**2. Breeding and selection of** *Rj* **gene-accumulated soybean**

### **2.1 Phenotypic analysis of "Bonminori" × "Fukuyutaka" F3 seeds**

To select *Rj* gene-accumulated soybean lines with the *Rj*2*Rj*3*Rj*4 genotype, 157 F3 seeds from the experimental field of the Faculty of Agriculture, University of Miyazaki (31°49′41″N, 131°24′45″E), were subjected to the inoculation test. *B*. *japonicum* Is−1 and *B*. *japonicum* Is−34, which exhibit incompatibility with the *Rj*<sup>2</sup> and *Rj*4 genotypes of soybean, respectively, were used as the inoculum strains. The strains were cultured in 1 mL of HEPES-MES (HM) medium [46] supplemented with 0.1% l-arabinose [47] for 3–5 days in the dark at 28°C. The bacterial cultures were then diluted with sterile distilled water to 106 cells mL−1 and mixed with the respective strain. Culture pots (1 L) were filled with vermiculite containing aqueous N-free nutrient solution (40% v/v) [48] and autoclaved at 121°C for 20 min. F3 seeds were sterilized using 70% ethanol for 30 s and diluted sodium hypochlorite solution (0.25% available chlorine) for 3 min and then washed with sterile distilled water. Five seeds of each line were sown per pot, inoculated with 1 mL of diluted bacterial culture per seed, and cultivated for 3–4 weeks in a growth chamber (day, 28°C for 16 h; night, 25°C for 8 h), with weekly supply of sterile distilled water. After cultivation for 3–4 weeks, the presence or absence of nodulation on soybean roots was observed.

### **2.2 Selection of non-nodulation phenotype using the inoculation test**

Theoretically, all F1 lines obtained by crossing "Bonminori" and "Fukuyutaka" should present the *Rj*2*Rj*2*Rj*4*Rj*4 genotype, and according to Mendel's law, the probability of F2 lines exhibiting this genotype is 1/16. In the F3 lines, individuals with the *Rj*2*Rj*2*Rj*4*Rj*4 genotype can be obtained by self-fertilization of F2 individuals with the *Rj*2*rj*2*Rj*4*Rj*4, *Rj*2*Rj*2*Rj*4*rj*4, and *Rj*2*rj*2*Rj*4*rj*4 genotypes. To select individuals with the *Rj*2*Rj*2*Rj*4*Rj*4 genotype, we subjected 153 lines of F3 seeds, excluding four lines of seeds that had decayed during storage in the refrigerator, to inoculation test. The appearance of soybean roots during the inoculation test is shown in **Figure 1**. Dominant homozygous plants, such as those with the *Rj*2*Rj*2*Rj*4*Rj*4 genotype, did not form root nodules (**Figure 1a**). We proceeded with the screening based on this phenotype and selected eight lines from the 153 lines (**Table 1**). These eight lines were grown for

### **Figure 1.**

*Appearance of nodulation following the inoculation of Bradyrhizobium japonicum Is−1 and Is−34. Soybean roots with (a) and without (b) nodulation restriction.*


*Among the five seeds sown for selection, lines that did not form root nodule on all plants were classified as "non-nodulation," lines that formed root nodule more than one of the five plants were classified as "non-nodulation or nodulation," and lines that formed root nodule all plants classified as "nodulation."*

### **Table 1.**

*Nodulation phenotypes of F3 seeds.*

several years. Three plants differing in terms of the flowering and ripening periods by approximately 1 week each were detected and selected for further cultivation.
