Field Crop 2025, Vol.8, No.2, 82-92 http://cropscipublisher.com/index.php/fc 87 5 Validation of Candidate QTLs and Gene Discovery 5.1 Validation of key QTLs across different environments and germplasm Verifying QTL, in essence, means repeatedly testing in different places and among different varieties before it can be effective. We have suffered-some QTLS that performed well in the laboratory group failed to adapt to the field (Martinez et al., 2016). Sites like those that control flower color and leaf characteristics were found to have their effects discounted when verified in tetraploid materials when the environment changed. Now I've learned my lesson. During the verification, I deliberately chose different materials ranging from diploid to tetraploid. New technologies such as high-throughput sequencing and BSA have indeed been of great help, especially for difficult traits like precocious puberty, which can quickly identify candidate regions. However, the most practical approach still requires more experiments. For instance, the same early-maturing QTL might perform outstandingly under long-day conditions in the north but be rather unremarkable in the south. So now when making mark-assisted selections, it is necessary to first ask clearly: In which environments has this QTL been verified? Otherwise, the bred varieties are very likely to suffer from "water and soil incompatibility". 5.2 Screening and annotation analysis of candidate genes in QTL-associated regions Identifying the candidate genes in the QTL region is like playing a detective game in the genome. We have tried various methods-the old technique of cDNA-AFLP can still bring surprises from time to time, especially when tracking transcripts that occur simultaneously with precocious traits. However, nowadays simplified genome sequencing combined with high-density mapping is more commonly used, which can narrow the QTL interval to a very small range (Yamakawa et al., 2021). The discovery of the PUB14 ubiquitin ligase gene that time was made possible by this combination of measures. The funniest thing is that sometimes the candidate genes are clearly within the interval and the functions are correct, but they are not responsible for the phenotype-at this time, new methods such as QTL-seq have to be relied on for re-verification (Saini et al., 2021). Nowadays, for precise positioning, it is often necessary to combine resequencing data and compress the interval to within tens of kilobytes before identifying candidate genes. However, to be honest, even if the gene is identified, to prove that it indeed controls the target trait, functional verification is still necessary. This entire process would take at least one or two years to complete. 5.3 Functional validation of key genes through transcriptomics and other analyses To verify the functions of candidate genes, in essence, it is about catching the "suspected genes" red-handed. The most commonly used method in our laboratory is to compare extreme materials-for example, placing the earliest maturing and the latest maturing potatoes together for transcriptome analysis, and as a result, several key genes were really identified (Sonsungsan et al., 2024). The bch gene that controls the yellow color of potato flesh is particularly interesting. I never thought that carotene metabolism is so important in potatoes. Verifying the TLRP gene was even more challenging. Just building the BAC library took more than half a year. However, it was eventually confirmed that it does affect the quality of cooking, and all the efforts were not in vain. Nowadays, functional verification increasingly emphasizes the combination of multiple omics. For instance, recently, cross-analysis of transcriptome data and proteome data was conducted, and it was found that although the transcriptional level of some genes remains unchanged, there are significant differences in protein activity. Although these discoveries are time-consuming and labor-intensive, they can provide solid targets for molecular breeding and are still better than blind screening. 6 Applications of QTL Mapping in Potato Breeding 6.1 Development of molecular markers based on QTLs and marker-assisted selection (MAS) QTL positioning technology has indeed brought new ideas to potato breeding. Now, variety improvement is much more precise than before. By analyzing those potato materials with high yield, disease resistance or outstanding quality, we found quite a few useful DNA markers (Kulkarni et al., 2020). Take the issue of nematode resistance as an example. The markers developed using the polyploid QTL-seq method have increased the efficiency of screening disease-resistant varieties several times over. There have also been breakthroughs in starch content and tuber quality-the QTL sites shown in Figure 2 have now all been transformed into practical molecular markers. However, in actual operation, it was found that tetraploid and hexaploid crops have different responses to the
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