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

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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Main Authors: Munegowda Manoj Gowda, Vignesh Muthusamy, Rashmi Chhabra, Hriipulou Duo, Saikat Pal, Nisrita Gain, Ashvinkumar Katral, Ravindra K. Kasana, Rajkumar U. Zunjare, Firoz Hossain
Format: Article
Language:English
Published: MDPI AG 2025-01-01
Series:Plants
Subjects:
maize
uniplex- and multiplex-PCR
cost and time
genomics-assisted breeding
biofortification
Online Access:https://www.mdpi.com/2223-7747/14/1/142
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Summary: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.
ISSN:2223-7747

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