Maize Genomics and Genetics 2024, Vol.15, No.3, 111-122 http://cropscipublisher.com/index.php/mgg 113 3 Techniques in Genomics-Assisted Breeding 3.1 Quantitative trait loci (QTL) mapping QTL mapping is a fundamental technique used to identify genomic regions associated with specific phenotypic traits. This method involves crossing two genetically distinct lines to produce a mapping population, which is then genotyped and phenotyped to detect QTLs. For instance, QTL mapping has been instrumental in identifying loci associated with yield-related traits in maize, such as kernel weight and ear length (Zhao and Su, 2019). Additionally, meta-analysis of QTLs has been used to identify stable QTLs for traits like popping quality and disease resistance, which are crucial for marker-assisted selection (Kaur et al., 2021; Akohoue and Miedaner, 2022). 3.2 Genome-wide association studies (GWAS) GWAS is a powerful approach that scans the entire genome to find genetic variants associated with traits of interest. This method utilizes high-density SNP arrays and large, diverse populations to detect associations between genetic markers and phenotypic traits. For example, GWAS has been used to identify SNPs linked to yield-related traits and kernel micronutrient concentrations in maize (Hindu et al., 2018; Zhang et al., 2020). Multi-trait GWAS approaches have also been effective in uncovering pleiotropic QTLs that influence multiple traits simultaneously, enhancing our understanding of complex trait architecture (Rice et al., 2020). 3.3 Transcriptomics and RNA-seq Transcriptomics, particularly RNA-Seq, provides insights into gene expression patterns and regulatory networks underlying phenotypic traits. This technique involves sequencing the RNA transcripts in a sample to quantify gene expression levels. RNA-Seq has been used to validate candidate genes within QTL regions and to understand the genetic basis of traits such as kernel width and disease resistance (Zhao et al., 2022). By integrating transcriptomic data with QTL mapping, researchers can identify differentially expressed genes that contribute to trait variation. 3.4 CRISPR/Cas9 and gene editing CRISPR/Cas9 is a revolutionary gene-editing technology that allows precise modifications of the genome. This technique has been applied to create targeted knockouts and insertions in maize, facilitating the study of gene function and the improvement of complex traits. For instance, the BREEDIT pipeline combines CRISPR/Cas9-mediated multiplex genome editing with traditional breeding to enhance traits like yield and drought tolerance (Lorenzo et al., 2022) (Figure 1). This approach accelerates the development of improved maize varieties by enabling the precise manipulation of multiple genes simultaneously. Lorenzo et al. (2022) presents an innovative CRISPR/Cas9 multiplex genome editing pipeline designed to enhance maize growth by targeting 48 growth-related genes (GRGs). It employs a sophisticated approach that combines multiple gRNAs into vectors (SCRIPTs) and transforms Cas9-expressing lines (EDITOR lines) to create supertransformed plants. These plants undergo various crossing schemes to maximize the diversity of gene edits. High-throughput sequencing and bioinformatics workflows monitor gene edits, classify them into loss-of-function (LOF) categories, and facilitate genotype-to-phenotype associations. This method enables the systematic evaluation of the effects of multiple gene edits on plant growth, ultimately identifying key genes that significantly influence growth traits. The study demonstrates the potential of this pipeline to generate a large collection of higher-order mutants, providing a valuable resource for future research and trait improvement in maize. 3.5 Epigenomics Epigenomics involves the study of heritable changes in gene expression that do not involve changes to the DNA sequence. These changes can be influenced by environmental factors and can affect traits such as stress tolerance and disease resistance. Epigenomic studies in maize have the potential to uncover novel regulatory mechanisms and epigenetic markers that can be used in breeding programs. Although specific studies on epigenomics in maize are less prevalent, integrating epigenomic data with other genomic techniques can provide a comprehensive understanding of trait regulation and inheritance. In summary, genomics-assisted breeding in maize employs a suite of advanced techniques to dissect the genetic basis of important traits and to accelerate the development of improved varieties. By integrating QTL mapping,
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