 5-6, 2016.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:PI Gore was an instructor at the Tucson Plant Breeding Institute (Tucson, Arizona; Piracicaba, Brazil; Seoul, South Korea) for the two modules: Introduction to Plant Quantitative Genetics and Advanced Statistical Plant Breeding. Classroom teaching examples included goals, background information, and preliminary results from this Hatch project and the 50-100 students in attendance were representing public institutions, private companies, and non-profit organizations from NY, the US and many other countries across 5 continents. While teaching at Cornell in 2015/16, Gore presented the project's sweet corn biofortification research efforts in several lectures to undergraduate and graduate students that included those from local farming communities and the New York metropolitan area over the semester in Nutritional Quality Improvement of Food Crops (PLBR/BIOPL/HORT 4070). It was the first introduction for many of them to biofortification and its role in addressing human nutritional deficiencies in New York State and beyond. In addition, PI Gore presented preliminary results from this Hatch project at several scientific conferences and seminars at the national and international levels. Changes/Problems:There are no major problems or delays to report that could have a significant impact on the rate of expenditure. There have been only minor modifications to the research procedure and none of which will cause significant deviations from the minorly revised research schedule or goals as stated in last year's annual report. What opportunities for training and professional development has the project provided?The Hatch project over the past year has been the cornerstone to training the next-generation of plant breeders in the Gore lab and helped to address the paucity of women and racial and ethnic minorities in agriculture. PI Gore (field, lab, and computational), Technician III Nicholas Kaczmar (field and lab), and Research Support Specialist III James Clohessy (computational) provided training in field (pollinations and phenotyping), lab (ear phenotyping and post-processing of fresh- harvested kernels), and computational (mobile applications and programming) research to Brazilian (1 male), Chinese (1 female), and American (1 female) graduate students and a visiting international scholar from China (1 female). In the past year, the Hatch project involved eight undergraduate students that participated in field and lab research and of which four were women. Additionally, the project involved the training of a high school student (1 male) in field, lab, and computational research. How have the results been disseminated to communities of interest?As an instructor at the Tucson Plant Breeding Institute (two modules: Introduction to Plant Quantitative Genetics and Advanced Statistical Plant Breeding) over the past year in Brazil (October), Arizona (January) and South Korea (September), PI Gore included classroom teaching examples on the Hatch project's sweetcorn biofortification research efforts to the more than 100 students in attendance from public institutions, private companies, and non-profit organizations. Furthermore, collectively, these students represented more than 15 countries from 5 continents. While teaching at Cornell in 2015/16, PI Gore presented the project's sweet corn biofortification research efforts in several lectures to undergraduate and graduate students over the semester in Nutritional Quality Improvement of Food Crops (PLBR/BIOPL/HORT 4070), as well as a lecture on biofortification to undergraduate and graduate students in Methods of Plant Breeding Laboratory (PLBR 4060). In terms of outreach activities, PI Gore discussed the biofortification research with SeedWorld (video and print) and Ezra Magazine (video and print). What do you plan to do during the next reporting period to accomplish the goals?(i) conduct a genome-wide association study (GWAS) to identify genes and favorable alleles responsible for quantitative variation in grain carotenoid and tocochromanol levels in a sweet corn diversity panel (years 1 and 2) Over the next reporting period, ~400 grain samples (sweet corn lines and checks) defining a single replicate (augmented incomplete block design) from the 2015 NY field location will be analyzed for their content and composition of carotenoids (provitamin A) and tocochromanols (vitamin E) by the MI collaborator (Dean DellaPenna) in spring 2017. The GBS data will be analyzed with the de novo SNP discovery pipeline to identify SNP additional markers that are potentially unique to the sweet corn germplasm pool. These generated and combined metabolite and SNP data sets will be used to conduct a more robust GWAS for the identification of causative genes and favorable alleles. (ii) development and validation of marker-based prediction models to convert locally adapted sweet corn germplasm to dark orange grain with high provitamin A and vitamin E content (years 1-3) The collected metabolite and SNP marker data from the 2014 and 2015 NY field seasons will be collectively used to retrain the whole-genome prediction models. Multi-trait WGP on correlated metabolite traits will be evaluated to further improve prediction accuracies. The segregating F2 families will be field evaluated in summer 2017, with the intent to select individuals from these families and validate WGP models.

Impacts
What was accomplished under these goals? Impact: The Hatch project includes research and extension components that are focused on innovative approaches to improve the NYS food system through the sustainable development and promotion of healthier food products. The intended outcomes of this new research program are the release of validated breeding methods to expedite the development of nutrient-dense sweet corn germplasm, as well as the availability of NY adapted, early generation sweet corn germplasm with enhanced levels of provitamin A (carotenoids) and vitamin E (tocochromanols). These intended outcomes directly address the priority of Food Security and Hunger (4.4) and contribute to priority 4.1 Healthy Eating and Active Living (adult and youth). To that end, a panel of more than 400 diverse sweet corn inbred lines from multiple breeding programs were assembled and evaluated in replicated field trials in WI and NY for a number of agronomically important traits related to local adaption, plant architecture, and productivity. The fresh-harvested kernels from these lines will be analyzed by an analytical chemistry method for levels of provitamin A and vitamin E at a laboratory in MI, thereby generating data that when combined with molecular marker information will be used in the identification of parents to construct sweet corn breeding populations. Such constructed populations will then be used for the genomics-assisted selection of progeny with higher nutritional value. In terms of intended impact, the to be developed nutrient-dense sweet corn germplasm will be adapted to local production in NY, thus building the capacity for large-scale commercial farms and single family households with small parcels of land to grow more nutritious sweet corn. Over the past year, the potential benefit of nutritionally enhanced fresh market sweet corn was highlighted to stakeholders through teaching and outreach activities, with its future availability conveyed as an approach for helping to address the nutritional deficiencies of vitamin E and provitamin A, including lutein and zeaxanthin, for vulnerable segments of the NY population such as the elderly. (i) Conduct a genome-wide association study (GWAS) to identify genes and favorable alleles responsible for quantitative variation in grain carotenoid and tocochromanol levels in a sweet corn diversity panel (years 1 and 2) A panel of ~400 sweet corn inbred lines was grown in replicated field trials at University of Wisconsin's West Madison Research Station in Verona, WI (collaborator William Tracy) and Cornell University's Musgrave Research Farm in Aurora, NY (PI Gore and Co-PI Smith) during summer 2014 and 2015. The sweet corn inbred lines were chosen to capture major levels of genetic diversity found in the U.S. sweet corn germplasm pool. The diversity panel was scored in both field locations for the following agronomic traits: emergence, plant height, ear height, lodging, flowering time, and tiller number. For each experimental plot, two self-pollinated ears were harvested at 400 growing-degree days [~21 days after pollination (DAP)] to represent the immature (fresh) stage of kernel development. The two fresh harvested ears were immediately frozen in liquid nitrogen, shelled, bulked, and stored at -80°C. An IKA tube mill control was used for grinding of frozen kernels and a custom protocol was develped to achieve a uniform fine powder. To date, 407 samples (Rep I from 2014 planted in NY) were analyzed using high-performance liquid chromatography (HPLC). Next, 474 samples (Rep I from 2015 planted in NY) were ground and sent to Michigan State University, with the plan for them to be analyzed for carotenoids and tocochromanols via HPLC in the first quarter of 2017. Genotyping-by-sequencing (GBS) data from 384 inbred lines were received in March 2016, followed by calling single-nucleotide polymorphism (SNP) markers using the Tassel 5 production pipeline. These SNPs were filtered using several quality control criteria, resulting in about 190,000 high quality markers. These resultant SNP markers were used for genome-wide association studies (GWAS) and whole-genome prediction (WGP). Next, we will work on SNP calling using the Tassel 5 discovery pipeline, allowing us to identify SNPs that might be unique but informative within sweet corn. Through GWAS, SNP markers were found to be significantly associated with the two following trait ratios: delta-tocopherol to alpha-tocopherol and delta-tocotrienol to gamma-tocotrienol. These SNP markers are in the proximity of gamma-tocopherol methyltransferase (ZmVTE4) and tocopherol cyclase (ZmVTE1), respectively, which are genes encoding enzymes that are core to the tocochromanol pathway. After we receive HPLC data from the 2015 replicate and GBS data from the remaining 51 lines, we expect to have enhanced statistical power to find additional marker-trait associations. For carotenoids, a significant association signal was found between the ratio of beta-xanthophylls to alpha-xanthophylls and a SNP located in the proximity of lycopene epsilon-cyclase (lycE), which encodes an enzyme responsible for converting lycopene to alpha-carotene. (ii) Development and validation of marker-based prediction models to convert locally adapted sweet corn germplasm to dark orange grain with high provitamin A and vitamin E content (years 1-3) Whole-genome prediction (WGP) via the genomic best linear unbiased prediction (GBLUP) method using all ~190,000 SNP markers resulted in relatively high accuracies, measured as the correlation (r2) between genomic estimated breeding values (GEBVs) and best linear unbiased predictors (BLUPs), for some of the metabolite traits. Average prediction accuracy using a 5-fold cross validation procedure ranged from 0.30 to 0.58 for gamma-tocopherol and delta-tocotrienol, respectively. Prediction of alpha-tocopherol, the compound with the greatest vitamin E activity, had an average accuracy of 0.43. In contrast, the accuracies were slightly lower for carotenoids, but total carotenoids and provitamin A (sum of beta-carotene + ½ alpha-carotene + ½ beta-cryptoxanthin) exhibited a mean accuracy of 0.51 and 0.41, respectively. The compound with the highest vitamin A activity, beta-carotene, had a prediction accuracy of 0.21. Nonetheless, the relatively high prediction accuracies for alpha-tocopherol and provitamin A are promising for developing sweet corn lines with improved nutritional quality. Through the implementation of trained WGP models, a set of 25 sweet corn inbred lines were selected based on their relatively larger GEBVs for the key traits alpha-tocopherol, beta- and alpha-carotene, and beta-cryptoxanthin. Next, the selected lines were crossed among themselves within each kernel type pool (su1 and sh2), prioritizing crosses between lines with complementary traits such as high alpha-tocopherol to high beta-carotene. Crosses were made in NY during summer 2016, producing 72 ears with sufficient seed. From each cross, 10 seeds were sent to Chile for planting and eventualy self-pollinating of the F1 plants. In 2017, the resultant F2 seeds will be planted, with the F2 plants evaluated in NY over summer 2017. The results from the 2017 field season will be used to prioritize the selection of individuals within breeding populations and validate WGP models.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Baseggio, M., Murray, M., Diepenbrock, C. H., Kandianis, C. B., Kaczmar, N., Magallanes-Lundback, M., Rocheford, T. R., Buckler, E. S., DellaPenna, D., Tracy, W., Smith, M. E., and Gore, M. A. Genome-wide association study of carotenoid and tocochromanol levels in sweet corn kernels. Poster# 289 presented at the 58th Annual Maize Genetics Conference in Jacksonville, Florida. March 17-20, 2016.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:PI Gore was an instructor at the Tucson Plant Breeding Institute in January (Tucson, AZ) and September (Kampala, Uganda) 2015 for the two modules: Introduction to Plant Quantitative Genetics and Advanced Statistical Plant Breeding. Classroom teaching examples included goals, background information, and preliminary results from this Hatch project and the 50-100 students in attendance were representing public institutions, private companies, and non-profit organizations from NY, the US and many other countries across 5 continents. While teaching at Cornell in 2014/15, Gore presented the project's sweet corn biofortification research efforts in several lectures to undergraduate and graduate students that included those from local farming communities and the New York metropolitan area over the semester in Nutritional Quality Improvement of Food Crops (PLBR/BIOPL/HORT 4070). It was the first introduction for many of them to biofortification and its role in addressing human nutritional deficiencies in New York State and beyond. Additionally, an in-field and video-recorded presentation on the genetic improvement of maize and sweet corn was given by PI Gore and members of his lab to members of the Bill and Melinda Gates Foundation including Mr. Bill Gates himself. Changes/Problems:There are no major problems or delays to report that could have a significant impact on the rate of expenditure. However, the newly realized need to analyze carotenoids and tocochromanols extracted from fresh, frozen, ground sweet corn grain (explained further below) and high volume of samples (internal and external to this project) simultaneously received for high-performance liquid chromatography (HPLC) analysis by the collaborator at MI has delayed the generation of HPLC data until spring 2016. In addition, the WI collaborator had multiple failures with isolating high quality genomic DNA from individual plants of the sweet corn diversity panel, thus the Genomic Diversity Facility (GDF) at Cornell University will now isolate the genomic DNA followed by sequencing of individuals with genotyping-by-sequencing (GBS). These delays are expected to influence the research schedule or goals. The completion of milestone 2) is expected now in year 2. The aggressive timeline to have model validation results with F2:3 families by the end of year 3 will likely not be met because the start of milestone 5) has been now shifted to year 2 due to the delay in generating marker and HPLC data. The project will still proceed with efforts to achieve model validation but with earlier validation at the F2 generation instead of with F2:3 families. The replication of alleles should still allow for robust validation results in the F2 generation, although the breeding cycle will still be carried forward as planned to validate at the F3 generation. There have been only minor modifications to the research procedure and none of which will cause significant deviations from the research schedule or goals. One of these minor modifications is the revised focus on only two research locations (WI and NY), given that the collaborator in IN was ultimately unable to commit in-kind resources to the field evaluation of the sweet corn diversity panel over two years. Additionally, a second minor modification, which was discussed as a likely possibility in the full proposal, is that the HPLC analysis now focuses only on the more biological and nutritionally relevant eating stage (400 growing degree days; ~20 days after pollination) of developing sweet corn and will not include the analysis of physiological mature sweet corn grain. The third and final modification is that carotenoids and tocochromaols will be extracted from fresh, frozen, ground sweet corn grain for analysis by HPLC, because lyophilization of grain harvested at the fresh eating stage resulted in the unfavorable oxidation of carotenoids and tocochromanols. What opportunities for training and professional development has the project provided?The Hatch project over the past year has been the cornerstone to training the next-generation of plant breeders in the Gore lab and helped to address the paucity of women and racial and ethnic minorities in agriculture. PI Gore (field, lab, and computational), Technician III Nicholas Kaczmar (field and lab), and Research Support Specialist III James Clohessy (computational) provided training in field (pollinations and phenotyping), lab (ear phenotyping and post-processing of fresh-harvested kernels), and computational (mobile applications and programming) research to Brazilian (1 male), Ugandan (1 female), Chinese (1 female), and American (1 female) graduate students. The Brazilian graduate student working full-time on this Hatch project, Mr. Matheus Baseggio, participated in several professional development training events critical to providing a foundation for conducting research on this project: the Tucson Plant Breeding Institute (Introduction to Plant Quantitative Genetics and Advanced Statistical Plant Breeding), Workshop in Field-based High-Throughput Phenotyping, and Cornell University's Institute of Biotechnology Workshops (Linux for Biologists, Genome assembly, and Variant Calling). This summer the Hatch project involved four undergraduate students that participated in field and lab research and of which three were women. Of the three women, one of them was an African-American Cornell undergraduate student majoring in Plant Science. The second undergraduate woman was a Cornell undergraduate student majoring in Food Science that is now undertaking a plant breeding/nutritional sciences internship in Germany this spring 2016. The third undergraduate women was a California Polytechnic State University undergraduate majoring in Kinesiology, with an interest geared towards health science. Additionally, the project involved the training of two high school students (1 male and 1 female) in field, lab, and computational research. How have the results been disseminated to communities of interest?As an instructor at the Tucson Plant Breeding Institute (two modules: Introduction to Plant Quantitative Genetics and Advanced Statistical Plant Breeding) over the past year in Arizona (January) and Uganda (September), PI Gore included classroom teaching examples on the Hatch project's sweetcorn biofortification research efforts to the more than 100 students in attendance from public institutions, private companies, and non-profit organizations. Furthermore, collectively, these students represented more than 15 countries from 5 continents. While teaching at Cornell in 2014/15, PI Gore presented the project's sweet corn biofortification research efforts in several lectures to undergraduate and graduate students over the semester in Nutritional Quality Improvement of Food Crops (PLBR/BIOPL/HORT 4070), as well as a lecture on biofortification to undergraduate and graduate students in Methods of Plant Breeding Laboratory (PLBR 4060). In terms of outreach activities PI Gore and several members of the Gore lab presented the research on biofortification to Gates Notes and the Bill and Melinda Gates Foundation including Mr. Bill Gates himself, as well as an age-appropriate interactive presentation on plant breeding and biofortification to more than 30 students ranging from 2-5 years old at the Community Nursery School in Ithaca, NY. What do you plan to do during the next reporting period to accomplish the goals?(i) conduct a genome-wide association study (GWAS) to identify genes and favorable alleles responsible for quantitative variation in grain carotenoid and tocochromanol levels in a sweet corn diversity panel (years 1 and 2) Over the next reporting period, ~400 grain samples (sweet corn lines and checks) from one replicate (augmented incomplete block design) of the 2014 NY field location will be analyzed for their content and composition of carotenoids (provitamin A) and tocochromanols (vitamin E) by the MI collaborator (Dean DellaPenna) in spring 2016. Additionally, sequencing of these ~400 lines using genotyping-by-sequencing (GBS) by the Genomic Diversity Facility (GDF) at Cornell University in collaboration with WI (William Tracy) and NY (Edward Buckler) collaborators is expected to generate 10,000s of SNP markers in spring 2016. Once collected, the marker-based heritability of the metabolite data will be estimated and for which will provide insight into if a second biological replicate of grain samples from NY 2014 needs to be analyzed in summer 2016. When combined with calculated best linear unbiased predictors (BLUPs) of these metabolite data in summer 2016, the resultant SNP markers will be used to conduct a GWAS for identifying causative genes and favorable alleles controlling levels of provitamin A carotenoids and vitamin E tocochromanols in sweet corn at the eating stage. Furthermore, these GWAS results will also help inform us as to whether it is advisable to only conduct HPLC analysis on grain samples from the 2014 and 2015 NY outgrowths of the sweet corn panel by the end of year 2. These metabolite traits have been previously shown and expected to have high heritability (low environmental influence), thus minimal benefit would likely be gained from analyzing the WI environments. If it is unexpectedly determined that HPLC analysis of grain samples from all the WI environments (2014 and 2015) will add exceptionally high value to the GWAS, then the collection of HPLC data will likely not be completed until the beginning of year 3. (ii) development and validation of marker-based prediction models to convert locally adapted sweet corn germplasm to dark orange grain with high provitamin A and vitamin E content (years 1-3) The collected metabolite and SNP marker data from the 2014 NY field season will be used to train genomic selection models of varying marker densities, which will then be used to select several founder lines to establish a breeding population and validation of the model predictions in summer 2016. The founder lines will not be selected on nutritional density alone but also in the light of available data for agronomic, kernel quality, and organoleptic traits.

Impacts
What was accomplished under these goals? Impact: The Hatch project includes research and extension components that are focused on innovative approaches to improve the NYS food system through the sustainable development and promotion of healthier food products. The intended outcomes of this new research program are the release of validated breeding methods to expedite the development of nutrient-dense sweet corn germplasm, as well as the availability of NY adapted, early generation sweet corn germplasm with enhanced levels of provitamin A (carotenoids) and vitamin E (tocochromanols). These intended outcomes directly address the priority of Food Security and Hunger (4.4) and contribute to priority 4.1 Healthy Eating and Active Living (adult and youth). To that end, a panel of more than 400 diverse sweet corn inbred lines from multiple breeding programs were assembled and evaluated in replicated field trials in WI and NY for a number of agronomically important traits related to local adaption, plant architecture, and productivity. The fresh-harvested kernels from these lines will be analyzed by an analytical chemistry method for levels of provitamin A and vitamin E at a laboratory in MI, thereby generating data that when combined with molecular marker information will be used in the identification of parents to construct sweet corn breeding populations. Such constructed populations will then be used for the genomics-assisted selection of progeny with higher nutritional value. In terms of intended impact, the to be developed nutrient-dense sweet corn germplasm will be adapted to local production in NY, thus building the capacity for large-scale commercial farms and single family households with small parcels of land to grow more nutritious sweet corn. Over the past year, the potential benefit of nutritionally enhanced fresh market sweet corn was highlighted to stakeholders through teaching and outreach activities, with its future availability conveyed as an approach for helping to address the nutritional deficiencies of vitamin E and provitamin A, including lutein and zeaxanthin, for vulnerable segments of the NY population such as the elderly. (i) Conduct a genome-wide association study (GWAS) to identify genes and favorable alleles responsible for quantitative variation in grain carotenoid and tocochromanol levels in a sweet corn diversity panel (years 1 and 2) In 2014, a diversity panel of ~400 sweet corn inbred lines was constructed to capture the common levels and patterns of genetic diversity in the germplasm pool, with its composition finalized in 2015. This sweet corn panel was evaluated in a replicated incomplete block design over the summers of 2014 and 2015 at University of Wisconsin-Madison's West Madison Research Station in Verona, WI (collaborator William Tracy) and Cornell University's Musgrave Research Farm in Aurora, NY (PI Gore and Co-PI Smith), for a total of four environments. Having the 2014 field season (NY field and labor costs covered by Cornell startup funds) before the official start of the Hatch project allowed elimination of the need for growing the panel in year 2 and as a result be further ahead in terms of the research timeline. The panel was scored across the four environments for the following traits: seedling emergence, plant height, ear height, lodging, flowering time, tiller number, kernel row number, kernel color, kernel type, ear and cob length and width, and 20-kernel dry weight. These ear and kernel traits were scored on open-pollinated ears harvested at physiological maturity. For the NY 2015 field season, a suite of non-contact sensors was used to phenotype the panel via proximal sensing nearly each week of the growing season from flowering to senescence for the following canopy traits: normalized difference and normalized difference red edge vegetation indices, relative leaf area index, chlorophyll content, height and temperature. A relational database was developed to store, manage, query, update, and export these collected phenotypic data. In each environment, two self-pollinated ears with fresh kernels were harvested from each plot at 400 growing degree days (GGD, ~20 days after pollination) to represent the immature (milk) stage of development when sweet corn is consumed. The two fresh harvested ears were immediately frozen in liquid nitrogen, shelled, and stored at -80°C. It is these grain samples that will be analyzed for carotenoid (provitamin A) and tocochromanol (vitamin E) levels. To help with the efficient fresh harvest of these samples, a mobile application for handheld devices (smartphones and tablet computers) was developed to indicate which plots had self-pollinated sweet corn that were ready to harvest at 400 GGD based on the pollination dates and weather data collected from a weather station located within the research field. The results of a pilot study in collaboration with Dean DellaPenna (Michigan State University) showed that lyophilization causes unacceptable levels of degradation via oxidation to carotenoids (provitamin A) and tocochromanols (vitamin E). Notably, lyophilization is the generally accepted approach for processing of fresh-harvested sweet corn grain prior to the extraction of lipid-soluble antioxidants. Therefore, a protocol was developed for grinding fresh, frozen sweet corn grain to a uniform fine powder. Grinding of the samples was performed in the presence of liquid nitrogen to avoid oxidation of the metabolites from thawing of the tissue. With this newly developed procedure, the ~400 grain samples from one outgrowth of the panel (NY 2014 Rep1) were ground and sent frozen on dry ice to Michigan State University for the analysis of carotenoid and tocochromanol levels via high-performance liquid chromatography (HPLC) in spring 2016. This generated data set along with the SNP marker data set will be used together for an initial genome-wide association study to identify the genes and favorable alleles responsible for quantitative variation in grain carotenoid and tocochromanol levels in a sweet corn diversity panel. (ii) Development and validation of marker-based prediction models to convert locally adapted sweet corn germplasm to dark orange grain with high provitamin A and vitamin E content (years 1-3) The first iterations of the genomic selection models to be tested were initially evaluated for grain carotenoids in a panel of diverse maize inbred lines (field corn) and the results of which were published in December 2014 (Owens et al. 2014. Genetics 198:1699-1716). While in the process of generating the genotypic and phenotypic data for this Hatch project, there has been additional exploration of genomic selection models for the prediction of carotenoid and tocochromanol traits in the maize nested association mapping population. The results from these efforts are helping to calibrate the approach for the sweet corn panel. Once we have collected the genotypic (samples submitted for SNP genotyping to Genomic Diversity Facility) and metabolite data in spring 2016, we will start selecting parents with these and as well as the agronomic data to construct breeding populations that will serve as the foundation for improving provitamin A and vitamin E content in sweet corn grain.

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