Legume Genomics and Genetics 2025, Vol.16, No.3, 128-134 http://cropscipublisher.com/index.php/lgg 129 2 Genome-Wide Identification of NBS-LRR Genes in Adzuki Bean 2.1 Genome data sources and analytical methods overview In fact, to identify a complete NBS-LRR gene family, the most crucial prerequisite is still to have a reliable draft genome. Without high-quality assembly, the subsequent annotations are basically useless. The common practice nowadays is basically to use tools like NLGenomeSweeper, along with homology alignment, to find sequences containing NB-ARC domains (Toda et al., 2020; Andolfo et al., 2022). The process is generally divided into two steps. First, Pfam (such as PF00931) is used to pull out a batch of candidates, and then a hidden Markov model trained for a specific species is used for further screening. Finally, annotation tools like InterProScan will come into play to do some manual organization and classification. Although this method involves many steps, its advantage is that it can also extract the functional NB-LRR genes hidden in the repetitive regions. 2.2 Identification criteria and classification of NBS, LRR, and associated domains Many people get a headache when they hear the term "domain", but in fact, the core structure of genes like NS-LRR is quite regular. In the middle is NB-ARC, followed by a leucine repeat LRR region (Andolfo et al., 2022). As for the front-end domains, different types have different configurations: those with the CC prefix are called CNL, those with the TIR prefix are called TNL, and another type with the RPW8 domain is classified as RNL (Toda et al., 2020). If any N-terminal domain is missing, there is also a classification - that is the NL class. When classifying, two lines are basically followed: one is that the NB-ARC must be complete, and the other is that at least one LRR motif should be included. Other domains are supplemented with annotations by tools such as InterProScan. This set of standards is not only for classification but also to provide a reference for subsequent analysis of their differences in evolutionary paths and functions. 2.3 Chromosomal distribution and genomic features of NBS-LRR genes in adzuki bean If we refer to the research on kidney beans or soybeans, we can roughly guess that the situation of adzuki beans won't be much different. The NS-LRR gene is generally not scattered but prefers to "cluster" - either in clusters or simply in tandem (Afzal et al., 2021). Take kidney beans as an example. The research found that a total of 178 complete NB-LRR genes and 145 partial genes were located on its 11 chromosomes, and they were all classified into the two major categories of TNL and CNL (Wu et al., 2017). The locations of these genes often overlap with the known disease-resistant QTL regions, indicating that they are likely related to resistance (Kang et al., 2012). As for the characteristic of cluster distribution, it is more the result of the continuous replication and expansion of NS-LRR during the evolutionary process. Precisely because of this, they are also more adaptable to the variable pathogen environment. Therefore, conducting a comprehensive genomic mapping of adzuki beans is not only fundamental research but may also provide direct clues for disease-resistant breeding. 3 Phylogenetic Analysis and Classification Characteristics 3.1 Construction of phylogenetic tree for NBS-LRR genes in adzuki bean Building a phylogenetic tree is a very standard thing: usually, it involves comparing the NB-ARC conserved domains in the NB-LRR gene, applying the adjacency method, running an evolutionary analysis program, and finally creating a tree with supporting values. But although it seems standardized, in fact, every step may affect the details of classification. In leguminous plants like kidney beans, this method can indeed divide NBS-LRR into two main categories: TNL (TIR type) and CNL (CC type), with clear classification and corresponding structures (Wu et al., 2017). But don't forget that such an analysis mainly provides a framework. To understand the function later, more levels of verification are still needed. 3.2 Evolutionary comparison with NBS-LRR genes in other legume species Not all plants have the same NS-LRR gene; the differences among leguminous plants are quite significant. For example, there are 178 full-length NBS-LRR genes in kidney beans, among which the CNL type is significantly more, while the TNL type is relatively less. A similar phenomenon also exists in plants such as chili peppers (Wu et al., 2017; Liu et al., 2025). But looking at Arabidopsis thaliana in reverse, the proportion of TNL is much higher on its side. This indicates that different species actually have their own evolutionary rhythms (Shao et al.,
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