Source: CORNELL UNIVERSITY submitted to
HULL-LESS SEEDS: A TRAIT IN CUCURBITA PEPO (SQUASH) THAT WOULD BENEFIT NUTRITION AND VEGETABLE SALES AND REDUCE WASTE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0219969
Grant No.
(N/A)
Project No.
NYC-149423
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2009
Project End Date
Sep 30, 2012
Grant Year
(N/A)
Project Director
Mazourek, M.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
Plant Breeding
Non Technical Summary
All of the squash we eat have edible seeds. These seeds are in fact an excellent source of vitamins, nutrients and omega-fatty acids. Pumpkin seeds are a common snack and appear in an increasing number of health foods. Pumpkin seed oil is readily pressed from these seeds and sold at a premium. Unfortunately, most of this source of nutrition is wasted as most consumers dispose of these seeds in their trash and the seed end up in landfills instead. Zucchini and summer squash that are over-mature are culled in grower fields and home gardens because of the development of woody seeds lessening their quality. A solution to this problem is already available in pumpkins, so called, naked, hull-less or shell-less seeds lack the usual woody seed coat. These seed are much larger and can be much more flavorful and nutritious than those with hulls and are the seed. Pumpkins are easily crossed with most other squash including zucchini and other summer squash as well as acorn, dumpling and delicata winter squash. Hubbard and butternut are different species, which restricts transferring the trait to these squashes, but would be future candidates for this approach. There are two reasons why consumers may not be eating squash seeds now. One is a lack of realization that the seeds are edible. The marketing of these new varieties would create this awareness. The other reason is likely to be the additional work in preparing the seeds and removing their shells, an issue directly addressed by our proposal. Our work would consist of reiteratively cross-pollinating existing Cornell varieties with pumpkins that lack a hull on their seeds and selecting for plants with this trait, superior performance and that resemble the target parent squash. Cornell has developed several squash varieties that are grown in New York and globally. Since we are breeding and selecting for plants that excel in our fields they have de facto adaptation to New York growing conditions and resistance to our major disease pressures. Natural disease resistance reduces crop losses, yields higher quality fruit, and reduces pesticide exposure and environmental harm. This benefit is further realized when grown organically, one of the focuses in our vegetable breeding efforts. The process to generate these squash is straightforward and the solution is simple, but outcomes and impacts would be realized on many levels. These squash would become a product that would increase grower and retailer vegetable sales. These edible seed would also offer ready access to nutrient rich food that could be purchased affordably or grown at home.
Animal Health Component
(N/A)
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2021429108050%
2121429108050%
Goals / Objectives
Objectives: Goal: This proposal seeks to deploy the hull-less trait found in pumpkins used for seed oil broadly into cucurbit species to increase utilization of their seeds, create a value added crop and serve as a germplasm base for variety development for vegetable and high quality oil seed production that are adapted to excel for growers in the Northeast. Specific objectives: 1. Create new C. pepo breeding lines with hull-less seed trait Breeding lines will be created from Cornell cultivars in combination with various hull-less cultivars. These will be near finished varieties in some cases but otherwise generally useful for breeding hull-less seeded squash. Each generation will be selected for resistance that is endemic to New York and for agronomic, yield and quality characteristics. 2. Create new germplasm for selection and interspecific crossing Butternut squash (C. moschata) can be crossed with C. pepo accessions, albeit with some difficulty. The genetic basis for this partial barrier is unknown, but parents with diverse backgrounds tend to hybridize more readily with C. moschata. The materials developed as part objective 1 will be used to attempt hybridization with C. moschata toward the goal of introgressing the hull-less trait into butternut-types. These hybrids can then be used as a bridge to transfer the hull-less trait into C. maxima cultivars. 3. Select hull-less C. pepo varieties with Phytophthora capsici tolerance Partial resistance to crown rot caused by P. capsici has been indentified, but fruit rot resistance seems dependent on thick, hard fruit rinds. A hard rind is a negative attribute for Halloween pumpkins and many other squashes. This is not a problem for pumpkins intended solely for oil seed production because the fruit is processed mechanically. Oil seed pumpkins represent an alternative crop, utilizing much of the same infrastructure, for pumpkin/squash farms whose production is threatened by P. capsici. 4. Employ molecular markers in the selection process In the course of a backcross breeding program, it is not phenotypically apparent which individuals carry one copy of the recessive allele. In addition to crosses to the recurrent parent, self pollinations must also be performed to test progeny for segregation. Linked molecular markers allow the elimination of individuals at the seedling stage that lack the hull-less allele. As new markers linked to useful traits such as disease resistances are developed, they will be integrated in to the molecular marker assays for seedlings.
Project Methods
A backcross approach will be used to transfer the hull-less trait into a variety of Cucurbita pepo cultivars as illustrated in Figure 2. These include: Romulus (powdery mildew resistant zucchini), Success PM (powdery mildew resistant yellow summer squash), Bush Delicata (All-American Selection and powdery mildew resistant) Sweet REBA (disease resistant, early, bush acorn), Cosatata Romanesco summer squash (breeding lines with powdery mildew resistance), pumpkins with powdery mildew resistance. We will grow two generations per year alternating between field and winters in the greenhouse. Three backcross generations will be employed to recover the recurrent parent type and these BC3F1 generations will then be self pollinated twice to yield stable hull-less seeded squash that have been selected for performance and adaptation to New York at each summer generation. SSR markers linked to the hull-less gene will be used for marker assisted selection. The initial hybrids and parents will be surveyed to assess polymorphism. RAPD markers also linked to the hull-less locus can be used to supplement the SSR markers. The BC1F2 generation will be grown to confirm cosegregation of the phenotype with the genetic marker the second summer. As more molecular markers are available in squash they will be incorporated into the breeding program and confirmed based on phenotypic screens, as appropriate for disease resistance, plant growth habit etc. Disease resistance will be evaluated based on phenotype. Powdery mildew is an endemic pathogen and susceptible individuals will be discarded. Statistical tests will not be possible because populations will be segregating and plants are best treated as individuals. The materials generated by this project will be subsequently evaluated in a rigorous statistical manner in the process of generating finished cultivars. Oil seed squash with hard rinds will be grown in fields infected with P. capsici to test resistance to fruit rot.

Progress 10/01/09 to 09/30/12

Outputs
OUTPUTS: The overall goal of our project was to create new breeding lines of squash with hull-less seeds, adapted to excel in New York and beyond, with the premise that this aspect of the seeds would increase consumption of squash seeds as nutritious food that is commonly discarded as waste. We intended to accelerate this process through the use of published molecular markers linked to the major gene controlling the presence or absence of seed coats in squash. However, due to technical difficulties we adopted a more conservative phenotype-based breeding strategy. Thirty one breeding lines were generated as a result of this project. We produced zucchini and summer squash breeding lines with hull-less seeds with a goal of them being able to be used for immature consumption as well as consumption of seed from over-mature fruit. Of these classes, zucchini lines are the most true to type. We produced acorn and delicata breeding lines for squash that could be consumed at the mature stage along with their seeds. The acorn class was the most true to type, but both had lines with good Brix of 10-12. Crosses between hull-less pumpkins and Romulus, a zucchini and source of powdery and downy mildew resistance, yielded oil seed pumpkins with much improved disease resistance. Similar crosses with Success PM summer squash yielded novel jack o'lantern type pumpkins. Due to the lack of transferability of published genetic markers for seed coat inheritance to our materials, we developed an acorn squash fruit and seed transcriptome from one of our parents in the breeding program, Sweet REBA toward the development of genetic markers more relevant to our materials. The transcriptome was derived from both pericarp and seed tissue of an acorn squash, sampled at five time points during fruit development. We assembled 141,838,600 high-quality paired-end Illumina reads into 99,839 unigenes using the program Trinity. More than 83% of these unigenes have homology with known genes and more than 69% could be functionally annotated. We have also identified fruit quality-related genes associated with carotenoid accumulation and carbohydrate metabolism. A fatty acid characterization of all the available Cucurbita pepo accessions in the USDA collection demonstrated that omega-3 fatty acids, often described as a primary pumpkin seed nutritive factor were found to be minor components of the seed and relatively non-variable in the germplasm. Phytophthora capsici foliar resistance was identified and the initial crosses were made to combine these genetics with the same elite parents that were intercrossed with hull-less lines. The combination of foliar and fruit resistance that is possible by intercrossing the progeny of these crosses with hard-rinded oil seed pumpkins will provide a particularly resistant crop. PARTICIPANTS: Six individuals worked on the project. Michael Mazourek, the PI, oversaw the project, interpreted results, and designed experiments. Maryann Fink-Brodnicki, a technician, was responsible for planting, pollinating, harvest and evaluations. Danny Fink, a technician, assisted with pollinations. Three training opportunities were realized as part of this work. Alex Noel, an undergraduate student, prepared samples for fatty acid analysis. Lindsay Wyatt, a graduate student, analyzed the fatty acid data and genotyped parents and populations with molecular markers and sequenced and assembled the transcriptome. James Keach, a graduate student, is attempting embryo rescue of interspecific crosses. TARGET AUDIENCES: The target audiences for our current work are primarily cucurbit growers, seed companies, and oil extractors. These audiences were engaged as described above. Materials from this project were also used to supplement a course material for Plant Genetics 2250 at Cornell. The end harvested products are intended to benefit the health of consumers. PROJECT MODIFICATIONS: There were significant changes during the course of the project. The breeding activity was slowed by the lack of polymorphic molecular markers. Nonetheless, for zucchini, acorn and oilseed pumpkin market classes progress was consistent with that which was originally targeted in terms of disease resistance, yield and quality. We were able to focus instead on oilseed quality and developing these resources. The creation of a winter squash fruit transcriptome, selection for seed eating quality and the analysis of seed oil composition activities were added as additional activities beyond the planned objectives.

Impacts
The breeding lines we have generated are being sampled by seed companies and are being made available for on farm trials and are likely to impact industry by their incorporation into commercial products and thereby impact human health by leading to the consumption of these squash and seeds thus improving nutrition. The discovery that the published markers were not useful in our germplasm prompted us to sequence a fruit transcriptome from a winter squash fruit and seeds, Sweet Reba, (see publications) that will provide information about the fruit and seed quality metabolism. The germplasm that can be created by combining the two types of Phytophthora capsici resistance will provide increased yield stability, reduce the need for fungicides and can provide growers with an alternative cucurbit crop if their cucurbit production fields become infested with Phytophthora blight. The knowledge that omega-3 fatty acids are scarce in pumpkin/Cucurbita pepo/squash seed oils informs future research that an alternate compound(s) is responsible for the health benefits of pumpkin seed oils (ex phytosterols) and should likely be focus of research and breeding efforts.

Publications

  • Wyatt, L., and Mazourek, M. (2012) De novo sequencing and assembly of an acorn squash (Cucurbita pepo ssp. ovifera) fruit and seed transcriptome. BMC Genomics. (Submitted).


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Our breeding program has continued to advance our hull-less lines of of acorn, delicata, Costata, zucchini and yellow summer squash. We have recovered the majority of morphological characteristics of the recurrent parent and are selecting for Brix, seed eating quality and other characteristics. PARTICIPANTS: Michael Mazourek, the PI, oversaw the project, interpreted results, and designed experiments. Maryann Fink-Brodnicki, a technician, was responsible for planting, pollinating, harvest and evaluations. Danny Fink, a technician, assisted with pollinations. TARGET AUDIENCES: The target audiences for our current work are primarily cucurbit growers, seed companies, and oil extractors. These audiences were engaged through field day events where they were presented with the new breeding lines and progress updates on the improvement processs. Materials from this project were also used to supplement a course material for Plant Genetics 2250 at Cornell. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Students and attendees at field days are often surprised to learn that seed of squash are edible. While many think of pumpkin seeds as edible, the horticultural distinction between squash and pumpkin is often the overriding concept. Sampling seed of zucchini has been an excellent way to discuss the taxonomic relationship of Cucurbita pepo and Cucurbita moschata cultivars.

Publications

  • No publications reported this period


Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: We had planned to use marker assisted selection to improve the efficiency of our breeding program for squash that produce hull-less seeds. Several molecular markers linked to the hull-less allele have been published by others. These markers were used to screen our breeding lines and hull-less seeded cultivars for polymorphisms that could be used to determine their genotype when used in breeding populations. The most promising were SSR markers that would be adaptable to a moderately high-throughput, fluorescently labeled fragment analysis system available on campus. Unfortunately, two complications were discovered. First, the markers tended to be monomorphic in our populations. Second, the polymorphisms tended to be single nucleotide indels which were not able to be scored at high confidence. Despite this setback, we have made significant progress in introgressing the hull-less trait into various squash backgrounds. We have hull-less examples of acorn, delicata, Costata, zucchini and yellow summer squash where we have recovered the majority of morphological characteristics of the recurrent parent. Quality characteristics will form the second selection criteria as the backcross program is continued. Given that oil quality is a major factor affecting the popularity of pumpkin seed oil, a survey of the fatty acid profile of all the USDA accessions of Cucurbita pepo was conducted. Omega-3 fatty acids are widely recognized as one of the most important healthful components of seed oils. However, this survey revealed that none of these 799 accessions contained more than 5% omega-3 fatty acids. We are actively engaged in embryo rescue for crosses between C. pepo cultivars with hull-less seeds and butternut cultivars of C. moschata to transfer the hull-less trait across the species barrier. Our results have been disseminated through presentations at the Cornell Seed Conference, to vegetable breeders in the seed industry at our annual field days, and to student groups that tour our fields. We have also had discussions with growers and processors interested in producing snack seed pumpkins or their oils. PARTICIPANTS: Three individuals worked on the project. Michael Mazourek, the PI, oversaw the project, interpreted results, and designed experiments. Maryann Fink-Brodnicki, a technician, was responsible for planting, pollinating, harvest and evaluations. Danny Fink, a technician, assisted with pollinations. Three training opportunities were realized as part of this work. Alex Noel, an undergraduate student, prepared samples for fatty acid analysis. Lindsay Wyatt, a graduate student, analyzed the fatty acid data and genotyped parents and populations with molecular markers. James Keach, a graduate student, is attempting embryo rescue of interspecific crosses. TARGET AUDIENCES: The target audiences for our current work are primarily cucurbit growers, seed companies, and oil extractors. These audiences were engaged as described above. Materials from this project were also used to supplement a course material for Plant Genetics 2250 at Cornell. PROJECT MODIFICATIONS: Due to the issues encountered with molecular markers in our populations, we are unable to utilize a blind backcross program as efficiently as we planned. As a result, we will rely on phenotypic selection. We will reduce the number of hull-less sources we use in our populations and will not be able to complete as many backcross generations. A benefit of utilizing phenotypic selection for the hull-less trait will be that these seed will be available for tasting evaluations. This quality component will be added.

Impacts
While the existing molecular markers were not generally useful in our populations, these populations will be excellent resources for the molecular characterization of the hull-less trait in squash using genomic resources in the Cucurbitaceae and molecular tools that are becoming more accessible. Rather than a single biparental cross, these populations feature the hull-less allele derived from multiple sources in multiple backgrounds. This history should significantly reduce linkage disequilibrium. The lack of C. pepo accessions in the USDA collection with significant levels of omega-3 fatty acids in their seed oil indicates that improving this oil component through conventional plant breeding techniques will likely not be straightforward. It also suggests that the observed health benefits of these oils derive from another less characterized component or components. Omega-3 content will likely not be something we address in future breeding efforts.

Publications

  • No publications reported this period