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Breeding of Rj Gene-Accumulated Soybean Genotypes and Their Availability for Improving Soybean Productivity

Written By

Sokichi Shiro and Yuichi Saeki

Submitted: January 3rd, 2022Reviewed: January 24th, 2022Published: March 22nd, 2022

DOI: 10.5772/intechopen.102833

IntechOpen
Soybean - Recent Advances in Research and ApplicationsEdited by Takuji Ohyama

From the Edited Volume

Soybean - Recent Advances in Research and Applications [Working Title]

Prof. Takuji Ohyama, Dr. Yoshihiko Takahashi, Dr. Norikuni Ohtake, Dr. Takashi Sato and Dr. Sayuri Tanabata

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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 Rj2Rj3Rj4 genotype by crossing the Japanese cultivars “Bonminori” (Rj2Rj3) and “Fukuyutaka” (Rj4) 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 Rj2Rj3Rj4 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 Rj2Rj3Rj4 genotype of soybean may be inoculated with B. diazoefficiens USDA 110T, which is not restricted by the Rj2 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, 3, 4, 5, 6, 7]. In addition to these, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense, Sinorhizobium xinjiangense, and Mesorhizobium tianshanensehave been reported as soybean-nodulating rhizobial species [8, 9, 10, 11, 12, 13, 14]. B. diazoefficiensUSDA 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, 16, 17]. The inoculation of bradyrhizobia possessing this system, such as B. diazoefficiensHup+ 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.

Rjor rjare the well-known host genes that regulate soybean nodulation, and non-Rj, rj1, Rj2, Rj3, Rj4, and Rfg1genotypes of soybean have been confirmed to exist naturally [19, 20, 21, 22, 23, 24]. In addition to these, Rjgenotypes, including rj5, rj6, and rj7, have been developed through experimental mutagenesis [25, 26, 27, 28, 29, 30, 31]. The Rj2, Rj3, Rj4, and Rfg1genotypes are known to restrict nodulation by specific strains of Bradyrhizobiumor Sinorhizobiumspecies. Meanwhile, the Rj2, Rj3, Rj4, and Rfg1genotypes restrict nodulation by B. diazoefficiensUSDA 122, B. elkaniiUSDA 33, B. elkaniiUSDA 61, and Sinorhizobium frediiUSDA 257. Furthermore, B. japonicumIs−1 and Is−34 exhibit incompatibility with the Rj2 and Rj4 genotypes, respectively [32]. The rj1, rj5, and rj6 genotypes restrict nodulation by all soybean-nodulating rhizobial strains. The rj7 genotype developed through ethyl methane sulfonate (EMS)-induced mutagenesis is a “hypernodulation” genotype, which can form abundant nodules [33]. The Rj2/Rfg1gene 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 Rj2 genotype in cultivated and wild soybeans has been reported [35]. The Rj4 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. elkaniiUSDA 61 and B. japonicumIs−34 is involved in the restriction of nodulation in the Rj4 genotype of soybean [38, 39].

The compatibility and preference for nodulation by bradyrhizobial strains of soybean cultivars and varieties exhibiting the Rjgenotype have been investigated [32], and the Rj2Rj3Rj4 genotype lines, in which the Rjgenes are accumulated, have been bred by crossing the soybean cultivars “IAC-2” (Rj2Rj3) and “Hill” (Rj4) [40]. The Rj2Rj3Rj4 genotype is superior to the non-Rj, Rj2Rj3, and Rj4 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 Rjgenotypes (non-Rj, Rj2Rj3, Rj3, Rj4, and Rj2Rj3Rj4) [42]. Furthermore, the Rj2Rj3 and Rj2Rj3Rj4 genotypes presented a higher occupancy of the indigenous soybean-nodulating bradyrhizobial cluster of B. diazoefficiensUSDA 110T than the non-Rj, Rj3, and Rj4 genotypes, regardless of the cultivation temperature [43]. Thus, the availability of the Rj2Rj3Rj4 genotype of soybean has been reported. Since the Rj2Rj3Rj4 genotype of soybean has been produced by crossing foreign cultivars, Rjgene-accumulated soybean cultivars that match the needs of Japanese consumers and producers must be developed. Therefore, we bred the Rj2Rj3Rj4 genotype of soybean by crossing the Japanese soybean cultivars “Bonminori” (Rj2Rj3) and “Fukuyutaka” (Rj4). 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 Rjgene-accumulated soybean genotypes produced by crossing Japanese cultivars.

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2. Breeding and selection of Rjgene-accumulated soybean

2.1 Phenotypic analysis of “Bonminori” × “Fukuyutaka” F3 seeds

To select Rjgene-accumulated soybean lines with the Rj2Rj3Rj4 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. japonicumIs−1 and B. japonicumIs−34, which exhibit incompatibility with the Rj2 and Rj4 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 Rj2Rj2Rj4Rj4 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 Rj2Rj2Rj4Rj4 genotype can be obtained by self-fertilization of F2 individuals with the Rj2rj2Rj4Rj4, Rj2Rj2Rj4rj4, and Rj2rj2Rj4rj4 genotypes. To select individuals with the Rj2Rj2Rj4Rj4 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 Rj2Rj2Rj4Rj4 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 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.

Figure 1.

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

Phenotypes of F3 seedsNumber of soybean lines
non-nodulation8
non-nodulation or nodulation63
nodulation82
not tested4
Total157

Table 1.

Nodulation phenotypes of F3 seeds.

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.”

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3. Evaluation of shoot growth and yield components of Rjgene-accumulated soybean

3.1 Experimental site, design, and tested soybean variety

A 2-year field trial was conducted in 2016 and 2017 in the experimental field of the Agricultural Science Section, Education and Research Center for Biological Resources, Faculty of Life and Environmental Science, Shimane University, Japan (35°30′55″N, 133°06′36″E). The experimental sites were located at 35°30′60″N, 133°06′35″E in 2016 and 35°31′02″N, 133°06′40″E in 2017. Both experimental fields had gray lowland soil (paddy conversion fields). Soil pH (H2O) and electrical conductivity (mS cm−1) were respectively 6.42 and 0.10 in 2016 and respectively 6.72 and 0.06 in 2017. Before sowing, nitrogen, potassium, and phosphorus were applied at doses of 40, 100, and 100 kg ha−1, respectively. To correct soil pH, magnesium lime was applied at the dose of 1000 kg ha−1. The experiment followed the split-plot design with three replicates. Three soybean cultivars, namely “Enrei,” “Sachiyutaka,” and “Fukuyutaka,” as well as F10 or F11 plants of three Rjgene-accumulated soybean lines with different flowering and ripening periods, namely B × F − E, B × F − M, and B × F − L, were tested. “Enrei” and “Sachiyutaka” were registered in 1971 and 2001, respectively [45]. “Enrei” presents the rj4 genotype [37]. “Sachiyutaka” may present the Rj4 genotype, because it is bred through backcrossing “Enrei” with F2 plants from a cross “Enrei” and “Fukuyutaka” [45]. All soybean seeds were sown at a depth of 3–4 cm on June 20, 2016, and June 21, 2017, respectively, and the planting density was 11.1 and 10.1 plants m−2 in 2016 and 2017, respectively.

3.2 Data collection and analysis

Soybean growth was evaluated during the flowering and harvest periods. Samples were collected by from 10 consecutive plots per replicate. During the flowering period, plant height, node number, branch number, stem and leaf dry weight, and main culm dry weight (2017 only) were measured. During the harvest period, plant height, node number, shoot dry weight, pod number, seed number, 100-seed weight, and yield were measured. Plant dry weight was measured after drying at 70°C for over 72 h in a drying apparatus. All statistical analyses were performed using R version 4.0.3 [49]. Soybean growth parameters during the flowering period were analyzed using Tukey’s honestly significant difference (HSD) test for multiple comparisons using the R package “multcomp.” Soybean yield components were subjected to two-way analysis of variance using anovakun version 4.8.5 [50]. Meteorological data during soybean cultivation were collected from past information provided by the Japan Weather Association (Table 2).

YearmonthTemperature (°C)Precipitation (mm)Sunshine duration (h)
minimummaximummean
2016Jun.19.026.822.3166.04.9
Jul.23.530.826.677.05.7
Aug.23.632.227.2140.57.7
Sep.20.726.723.3293.02.8
Oct.15.322.318.5103.53.5
Nov.8.816.212.2120.03.2
2017Jun.16.525.920.986.57.3
Jul.24.831.527.6168.55.6
Aug.24.431.527.4141.56.8
Sep.18.526.322.1214.54.9
Oct.14.321.017.5358.03.6
Nov.6.715.711.193.04.6

Table 2.

Meteorological data during soybean cultivation in 2016 and 2017.

Values indicate monthly averages, and each value was calculated based on meteorological data provided by the Japan Weather Association.

3.3 Growth and yield of Rjgene-accumulated soybean

The results of soybean growth during the flowering period in 2017 are presented in Table 3. The measurements during the flowering period were conducted on August 10, 2017, for B × F − E and “Enrei”; August 17, 2017, for B × F − M and “Sachiyutaka”; and August 26, 2017, for B × F − L and “Fukuyutaka.” The plant height of Rjgene-accumulated soybean lines tended to increase in the order B × F − E < B × F − M < B × F − L, indicating dependence on the lateness of the flowering period. Similarly, the plant height of other soybean cultivars tended to increase in the order of “Enrei” < “Sachiyutaka” < “Fukuyutaka,” indicating dependence on the lateness of the flowering period. Branch number was the highest in “Fukuyutaka” and the lowest in “Enrei.” In addition, among the Rjgene-accumulated soybean lines, plant height tended to be higher in B × F − L than in the other lines, although the difference was not significant. There were no significant differences in shoot dry weight among the cultivars, although “Fukuyutaka,” B × F − M, and “Enrei” showed higher values in that order.

CultivarPlant height (cm plant−1)Node number (No. plant−1)Branching number (No. plant−1)Main culm dry weight (g m−2)Stem and leaf dry weight (g m−2)Shoot dry weight (g m−2)
BxF–E66.2 ab13.56.1 ab13.023.136.1
BxF–M67.9 ab13.16.5 ab15.325.140.4
BxF–L74.9 b13.67.2 bc16.722.439.0
Enrei53.7 a11.54.5 a12.227.039.2
Sachiyutaka57.3 a12.36.4 ab12.722.635.4
Fukuyutaka64.5 ab12.29.2 c17.423.841.2
ANOVA*ns***nsnsns

Table 3.

Growth of soybean cultivars during the flowering period in 2017.

Values are presented as the means of three replicates. *p < 0.05, ***p < 0.001, and ns = not significant. Different letters indicate significant differences (Tukey’s HSD test) at p < 0.05 for different soybean cultivars.

The results of yield components of soybean cultivars during the harvest period in 2016 and 2017 are presented in Table 4. In ANOVA, all yield components, except 100-seed weight, significantly differed between years and among cultivars. Specifically, pod and seed number and yield were significantly higher in 2016 than in 2017. Conversely, plant height, node number, and shoot dry weight were significantly higher in 2017 than in 2016. Based on the average values of the 2 years, pod and seed number in B × F − M was significantly higher than that in the other cultivars. Moreover, the yield of B × F − M and “Sachiyutaka” was significantly higher than that of B × F − E, B × F − L, and “Enrei.” Furthermore, 100-seed weight of “Sachiyutaka” was significantly higher than that of the other cultivars, except “Fukuyutaka.” Plant height and shoot dry weight of B × F − L tended to be higher than those of the other cultivars. The interaction between year and cultivar was detected for all test parameters, except seed number and yield. Therefore, multiple comparison analysis was performed among 12 cohorts for each test item, and the results are shown in Figure 2. Briefly, pod and seed number and yield were lower in all soybean cultivars in 2017 than in 2016. Furthermore, pod and seed number of B × F − E, B × F − M, “Enrei,” and “Sachiyutaka” decreased significantly. While the yield of “Sachiyutaka” decreased significantly, that of B × F − E, B × F − M, B × F − L, “Enrei,” and “Fukuyutaka” tended to decrease, albeit without significant differences. “Enrei,” “Sachiyutaka,” and “Fukuyutaka” are soybean cultivars that are suitable or possible to cultivate in the Chugoku region of Japan, including Shimane prefecture, where the cultivation test was conducted in the present study [51, 52]. Additionally, pod and seed numbers are the most important soybean yield components, which are primarily determined during the period from before and after flowering to pod set, including the beginning of the seed filling period [51]. However, increasing temperature during the growing season can negatively affect soybean leaf photosynthesis, growth, flowering, pod and seed number, and yield [52, 53]. In the present study, the monthly mean maximum temperature in August during the flowering period of soybean was 32.2°C in 2016 and 31.5°C in 2017 (Table 2). Specifically, in early August of 2017, when B × F − E and “Enrei” were flowering, the temperature remained above 35°C for 3 consecutive days. Furthermore, in late August of 2017, the temperature remained above 32°C for 4 consecutive days. Additionally, in October 2017, nearly 3.5 times the amount of precipitation in 2016 was recorded (Table 2). Soybean pod and seed number and yield in 2017 were significantly lower than the values in 2016 due to the effects of these meteorological factors (Table 4). Moreover, the 100-seed weight of B × F − M was lower than that of “Sachiyutaka” and “Fukuyutaka” (Table 4). Therefore, backcrossing with these cultivars is expected to produce soybean cultivars with larger seeds and higher yield.

YearCultivarPlant height (cm plant−1)Node number (No. plant−1)Shoot dry weight (g m−2)Pod number (No. m−2)seed number (No. m−2)100-seed weight (g)Yield (g m−2)
2016BxF–E50.311.1251.61426.7937.023.9214.4
BxF–M51.111.3298.51927.02276.822.9514.5
BxF–L71.112.1336.61036.01372.923.5325.8
Enrei34.39.5215.7542.6421.422.696.1
Sachiyutaka46.112.1300.31531.11742.933.8584.6
Fukuyutaka56.312.1267.11057.31377.329.8407.4
2017BxF–E67.413.4251.8318.8218.924.653.8
BxF–M69.413.8432.81180.71477.423.6348.2
BxF–L71.013.3424.1784.01037.720.7214.6
Enrei56.212.8340.0101.056.229.716.7
Sachiyutaka55.212.1344.2704.1974.429.3285.8
Fukuyutaka70.516.1467.9934.11245.625.0311.7
201651.511.4278.31253.41354.726.1357.1
201764.913.6376.8670.4835.025.5205.1
BxF–E58.8 bc12.2 a251.7 a872.7 b578.0 a24.2 a134.1 a
BxF–M60.3 bc12.5 ab365.7 b1553.8 c1877.1 c23.2 a431.4 c
BxF–L71.0 d12.7 ab380.4 b910.0 b1205.3 b22.1 a270.2 b
Enrei45.2 a11.1 a277.8 a321.8 a238.8 a26.1 a56.4 a
Sachiyutaka50.6 ab12.1 a322.3 ab1117.6 b1358.6 b31.6 b435.2 c
Fukuyutaka63.4 cd14.1 b367.5 b995.7 b1311.5 b27.4 ab359.6 bc
ANOVAYear (Y)***************ns***
Cultivar (C)*********************
Y x C*******ns**ns

Table 4.

Yield components of soybean cultivars in 2016 and 2017.

Values are presented as the means of three replicates. *p < 0.05, **p < 0.01, ***p < 0.001, and ns = not significant. Different letters indicate significant differences (Tukey’s HSD test) at p < 0.05 for different soybean cultivars.

Figure 2.

Yield components for each soybean cultivar in 2016 and 2017 growing seasons. Values are presented as the mean ± SE of three replicates. Different letters indicate significant differences (Tukey’s HSD test) atp < 0.05. (a) Plant height. (b) Node number. (c) Shoot dry weight. (d) Pod number. (e) Seed number. (f) 100-seed weight. (g) Yield.

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4. Occupancy of indigenous soybean-nodulating bradyrhizobia

4.1 Sampling of soybean root nodules and isolation of nodulating bradyrhizobia

The nodules used in the present experiment were collected from soybean roots at the flowering stage in 2017, as described in Section 3.2. The nodules were surface-sterilized with 70% ethanol for 3 min and diluted sodium hypochlorite solution (0.25% available chlorine) for 30 min, followed by washing with sterile distilled water. After washing, 24 nodules were randomly collected and transferred to 1.5 mL microcentrifuge tubes. Each nodule was homogenized in sterile distilled water and streaked onto a yeast extract–mannitol agar (YMA) plate [54]; to isolate a single colony per nodule, the plates were incubated for 5–7 days in the dark at 28°C. A total of 144 isolates were obtained from six soybean plants and used for PCR-restriction fragment length polymorphism (RFLP) analysis of the 16S–23S rRNA gene internal transcribed spacer (ITS) region.

4.2 PCR-RFLP analysis of the 16S: 23S rRNA gene ITS region

For DNA extraction, each isolate was cultured in 1.5 mL HM medium supplemented with 0.1% l-arabinose for 5–7 days in the dark at 28°C. Total DNA for use as the PCR template was extracted from the isolates in BL extraction buffer, as described previously [42] based on the method reported by Hiraishi et al. [55]. As reference strains, B. japonicumUSDA 6T; B. diazoefficiensUSDA 110T; and B. elkaniiUSDA 46, 76T, and 94 were used [16]. Total DNA of the reference strains for use as the PCR templates was also extracted using the same method [42, 55].

The 16S–23S rRNA gene ITS region was PCR-amplified using TaKaRa Ex Taq® Hot Start Version (TaKaRa Bio, Shiga, Japan) and the ITS primer set (BraITS-F: 5′-GACTGGGGTGAAGTCGTAAC-3′ and BraITS-R1: 5′-ACGTCCTTCATCGCCTC-3′) [56]. The PCR cycle comprised a pre-run at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min. This temperature control sequence was repeated for 30 cycles, followed by a final run at 72°C for 10 min. RFLP analysis of the 16S–23S rRNA gene ITS region was performed using the restriction enzyme MspI (TaKaRa Bio, Shiga, Japan). The PCR product was digested with the restriction enzyme at 37°C for 16 h, and the restricted fragments were separated using 3% agarose gel electrophoresis.

4.3 Occupancy of infected indigenous bradyrhizobia carrying hupSLgenes

Eighty-seven indigenous soybean-nodulating bradyrhizobial isolates belonging to the cluster B. diazoefficiensUSDA 110T were investigated for the presence of hupSLgenes. PCR templates of the test isolates, obtained as described in Section 4.2, were used. The PCR amplification for hupSLwas performed using KAPA Taq® Extra Hot Start ReadyMix with dye (Kapa Biosystems, USA) and the hupSLprimer set (hupS-F261: 5′-TCGAACAGGCGTTGTAAGTG-3′, hupS-R830: 5′-TCGACTACGACGACACCATC-3′, hupL-F962:5′-TCGGGCAGATAGACCATTTC-3′ and hupL-R1632: 5′-GGGATCGAAGTGATCCTGAA-3′). The PCR cycle comprised a pre-run at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 1 min. This temperature control sequence was repeated for 30 cycles, followed by a final run at 72°C for 1 min. The PCR products were electrophoresed on a 2% agarose gel to confirm amplification.

4.4 Occupancy of soybean-nodulating bradyrhizobia carrying hupSLgenes

The occupancy rate of indigenous bradyrhizobia infecting each soybean cultivar is presented in Table 5. Based on the fragment patterns obtained from PCR–RFLP analysis, the indigenous isolates with the similar patterns as the five reference strains, namely B. japonicumUSDA 6T; B. diazoefficiensUSDA 110T; and B. elkaniiUSDA 46, 76T, and 94 were defined as Bj6, Bd110, Be46, Be76, and Be94, respectively. Since cluster Bd110 includes isolates carrying the hupSLgenes, such as B. diazoefficiensUSDA 110T and 122 [16], the occupancy rate of these isolates was also determined. PCR analysis targeting the hupSLgenes revealed amplicons exhibiting zero to two bands. The isolates exhibiting two amplification products corresponding to hupSand hupLwere defined as hupS+L+, those exhibiting a single amplification product corresponding to hupLwere defined as hupSL+, and those exhibiting no amplification products were defined as hupSL. In B × F − E, Bd110 isolates exhibiting hupSL+ were the most dominant (62.5%), followed by Bd110 isolates exhibiting hupS+L+ (12.5%) and Bj6 isolates (12.5%). In B × F − M, Bj6 isolates were the most dominant (70.8%), followed by Bd110 isolates exhibiting hupSL+ (16.7%). In B × F − L, Bd110 isolates exhibiting hupSL+ (45.8%) were the most dominant, followed by Bd110 isolates exhibiting hupS+L+ (25.0%). In “Enrei,” Bj6 isolates were the most dominant (83.4%), followed by Bd110 isolates exhibiting hupSL+ (8.3%) or hupS+L+ (8.3%). In “Sachiyutaka,” Bd110 isolates exhibiting hupS+L+ (39.1%) were the most dominant, followed by Bd110 isolates exhibiting hupSL+ (34.8%). In “Fukuyutaka,” Bd110 isolates exhibiting hupS+L+ (37.5%) were the most dominant, followed by Bj6 (25.0%) isolates. “Sachiyutaka” and “Fukuyutaka” with the Rj4 genotype tended to present a higher occupancy rate of Bd110 isolates exhibiting hupS+L+. In contrast, soybean lines with the Rj2Rj3Rj4 genotype tended to present a lower occupancy rate of Bd110 isolates exhibiting hupS+L+. These results may be explained by the effect of the presence of Rj2. Rj2 restricts B. diazoefficiensUSDA 122 [32]. Indigenous bradyrhizobial isolates, such as B. diazoefficiensUSDA 110 T, which are not restricted by the Rj2 gene, can infect Rjgene-accumulated soybean lines. To solve this problem, the occupancy rate of inocula carrying the hupSLgenes may be improved by inoculating Rjgene-accumulated soybean with B. diazoefficiensUSDA 110T during cultivation. To test this hypothesis, we are currently investigating the effect of B. diazoefficiensUSDA 110T inoculation on the growth and yield of various soybean genotypes, including Rjgene-accumulated ones.

CultivarBj6Bd110Be46Be76Be94
hupSLhupSL+hupS+L+
BxF–E12.58.362.512.50.00.04.2
BxF–M70.80.016.712.50.00.00.0
BxF–L0.08.345.825.04.216.70.0
Enrei83.40.08.38.30.00.00.0
Sachiyutaka13.08.734.839.10.04.40.0
Fukuyutaka25.020.816.737.50.00.00.0

Table 5.

Occupancy rate (%) of indigenous soybean-nodulating bradyrhizobia in each soybean cultivar in 2017.

Bj6, Bd110, Be46, Be76, and Be94 showed RFLP patterns similar to those of B. japonicumUSDA 6T, B. diazoefficiensUSDA 110T, B. elkaniiUSDA 46, B. elkaniiUSDA 76T, and B. elkaniiUSDA 94, respectively. hupSL, hupSL+, and hupS+L+ indicate isolates carrying or not carrying the hupSand/or hupLgenes.

4.5 Correlation between the occupancy rate of indigenous bradyrhizobial strains and yield components of soybean

Correlation analysis was used to evaluate the association between the occupancy rate of indigenous bradyrhizobial strains and yield components. Correlation coefficients were computed based on data obtained from the measurement of yield components and occupancy rate of indigenous soybean-nodulating bradyrhizobia. The R package “psych” was used to compute and plot the correlations. Additionally, the significance of the correlations was tested using the “cor.test” function in R.

The results of correlation analysis between the occupancy rate of indigenous bradyrhizobial strains and yield components of soybean are presented in Figure 3. The correlation coefficients of the occupancy rate of Bj6 isolates with plant height, node number, shoot dry weight, pod number, seed number, 100-seed weight, and yield were − 0.30, −0.04, 0.09, −0.13, −0.15, 0.41, and − 0.16, respectively. The correlation coefficients of the occupancy rate of Bd110 isolates exhibiting hupSL with plant height, node number, shoot dry weight, pod number, seed number, 100-seed weight, and yield were 0.36, 0.68, 0.32, 0.25, 0.29, −0.18, and 0.33, respectively. The correlation coefficients of the occupancy rate of Bd110 isolates exhibiting hupSL+ with plant height, node number, shoot dry weight, pod number, seed number, 100-seed weight, and yield were 0.25, −0.26, −0.56, −0.18, −0.22, −0.41, and − 0.25, respectively. The correlation coefficients of the occupancy rate of Bd110 isolates exhibiting hupS+L+ with plant height, node number, shoot dry weight, pod number, seed number, 100-seed weight, and yield were − 0.02, 0.25, 0.40, 0.42, 0.51, 0.05, and 0.61, respectively. Among correlations between the occupancy rate of each indigenous bradyrhizobial strain and soybean yield, Bd110 isolates exhibiting hupS+L+ presented a strong positive correlation (r = 0.61), albeit without significant differences. Additionally, in another experiment, which revealed different results from the present findings, showed that the occupancy rate of B. diazoefficiensUSDA 110T was correlated with the shoot dry weight of soybean (Figure 4). This result was obtained in the correlation analysis between the growth and the occupancy rate of B. diazoefficiensUSDA 110T, B. japonicumUSDA 6T and 123, and B. elkaniiUSDA 31 in soybean inoculated at the same bacterial density (106 cells mL−1) and cultivated for 5 weeks. Although this experiment was set in a greenhouse using cultivation pots, a significant positive correlation between the occupancy rate of USDA 110 and shoot dry weight of soybean plants was noted (r = 0.86, p = 0.03). A positive correlation between soybean growth and yield has been reported in previous study [57, 58, 59, 60]. In the present study, yield was positively correlated with plant height (r = 0.35) and shoot dry weight (r = 0.75), albeit without significant differences (Figure 3). Therefore, enhancing the infection rate of bradyrhizobial strains, such as B. diazoefficiensUSDA 110T carrying the hupSLgenes, may promote the growth of soybean and consequently increase yield.

Figure 3.

Correlation coefficient between the occupancy rate of Bd110 isolates carrying thehupSLgene and yield components. The correlation coefficients were computed based on data inTables 4and5(n = 6). A, B, C, D, E, F, G, H, I, J, and K indicate the occupancy rate (%) of Bj6, occupancy rate (%) of Bd110 exhibitinghupSL, occupancy rate (%) of Bd110 exhibitinghupSL+, occupancy rate (%) of Bd110 exhibitinghupS+L+, plant height, node number, shoot dry weight, pod number, seed number, 100-seed weight, and yield, respectively. *p < 0.01, **p < 0.001, respectively.

Figure 4.

Correlation between the occupancy rate ofBradyrhizobium diazoefficiensUSDA 110T and shoot dry weight of soybean. The values were obtained based on the growth investigation of soybean inoculated withB.diazoefficiensUSDA 110T, B.japonicumUSDA 6T,B.japonicumUSDA 123, andBradyrhizobium elkaniiUSDA 31 at the same bacterial density (106 cells mL−1) and then cultivated for 5 weeks. Values are presented as the mean of three replicates.

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5. Conclusion

In the present chapter, we described the breeding and selection processes, shoot growth, yield components, and infection tendency of useful bradyrhizobial strains carrying the hupSLgenes of Rjgene-accumulated soybean lines exhibiting the Rj2Rj3Rj4 genotype, obtained by crossing the Japanese soybean cultivars “Bonminori” (Rj2Rj3) and “Fukuyutaka” (Rj4).

First, we selected eight lines exhibiting the characteristics of the Rj2Rj2Rj4Rj4 genotype from 153 lines of F3 seeds following inoculation with B. japonicumIs−1 and B. japonicumIs−34 (Table 1). These eight lines were grown for several years, and three groups (B × F − E, B × F − M, and B × F − L) differing in terms of the flowering and ripening periods by approximately a week each were detected, which were cultivated further. Second, to investigate yield components, three Rjgene-accumulated soybean lines (B × F − E, B × F − M, and B × F − L) of F10 or F11 plant and three soybean cultivars (“Enrei,” “Sachiyutaka,” and “Fukuyutaka”) were cultivated in the 2016 and 2017 growing seasons. The yield of B × F − M was equivalent to that of “Sachiyutaka,” and this genotype likely possesses a greater yield potential than the parent soybean cultivar “Fukuyutaka,” among the Rjgene-accumulated soybean lines. However, the 100-seed weight of B × F − M was lower than that of “Sachiyutaka” and “Fukuyutaka.” Therefore, backcrossing with these cultivars is expected to produce soybean cultivars with larger seeds and higher yield potential. Third, to assess the occupancy rate of infected indigenous soybean-nodulating bradyrhizobia carrying the hupSLgenes, we collected nodules from soybean roots and performed PCR-RFLP analysis of the 16S–23S rRNA gene ITS region. Furthermore, 87 indigenous soybean-nodulating bradyrhizobial strains belonging to the B. diazoefficiensUSDA 110T cluster were investigated for the presence of the hupSLgenes using PCR. The occupancy rate of Bd110 isolates carrying the hupSLgenes tended to be lower in the Rjgene-accumulated soybean lines than in “Sachiyutaka” and “Fukuyutaka.” In addition, among the Rjgene-accumulated soybean lines, B × F − L presented the highest occupancy rate of Bd110 isolates carrying the hupSLgenes. Based on these results, during the cultivation of Rjgene-accumulated soybean, the occupancy rate of inocula carrying the hupSLgenes can be improved by inoculating B. diazoefficiensUSDA 110T, which is not restricted by the Rj2 gene.

Finally, to evaluate the association between the occupancy rate of indigenous bradyrhizobial strains and yield components of soybean, correlation analysis was performed. Correlation coefficients of the occupancy rate of Bd110 isolates exhibiting hupS+L+ with plant height, node number, shoot dry weight, pod number, seed number, 100-seed weight, and yield were − 0.02, 0.25, 0.40, 0.42, 0.51, 0.05, and 0.61, respectively, and the occupancy rate of Bd110 isolates exhibiting hupS+L+ was strongly and positively correlated with yield components (r = 0.61), albeit without significant differences. Furthermore, soybean yield was positively correlated with plant height (r = 0.35) and shoot dry weight (r = 0.75), albeit without significant differences. Therefore, enhancing the infection rate of bradyrhizobial strains, such as B. diazoefficiensUSDA 110T carrying the hupSLgenes, may promote the growth of soybean and consequently increase its yield. In the future, we intend to conduct further inoculation tests with useful strains, such as B. diazoefficiensUSDA 110T carrying the hupSLgenes, to evaluate in greater detail the availability of Rjgene-accumulated soybean lines with the Rj2Rj3Rj4 genotype for improving productivity.

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Acknowledgments

The authors thank the members of the laboratories of Shimane University and University of Miyazaki involved in the present study. Additionally, the authors thank the technical staff of the Agricultural Science Section, Education and Research Center for Biological Resources, Faculty of Life and Environmental Science, Shimane University for their support in managing soybean cultivation. The authors also thank the Faculty of Life and Environmental Sciences at Shimane University for financial support to publish this chapter.

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Conflict of interest

The authors declare no conflict of interest.

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Written By

Sokichi Shiro and Yuichi Saeki

Submitted: January 3rd, 2022Reviewed: January 24th, 2022Published: March 22nd, 2022