TILLING-by-Sequencing<sup>+</sup> to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis
Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members th...
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| Format: | Article |
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MDPI AG
2021-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/8/4219 |
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| author | Naoufal Lakhssassi Zhou Zhou Mallory A. Cullen Oussama Badad Abdelhalim El Baze Oumaima Chetto Mohamed G. Embaby Dounya Knizia Shiming Liu Leandro G. Neves Khalid Meksem |
| author_facet | Naoufal Lakhssassi Zhou Zhou Mallory A. Cullen Oussama Badad Abdelhalim El Baze Oumaima Chetto Mohamed G. Embaby Dounya Knizia Shiming Liu Leandro G. Neves Khalid Meksem |
| author_sort | Naoufal Lakhssassi |
| collection | DOAJ |
| description | Reverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing<sup>+</sup> (TbyS<sup>+</sup>), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS<sup>+</sup> in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, <i>GmSACPD</i>, <i>GmFAD2</i>, and <i>GmFAD3</i>, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS<sup>+</sup>, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits. |
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| format | Article |
| id | doaj.art-52b916f9845145ccb68e83d747a56551 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T12:11:26Z |
| publishDate | 2021-04-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-52b916f9845145ccb68e83d747a565512023-11-21T16:10:31ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-04-01228421910.3390/ijms22084219TILLING-by-Sequencing<sup>+</sup> to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional AnalysisNaoufal Lakhssassi0Zhou Zhou1Mallory A. Cullen2Oussama Badad3Abdelhalim El Baze4Oumaima Chetto5Mohamed G. Embaby6Dounya Knizia7Shiming Liu8Leandro G. Neves9Khalid Meksem10Department of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USADepartment of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USADepartment of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USADepartment of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USADepartment of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USADepartment of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USADepartment of Animal Science, Food, and Nutrition, Southern Illinois University, Carbondale, IL 62901, USADepartment of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USADepartment of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USARAPiD Genomics, Gainesville, FL 32601, USADepartment of Plant, Soil, and Agricultural Systems, Southern Illinois University, Carbondale, IL 62901, USAReverse genetic approaches have been widely applied to study gene function in crop species; however, these techniques, including gel-based TILLING, present low efficiency to characterize genes in soybeans due to genome complexity, gene duplication, and the presence of multiple gene family members that share high homology in their DNA sequence. Chemical mutagenesis emerges as a genetically modified-free strategy to produce large-scale soybean mutants for economically important traits improvement. The current study uses an optimized high-throughput TILLING by target capture sequencing technology, or TILLING-by-Sequencing<sup>+</sup> (TbyS<sup>+</sup>), coupled with universal bioinformatic tools to identify population-wide mutations in soybeans. Four ethyl methanesulfonate mutagenized populations (4032 mutant families) have been screened for the presence of induced mutations in targeted genes. The mutation types and effects have been characterized for a total of 138 soybean genes involved in soybean seed composition, disease resistance, and many other quality traits. To test the efficiency of TbyS<sup>+</sup> in complex genomes, we used soybeans as a model with a focus on three desaturase gene families, <i>GmSACPD</i>, <i>GmFAD2</i>, and <i>GmFAD3</i>, that are involved in the soybean fatty acid biosynthesis pathway. We successfully isolated mutants from all the six gene family members. Unsurprisingly, most of the characterized mutants showed significant changes either in their stearic, oleic, or linolenic acids. By using TbyS<sup>+</sup>, we discovered novel sources of soybean oil traits, including high saturated and monosaturated fatty acids in addition to low polyunsaturated fatty acid contents. This technology provides an unprecedented platform for highly effective screening of polyploid mutant populations and functional gene analysis. The obtained soybean mutants from this study can be used in subsequent soybean breeding programs for improved oil composition traits.https://www.mdpi.com/1422-0067/22/8/4219TILLING by target capture sequencingTILLING-by-sequencing<sup>+</sup>oil biosynthesis pathwayfatty acid desaturases<i>GmSACPD-C</i><i>GmFAD2-1A/1B</i> |
| spellingShingle | Naoufal Lakhssassi Zhou Zhou Mallory A. Cullen Oussama Badad Abdelhalim El Baze Oumaima Chetto Mohamed G. Embaby Dounya Knizia Shiming Liu Leandro G. Neves Khalid Meksem TILLING-by-Sequencing<sup>+</sup> to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis International Journal of Molecular Sciences TILLING by target capture sequencing TILLING-by-sequencing<sup>+</sup> oil biosynthesis pathway fatty acid desaturases <i>GmSACPD-C</i> <i>GmFAD2-1A/1B</i> |
| title | TILLING-by-Sequencing<sup>+</sup> to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis |
| title_full | TILLING-by-Sequencing<sup>+</sup> to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis |
| title_fullStr | TILLING-by-Sequencing<sup>+</sup> to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis |
| title_full_unstemmed | TILLING-by-Sequencing<sup>+</sup> to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis |
| title_short | TILLING-by-Sequencing<sup>+</sup> to Decipher Oil Biosynthesis Pathway in Soybeans: A New and Effective Platform for High-Throughput Gene Functional Analysis |
| title_sort | tilling by sequencing sup sup to decipher oil biosynthesis pathway in soybeans a new and effective platform for high throughput gene functional analysis |
| topic | TILLING by target capture sequencing TILLING-by-sequencing<sup>+</sup> oil biosynthesis pathway fatty acid desaturases <i>GmSACPD-C</i> <i>GmFAD2-1A/1B</i> |
| url | https://www.mdpi.com/1422-0067/22/8/4219 |
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