**Abstract**

Some soybean varieties harbor the *Rj* genes, which regulate nodulation by preventing infection and nodulation by specific rhizobial strains. Soybean genotypes carrying several *Rj* genes may enhance the occupancy of useful bradyrhizobia, which exhibit potent nitrogen fixation ability and exhibit nodulation compatible with the *Rj* genotype of soybean. Therefore, we bred soybean lines presenting the *Rj*2*Rj*3*Rj*4 genotype by crossing the Japanese cultivars "Bonminori" (*Rj*2*Rj*3) and "Fukuyutaka" (*Rj*4) and studied the effects of *Rj* gene accumulation on productivity. To investigate yield components, three *Rj* gene-accumulated soybean lines (B × F − E, B × F − M, and B × F − L) and three soybean cultivars ("Enrei," "Sachiyutaka," and "Fukuyutaka") were cultivated in 2016 and 2017. Pod and seed number and yield were the highest in B × F − M. The occupancy rate of isolates in cluster of *Bradyrhizobium diazoefficiens* USDA 110T carrying the hydrogen uptake genes tended to be lower in the *Rj*2*Rj*3*Rj*<sup>4</sup> soybean lines than in "Sachiyutaka" and "Fukuyutaka." Additionally, the occupancy rate of this cluster was positively correlated with yield. Therefore, promoting infection by bradyrhizobial strains carrying the hydrogen uptake genes may improve soybean productivity. Moreover, the *Rj*2*Rj*3*Rj*4 genotype of soybean may be inoculated with *B*. *diazoefficiens* USDA 110T , which is not restricted by the *Rj*2 gene, to further enhance soybean productivity.

**Keywords:** soybean, *Rj* gene, breeding, yield components, infection tendency

### **1. Introduction**

Soybean (*Glycine max* (L.) Merr.) is one of the most important legume crops in the world, including Japan. According to the information on soybean production and consumption published by the Japanese Ministry of Agriculture, Forestry and Fisheries (MAFF), soybean yield in the country is 166 kg 10 a−1, which is lower than those in major producing countries, including the United States (358 kg 10 a−1), Brazil (342 kg 10 a−1), Argentina (309 kg 10 a−1), and China (188 kg 10 a−1) [1]. To improve this lower productivity, breeding of high-yielding soybean and improvement of cultivation

techniques, such as pest control, field management, and plantation methods, have been extensively studied. One of the cultivation techniques is the inoculation of rhizobia, which exhibit potent nitrogen-fixing capacity, during soybean plantation.

As a leguminous plant, soybean roots bear nodules formed as a result of infection by nodulating rhizobia, which perform symbiotic nitrogen fixation, and the plant acquires atmospheric nitrogen in the form of ammonia through these root nodules. Major soybean-nodulating rhizobia include *Bradyrhizobium japonicum*, *Bradyrhizobium diazoefficiens*, *Bradyrhizobium elkanii*, and *Sinorhizobium* (=*Ensifer*) *fredii* [2–7]. In addition to these, *Bradyrhizobium yuanmingense*, *Bradyrhizobium liaoningense, Sinorhizobium xinjiangense*, and *Mesorhizobium tianshanense* have been reported as soybean-nodulating rhizobial species [8–14]. *B*. *diazoefficiens* USDA 110T is a symbiont possessing a hydrogen uptake (Hup) system that recycles H2 produced as a by-product of nitrogenase activity, thereby increasing nitrogen fixation efficiency [15–17]. The inoculation of bradyrhizobia possessing this system, such as *B*. *diazoefficiens* Hup+ strains, enhances the productivity of legume crops [18]. However, the efficiency of inoculated rhizobia with high nitrogen fixation ability remains poor in the field, because they cannot compete with indigenous soybean-nodulating rhizobia in the soil. To solve this problem, the ecology of indigenous soybean-nodulating rhizobia in terms of genetic diversity and compatibility with the host soybean must be elucidated.

*Rj* or *rj* are the well-known host genes that regulate soybean nodulation, and non-*Rj*, *rj*1, *Rj*2, *Rj*3, *Rj*4, and *Rfg1* genotypes of soybean have been confirmed to exist naturally [19–24]. In addition to these, *Rj* genotypes, including *rj*5, *rj*6, and *rj*7, have been developed through experimental mutagenesis [25–31]. The *Rj*2, *Rj*3, *Rj*4, and *Rfg1* genotypes are known to restrict nodulation by specific strains of *Bradyrhizobium* or *Sinorhizobium* species. Meanwhile, the *Rj*2, *Rj*3, *Rj*4, and *Rfg1* genotypes restrict nodulation by *B*. *diazoefficiens* USDA 122, *B*. *elkanii* USDA 33, *B*. *elkanii* USDA 61, and *Sinorhizobium fredii* USDA 257. Furthermore, *B. japonicum* Is−1 and Is−34 exhibit incompatibility with the *Rj*2 and *Rj*4 genotypes, respectively [32]. The *rj*1, *rj*5, and *rj*6 genotypes restrict nodulation by all soybean-nodulating rhizobial strains. The *rj*7 genotype developed through ethyl methane sulfonate (EMS)-induced mutagenesis is a "hypernodulation" genotype, which can form abundant nodules [33]. The *Rj*2/*Rfg1* gene encodes a member of the Toll-interleukin receptor–nucleotide-binding site–leucine-rich repeat (TIR–NBS–LRR) class of plant resistance (R) proteins, which confer resistance against microbial pathogens through an effector-triggered immune (ETI) response [34]. Furthermore, the amino acid determinant of the *Rj*2 genotype in cultivated and wild soybeans has been reported [35]. The *Rj*4 gene encodes a thaumatin-like protein (TLP), classified as pathogenesis-related protein 5 (PR5). PR proteins are induced by pathogen attack and involved in host resistance [36, 37]. In addition, the type III secretion system (T3SS) structural gene in *B*. *elkanii* USDA 61 and *B*. *japonicum* Is−34 is involved in the restriction of nodulation in the *Rj*4 genotype of soybean [38, 39].

The compatibility and preference for nodulation by bradyrhizobial strains of soybean cultivars and varieties exhibiting the *Rj* genotype have been investigated [32], and the *Rj*2*Rj*3*Rj*4 genotype lines, in which the *Rj* genes are accumulated, have been bred by crossing the soybean cultivars "IAC-2" (*Rj*2*Rj*3) and "Hill" (*Rj*4) [40]. The *Rj*2*Rj*3*Rj*4 genotype is superior to the non-*Rj*, *Rj*2*Rj*3, and *Rj*4 genotypes in terms of the efficiency of nodulation by inocula with potent nitrogen fixation ability [41]. In addition, the community structure of indigenous soybean-nodulating bradyrhizobia was significantly different across five *Rj* genotypes (non-*Rj*, *Rj*2*Rj*3, *Rj*3, *Rj*4, and *Rj*2*Rj*3*Rj*4) [42]. Furthermore, the *Rj*2*Rj*3 and *Rj*2*Rj*3*Rj*4 genotypes presented a

*Breeding of* Rj *Gene-Accumulated Soybean Genotypes and Their Availability for Improving… DOI: http://dx.doi.org/10.5772/intechopen.102833*

higher occupancy of the indigenous soybean-nodulating bradyrhizobial cluster of *B*. *diazoefficiens* USDA 110T than the non-*Rj*, *Rj*3, and *Rj*4 genotypes, regardless of the cultivation temperature [43]. Thus, the availability of the *Rj*2*Rj*3*Rj*4 genotype of soybean has been reported. Since the *Rj*2*Rj*3*Rj*4 genotype of soybean has been produced by crossing foreign cultivars, *Rj* gene-accumulated soybean cultivars that match the needs of Japanese consumers and producers must be developed. Therefore, we bred the *Rj*2*Rj*3*Rj*4 genotype of soybean by crossing the Japanese soybean cultivars "Bonminori" (*Rj*2*Rj*3) and "Fukuyutaka" (*Rj*4). According to the information on the development and diffusion of new soybean cultivars published by the Japanese MAFF, "Fukuyutaka" is the most cultivated soybean cultivar in the country [44], and this cultivar was registered in 1980 [45].

In this chapter, we describe breeding and selection processes, shoot growth, yield components, and infection tendency of useful bradyrhizobia of *Rj* gene-accumulated soybean genotypes produced by crossing Japanese cultivars.
