Cotton Genomics and Genetics 2025, Vol.16, No.5, 249-258 253 5.2 Integration and validation of genotyping methods (KASP, CAPS, SSR, etc.) Nowadays, cotton breeding rarely relies solely on a single typing method. KASP is currently the most widely used SNP detection method. The detection effect is stable and it is not troublesome to use. Many alleles of key traits are rapidly screened out by it (Li et al., 2022). Although SSR markers are more traditional, they have now been optimized to meet the requirements of high-throughput and even multi-analysis, such as for rapid variety identification (Kuang et al., 2022). Furthermore, more functional markers such as InDel and CAPS have also begun to be used for screening specific target traits such as fertility, disease resistance and stress tolerance (Feng et al., 2020; Dwivedi et al., 2025). The combined use of these methods not only enhances the screening efficiency but also ensures their universality in different breeding scenarios. 5.3 Practical effectiveness of marker-assisted selection (MAS) in breeding The benefits of MAS have already been experienced by many breeders-there is no need to wait for the harvest in the field to judge the quality, but rather to screen in advance at the molecular level, effectively saving time and cost. Especially those markers derived from functional areas are more predictive and indeed more efficient in the screening of varieties. However, it is not to say that MAS has no shortcomings. Its effect is often related to the distance between the marker and the target gene, and the stability in different environments is also a variable (Bolek et al., 2016). Even so, its advantages in multi-gene complex traits remain obvious, at least in terms of accuracy and speed, which are beyond the reach of traditional breeding (Ijaz et al., 2019; Wang and Zhang, 2024). 6 Case Studies: Representative Applications of Genotyping in Cotton Molecular Breeding 6.1 Application of GBS in breeding for Fusarium wilt resistance in China The genetic diversity of upland cotton is not wide, but this has not hindered the extensive use of sequencing-type genotyping (GBS) technology in the development of SNP markers. The research team attempted to apply GBS to different varieties and their hybrid offspring, and as a result, they found many SNP markers with strong polymorphism in the main cotton varieties cultivated in China. These markers can also be transformed into functional detection tools like KASP, facilitating their implementation in actual breeding projects. Especially in the breeding of Fusarium wilt resistance, the efficiency of locating resistance sites and screening candidate materials has significantly improved, and the breeding of disease-resistant varieties has also accelerated accordingly (Islam et al., 2015). 6.2 GWAS and SNP array-based strategies for yield improvement in American cotton The approach in the United States is slightly different. The breeding team is more inclined to use genome-wide association analysis (GWAS) in combination with high-density chips such as CottonSNP63K or CottonSNP80K. These platforms have shown stable performance in screening QTLS related to yield or fiber quality traits, and the results can be reproduced in different germplasm populations. For instance, CottonSNP63K has been utilized to construct genetic maps and conduct GWAS, capable of simultaneously covering intraspecspecific and interspecific differences, and has a very strong localization ability. These achievements do not remain at the theoretical level either. They directly support marker-assisted selection and genomic prediction, and are also common in practical breeding work for yield improvement (Si et al., 2022). 6.3 Multi-environment validation of high-throughput genotyping in transgenic and conventional materials It is worth noting that, for both conventional materials and transgenic strains, a considerable amount of adaptability verification of high-throughput genotyping platforms in different breeding contexts has been carried out. Multi-environment tests conducted using SNP chips or GBS not only have good repeatability in trait localization and variety identification, but also ensure that the association relationship between markers and traits remains stable under different environments. The CottonSNP80K chip is a typical example. Its polymorphism and detection rate in hundreds of germplasm materials indicate that it can basically meet the diverse breeding demands (Figure 2) (Billings et al., 2022; Chen et al., 2022).
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