Legume Genomics and Genetics 2025, Vol.16, No.5, 234-244 http://cropscipublisher.com/index.php/lgg 238 "escape silencing". This phenomenon has also been observed in wheat and grapevines (Lu et al., 2020). In contrast, circRNA research has not kept pace yet, but some data have suggested that they may also function in the form of "miRNA sponges" (Wang et al., 2019). Overall, although lncRNA and circRNA are not as "conspicuous" as mRNA, it might be they that maintain the response rhythm of adzuki beans behind the scenes during cold adaptation. 5 Case Study: Identification and Functional Validation of Key Cold-Responsive Genes 5.1 RNA-Seq-based analysis of adzuki bean under cold stress conditions Not all transcriptome studies were initially aimed at low temperatures. Gene families involved in RNA silencing, such as DCL, AGO and RDR, were actually first systematically organized during the study of drought or disease stress (Li et al., 2023). However, it was precisely these studies that provided methods and ideas, and also enabled us to later identify some differentially expressed genes and regulatory networks more quickly under the cold stress conditions of adzuki beans using RNA-Seq. Looking back now, these classified gene families might also be an indispensable part of the cold adaptation mechanism. 5.2 qRT-PCR validation and expression profiling of candidate genes The results of RNA-Seq cannot be used directly and need to be further confirmed by qRT-PCR. In the case of adzuki beans, previous studies have analyzed the expression patterns of stress-related genes such as DCL, AGO, and RDR using qRT-PCR and found that their expression changes under adverse stress were indeed quite obvious (Figure 2) (Li et al., 2023). However, to be more precise, it still depends on whether the reference genes are well selected. Some studies specifically selected and verified reference genes applicable to adzuki beans, so that expression profiling analysis under different adverse conditions (including low temperature) would not be biased (Imoto et al., 2022). 5.3 Functional analysis in heterologous systems Not all genes can be verified in adzuki beans before discussing their functions. At this point, model plants need to be used for assistance. Laboratory "veterans" like Arabidopsis thaliana and tobacco are often used in functional verification to overexpress candidate genes to see if they can enhance stress tolerance. The previously mentioned DCL, AGO and RDR genes, their related miRNA targets and feedback regulatory circuits have obtained preliminary clues in adzuki beans, and also provide good candidates for subsequent functional verification in these heterologous systems (Li et al., 2023). 6 Breeding Potential of Cold-Responsive Genes in Adzuki Bean 6.1 Development of molecular markers and MAS strategies for cold tolerance Nowadays, in breeding, relying on intuition or field experience is no longer sufficient. Especially when it comes to complex traits such as cold resistance, traditional methods are often inefficient and time-consuming. As a result, breeders have increasingly relied on molecular markers to assist in screening (MAS). Of course, this is not something that can be accomplished overnight - a reliable high-density genetic map is needed first, along with QTL mapping, to provide technical support. For crops like adzuki beans, some QTLS related to abiotic stress have been identified. It is precisely these fundamental studies that have made it possible for MAS to be applied in cold-resistant breeding (Jha et al., 2016; Dwivedi et al., 2023). 6.2 Association studies between cold-responsive genes and QTLs Not all genes can be associated with low-temperature response. To figure out who is related to whom, QTL analysis and association plotting are required. In recent years, researchers have identified some QTL regions related to stress such as cold resistance and drought, among which there are many hotspots related to domesticated traits. Some candidate genes were also proposed and entered the subsequent functional verification process (Wang et al., 2025). From a practical perspective, this approach of integrating multiple stress-response traits helps enhance the environmental adaptability of varieties, not only focusing on cold but also coping with other stresses simultaneously.
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