Development and Validation of Multiplex-PCR Assay for <i>β-Carotene hydroxylase</i> and <i>γ-Tocopherol methyl transferase</i> Genes Governing Enhanced Multivitamins in Maize for Its Application in Genomics-Assisted Breeding
Traditional maize possesses low concentrations of provitamin-A and vitamin-E, leading to various health concerns. Mutant alleles of <i>crtRB1</i> and <i>vte4</i> that enhance β-carotene (provitamin-A) and α-tocopherol (vitamin-E), respectively, in maize kernels have been expl...
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2025-01-01
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| author | Munegowda Manoj Gowda Vignesh Muthusamy Rashmi Chhabra Hriipulou Duo Saikat Pal Nisrita Gain Ashvinkumar Katral Ravindra K. Kasana Rajkumar U. Zunjare Firoz Hossain |
| author_facet | Munegowda Manoj Gowda Vignesh Muthusamy Rashmi Chhabra Hriipulou Duo Saikat Pal Nisrita Gain Ashvinkumar Katral Ravindra K. Kasana Rajkumar U. Zunjare Firoz Hossain |
| author_sort | Munegowda Manoj Gowda |
| collection | DOAJ |
| description | Traditional maize possesses low concentrations of provitamin-A and vitamin-E, leading to various health concerns. Mutant alleles of <i>crtRB1</i> and <i>vte4</i> that enhance β-carotene (provitamin-A) and α-tocopherol (vitamin-E), respectively, in maize kernels have been explored in several biofortification programs. For genetic improvement of these target nutrients, uniplex-PCR assays are routinely used in marker-assisted selection. However, due to back-to-back breeding seasons, the time required for genotyping individually for each target gene in large backcross populations becomes a constraint for advancing the generations. Additionally, multiple PCR assays for various genes increase the required costs and resources. Here, we aimed to develop a multiplex-PCR assay to simultaneously identify different allelic forms of <i>crtRB1</i> and <i>vte4</i> genes and validate them in a backcross-based segregating population. The PCR assay was carried out using newly developed primers for <i>crtRB1</i> and a gene-specific primer for <i>vte4</i>. The uniplex-PCR assay was standardized for selected primer pairs in the BC<sub>1</sub>F<sub>1</sub> population segregating for <i>crtRB1</i> and <i>vte4</i> genes. Subsequently, a multiplex-PCR assay for <i>crtRB1</i> and <i>vte4</i> genes was developed and employed for genotyping in the BC<sub>1</sub>F<sub>1</sub> population. The assay differentiated among four possible genotypic classes, namely <i>crtRB1<sup>+</sup>crtRB1/vte4<sup>+</sup>vte4</i>, <i>crtRB1crtRB1/vte4<sup>+</sup>vte4</i>, <i>crtRB1<sup>+</sup>crtRB1/vte4<sup>+</sup>vte4<sup>+</sup></i>, and <i>crtRB1crtRB1/vte4<sup>+</sup>vte4<sup>+</sup>.</i> This newly developed multiplex-PCR assay saved 41.7% of the cost and 35.6% of the time compared to two individual uniplex-PCR assays. The developed assay could accelerate maize nutritional quality breeding programs through rapid and cost-effective genotyping for the target genes. This is the first report of a multiplex-PCR assay specific to <i>crtRB1</i> and <i>vte4</i> genes for its use in genomics-assisted breeding in maize. |
| format | Article |
| id | doaj-art-1b853aaeca64485ba87cea69e68015f9 |
| institution | Kabale University |
| issn | 2223-7747 |
| language | English |
| publishDate | 2025-01-01 |
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| series | Plants |
| spelling | doaj-art-1b853aaeca64485ba87cea69e68015f92025-01-10T13:19:53ZengMDPI AGPlants2223-77472025-01-0114114210.3390/plants14010142Development and Validation of Multiplex-PCR Assay for <i>β-Carotene hydroxylase</i> and <i>γ-Tocopherol methyl transferase</i> Genes Governing Enhanced Multivitamins in Maize for Its Application in Genomics-Assisted BreedingMunegowda Manoj Gowda0Vignesh Muthusamy1Rashmi Chhabra2Hriipulou Duo3Saikat Pal4Nisrita Gain5Ashvinkumar Katral6Ravindra K. Kasana7Rajkumar U. Zunjare8Firoz Hossain9Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaDivision of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaDivision of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaDivision of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaDivision of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaDivision of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaDivision of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaDivision of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaDivision of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaDivision of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi 110012, IndiaTraditional maize possesses low concentrations of provitamin-A and vitamin-E, leading to various health concerns. Mutant alleles of <i>crtRB1</i> and <i>vte4</i> that enhance β-carotene (provitamin-A) and α-tocopherol (vitamin-E), respectively, in maize kernels have been explored in several biofortification programs. For genetic improvement of these target nutrients, uniplex-PCR assays are routinely used in marker-assisted selection. However, due to back-to-back breeding seasons, the time required for genotyping individually for each target gene in large backcross populations becomes a constraint for advancing the generations. Additionally, multiple PCR assays for various genes increase the required costs and resources. Here, we aimed to develop a multiplex-PCR assay to simultaneously identify different allelic forms of <i>crtRB1</i> and <i>vte4</i> genes and validate them in a backcross-based segregating population. The PCR assay was carried out using newly developed primers for <i>crtRB1</i> and a gene-specific primer for <i>vte4</i>. The uniplex-PCR assay was standardized for selected primer pairs in the BC<sub>1</sub>F<sub>1</sub> population segregating for <i>crtRB1</i> and <i>vte4</i> genes. Subsequently, a multiplex-PCR assay for <i>crtRB1</i> and <i>vte4</i> genes was developed and employed for genotyping in the BC<sub>1</sub>F<sub>1</sub> population. The assay differentiated among four possible genotypic classes, namely <i>crtRB1<sup>+</sup>crtRB1/vte4<sup>+</sup>vte4</i>, <i>crtRB1crtRB1/vte4<sup>+</sup>vte4</i>, <i>crtRB1<sup>+</sup>crtRB1/vte4<sup>+</sup>vte4<sup>+</sup></i>, and <i>crtRB1crtRB1/vte4<sup>+</sup>vte4<sup>+</sup>.</i> This newly developed multiplex-PCR assay saved 41.7% of the cost and 35.6% of the time compared to two individual uniplex-PCR assays. The developed assay could accelerate maize nutritional quality breeding programs through rapid and cost-effective genotyping for the target genes. This is the first report of a multiplex-PCR assay specific to <i>crtRB1</i> and <i>vte4</i> genes for its use in genomics-assisted breeding in maize.https://www.mdpi.com/2223-7747/14/1/142maizeuniplex- and multiplex-PCRcost and timegenomics-assisted breedingbiofortification |
| spellingShingle | Munegowda Manoj Gowda Vignesh Muthusamy Rashmi Chhabra Hriipulou Duo Saikat Pal Nisrita Gain Ashvinkumar Katral Ravindra K. Kasana Rajkumar U. Zunjare Firoz Hossain Development and Validation of Multiplex-PCR Assay for <i>β-Carotene hydroxylase</i> and <i>γ-Tocopherol methyl transferase</i> Genes Governing Enhanced Multivitamins in Maize for Its Application in Genomics-Assisted Breeding Plants maize uniplex- and multiplex-PCR cost and time genomics-assisted breeding biofortification |
| title | Development and Validation of Multiplex-PCR Assay for <i>β-Carotene hydroxylase</i> and <i>γ-Tocopherol methyl transferase</i> Genes Governing Enhanced Multivitamins in Maize for Its Application in Genomics-Assisted Breeding |
| title_full | Development and Validation of Multiplex-PCR Assay for <i>β-Carotene hydroxylase</i> and <i>γ-Tocopherol methyl transferase</i> Genes Governing Enhanced Multivitamins in Maize for Its Application in Genomics-Assisted Breeding |
| title_fullStr | Development and Validation of Multiplex-PCR Assay for <i>β-Carotene hydroxylase</i> and <i>γ-Tocopherol methyl transferase</i> Genes Governing Enhanced Multivitamins in Maize for Its Application in Genomics-Assisted Breeding |
| title_full_unstemmed | Development and Validation of Multiplex-PCR Assay for <i>β-Carotene hydroxylase</i> and <i>γ-Tocopherol methyl transferase</i> Genes Governing Enhanced Multivitamins in Maize for Its Application in Genomics-Assisted Breeding |
| title_short | Development and Validation of Multiplex-PCR Assay for <i>β-Carotene hydroxylase</i> and <i>γ-Tocopherol methyl transferase</i> Genes Governing Enhanced Multivitamins in Maize for Its Application in Genomics-Assisted Breeding |
| title_sort | development and validation of multiplex pcr assay for i β carotene hydroxylase i and i γ tocopherol methyl transferase i genes governing enhanced multivitamins in maize for its application in genomics assisted breeding |
| topic | maize uniplex- and multiplex-PCR cost and time genomics-assisted breeding biofortification |
| url | https://www.mdpi.com/2223-7747/14/1/142 |
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