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%
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.