Soybean is an integral oilseed crop that is used all over the world. Despite this, soybean has been experiencing a decrease in seed quality seen by the decreasing trend in test weight, and is limited in its uses due to the antinutritional factor, trypsin inhibitor (TI). This project first worked to identify single nucleotide polymorphisms (SNPs) associated with high test weight through a genome-wide association study (GWAS) as well as determine the relationship between important seed composition traits and test weight. Additionally, it focused on identifying and knocking out KTI genes specifically expressed in soybean seed tissue through CRISPR/Cas9. Lastly, this project investigated further use of soybean in aquaculture by evaluating the performance of Rainbow trout fed diets with low-TI, 'VT Barrack' soy meal. Nine SNPs on chromosome 15 were found to be significantly associated with high test weight as and eight potential gene candidates were identified. Test weight was found to be significantly, and negatively related to seed oil content and had inconsistent correlations with protein and sugar content. The KTI genes KTI1, Glyma01g095000, and KTI3, Glyma08g341500, were found to be only expressed in seed tissues and multiple KTI knock-out soybean plants with decreased seed TI content and activity were developed. Lastly, we found that our low-TI soymeal could replace 30% of fish meal in aquafeeds with no negative effects on trout growth and health. / Doctor of Philosophy / Soybean is an essential and lucrative crop due to its countless uses. Despite its importance and flexibility, it contains antinutritional factors, such as trypsin inhibitors (TI), and has been experiencing a decline in test weight, the amount of soybean seed in kilograms per hectoliter, that can limit the profits that growers receive. This dissertation seeks to contribute to the development of improved soybean varieties in addition to evaluating current low-TI varieties' application in aquaculture as a substitute for fish meal. A genome-wide association study (GWAS) was performed to identify molecular markers associated with high test weight and the relationship between high test weight and important seed composition traits was determined. Gene editing was conducted to knock-out expression of Kunitz trypsin inhibitor (KTI) genes specifically expressed in seed tissue. A fish feeding trial was conducted to evaluate the performance of Rainbow trout-fed diets using our low-TI soybean cultivar, 'VT Barrack'. We were able to successfully identify multiple molecular markers associated with high test weight as well as identify multiple candidate genes. We were able to determine that high test weight has a strong, negative relationship with seed oil content. We identified two KTI genes for gene knockout and were able to successfully develop a low-TI soybean line that has limited TI in just seed tissue. Lastly, we found that our low-TI soymeal could replace 30% of fish meal in aquafeeds with no negative effects on trout growth and health.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/113760 |
| Date | 09 February 2023 |
| Creators | Shea, Zachary |
| Contributors | Crop and Soil Environmental Sciences, Zhang, Bo, Ovissipour, Reza, Saghai-Maroof, Mohammad A., Zhao, Bingyu, Schwarz, Michael H. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/vnd.openxmlformats-officedocument.wordprocessingml.document |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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