Molecular Plant Breeding 2024, Vol.15, No.4, 187-197 http://genbreedpublisher.com/index.php/mpb 189 3.1.2 Biochemical markers Biochemical markers involve the analysis of proteins and other metabolites. Isozymes, which are different forms of an enzyme that catalyze the same reaction, are commonly used biochemical markers. These markers can provide more precise genetic information compared to morphological markers but are still limited by environmental influences and the complexity of protein analysis. 3.1.3 Molecular markers Molecular markers are DNA sequences that can be used to identify genetic differences between individuals. They are highly reliable and not influenced by environmental conditions. Several types of molecular markers are used in plant breeding. Restriction fragment length polymorphisms (RFLP): this technique involves the digestion of DNA with restriction enzymes and the separation of resulting fragments by gel electrophoresis. RFLPs are highly polymorphic and co-dominant, making them useful for genetic mapping and diversity studies (Ma et al., 2010). Amplified fragment length polymorphisms (AFLP): AFLP combines the principles of RFLP and PCR to amplify DNA fragments. It is a highly sensitive method that can detect a large number of polymorphisms, making it suitable for constructing genetic linkage maps (Chang et al., 2017). Simple sequence repeats (SSR): also known as microsatellites, SSRs are short, repetitive DNA sequences that are highly polymorphic. They are widely used in genetic diversity studies, linkage mapping, and marker-assisted selection due to their high reproducibility and co-dominant inheritance (Sharma et al., 2009; Li et al., 2021; Tian et al., 2022). Single nucleotide polymorphisms (SNP): SNPs are single base pair variations in the DNA sequence. They are the most abundant type of genetic variation and can be used for high-resolution mapping and association studies. SNP markers are increasingly used in plant breeding due to advances in next-generation sequencing technologies (Yang et al., 2012). 3.2 Development of genetic markers for Camellia 3.2.1 Marker discovery and validation The discovery of genetic markers in Camellia involves the identification of polymorphic DNA sequences that can be used to differentiate between genotypes. Techniques such as RNA sequencing (RNA-seq) and expressed sequence tag (EST) analysis have been employed to identify SSR markers in various Camellia species (Liu et al., 2018; Li et al., 2021; Tian et al., 2022). For instance, in Camellia japonica, a total of 28 854 potential SSRs were identified, and 172 primer pairs were synthesized, with 111 found to be polymorphic (Li et al., 2021). Similarly, in Camellia chekiangoleosa, 97 510 SSR loci were identified, and the development efficiency of polymorphic SSR primers was 26.72% (Tian et al., 2022). Validation of these markers involves testing their polymorphism and transferability across different Camellia species and accessions. For example, 96 SSR markers were developed for Camellia sinensis, and their polymorphism was assessed in 47 tea cultivars, demonstrating high levels of genetic diversity (Liu et al., 2018). Additionally, 74 novel polymorphic EST-SSR markers were developed for Camellia sinensis, with observed heterozygosity ranging from 0.000 to 1.000 (Ma et al., 2010). 3.2.2 Linkage mapping and QTL analysis Linkage mapping involves the construction of genetic maps that show the relative positions of genetic markers on chromosomes. These maps are essential for identifying quantitative trait loci (QTLs) associated with important agronomic traits. In Camellia sinensis, a genetic linkage map was constructed using RAPD, AFLP, and SSR markers, covering 1441.6 cM with an average distance of 4.7 cM between markers (Chang et al., 2017). This map provides a foundation for QTL analysis and marker-assisted selection in tea breeding programs. QTL analysis involves the identification of genomic regions associated with specific traits. For example, in a study on Lupinus angustifolius, next-generation sequencing was used to identify SNP markers linked to disease resistance genes, demonstrating the potential of NGS for rapid marker development and QTL mapping (Yang et al., 2012). Similar approaches can be applied to Camellia breeding programs to enhance the efficiency of selecting desirable traits.
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