Cotton Genomics and Genetics 2025, Vol.16, No.1, 39-47 http://cropscipublisher.com/index.php/cgg 44 6 Challenges and Future Directions 6.1 Limitations of current comparative genomics approaches Although comparative genomics of cotton has made a lot of progress, there are still many problems. The polyploid genome of cotton is large and complex, with many repeated genes and similar sequences, which makes it difficult to assemble, annotate and study the function of the genome (Yang et al., 2020). At present, it is still not easy to accurately determine the function of a gene, and the speed is not as fast as genome sequencing. On the one hand, this is because a more refined reference genome is needed, and on the other hand, the diversity materials are not rich enough. In addition, current breeding methods rely more on varieties with a narrow genetic background, which may affect future yield increases and may miss some new useful genes (Kumar et al., 2024). There are also some technical problems, such as the CRISPR/Cas system sometimes has off-target effects, and the design of guide RNA also needs to be optimized, which limits its use in actual breeding. 6.2 Integration with other omics and breeding tools Comparative genomics alone is far from enough. Especially when faced with complex traits that are easily affected by multiple factors, it is difficult to see the whole picture by studying gene structure alone. Sometimes, phenotypic changes are not a problem with the gene sequence itself, but the "unspoken rules" at the regulatory level. This requires the use of other "omics" together - such as transcriptome, metabolome, proteome, and epigenetic data (Yang et al., 2022a). Although these contents look more complicated when put together, the advantage is that the prediction model can be made more accurate and key pathways and functional genes can be targeted more specifically (Billings et al., 2022). Of course, no matter how much data there is, it is difficult to implement without breeding methods. In order to turn this information into truly useful varieties, it is necessary to combine rapid breeding, high-throughput phenotyping technology, and pan-genomic research. Especially in the current context of the continuous compression of genetic diversity, collecting different germplasm resources may be the only way for us to find those "lost genetic fragments" (Khalilisamani et al., 2024). This is not addition, but a necessary condition. 6.3 Future perspectives and research needs What should we do next? The direction is not difficult to guess. The foundation needs to be filled first, for example, the genome assembly can be more refined, and the data coverage of resequencing still has a lot of room for improvement. At present, many germplasm materials have not been included in the big data platform. If this gap is not filled, the subsequent analysis will not be able to achieve a "global perspective". In addition, the functional verification step can no longer be delayed - we can't wait for generations of experiments to verify, and the development of a set of high-throughput, fast and effective functional omics tools is the current priority (Ashraf et al., 2018). However, it is not realistic to rely on a single breakthrough. Omics must be interoperable, and breeding technology must be upgraded. This involves the update of tools such as CRISPR, such as improving specificity, reducing off-targets, and editing multiple targets at once (Khan et al., 2023). If we want these studies to not just stay in papers, we must promote the construction of a sharing mechanism - data can be interoperable, resources can be used interchangeably, and ideas can be jointly built. In this way, the public breeding platform will have a real chance to take off, and the sustainable development of cotton will not just be a vision written in a report (Sreedasyam et al., 2024). 7 Concluding Remarks In the past, our understanding of cotton often stayed at the level of trait performance and breeding experience. Now it is different. With the emergence of comparative genomics, many things began to have an "inside perspective". After the reference genome, pan-genome, and various variant databases were established, the genetic diversity that was originally "hidden" during the domestication or selection process was slowly found. Indeed, not every variation is important, but some newly discovered sites, missing variants, and potential candidate genes are gradually rewriting our understanding of the control of cotton traits. Methods such as whole-genome association analysis, gene introgression maps, and functional verification have made many things that used to rely on guesses have a hammer.
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