Euryale ferox (prickly waterlily) is an annual aquatic plant in tropical and subtropical regions of Southeast and East Asia and is the only species in the Euryale genus in the Nymphaeaceae family. It is cultivated as a nutritious food due to its high starch content; additionally it is also a common Chinese traditional medicine which can treat depression and diabetes mellitus (Ahmed et al., 2015; Wu et al., 2017; Huang et al., 2018; Liu et al., 2018). However, the productivity of E. ferox is threatened by various pests and microbial pathogens, including fungi, bacteria, and viruses. The diseases caused by these pathogens pose serious threats to not only plant growth and productivity but also the quality of the edible tissues. However, up to now, no functional disease resistance genes (R genes) have been cloned from E. ferox.

In order to cope with pathogen invasion, plants have developed sophisticated immune systems during the long-term evolutionary process. R genes in plants, which play a core role of the immune system, detect pathogens and trigger downstream resistance response, frequently accompanied by hypersensitive reactions (HR) (Cui et al., 2015; Jones et al., 2016; Wang and Chai, 2020). The nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes are the largest R gene family in plants. To date, more than 60% of the functional resistance genes cloned from angiosperms belong to the NBS-LRR gene family, which can provide resistance to bacteria, fungi, viruses, nematodes, and other pathogens (Kourelis and van der Hoorn, 2018). NBS-LRR genes originated in the common ancestor of all green plants, and diverged early into different subclasses (subfamilies) with different domain combinations along plant evolution (Xue et al., 2012; Shao et al., 2019). According to the N-terminal domain, angiosperm NBS-LRR genes can be divided into three subclasses: TIR-NBS-LRR (TNL), CC-NBS-LRR (CNL), and RPW8-NBS-LRR (RNL) (Shao et al., 2016). The majority of TNL and CNL proteins usually serve as pathogen detectors (Kourelis and van der Hoorn, 2018). NBS-LRR proteins are usually in a signaling-competent yet autoinhibited state, with LRR domain folding back onto the central NACHT domain (Hu et al., 2013). After recognizing pathogen effectors injected into the host cell, NBS-LRR proteins are activated, and NBS domain underwent conformational alterations, with exposed N-terminal domains to trigger downstream hypersensitive reactions, finally eliciting apoptosis of infected cells to suppress the pathogens transmission and proliferation (Andersen et al., 2018). The low expression of NBS-LRR genes are considered a normal stage and logically makes sense, otherwise activated HR would cause apoptosis all over plant organs and tissues. The RNL subclass can be divided into two small and ancient subclasses, namely ADR1 and NRG1, which can transduce immune signals and function downstream of ‘sensor NBS-LRR’ (sNLR) activation, so it was called ‘helper NBS-LRR’ (hNLR) (Collier et al., 2011; Shao et al., 2016; Zhong and Cheng, 2016). Furthermore, NRG1 proteins have advantages in TNL-mediated immune signaling (Peart et al., 2005; Qi et al., 2018; Castel et al., 2019; Wu et al., 2019). Recent studies indicated that as Ca2+-permeable channels, CNL and RNL proteins provoked immune response and cell death (Bi et al., 2021; Jacob et al., 2021).

Since the genome-wide study of NBS-LRR in Arabidopsis thaliana and rice early in the 21st century, NBS-LRR genes have been identified and analyzed in dozens of plant genomes (Meyers et al., 2003). These studies have found that the number of NBS-LRR genes varies greatly among different species, ranging from dozens to hundreds (Liu et al., 2021; Luo et al., 2012; Qian et al., 2017; Shao et al., 2014; Xue et al., 2020; Zhang et al., 2016; Zhou et al., 2020). These results provided abundant data resources for studying the structure, mechanism, and evolution of NBS-LRR genes. However, the current understanding of NBS-LRR gene evolution in angiosperms is mainly derived from studies of monocots and dicots, and the research on NBS-LRR genes in the basal angiosperms are limited to Amborella trichopoda (Shao et al., 2016). Recently, the genomic data of E. ferox provided not only important data resources for the study of disease resistance genes in this species but also a new research object for the in-depth study of the characteristics and evolution pattern of NBS-LRR genes in basal angiosperm species (Yang et al., 2020). In this study, NBS-LRR genes in the E. ferox genome were comprehensively identified and analyzed, which laid a foundation for the subsequent screening of disease resistance resources and the cloning of disease resistance genes and provided a theoretical basis for the evolution of NBS-LRR genes in angiosperms.

NBS-LRR genes consist of hundreds of members and are the largest family among R genes. In two-tier immune systems, the NBS-LRR gene family plays important roles in effector-triggered immunity. Revealing the profile of NBS-LRR genes in a species is very important, and by exploring the evolutionary patterns of NBS-LRR genes in the species and its close relatives, we can put these conclusions into practice (Meyers et al., 2003; Zhang et al., 2016). In the past 20 years, genome-wide identification and evolutionary analysis have been conducted in many angiosperms (Liu et al., 2021; Luo et al., 2012; Qian et al., 2017; Shao et al., 2014; Xue et al., 2020; Zhang et al., 2016; Zhou et al., 2020). It is noteworthy that of the more than 300 disease resistance genes, over 60% that have been cloned from different plants belong to the NBS-LRR gene family (Kourelis and van der Hoorn, 2018). Angiosperms inherited three subclasses of NBS-LRR genes: TNLs, CNLs, and RNLs. Studies have shown that proteins encoded by CNLs and TNLs function as receptors for pathogen recognition, and these two subclasses have expanded to various degrees during the long-term ‘arms race’ with pathogens (Shao et al., 2014).

In this study, the E. ferox genome was found to harbor only 131 NBS-LRR genes, consistent with a recent report. Liu et al. (2021) proposed that a convergent contraction of NBS-LRR genes occurred during the evolution of adaptation to an aquatic lifestyle, which may be the reason for the fewer NBS-LRR genes in E. ferox compared to other angiosperms. The structures of NBS-LRR genes are relatively diverse. Among four different IDs, MARCH1, also known as membrane-associated RING finger protein 1, is a membrane-anchored E3 ubiquitin ligase that mainly expressed in cells of the immune system (Bartee et al., 2004). MARCH1 also plays a regulatory role in T cell activation during immune responses, thus, we speculated that it regulates immune responses in plants.

NBS-LRR genes can expand via different duplication methods. Many studies have shown that tandem duplication is dominant in NBS-LRR gene expansion in most angiosperms (Shao et al., 2014; Qian et al., 2017; Zhou et al., 2020). We analyzed the duplication types of NBS-LRR genes in the E. ferox genome and found that there were 28 NBS-LRR genes generated by tandem duplication in the E. ferox genome, accounting for only 21% in total. This proportion is much lower than the 70–80% in the legume family (Shao et al., 2014). Furthermore, it was documented that E. ferox experienced two rounds of WGDs (Yang et al., 2020). Thus, we thought since tandem duplications were rare in its evolutionary history, the majority of E. ferox NBS-LRR genes are derived from segmental duplications, which is different from the situation in most other angiosperms, and the fewer number of tandemly duplicated genes may also be one of the reasons for the relatively fewer NBS-LRR genes in E. ferox. Additionally, the phylogenetic analysis of NBS-LRR genes in E. ferox and two other Nymphaeaceae species revealed that there were at least 122 ancestral NBS-LRR genes in the common ancestor of the three species, whereas only 131 NBS-LRR genes were found in the E. ferox genome, which further indicated a low level of NBS-LRR gene expansion in the E. ferox genome during its long-term evolution.

The comprehensive identification of NBS-LRR genes from genomic data is not only important for revealing the evolution of this important disease resistance gene family but also serves as an important basis for discovering and utilizing functional disease resistance genes in crops. Based on the analysis of NBS-LRR genes in the rice genome, Zhang et al. (2015) used targeted cloning of NBS-LRR genes in resistant rice varieties, and verified dozens of NBS-LRR genes that play important roles in blast resistance in rice. Witek et al. (2016) successfully cloned the late blight resistance gene Rpi-AMR3I from the resistance locus of Solanum americanum by target region sequencing and third-generation sequencing based on the analysis of NBS-LRR genes in the potato genome. These studies indicate that the genome-wide identification and analysis of NBS-LRR genes promotes the cloning of functional resistance genes in the studied species and related species. E. ferox is an important functional food and medicinal plant that has been widely cultivated in China since time immemorial. However, due to its characteristics of underwater flowering and pollination, research on germplasm innovation and disease resistance breeding has lagged behind research in other species. With the breakthrough of artificial hybrid technology for E. ferox in the 1980s and the development of tissue culture technology in recent years, it is possible to breed superior varieties of E. ferox by molecular breeding. E. ferox is vulnerable to a large number of diseases, so it is particularly important to develop molecular breeding in E. ferox.

In summary, the present study identified a complete set of 131 NBS-LRR genes in the genome of E. ferox. Although the quantity is significantly lower than that of most species in Solanaceae, Leguminosae, and Poaceae, it is roughly equivalent to that of Brassicaceae crops, and higher than those of Cucurbitaceae crops (Luo et al., 2012; Lin et al., 2013; Shao et al., 2014; Morata and Puigdomènech, 2017; Qian et al., 2017). The identification of NBS-LRR genes, analysis of sequence features, and revelation of chromosomal distribution pattern will provide important references for the molecular breeding of E. ferox. In addition, Nymphaeaceae contains many kinds of important flowering plants. The comparative analysis of NBS-LRR genes in these plants will also facilitate the molecular breeding of different species in the same genus.