Source: CORNELL UNIVERSITY submitted to NRP
DISCOVERING GENETIC BASIS OF HIGH CAROTENOID ACCUMULATION FROM SQUASH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
Reporting Frequency
Annual
Accession No.
1028423
Grant No.
2022-67013-37048
Cumulative Award Amt.
$649,000.00
Proposal No.
2021-07834
Multistate No.
(N/A)
Project Start Date
Apr 15, 2022
Project End Date
Apr 14, 2026
Grant Year
2022
Program Code
[A1103]- Foundational Knowledge of Plant Products
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Plant Breeding
Non Technical Summary
Winter squash and pumpkin are among the richest sources of carotenoids in our diet. Understanding the genetic and molecular basis underlying carotenoid accumulation in these crops, which provide up to 250% RDA per serving, is important for improving human health and nutrition. One gene pair in particular, B and L-2, have long been known to greatly boost squash carotenoid content. However, their identity and mechanisms remain unknown. Genomic resources have emerged in squash along with breakthroughs in understanding of chromoplast development and carotenoid accumulation. It's important to characterize established genetic systems that underlie carotenoid accumulation beyond model species, to uncover new paths to improve the quality of our food. Our objectives are to 1) understand the molecular mechanisms of the B gene in squash that induces precocious chromoplast development, 2) unravel the identity of L-2 which interacts with B to greatly increase carotenoid accumulation by an order of magnitude, and 3) characterize a second B locus in another squash species with a more stable expression. Outcomes and impacts will be the elucidation and informed deployment of the B and L-2 interacting gene pair. This knowledge will apply directly to increase nutrient density within squash. It will also inform approaches and future hypotheses for carotenoid biofortification in other crops.
Animal Health Component
30%
Research Effort Categories
Basic
60%
Applied
30%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20114291080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1429 - Cucurbits, other;

Field Of Science
1080 - Genetics;
Goals / Objectives
Our overall goals are to understand the B and L-2 loci of Cucurbita which together dramatically increase carotenoid content. Toward this we need to confirm the candidate for the B gene in C. pepo (Bpepo), and identify the B gene in C. maxima (Bmax) and the L-2 gene in C. pepo. Further, we will explore their molecular mechanisms.Objective 1. Elucidation of the molecular basis of the B gene in C. pepo 1.1. Confirm the B gene function in weakening chlorophyll biosynthesis and promoting chromoplast formation1.2. Examine the effect of B on enzyme complex formation and on its interactions with known regulators in regulation of chlorophyll biosynthesis1.3. Identify new interacting partners of B for carotenoid accumulation in chromoplastsObjective 2. Identify L-2, the synergistic partner of the B gene2.1 Identify the L-2 region with QTL-Seq2.2 Fine mapping of L-2 to identify candidate genes2.3 Functional confirmation of candidates for L-22.4. Characterization of the L-2 geneObjective 3. Characterize Bmax, a second potentially superior B locus3.1 Test Bmax and Bpepo allelism in conspecific C. moschata background3.2 Examine whether Bmax codes a paralog in C. maxima
Project Methods
We will use a callus culture system to explore the identity and function of the loci under investigation. Calli present a relatively fast approach to examine chlorophyll biosynthesis and carotenoid accumulation in squash. While the callus cells without carotenoid accumulation are white when grown in the dark, they are green under light due to chloroplast development from proplastids and can be yellow/orange color when carotenoid accumulation precede in chromoplasts. Overexpression constructs will be transformed into squash explants and infected explants will be induced for callus formation under light. The calli will be visually examined for color changes and analyzed for carotenoid content. Plastids in callus cells will be examined under both light and confocal microscopy. In addition, for a definitive proof of function, stable transgenic plants will be generated to examine whether candidate genes reproduce the expected phenotypes in the squash.To isolate the B interacting proteins, we will use complementary strategies of yeast two hybrid (Y2H) cDNA library screening and co-immunoprecipitation coupled with mass spectrometry (co-IP/MS) analysis. For Y2H library screening, the B candidate will be used to screen a Y2H library constructed from young squash fruit with BB. The plasmids from positives will be extracted and sequenced. In addition, we will isolate potential B interacting proteins via co-IP/MS. The overexpressing transgenic squash calli mimic pigment and plastid development of young fruit cells and can be generated quickly. The Myc-tagged B and b proteins from the calli will be immunoprecipitated with anti-Myc agarose beads. The co-immunoprecipitated proteins will be identified at Cornell Proteomics Facility. Since callus transformation can be fast and easily accommodates a few genes, we will again take advantage of the callus system to functionally confirm the possible involvements of the confirmed B interacting proteins on carotenoid biosynthesis in chromoplast or chlorophyll accumulation.To isolate L-2, we propose to utilize the QTL-seq strategy to rapidly identify the genomic region harboring this dominant allele. We are generating a mapping population that is fixed for the Bpepo allele and segregating for L-2 by selfing a B/B L-2/l-2 hybrid. An F2 population of 300 individuals will be phenotyped for L-2 by visual assessment of orange fruit flesh. This will be corroborated with measuring carotenoid content of flesh. Following blue pippin size selection and quantification, bulked libraries will be barcoded and pooled for whole-genome resequencing.The locus associated with the L-2 gene will be identified based on its p value for the Δ(SNP index) under the null hypothesis of no QTLs following the described method. For fine mapping, SNPs in the target region will be converted into CAPS markers. CAPS markers equally spacing the L-2 genomic region will be genotyped to identify recombinant individuals using the remnant F2. Two flanking markers of L-2 will be used to screen additional F2 plants of a large population for recombinants.To functionally confirm the identity of the best candidates for L-2, we will first perform phenotypic complementation in the callus system. The candidates from L-2 expressing fruit tissue will be overexpressed in squash calli with the BB and l-2/l-2 alleles as described above. In addition, we will knockout the endogenous genes in squash calli with the BB and L-2/L-2 alleles. The calli will be visually examined for color changes and analyzed for carotenoid content. Once a candidate gene is confirmed to be L-2 in the callus system, the overexpression construct will be used to generate transgenic squash in the BB and l-2/l-2 background to examine its effect on carotenoid accumulation in fruit as described above.We will perform molecular and biochemical characterization of this L-2 gene to gain a better understanding of its mode of action. These experiments include investigation of L-2 gene and protein expression patterns in squash fruit by RT-qPCR and western blot analysis, L-2 promoter activity by fusing to β-glucuronidase (GUS) gene and transforming into Arabidopsis, and L-2 protein subcellular localization by fusing with GFP and transiently transforming in tobacco leaves. We will also examine transgenic squash calli and plants to see how L-2 together with B to affect chromoplast development and carotenoid contentAllelism tests will be performed with C. moschata lines with Bpepo and Bmax independently introgressed. The C. moschata B lines will be intermated to create an F2 population segregating for both loci. For this study, we will also backcross the F1 to each parent. We will analyze the populations by classifying phenotypically, then applying the molecular markers developed from the fine mapping work to track Bpepo and Bmax. Further we will explore the genomic positions of these loci directly on the Cucurbita genome. We will add the introgression lines to the QTL-seq pipeline to take advantage of leftover sequencing capacity to identify introgressed interspecific regions and align these with the synteny viewer at CuGenDB (http://cucurbitgenomics.org/synview/search). To test out whether Bmax encodes a paralog of Bpepo or another gene in C. maxima, we will clone the genes and cDNAs of these three candidates from C. maxima varieties with and without the Bmax allele and compare their sequences.

Progress 04/15/23 to 04/14/24

Outputs
Target Audience:Our primary audience again was other scientists and plant breeders. We reached them through presentations at scientific conferences and industry field days. Given the technical nature of the work, the results are most relevant to those seeking to understand the mechanisms of carotenoid accumulation to apply our results to their systems and plant breeders seeking tools to track the genes underlying key phenotypes. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Casey Wilson earned his MS under the mentorship of Jim Myers at OSU. Xuesong Xu served as a postdoctoral researcher and provided mentorship in Li Li's lab. Emalee Wrightstone and Jack Fabrizio performed a portion of their doctoral research and presented on their findings. Both mentored undergraduates in projects connected to this grant. An undergrad was trained in molecular biology by working on the project: Kyle Gisworld, 2024 Plant Science Major (undergraduate at Cornell), August 2023 - May 2024 How have the results been disseminated to communities of interest?Results have been disseminated through presentations and publications. These have included a dissertation, graduate student presentations and presentations to seed industry plant breeders at field days. What do you plan to do during the next reporting period to accomplish the goals?We will continue to identify interacting partners of Bpepo and its mechanism of expression. L-2 candidate genes will be tested for function. To date we have relied on introgression lines for Bmax characterisation, taking advantage of early ectopic expression in seedling stems as a proxy for expression in C. moschata. We now have the opportunity to leverage other projects in C. maxima to work with the native Bmax allele. CucCAP resequenced our MicroMax panel representing the diversity of the C. maxima species in a minimalist way. A Hatch award is allowing us to explore the species more deeply which will result in an opportunity to examine the genomic context of Bmax without interspecific recombination suppression.

Impacts
What was accomplished under these goals? Objective 1. During this reporting period, we characterized the B gene in different varieties of C. pepo. The B gene is a chlorophyll biosynthesis pathway gene and hypothesized to be non-functional in chlorophyll biosynthesis to produce yellow squash fruit. We found that the B gene shows much lower expression in yellow squash than green ones. The B gene has a larger size of transcript from yellow squash fruit than green fruit due to intron retention. As a result, the B gene encodes a truncated enzyme protein. Consistently, western blot analysis of B protein reveals that the yellow fruit has non-detectable protein band but the green fruit has detectable band. These results support the role of the B gene function in conferring carotenoid accumulation by impairing chlorophyll biosynthesis in squash fruit. We are in the process to examine whether other genes associated with B in chlorophyll biosynthesis also exhibit the same function as B in promoting carotenoid accumulation in chromoplasts. Objective 2. 2.1. To identify L-2, we performed QTL-seq analysis that combines bulked segregant analysis and high-throughput whole-genome re-sequencing of pale yellow l-2/l-2 and dark orange L-2/L-2 plants. This analysis detected a single QTL associated with L-2. 2.2. To validate the identified QTL, genetic mapping was carried out. InDel markers equally distributed within the QTL region were developed and used to generate a low-resolution genetic linkage map. Two flanking markers closely linked to L2 were utilized to screen ~500 F2 plants for recombinants. Additional InDel markers were developed and used to genotype the recombinants. A high-resolution linkage map was constructed and the L-2 was delimited in a physical interval of 55 Kbp. There are nine predicted genes in this region. Among them, only one gene shows a high level of expression in fruit tissue, which represents the best candidate for L-2 gene. 2.3. To functional confirm the candidate gene for L-2, we first cloned it from the L-2 and l-2 backgrounds. Sequence and protein structure analyses show differences for the candidate from L-2 and l-2 backgrounds. We are performing functional confirmation in squash plants now. 2.4. We are also in the process to characterize the candidate and found that L-2 has higher activity in affecting carotenoid levels while l-2 has much weaker function. Analysis of various squash inbred lines shows that L-2 can greatly increase the carotenoid level in the B background. Objective 3. Allelism tests of Bmax and Bpepo suggests that Bmax is a second potentially superior B locus. DNA bulks created from a F2 population were used for QTL-seq analysis. The analysis identified a single QTL, which is localized in a different chromosome from the Bpepo studied in Objective 1, supporting that Bmax and Bpepo are different B genes. We developed six InDel markers closely linked to Bmax and constructed a low-resolution linkage map. The Bmax locus was flanked by two markers with a genetic distance of 6.4 cM. Currently we are performing high-resolution mapping to narrow down Bmax interval to a region with a small number of candidate genes.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Wilson, Casey. 2023. "Inheritance and Allelism Among the B genes in Cucurbita moschata". Thesis.
  • Type: Other Status: Other Year Published: 2024 Citation: Wrightstone, Emalee, Li, Li. "Many ways to orange: my approaches towards uncovering novel carotenoid regulators in plants", Plant Breeding & Genetics Graduate Student Seminars, April 30, 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Fabrizio, Jack. "A Genomic Approach for a More Sustainable Pumpkin". Plant and Animal Genome Conference, San Diego, CA, January 12, 2024


Progress 04/15/22 to 04/14/23

Outputs
Target Audience:Our target audience during this period have been other Cucurbita researchers and public and private plant breeders. Until recently, Mazourek and Myers were the only public sector squash breeders in the United States (US) and Mazourek was the only breeder doing genomics work in squash. Now, there are two additional public sector faculty doing breeding and genomics and we are interacting with them frequently. Further, this project has been a bridge to our colleagues in Israel. Our progress is also shared with the seed industry at industry focused field days. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?An undergraduate student was trained on how to set up agricultural fields for genetic studies and to record accurate phenotypic data to differentiate between Bmax and Bpepo. Additional trainings included techniques for manual squash pollination and taking squash leaf samples for DNA extraction. How have the results been disseminated to communities of interest?Mazourek presented on the project at the Vegetable Breeding Institute Field Days to seed company plant breeders. We have also been in contact with collaborators in Israel that we originally connected with via a BARD project on carbohydrates. What do you plan to do during the next reporting period to accomplish the goals?We will proceed with the characterization of Bpepo as planned. Our most immediate steps will be to use RNASeq data and Western analysis to verify the nature of the mutation. We will also proceed to fine map the L-2 locus. We are hopeful that near isogenic lines generously shared by a collaborator will allow us to rapidly narrow down the target region. Depending on the timeline of QTL-seq analysis it may be necessary to grow out F3 progeny from the (CN1-7 x TG1-2 & CN2-3 x TG1-1) populations for fine mapping. In addition, select F3 progeny from the (AC1-3/CN1-5 & CN1-5/CN3) allelism test populations can be grown out for phenotypic/genotypic validation. We currently have BC3F1 individuals segregating for Bmax that are a promising alternative to the F3 families.

Impacts
What was accomplished under these goals? Objective 1. As the first step toward the goal of elucidation of the molecular basis of the B gene in C. pepo, we have grown and collected the fruit tissues of C. pepo varieties. We examined the B gene alleles from green and yellow fruit. We also cloned one allele of the B gene for phenotypical complementation for potentially testing out whether the B gene can reduce chlorophyll biosynthesis. Objective 2. To identify L-2, the synergistic partner of the B gene, we grew a F2 population that is homozygous for B gene and segregates for L-2. Fruit rind and flesh color from three fruit per plant were visually assessed. Carotenoid levels of flesh of three fruit per plant were measured by UPC2. Phenotypic bulks were created by selecting 21 pale yellow l-2/l-2 and 25 dark orange L-2/L-2 plants. Genomic DNA from the bulks were extracted and submitted for library constructions and the whole-genome sequencing using an Illumina HiSeq 2500 system with paired-end 2 × 150 bp run. Raw sequencing reads were obtained. One region, ~1Mb, has been identified as clearly harboring the L-2 locus. Objective 3.1, to test Bmax and Bpepo allelism in a conspecific C. moschata background, was completed. From 2019-2022 C. moschata lines with Bpepo and Bmax independently introgressed were established and intermated to create F2 and backcross populations segregating for both loci. In the summer of 2022 two populations (AC1-3/CN1-5 & CN1-5/CN3) totaling 1600 plants were grown and allelism tests were performed. Plants were phenotyped for Bmax and Bpepo by visual assessment of fruit and foliage and segregating classes were self-pollinated for future phenotypic/genotypic validation. Segregation data was analyzed for both one and two gene hypothesis. In addition, we began work on Objective 3.2, to examine Bmax candidates. From 2019-2021 another C. moschata population was generated segregating for only Bmax. In the summer of 2021 two F2 populations (CN1-7 x TG1-2 & CN2-3 x TG1-1) totaling 487 plants were phenotyped for Bmax and self-pollinated for further genotyping. Phenotypic bulks were created by selecting 20 orange Bmax/Bmax plants and 20 green Bmax+/Bmax+ plants from each population. In the winter of 2023 leaf samples of these phenotypic bulks were sent to Cornell for QTL-seq analysis.

Publications