Source: UNIVERSITY OF NEBRASKA submitted to NRP
BREEDING POPCORN AND SWEET CORN FOR DIVERSE NUTRITIONAL AND AESTHETIC OUTCOMES AND NEW OPPORTUNITIES FOR UNDERGRADUATES IN THE FIELD AND LAB
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
Reporting Frequency
Annual
Accession No.
1026071
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Mar 4, 2021
Project End Date
Sep 30, 2025
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
Agronomy & Horticulture
Non Technical Summary
Popcorn and sweet corn are highly consumed in the U.S. being nutritious snack and vegetable forms of the most highly grown U.S. crop, Zea mays. Breeding of popcorn has largely focused on improving profitability by maximizing flake expansion volume. Different flake types have been developed that best suit the mode of distribution and addition of various flavor components. Little attention has been given to investigating or improving the innate flavor, texture and nutritional characteristics of the popcorn itself. Sweet corn breeding has focused mostly on kernel sugar content and agronomic improvement but less attention has been paid to improving other macro- and micronutrients as well as aesthetic diversity of the kernels. There is considerable scope for combining genotypes and phenotypes between field corn, popcorn and sweet corn in order to improve both popcorn and sweet corn in terms of color diversity for enhanced consumer interest, macro nutrition in terms of essential amino acids, and micronutrition in terms of carotenoids and anthocyanins. Popcorn and sweet corn are both well suited to smaller-scale organic production and marketing of diverse varieties with improved nutrition, texture, flavor and aesthetics.To exploit popcorn and sweet corn diversity in developing new and exciting varieties, the goals of the project are as follows. First, the work will develop colored sugary-1 and shrunken-2 sweet corn varieties. This will explore the potential of sweet corn kernels to develop carotenoid and anthocyanin pigmentation by the prime eating stage and will test color combinations in established sweet corn hybrid combinations including Golden Cross and Iowa Chief sugary-1 and shrunken-2 hybrids. Second, the work will test the effects of opaque-2 induced high-lysine endosperm on eating stage sweet corn kernels. Sweet corn is a surprisingly good but incomplete protein source as a vegetable, but by rebalancing the proteome away from zeins and towards non-zein proteins, it will be established if sweet corn can be transformed into a more complete protein source. Third, the work will build on the knowledge generated by the recent development of elite Quality Protein Popcorn hybrids by generating an array for new, colored QPP varieties and testing their agronomic, biochemical, biophysical and consumer preference characteristics.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5021480108160%
5021511108140%
Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
1480 - Sweetcorn; 1511 - Popcorn;

Field Of Science
1081 - Breeding;
Goals / Objectives
In the broadest terms, the goals of this project are to breed new sweet corn and popcorn varieties with improved diversity for nutrition, taste and aesthetics. The project intends to develop products for small-scale organic production in a way that maximizes opportunities for undergraduates in the field and lab. The work leverages the knowledge and expertise of the PI in maize and popcorn breeding and undergraduate and graduate education as well as the infrastructure available at UNL for achieving the goals. The specific objectives are as follows:Breed colored sugary-1 and shrunken-2 sweet corn hybridsBreed and assess the potential of Quality Protein Sweet cornBreed novel Quality Protein Popcorn with diverse color and flavor
Project Methods
1. Breed colored sugary-1 and shrunken-2 sweet corn hybridsVarious Shrunken-2 (sh2) and Sugary-1 type sweet corn (su1) lines were acquired from the stock center and grown in the field at UNL East Campus in 2020. These included female and male parents in the sh2 Iowa chief hybrid (IA453 and IA5125) and the female and male parents in the su1 Golden Cross hybrid (P39 and P51) as well as the highly robust and large eared NE-EDR sh2 and su1 lines. In summer 2020, these and other sweet corn lines were crossed, in either direction, to series of dent and popcorn lines with various colored kernels. Color donors included dent and flint corns Coburns Early Red, Rainbow Flint Red, Bloody Butcher, Maize Azul, and Blue Indian Corn. Color was also mined from popcorn lines including Black Jewel, Red Popcorn, Glass Gem (many colors), Strawberry Popcorn, Cochiti Pueblo (Red, Blue), and Cherokee Long Ear (red, blue, purple). F1 kernels from all combinations obtained were planted in the greenhouse in the fall of 2020, self-pollinated and F2 ears show the predictable ¼ shrunken kernels. The F2 ears also segregate independently for the large array of reds, purples, blues and a large variety of lighter shades of mauve, pink and orange. Given the color diversity and the uncertainty of whether the various colors will develop by the sweetcorn eating stage, selection of mature, colored sweetcorn kernels will be done entirely by visual selection. Whole rows of selected colored sweetcorn F2 kernels will be planted and self-pollinated. F3 ears will be examined between 18 and 26 days after pollination to determine when colors first appear during the prime sweet corn eating stages. With the Golden Cross and Iowa Chief hybrid pairs, there is the possibility to make the colored hybrids from introgressions from the same or different colored parents thus expanding the possible color combinations.The anthocyanin and carotenoid pathways are responsible for a large repertoire of different colored pigments associated with beneficial antioxidant properties. The pathways are both complex involving multiple biosynthetic enzymes and transcription factors. All varieties used are naturally existing and so breeding with them is unlikely to result in unintended negative metabolic changes. However, colored sweet corn inbreds and hybrids will be compared to their uncolored counterparts to detect any negative effects plant performance, ear size, kernel size, sugar content and consumer preference. Any lines that ultimately show desirable color accumulation as early as the prime eating stage, with be subjected to quantitative measurements of carotenoids and anthocyanin pigments using HPLC and spectrophotometric methodology.2. Breed and assess the potential of Quality Protein Sweet corn Though sweet corn is not a primary protein source in human diets, as a vegetable, it has one of the higher protein contents at around 3.5% fresh weight. It therefore has the potential to contribute to increasing human consumption of plant-derived protein. This potential could be increased if the limiting and essential amino acid lysine could be increased in both free- and protein-bound forms. Although the lysine-devoid zein protein are abundantly accumulating by the prime sweet corn eating stage (18-21 DAP), their relative proportion with non-zein proteins and their consequent effect on protein quality at that stage is unclear. We will thus investigate the potential for rebalancing the sweet corn proteome by reducing zeins and increasing non-zeins.In Summer 2020, two QPM lines, CML154Q and Tx807 were crossed with all the sweet corn lines described above including the Su1 Golden Cross Hybrid and Sh2 Iowa Chief Hybrid parents and NeEDR Su1 and Sh2. F1 plants have been selfed in the greenhouse and the F2 ears are being screened in the spring of 2021. All F2 ears clearly segregate for ¼ shrunken kernels in all the F2 ears so their selection for replanting is easy. Of these sweetcorn kernels, ¼ are QPM and carry the homozygous o2 mutation (1/16 of F2 kernels). Whole rows of sweetcorn F2 seed will be planted in the summer 2021 field and genotyped using SSR marker UMC1066 to find the QPM plants and only this fraction will be self-pollinated to generate F3 ears. These F3 ears carry both the sweetcorn allele and opaque2 allele specifying quality protein in homozygous form.Since the sh2 and su1 sweetcorn variants are well characterized and produce no starch and much reduced starch respectively, and they are homozygous by the F3, it will not be necessary to test for sugar content at this early stage. Since they are also opaque-2, they have a rebalanced proteome with less zeins and more (lysine-rich) non-zeins and are, by definition, high lysine. To confirm this, we will measure free and protein bound amino acids at 20-25 days after pollination (sweetcorn eating stage) kernels comparing opaque-2 (QPM) sweetcorn to regular sweetcorn kernels. To do this, kernels will be flask frozen, lyophilized and ground into a powder and amino acids analysis will be performed. If successful, later efforts will focus on determining agronomic performance and consumer preference compared to the parental hybrids.3. Breed novel Quality Protein Popcorn with diverse color and flavorPrevious work by the PI's lab has shown the potential of popcorn subspecies introgressed with the o2 mutation and modifier genes to produced fully popable and high-lysine, Quality Protein Popcorn. While selecting for a QPM-like proteome, key popcorn traits such as popability, kernel morphology, and kernel size were selected throughout the breeding program. Hybrids produced from these QPP inbreds performed comparably with elite commercial popcorn hybrids in terms of agronomic performance, yield and popcorn flake expansion volume while manifesting lysine contents similar to QPM dent corn. Recent consumer testing has indicated that QPP flakes possess a more diverse array of taste and texture profiles which, in many cases, are preferable to elite conventional popcorn flakes. This project seeks to take advantage of this diversity in taste and texture by using our knowledge and methodology to breed new QPP varieties using public popcorn and QPM germplasm. The colored popcorn lines selected will also add desirable aesthetic diversity to the products.The colored popcorn lines described in Aim 1 were crossed to CML154 QPM and Tx807 QPM in Summer 2020. F1 plants are being selfed in the green house to produce segregating F2 ears on which phenotypic selections will commence. Since crosses between dent corn and popcorn segregate for kernel dent and popcorn phenotypes as early as the F2, we will select popcorn phenotype F2 kernels bearing desirable colors for propagation. The 1/4 o2/o2 fraction of each introgression will be germinated for DNA extraction from seedling leaf tissue followed by PCR genotyping. Markers will first need to be tested to find ones showing polymorphisms between QPM and popcorn parents. Alternatively, biochemical selection using SDS-PAGE can be applied to the F3 kernels to identify ears that are 100% modified o2 (low alpha-zein and carrying the gamma zein duplication). The percentage popcorn genome will be increased by backcrossing to the recurrent popcorn parent or a different popcorn parent followed by selfing to again produce segregating F2s. BC2 and BC3 F4 ears have been shown to be 100% popable popcorn in our previous QPP work. Increasing the popcorn genome makes this aim more long term than aim 1 and 2 but should result in novel QPP inbreds within four years. Longer-term work will be to generate and test different QPP hybrid combinations, and assess their yield, flake type, texture, taste and color diversity. Since all hybrids will be derived from modified o2, they are all expected to have the same elevated lysine content. Work will be completed by the PI, graduate student and Plant Biology and other selected undergraduates.

Progress 03/04/21 to 09/30/21

Outputs
Target Audience:The basic breeding goals and projected products of this project are all novel and are of interest to the maize genetics community. Interest in improving maize quality and diversity, especially by Project Narrative mining natural genetic diversity is continually increasing. New germplasm will be of interest to the maize seed bank where lines will be registered and deposited. The new lines resulting from this project as well as the technology used to create them will be of interest to large and specialist seed companies, popcorn and sweet corn breeders and food companies. The PI will reach out to specialist growers to devise and test methods for organic production. The educational opportunities afforded by the work are relevant to both the undergraduate and graduate level agronomy, horticulture and plant biology programs at UNL and will be showcased and incorporated as part of the improved content and visibility of both. Changes/Problems:The only problems relate to fewer than anticipated undergraduates being involved. This is as a result of covid restrictions as well as the PI not yet possessing dedicated funding to support this project. However, the project is very flexible and can be run minimally or much more expanded based on undergraduate and graduate student availability and funding. The PI is part of a recently funded REU proposal which will support several undergraduate students in the field and lab in the summer of 2022. Various other proposals are currently being prepared. What opportunities for training and professional development has the project provided?The project is training one graduate student (starting fall 2021) in field and lab teachniques in maize breeding. One undergraduate has been trained as part of the UNL McNair program which gives first generation and minority students research experiences. That student is subsequently funded by the UNL UCARE program until May 2022. Two further undergraduates are currently being employed on an hourly basis. How have the results been disseminated to communities of interest?The PI has presented the progress to Norm Krug, the CEO of preferred popcorn and talks are underway to develop and breed some of the QPP varieties for their export market in the developingworld. The colored and high lysine varieties in development have not yet been disseminated to communities of interest but will be in the next two years What do you plan to do during the next reporting period to accomplish the goals?Colored sweet corn. In summer 2022 we will grow four-row, open pollinated blocks in random block design of the best colored sweetcorn F3 families, selected on the basis of color, sweetness and texture from 2021. Sweet corn yield is not typically measured in bushels but simulation models can be used to predict high yield lines from fresh weight, ear number and ear size (Lizaso et al. 2007). From two rows we will estimate the yield potential from average ear size, ears per plant, row number and kernel size. Using the other two rows, will employ panels volunteer testers who will be asked to rank sweetcorn for visual appeal, and separately, for sweetness and texture of steamed sweet corn ears. We will also flash freeze at least five ears for each and store at -80 ?C for later use in pigment analysis, sugar measurement and microbiome analyses. Plans will be made for hybrid production from the best inbreds starting in 2023. High lysine sweetcorn (QPS). We have already identified true breeding sweetcorn ears for all introgressions that are either segregating or 100% o2. These will be confirmed with SDS-PAGE analysis in the spring of 2022. Then in the 2022 field, four-row blocks will be planted and used in the same way as for colored sweet-core for yield estimation as well as sweetness and texture sensory testing, and later, amino acid profiling and microbiome assays on frozen kernels. To combine the color traits with the high lysine trait in sweetcorn, we will cross the best colored sweetcorn lines with high lysine sweetcorn, to make new F1s which will already be true breeding for sweet corn but heterozygous for o2. By selfing the F1s in the winter greenhouse, we will generate the segregating F2s from which true breeding colored QPS will be selected for testing in 2023. Plans will be made for hybrid production from the best inbreds starting in 2023. Colored QPP. Colored and high lysine sweet corns do not require backcrossing because as long as the sweetcorn and o2 traits are fixed, the relative contributions of the parental genomes does not matter. On the contrary, popcorn breeding to a non-popcorn parent is much longer term since at least three doses of popcorn and extensive selfing are needed (BC2F5) to recover sufficient popcorn genome for good popping in the inbreds even prior to hybrid production (Ren et al. 2018). Consequently, summers 2022 and 2023 will be used to propagate the BC1F2 and BC2F2 after which o2 is fixed selfing generations are used to generate the finished colored QPP inbreds (Ren et al. 2018). At each generation, SDS PAGE is used to verify the correct proteome rebalanced phenotype and PCR is use to select for the presence of the 27 kD γ-zein duplication allele (the dominant QPM modifier gene). In the long term, hybrids will be made and tested by combining inbreds from the various QPM and popcorn parents. Amino acid profiling, popping measurements and microbiome assays, will be carried out both at the inbred and later, at the hybrid stages. Amino acid profiling will be carried out by collaborator Dr. Ruthie Angelovici at University of Missouri Columbia and Microbiome assays by Dr. Andy Benson of the Nebraska Food for Health Center. The PI is part of a recently funded REU proposal which will support several undergraduate students in the field and lab in the summer of 2022. Various other proposals are currently being prepared.

Impacts
What was accomplished under these goals? Breed coloredsugary-1andshrunken-2sweet corn hybrids Various (shrunken-2) sh2 and (sugary-1) su1 sweet corn lines were grown at UNL East Campus in 2020 including female and male parents in the sh2 Iowa chief hybrid (IA453 and IA5125) and the female and male parents in the su1 Golden Cross hybrid (P39 and P51) as well as the highly robust and large eared NE-EDR sh2 and su1 lines .In summer 2020, these and other sweet corn lines were crossed to colored kernel corn varieties. Color donors included dent, flint and popcorns including Coburns Early Red, Rainbow Flint Red, Bloody Butcher, Maize Azul, and Blue Indian Black Jewel popcorn, Red Popcorn, Glass Gem (many colors), Strawberry Popcorn, Cochiti Pueblo (Red, Blue), and Cherokee Long Ear (red, blue, purple). F1 kernels from all combinations obtained were planted in the greenhouse in the fall of 2020, self-pollinated and F2 ears show the predictable ¼ shrunken kernels. The F2 ears also segregated independently for the large array of reds, purples, blues and a large variety of lighter shades of mauve, pink and oranges. Given the color diversity and the uncertainty of whether the various colors will develop by the sweetcorn eating stage, selection of mature, colored sweetcorn kernels will be done entirely by visual selection. Whole rows of selected colored sweetcorn F2 kernels were planted and self-pollinated in Summer 2021. F3 ears were scored for color, sweetness and texture at the prime eating stage (20 DAP). Highly variable coloration in terms of shade, pattern and intensity were manifest in most F3 families at the prime eating stage. Sweetness and texture tasting on raw ears allowed families to be provisionally ranked, from tough to tender and from dull to sweet with scores of 1-5 for each criterion. Scores of 4/5 will be required for further larger scale testing. 2. Breed and assess the potential of Quality Protein Sweet corn In Summer 2020, CML154 QPM and Tx807 QPM were crossed with all the sweet corn lines described above including the Su1 Golden Cross Hybrid and Sh2 Iowa Chief Hybrid parents and NeEDR Su1 and Sh2. F1 plants were selfed spring 2021and the F2 ears screened. All F2 ears clearly segregated for ¼ shrunken kernels in F2 ears making selection for replanting unequivocal. Of these sweetcorn kernels, ¼ carry the homozygous o2 mutation (1/16 of F2 kernels). Whole rows of sweetcorn F2 seed were grown in the summer 2021. These F3 ears are homozygous for sweetcorn and 1:2:1 wild type: heterozygous: mutant for the o2 high lysine trait which is clearly visible in the F3 kernels. o2 sweetcorn F3 kernels from all QPM families will be planted in summer 2022 for prime eating stage sweetness and texture scoring and amino acid profiling. 3. Breed novel Quality Protein Popcorn with diverse color and flavor The colored popcorn parents were crossed to CML154 QPM and Tx807 QPM in Summer 2020. F1 plants were selfed to produce segregating F2 ears which showed highly variable coloration. Rows of F2 kernels were planted self-pollinated for each family of which ¼ were homozygous o2. All plants were also backcrossed to the recurrent popcorn parent to increase the popcorn genome content. Leaf DNA from all F2 plants was extracted and stored for later genotyping using UMC1066 and other O2 gene SSR markers. We are also using SDS-PAGE on F3 kernels to identify ears that are true breeding modified o2 (QPP). F3 ears have been screened to identify true breeding o2 and the corresponding BC1 kernel are now being selfed to generate the BC1F2 which will again segregate for o2. BC1F2 kernels that are modified o2 will be grown, selfed and backcrossed again in Summer 2022 to produce the BC2.

Publications