Nitrogen is the main nutrient element that restricts the growth of crops and is one of the indispensable components in the synthesis of amino acids and proteins. Although the content of nitrogen in the air is as high as 78%, except for a few bacteria with nitrogenase, plants lacking nitrogenase do not have the ability to directly fix nitrogen. Therefore, it is usually necessary to apply chemical nitrogen fertilizer to provide nitrogen for plants in agriculture in order to maintain or increase crop yield. However, excessive use of nitrogen fertilizer often leads to a series of environmental problems, such as land degradation, groundwater pollution, river eutrophication and so on (Galloway et al., 2008); in undeveloped areas, the lack of chemical fertilizer leads to persistent low crop yield and quality, and even leads to population malnutrition (Good, 2018). With the rapid development of genomics and genome editing technology in recent years, scientist try to transfer the nitrogenase system in bacteria to crops through genetic engineering, so that plants can directly use elemental nitrogen in the air. However, transferring the nitrogenase system of prokaryotic bacteria into plants (such as chloroplasts or mitochondrial genomes) by engineering means still faces a series of challenges (Good, 2018).

Another way to achieve artificial nitrogen fixation is to utilize the symbiosis of plants and nitrogen-fixing bacteria. In angiosperms, some plants can symbiosis with nitrogen-fixing bacteria, which are phylogenic-distributed in four orders of Rosid I - Fabales, Fagales, Rosales and Cucurbitales, namely N-fixing Nodulation Clade (NFC) (Griesmann et al., 2018). The roots of these plants can interact with symbiotic nitrogen-fixing bacteria to form root nodules: plants supply carbon sources and other photosynthetic products to bacteria; bacteria fix nitrogen in the atmosphere and provide nitrogen sources and other nutrients for plants. The nodulation and nitrogen fixation system of angiosperms mainly includes legume-rhizobium nodulation symbiotic system represented by legume (Fabales), non-leguminous-actinomycete symbiotic nodulation nitrogen fixation system (Fagales, Rosales and Cucurbitales) and non-legume-rhizobium symbiotic system (Parasponia) (Paramasivan et al., 2001). Among them, legume-rhizobium symbiosis system is the most deeply studied for about 35 years, a series of genetic systems and functional gene have been established in model legume Medicago truncatula, Lotus japonicus and soybean Glycine max (Roy, Liu, Sekhar, & Crook, 2019). The elucidated biological process of model legume is nodulation is as follows: firstly, the free rhizobia in the soil recognize the flavonoids produced by plant roots, which stimulate the binding of NodD transcription factors to Nod gene promoter 5, thus producing specific lipid chitin oligosaccharides (Nod factor) as bacterial nodulation signals (Peck, Fisher, & Long, 2006). Subsequently, the plant senses the bacterial nodulation signal, which triggers the symbiotic signal related to the formation of nodule organs and the regulation of rhizobium (Suzaki & Kawaguchi, 2014). In the epidermis, symbiotic signals guide the curl of root hairs, and then the division of root cortical cells induces the formation of nodule primordia. The rhizobium at the end of the root hair invades the cortical cells through the specialized intracellular tube-like structure of the plant, and then differentiates into bacteroides, which can fix nitrogen (Roux & Rodde, 2014). As the root nodulation nitrogen fixation system belongs to a single origin, comparing with legume-rhizobium symbiotic system, non-leguminous symbiotic system shared many similar genetic symbiosis factors. In addition, each of the four orders of NFC has nodulating plant and non-nodulating plants, which are dispersed in the evolutionary tree, forming convergent evolutionary events of multiple loss after a single origin (Griesmann et al., 2018).

Nodulation plants and nitrogen-fixing microorganisms have formed a complex and precise regulation system in the long-term interaction and co-evolution compared with non-nodulating plants. First, the regulation of nitrogen fixation process is closely related to the content of nitrogen in soil. The process of nitrogen fixation consumes a lot of energy, so when there is sufficient nitrogen in the soil, plants inhibit its nitrogen fixation so as to reduce energy waste. Excessive nitrogen salts in soil can affect the whole process of nodulation and nitrogen fixation, including inhibition of flavonoid production, rhizobium infection, nodule occurrence and growth, and nitrogen fixation; high nitrogen can also accelerate nodule senescence (Nishida & Suzaki, 2018). Secondly, the process of nitrogen fixation is regulated by oxygen, which will cause damage to nitrogenase, and nitrogen fixation requires oxygen to support respiration and provide a lot of energy. The endodermis of plant nodules evolves Oxygen Diffusion Barriers (OBD), its strong respiration can keep the central cells of root nodules in a state of hypoxia and protect nitrogenase from damage. When the transient oxygen content in roots increased, OBD reacted quickly through a series of molecular regulation to restore the nitrogen fixation activity of nodules and nitrogenase (Avenhaus et al., 2016). Finally, the interaction between nitrogen-fixing bacteria and plants needs to overcome the plant immune system (Zipfel & Oldroyd, 2017). Plants have evolved special receptor proteins to recognize nodulation factors of rhizobium; nodulation factors produced by rhizobium can inhibit plant immune system (Liang et al., 2013); and some immune elements of plants are involved in both immune response and nitrogen fixation process. and then play a role in balancing the two systems (Pan & Wang, 2017). Thus, function element of nitrogen fixing can be mined through the systematic design of comparative transcriptomes in roots and nodules with different nitrogen or rhizobium treatments.

For a long time, the research on N-fixing nodulation has mainly focused on a few key genes in genomic coding regions such as nodulation factor recognition or nitrogen fixation regulation (only accounting for about 3% of the genome). The acquisition of these key genes alone can not guarantee non-nodulating plants obtain the ability of nitrogen fixation. With the rapid development of comparative and functional genomics, more and more studies have found that non-coding sequences play a key role in the process of biological nitrogen fixation, such as promoters , intronic cis-regulatory element, small RNAs and lincRNAs. However, the functional association of these non-coding regulatory elements with known regulatory systems is still unclear. Thus, "deep annotation" and large-scale comparative evolutionary genomics research will bring us a systematic understanding of the genetic regulatory network and evolution mechanism of nodulating plant. In this project, we are going to use multi-layer comparative genomics to study how variance of genes, expression and non-coding regulatory elements contribute to the diversity of plant nodulation. which will provide a theoretical basis for genetic engineering of non-nitrogen-fixing organisms in the future.