Maize Genomics and Genetics 2024, Vol.15, No.5, 247-256 http://cropscipublisher.com/index.php/mgg 249 3 Applications of High-Throughput Sequencing in Maize Precision Breeding 3.1 Genomic selection High-throughput sequencing (HTS) has significantly enhanced the efficiency of genomic selection (GS) in maize breeding by enabling the rapid and accurate identification of genetic markers associated with desirable traits. The integration of HTS with GS allows breeders to predict the performance of breeding lines based on their genomic profiles, thus accelerating the breeding cycle and increasing genetic gains. For instance, the development of genotyping by target sequencing (GBTS) platforms has provided affordable and high-quality genotyping options, which are crucial for marker-assisted selection and genomic selection in maize (Guo et al., 2019). Additionally, HTS facilitates the collection of comprehensive genomic data, which can be used to improve the accuracy of genomic predictions and optimize breeding strategies (Cabrera-Bosquet et al., 2012; Wang and Zhang, 2024). Several case studies have demonstrated the effectiveness of HTS in improving maize yield and resistance to various stresses through genomic selection. For example, the integration of genomic-enabled prediction and high-throughput phenotyping has been shown to enhance the prediction accuracy for grain yield in drought-stressed and heat-stressed environments, thereby aiding the development of climate-resilient maize varieties (Juliana et al., 2018). Another study highlighted the use of HTS in developing SNP marker panels, which were validated and used for genomic selection to improve traits such as yield and stress resistance in maize breeding programs (Guo et al., 2019). 3.2 Integration of genome editing and HTS HTS plays a crucial role in detecting and validating genome editing events in maize. By providing detailed genomic information, HTS enables the precise identification of edits made by genome editing tools such as CRISPR/Cas9. This technology allows researchers to confirm the presence of intended modifications and assess any off-target effects, ensuring the accuracy and safety of genome editing applications (Figure 1) (Liu et al., 2020). The use of HTS in pre- and post-editing analysis ensures that only the desired genetic changes are retained, thereby streamlining the breeding process. Figure 1 Pipeline of High-Throughput Genome-Editing Design (Adopted from Liu et al., 2020) Image caption: (A) Candidates selected from QTL fine mapping, genome-wide association mapping studies (GWAS), and comparative genomics; (B) Line-specific sgRNA filtering based on assembled pseudo-genome of the receptor line KN5585; (C) Different vector construction approaches of double sgRNA pool (DSP) and single sgRNA pool (SSP); (D) Measuring the coverage and uniformity during plasmid pool by deep-sequencing; (E) to (G) Transformation and assignment of targets to each T0 individual by barcode-based sequencing. (H) to (J) Identification of mutant sequences by Sanger sequencing; (K) and (L) Identification of mutant sequences by Capture-based deep-sequencing; (M) Measuring phenotype changes and identification of functional genes (Adopted from Liu et al., 2020) HTS is instrumental in verifying the effects of CRISPR/Cas9 and other genome editing techniques in maize. By sequencing the edited regions, researchers can determine the efficiency and specificity of the editing process. For instance, a study demonstrated the use of HTS to identify and characterize gene-editing events in maize, revealing
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