Soybean (Glycine max L.) plays an essential role in human nutrition as a protein source, and in plant nutrition as a N source. The rate of N fixation varies depending on the cultivars and compatibility between the inoculated Rhizobium strain and the host cultivar. Characterizing the nodulation regulatory (Rj) genes is necessary to determine the compatibility of cultivars and Rhizobium strains. Rj genes were previously identified based on inoculation tests and PCR analyses. The six cultivars Yezin-3, Yezin-7, Yezin-11, Shan Seine (Local), Madaya (Local), and Hinthada (Local) were identified as harboring the Rj4 gene. Two cultivars, Yezin-6 and Yezin-8, were classified as non-Rj-gene harboring. Two other cultivars, Yezin-9 and Yezin-10, were identified as Rj3- and Rj2Rj3-gene harboring, respectively. Ours is the first report on Rj3- and Rj2Rj3-gene harboring cultivars in Myanmar. We evaluated Myanmar soybean cultivars for symbiotic effectiveness, relying on the standard strain Bradyrhizobium japonicum USDA110. In our first experiment, the soybean cultivar Yezin-11 (Rj4) showed the highest N fixing potential. Based on their potential for fixing N and nodulation, the top six soybean cultivars were Yezin-11 (Rj4), Yezin-9 (Rj3), Yezin-6 (non-Rj), Yezin-8 (non-Rj), Yezin-3 (Rj4) and Yezin-10 (Rj2Rj3). These cultivars were selected for a second experiment, which revealed that the N fixation, nodulation, and plant growth of Yezin-11 (Rj4) *Corresponding author. A. Z. Htwe et al. 2800 were superior to the other cultivars. We conclude that Yezin-11 (Rj4) is the most efficient cultivar for nodulation and N fixation when inoculated with B. japonicum USDA110.
B. japonicum USDA110 Inoculation Test PCR Analysis Nodulation Regulatory Genes (Rj Gene) Symbiotic Effectiveness1. Introduction
Myanmar is an agricultural country, as agriculture is the backbone of its economy. Legumes are the second largest crops in Myanmar, following rice (Oryza sativa L.), in terms of cultivated hectares. Due to the relatively low cost of cultivation and increasing demand for domestic consumption and export, the total cultivated area of pulses has increased from 0.73 million hectares in 1988-89 to 4.4 million hectares in 2011-12 [1] . Soybean (Glycine max L.) is an important cash crop and the second largest cultivated crop after rice in Myanmar [2] . Soybean is also one of the most efficient leguminous crops in terms of fixing N [3] [4] .
Nodule formation by a cultivar is often dependent on a specific Rhizobium strain [5] , which may be attributed to nodulation regulatory genes called Rj genes. Different soybean cultivars possess different nodulation Rj genes. In soybean, the alleles Rj(s) and rj(s) are dependent on their compatibility with Bradyrhizobium and Ensifer/Si- norhizobium species [6] . Some nodulation Rj genes are found in nature, while others resulted from artificially induced mutations [6] . Williams and Lynch [7] found a non-nodulating soybean line, the rj1 genotype, which resulted from a cross between the cultivars Lincoln and Richard. The Rj genes Rj2, Rj3 and Rj4 inhibit the formation of functional nodules by certain Bradyrhizobium strains [8] - [11] . The regulatory gene Rfg1 restricts nodulation by the fast-growing strain Sinorhizobium fredii USDA257 [12] .
Ishizuka et al. [13] [14] tested the compatibility and preference of the Rj-genotype with specific Bradyrhizo- bium strains. Bradyrhizobium strains are classified into nodulation Types A, B, and C based on their compatibility with Rj cultivars. Type A strains are capable of forming nodules on all Rj genotype cultivars. Type B strains cannot form nodules on the Rj2Rj3-gene harboring cultivars. Type C strains are inhibited from nodule formation by Rj4 genotype cultivars. When different Rj-gene harboring cultivars are planted in the same field, non-Rj, Rj4 and Rj2Rj3 cultivars selectively form nodules with the Types A, B and C strains, respectively. Many scientists reported that the indigenous Bradyrhizobium strains in the soil exhibited preferences for nodulation on compatible Rj genotypes [15] - [18] . Recently, Soe et al. [19] identified Myanmar soybean cultivars with non-Rj and Rj4 genotypes. Soe et al. [20] [21] pointed out that the cultivars Yezin-6, harboring the non-Rj gene, and Yezin-3, harboring the Rj4 gene, had enhanced nodulation and N fixation when inoculated with B. japonicum USDA110 and indigenous strains. Yamakawa et al. [22] [23] stated that Rj2Rj3Rj4 conferred improved nodulation when inoculated with B. japonicum USDA110. Bradyrhizobium japonicum USDA110, which is a Type A strain, could form functional nodules in all dominant Rj genotypes. Therefore, B. japonicum USDA110 is used in many countries as an inoculant to increase soybean yield.
The Rj genes are identified by an inoculation test, which uses strains that restrict nodulation on specific Rj genotype soybean cultivars. As an accelerated method for molecularly identifying Rj genes, Yang et al. [24] , Tang et al. [25] and Hayashi et al. [26] used cloning to identify the genes Rj2, Rfg1 and Rj4. Yang et al. [24] classified the Rj2 and Rfg1 genes that encoded a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat (TIRNBS- LRR) class of plant resistance (R) proteins involved in host resistance to microbial pathogens through an effector-triggered immune (ETI) response. Recently, Hayashi et al. [26] described the molecular identification of the Rj4 gene based on map-based cloning of several mapping populations. They identified the Rj4 genes that encoded a thaumatin-like protein (TLP) belonging to the pathogenesis-related (PR) protein family 5, which was involved in inhibition of nodulation with specific Rhizobia strains. Cloning of the Rj3 gene has not been reported, so its identification is only based on inoculation test results.
In Myanmar, many researchers have been focusing on selecting strains to increase soybean N fixation. Recently, the Department of Agricultural Research (DAR) has developed improved soybean varieties, such as Yezin-9, Yezin-10, and Yezin-11. However, Rj genes have not been identified in some of the released cultivars. To recommend the most efficient N-fixing cultivars, it is necessary to evaluate symbiotic effectiveness with inoculated strains and identify the nodulation Rj genes. In the past, Rj genes were identified based on inoculation tests. Therefore, our goal in this study was to identify Rj genes of Myanmar soybean cultivars based on inoculation tests and multiplex PCR analysis and to screen the cultivars for N-fixing efficiency by using the standard strain B. japonicum USDA 110.
2. Materials and methods2.1. Origin of Soybean varieties
Ten soybean varieties (Shan Seine [local], Hinthada [local], Madaya [local], Yezin-3, Yezin-6, Yezin-7, Yezin-8, Yezin-9, Yezin-10, Yezin-11) were collected from the Food Legume Section, Department of Agricultural Research, Yezin, Myanmar. These varieties were grown in the glasshouse of the Plant Nutrition Laboratory, Kyushu University, Japan from July to November 2013 to obtain genetically pure and viable seeds. The focus was to study the ability of cultivars to adapt to weather in Japan. Shan Seine [local], Hinthada [local], Madaya [local] were widely grown in Shan State, Ayeyawaddy Region, Mandalay and Sagaing Regions, respectively. Yezin cultivars used in this experiment were mainly grown in Yezin, Mandalya Region and Shan State, and recommended for farmers to improve soybean production. Flower color, days to maturity and origin of these varieties are shown in Table 1.
2.2. Determination of nodulation regulatory Genes by Inoculation test
The Rj genotypes of 10 soybean cultivars, including three reference cultivars D51 (Rj3), CNS (Rj2Rj3) and Hill (Rj4), were investigated to estimate their compatibility with native bradyrhizobia. These varieties were inoculated with the three bradyrhizobial strains B. japonicum Is-1, B. elkanii USDA33 and B. japonicum Is-34 [13] . The strains Is-1, USDA33 and Is-34 failed to produce nodules on the roots of soybean cultivars harboring the Rj2Rj3, Rj3 and Rj4 genes, respectively [10] [27] .
The seeds were sterilized by soaking them in 2.5% sodium hypochlorite solution for 5 min, rinsing five times with 10 mL of 99.5% ethanol, and washing five times with sterilized half-strength modified Hoagland Nutrient (MHN) solution [28] . Five surface-sterilized seeds were sown in pots filled with 1 L of vermiculite and 0.6 L of N-free MHN solution. The strains mentioned above were cultured in A1E liquid media [29] and incubated on a rotary shaker at 30˚C for 7 days. Inoculant was prepared by diluting 1 mL of liquid bacterial culture with 99 mL of sterilized MHN solution to obtain a bacterial suspension of about 107 cells∙mL−1. Seeds were inoculated with the bacterial suspension at 5 mL per seed.
Origin of Myanmar soybean varieties
Variety
Flower color
Days to maturity
Origin
Yezin-3
Violet
115
DAR, Yezin, MR
Yezin-6
Violet
115
DAR, Yezin, MR
Yezin-7
Violet
100
DAR, Yezin, MR
Yezin-8
Violet
115
DAR, Yezin, MR
Yezin-9
White
120
DAR, Yezin, MR
Yezin-10
White
120
DAR, Yezin, MR
Yezin-11
Violet
110
DAR, Yezin, MR
Shan Seine
Violet
120
Shan State
Hinthada
Violet
120
Hinthada, AR
Madaya
Violet
130
Madaya, MR
DAR: Department of Agricultural Research; AR: Ayeyawaddy Region; MR: Mandalay Region.
The plants were cultivated in an environmentally-controlled room (25˚C and 75% Relative Humidity) under natural light for 4 weeks. Control pots were used to check for contamination by non-relevant strains and inoculated strains used in this experiment. Watering was done weekly with autoclaved deionized water. After 1 month, the formation of effective nodules was checked to identify nodulation types of all isolates being tested. This experiment was conducted three times, from January to June 2015.
2.3. Determination of nodulation regulatory Genes by PCR Analysis
Multiplex PCR analysis was used to identify Rj genes and confirm the Rj2 and Rj4 alleles, but it could not detect the Rj3 allele. For DNA extraction, the plants were cultivated in a growth chamber (28˚C for 16 hours for the light condition and 23˚C for 8 hours for the dark condition). Genomic DNA for PCR templates was extracted from the leaves of seedlings using Takara Bio, following the manufacturer’s instructions. Primers were designated from sequence information in reports identifying the Rj2 and Rj4 genes [24] - [26] . The primers are described in Table 2. The PCR reaction consisted of a pre-run at 94˚C for 5 min, denaturation at 94˚C for 30 s, annealing at 65˚C for 30 s, and extension at 72˚C for 30 s for the first 10 cycles, with a decrease in annealing temperature of 1˚C per cycle. The remaining 20 cycles were repeated at the same temperatures for denaturing and annealing, annealing at 55˚C, and extension at 72˚C for 30 s, followed by the final extension at 72˚C for 10 min and preservation at 4˚C. The reaction producer of PCR analysis of Rj genes was innovated by Dr. Yuichi Saeki (Professor, Department of Biochemistry and Applied Biosciences, Miyazaki University). Photos of PCR products were taken after agarose gel electrophoresis (3% agarose gel in 1x TAE buffer) to check the band placement and identify the Rj genes.
2.4. Evaluation of symbiotic Effectiveness of Myanmar Soybean cultivars
The seeds were surface-sterilized as described above. Six surface-sterilized seeds were sown in pots filled with 1 L of vermiculite and 0.6 L of N-free MHN solution. Bradyrhizobium japonicum USDA110 was cultured in A1E liquid media and incubated on a rotary shaker at 30˚C for 7 days. Inoculant was prepared as described above. Seeds were inoculated with the bacterial suspension at 5 mL per seed. The cultivation conditions were the same as described above. Three plants were chosen from each pot for data collection in the first experiment. Six plants were taken from three different pots for the second experiment.
For the acetylene reduction assay (ARA), the soybean plants with intact nodules were placed in 100-mL conical flasks, sealed with a serum stopper and injected with 12 mL of acetylene (C2H2) gas to replace air with acetylene.
Primers sets and amplification of Multiplex PCR analysis
Primer sets
Primer sequences
Annealing temperature (˚C)
Amplification site (bp)
Rj2 specific primers
K452E-F
(5'-GCTTCAATAGATATGACTTGACAG-3')
58.5
161
R490I-R
(5'-AATCAAGTCATGCATTGTAACTA-3')
57.8
rj2 specific primers
K452-F
(5'-GCTTCAATAGATATGACTTGACAA-3')
58.9
161
R490-R
(5'-ATCAAGTCATGCATTGTAACTC-3')
58.2
Rj4 specific primers
T107A-F
(5'-TTGGAGGAAACGCCG-3')
62.2
324
202-203AY-R
(5'-AATCATGAGAAGAACAAGTATAAGC-3')
58.5
rj4 specific primers
T107-F
(5'-TTGGAGGAAACGCCA-3')
59.9
318
202-203del-R
(5'-AATCATGAGAAGAACAAGTATGGA-3')
60.4
The nitrogenase activity, in terms of ethylene (C2H4) concentration of the plants, was measured using a flame ionization gas chromatograph (GC-14A, Shimadzu, Kyoto, Japan) at 5 and 65 min after injecting with C2H2 gas as described by Soe et al. [20] . After completing the assay, nodules were counted by removing them from the roots. Shoots, roots, and nodules were collected separately and oven dried at 70˚C for 24 hours to record their dry weights. STATISTIX 8 was used for data analysis (Analytical Software, Tallahassee, FL, USA). Means were compared by using Tukey’s HSD test at P < 0.05.
3. Results3.1. Nodulation regulatory (Rj) genes of Myanmar soybean cultivars Identified by an Inoculation test
Identifying the Rj genes of soybean varieties is important to determine their host specificity and compatibility with specific bradyrhizobia. We evaluated the Rj genotypes of soybean cultivars from Myanmar to identify their nodulation Rj genes and estimate their compatibility with strains to be inoculated. Among the tested cultivars, six (Shan Seine [local], Hinthada [local], Madaya [local], Yezin-3, Yezin-7 and Yezin-11) were identified as harboring the Rj4-gene. Only two cultivars, Yezin-6, Yezin-8, were classified as non-Rj-gene harboring cultivars. Yezin-9 and Yezin-10 were identified as Rj3- and Rj2Rj3-gene harboring cultivars, respectively. The results of inoculation testing are shown in Table 3.
3.2. Nodulation Rj genes of Myanmar soybean cultivars Identified by PCR Analysis
Among the tested cultivars, six (Shan Seine [local], Hinthada [local], Madaya [local], Yezin-3, Yezin-7 and Yezin-11) harbored Rj4 gene alleles. Yezin-6, Yezin-8 and Yezin-9 did not harbor Rj4 or Rj2 genes, although Yezin-6 and Yezin-8 harbored the recessive alleles rj4 and rj2, and Yezin-9 and Yezion-10 harbored the recessive allele rj4. We found the Rj2 gene in Yezin-10. The results from the inoculation test and PCR analysis are shown in Figure 1.
3.3. Symbiotic effectiveness of USDA 110 on Myanmar soybean Cultivars
The number of nodules produced was significantly different when inoculated with B. japonicum USDA 110 (Table 4). The numbers of nodules ranged from 5 to 13 per plant. The most nodules were obtained from Yezin- 10 (Rj2Rj3), but it was not significantly different from other cultivars, except for Madaya Local (Rj4), which
Nodulation regulatory genes (Rj genes) of cultivars
Cultivar
Nodule No. plant−1 on inoculated strains
Rj gene
USDA 33
Is-1
Is-34
Shan Seine
Medium
Medium
None
Rj4
Madaya
Medium
Medium
Low
Rj4
Hinthada
Medium
High
None
Rj4
Yezin-3
Medium
High
Low
Rj4
Yezin-6
Medium
Medium
Medium
non-Rj
Yezin-7
Medium
High
None
Rj4
Yezin-8
Medium
Medium
Medium
non-Rj
Yezin-9
Low
Medium
Medium
Rj3
Yezin-10
None
None
High
Rj2Rj3
Yezin-11
Medium
Medium
None
Rj4
High = 10 - 15 nodules plant−1; Medium = 4 - 9 nodules plant−1; Low = 1 - 3 nodules plant−1; None = No nodulation. This division was based on Htwe et al. [43] .
Expression of rj or Rj gene alleles amplified by multiplex PCR with Rj<sub>2</sub> and Rj<sub>4</sub>, and rj<sub>2</sub> and rj<sub>4 </sub>specific primers. S: Shan Seine; M: Madaya; H: Hinthada; 3: Yezin-3; 6: Yezin-6; 7: Yezin-7; 8: Yezin-8; 9: Yezin-9; 10: Yezin-10; 11: Yezin-11
Effect of B. japonicum USDA 110 strain on acetylene reduction activity, nodulation and plant growth of Myanmar soybean cultivars at 28 DAS
Cultivars
NN (No. plant−1)
NDW (mg∙plant−1)
SDW (g∙plant−1)
RDW (g∙plant−1)
ARA (μmol C2H4 h−1 plant−1)
Yezin-3
10.67 ab
15.70 ab
0.22 ab
0.09 cde
0.49 ab
Yezin-6
9.67 ab
15.40 ab
0.23 ab
0.13 ab
0.52 ab
Yezin-7
10.67 ab
12.60 ab
0.16 bc
0.08 de
0.35 ab
Yezin-8
9.00 ab
19.20 a
0.23 ab
0.11 bcd
0.51 ab
Yezin-9
5.33 ab
11.40 ab
0.21 ab
0.12 abc
0.58 ab
Yezin-10
13.00 a
10.00 ab
0.20 ab
0.11 bcd
0.43 ab
Yezin-11
12.00 ab
23.80 a
0.29 a
0.15 a
0.65 a
Shane Seine
8.00 ab
11.80 ab
0.14 bc
0.08 de
0.11 ab
Hinthada
10.67 ab
16.30 ab
0.27 a
0.12 abc
0.41 ab
Madaya
5.00 b
2.80 b
0.09 c
0.05 e
0.08 b
Mean values in each column followed by the same letters are not significantly different at P > 0.05 (Tukey’s test). NN: nodule number; NDW: nodule dry weight; SDW: shoot dry weight; RDW: root dry weight; ARA: acetylene reduction activity. Yezin-6 was used as control. Nodule number, nodule dry weight and ARA value for control were zero. Shoot and root dry weight of control was 0.20 and 0.12 g∙plant−1, respectively.
produced the fewest nodules. Nodule dry weights also differed significantly among the cultivars. The nodule dry weights of Yezin-11 (Rj4) and Yezin-8 (non-Rj) were greater than weights of the other cultivars but, with the exception of Madaya Local (Rj4), these differences were not statistically significant.
Shoot dry weights were significantly different between some of the cultivars. The highest shoot biomass (0.29 g∙plant−1) was obtained from Yezin-11 (Rj4), but this did not differ significantly from that of Yezin-3, Yezin-6, Yezin-8, Yezin-9, Yezin-10, or Hinthada local cultivars. Significantly greater root dry weight was recorded for Yezin-11 (Rj4), but it was not significantly different from Yezin-6, Yezin-9, or Hinthada local cultivars. The nitrogenase activities varied significant among soybean cultivars when inoculated with B. japonicum USDA110 (Table 4). The highest ARA values were obtained from Yezin-11 (Rj4), at 0.65 mmol∙h−1∙plant−1, but this did not differ significantly from that of the other cultivars, except for the Madaya local cultivar with the lowest nitrogenase activity. These results indicated that nodulation, N fixation, and plant growth of soybean cultivars differed when inoculated with B. japonicum USDA110. Higher N-fixing cultivars, in terms of ARA per plant, were Yezin-11 (Rj4), Yezin-9 (Rj3), Yezin-6 (non-Rj), Yezin-8 (non-Rj), Yezin-3 (Rj4), and Yezin-10 (Rj2Rj3). These top six cultivars, with higher N fixing potential, were selected for the next experiment.
3.4. Symbiotic effectiveness of USDA 110 on selected Soybean Cultivars
The number of nodules, ranging from 10 to 17.67 per plant, differed significantly among cultivars when inoculated with B. japonicum USDA 110 (Figure 2(a)). We observed a significant increase in the number of nodules in Yezin-11, followed by Yezin-10. Nodule dry weight was also significantly different among treatments, due to inoculation with B. japonicum USDA 110 (Figure 2(b)). The highest nodule dry weight was observed for Yezin-11, but it was not statistically different from that of other cultivars, except for Yezin-10 with the lowest nodule dry weight. Shoot dry weights varied significantly among cultivars, ranging from 0.45 to 0.63 g∙plant−1 (Figure 3(a)). The highest shoot dry weight was obtained for Yezin-11 (Rj4), but it was not statistically different from that of other cultivars, except for Yezin-6 (non-Rj). Root dry weight differed significantly among the cultivars (Figure 3(b)). The highest root dry weight was obtained from Yezin-11 (Rj4). There were significant differences between the cultivars in nitrogenase activity, which ranged from 0.36 to 1.49 μmol∙h−1∙plant−1 (Figure 4). The highest ARA value was obtained from Yezin-11 (Rj4). Although the lowest ARA value was obtained from Yezin-8 (non-Rj), it did not differ statistically from Yezin-3 (Rj4), Yezin-6 (non-Rj), Yezin-9 (Rj3), or Yezin-10 (Rj2Rj3). These results indicated that Yezin-11 (Rj4) was the most efficient cultivar, with the most nodules, the highest nodule, shoot, and root dry weights, and the greatest nitrogenase activity.
Effect of B. japonicum USDA 110 strain on (a) Nodule number and (b) Nodule dry weight of selected Myanmar soybean cultivars at 28 DAS. Mean values followed by same letters are not significantly different at P > 0.05 (Tukey’s test). Yezin-6 and Yezin-8 were used as control treatments. Nodule number and nodule dry weight for controls were zero.
Effect of B. japonicum USDA 110 strain on (a) Shoot dry weight (SDW) and (b) Root dry weight of selected Myanmar soybean cultivars at 28 DAS. Mean values followed by same letters are not significantly different at P > 0.05 (Tukey’s test). Yezin-6 and Yezin-8 was used as control treatments. Shoot and root dry weight of controls were 0.37 and 0.19 g∙plant<sup>−1</sup>, and 0.46 and 0.22 g∙plant<sup>−1</sup>, respectively.
Effect of B. japonicum USDA 110 strain on Acetylene reduction activity (ARA) of Selected Myanmar soybean cultivars at 28 DAS. Mean values followed by same letters are not significantly different at P > 0.05 (Tukey’s test). Yezin-6 and Yezin-8 were used as control treatments. ARAs for each control were zero
4. Discussion
Symbiotic N fixation of soybean could provide 65 to more than 160 kg fixed N∙ha−1 [30] , accounting for 40 to 70% of the total N requirement. This symbiosis is highly specific, as a particular species or strain of Rhizobia could induce a symbiotic association with only a specific leguminous species or cultivars [31] . This specificity involves molecular recognition between the host and the bacterium, through exchange of compound signals that induce nodulation and N fixation [32] [33] . A Rhizobium strain that is effective on one legume might not be highly effective on other legumes [34] , as the host legume has a dominant role in determining the nodule forming strain. Saeki et al. [17] stated that Rj genotypes of soybean cultivars have the ability to affect both compatibility and preference for nodulation between the host cultivar and soybean Rhizobia.
Compatibility between Rj soybean genotypes and soybean-nodulating bradyrhizobia must be considered in selecting the best varieties and strains for soybean cultivation. Therefore, nodulation Rj genes collected from soybean cultivars in Myanmar were evaluated to determine compatibility and preference between strains and cultivars. The Rj genes could be identified by inoculating with specific strains of Bradyrhizobium, such as B. japonicum Is-1, B. elkanii USDA33 and B. japonicum Is-34 [13] . The strains Is-1, USDA33, and Is-34 failed to form nodules on the roots of soybean cultivars harboring the Rj2Rj3, Rj3 and Rj4 genes, respectively [10] [27] . In this study, Yezin-6 and Yezin-8 formed nodules with all inoculated strains. Therefore, we identified them as non-Rj-gene harboring cultivars. The soybean cultivars Shan Seine (local), Hinthada (local), Yezin-7 and Yezin- 11 were restricted in nodule formation when inoculated with B. japonicum Is-34. Therefore, we classified them as Rj4 genotype cultivars. In the inoculation test, a few nodules were produced on the roots of Yezin-3 and Madaya (local) when inoculated with Is-34, but we assumed that they harbored the Rj4 gene. One or two nodules were formed on the roots of Yezin-9 and D51 when inoculated with USDA33. Yamakawa et al. [22] stated that a few effective nodules and numerous ineffective nodules were produced by D51 when inoculated with USDA33. This agrees with our findings. Therefore, we assumed that Yezin-9 was an Rj3 gene-harboring cultivar. Soe et al. [19] identified the Rj genes of some Myanmar cultivars. They reported that Hinthada, Southern Shan local, Northern Shan local, Yezin-3, and Yezin-11 cultivars harbored the Rj4-gene, whereas Shan Sein, Shan Wha, Yezin-6, Yezin-8, and Yezin-14 cultivars did not harbor Rj genes.
In this study, we performed PCR analysis to confirm the Rj genotypes. Although we could not identify Rj3, the analysis was very useful in identifying the Rj4 and Rj2 alleles. The cultivars Shan Seine (local), Hinthada (local), Madaya (local), Yezin-3, Yezin-7 and Yezin-11 harbored Rj4 alleles. Yezin-6, Yezin-8, and Yezin-9 cultivars did not harbor other dominant Rj alleles, although they harbored the recessive alleles rj2 and rj4. Only Yezin-9 was identified as an Rj2-gene harboring cultivar. Based on PCR analysis, we confirmed that Yezin-3 and Madaya (local) were Rj4-genotype soybean cultivars, although they could also form a few nodules with their nodulation restricting strain Is-34. Contrary to Soe et al. [19] , the Rj genotypes of Hinthada (local), Yezin-3, Yezin-6, Yezin-8 and Yezin-11 were the same and the results for Shan Seine (local) differed from those reported by Soe et al. [19] . In this study, Shan Seine (local) was identified as an Rj4-gene harboring cultivar, according to the inoculation test and PCR analysis results. Therefore, PCR analysis for Rj gene determination was deemed necessary if the inoculation test results were uncertain.
According to the inoculation test and PCR results, the cultivars Shan Seine (local), Hinthada (local), Madaya (local), Yezin-3, Yezin-7 and Yezin-11 were identified as Rj4-genotype cultivars. Yezin-6 and Yezin-8 were identified as non-Rj genotype cultivars. Yezin-9 and Yezin-10 were identified as harboring the Rj3 and Rj2Rj3 genes, respectively.In Myanmar, the Rj4 genotype cultivars were the most widely grown cultivars, accounting for 60% of the tested cultivars. Devine and Kuykendall [35] reported the Rj4 genotype cultivars as most frequently found in Southeast Asia, but not common in North Asia. Devine and Breithaupt [36] also reported that 60% of the cultivars from Southeast Asia and 71.2% of those from Myanmar harbored Rj4 genes.
In Myanmar, DAR has developed improved soybean cultivars to replace local cultivars. However, some Myanmar farmers have continued to grow local soybean cultivars, such as Shan Seine (local), Madaya (local) and Hinthada (local). Proper matching of soybean cultivars and Rhizobia strains optimizes performance through enhanced N fixation. In this study, we screened improved and local cultivars using the strain B. japonicum USDA110, as several studies have reported significant increases in growth, yield, nodulation, and N fixation of Myanmar soybean cultivars due to inoculation with this strain [20] [37] [38] .
We found that local cultivars such as Madaya (local) (Rj4), Shan Seine (local) (Rj4) and Yezin-7 (Rj4) showed lower nitrogenase activity, nodulation and plant growth. Hinthada (local) produced an increased number of nodules and nodule dry weight, but its N fixation was lower than other improved cultivars such as Yezin-11 and Yezin-3, despite having the Rj4 genes in common. Soe et al. [20] also reported that improved Yezin cultivars were more efficient for N fixation compared with local cultivars. We also discovered that the improved Yezin cultivars Yezin-11 (Rj4), Yezin-9 (Rj3), Yezin-6 (non-Rj), Yezin-8 (non-Rj), Yezin-3 (Rj4), and Yezin-10 (Rj2Rj3) were superior in N fixing capacity. These top six cultivars, showing higher nitrogenase activity, were selected for the second screening experiment. Selection of cultivars was based on nitrogenase activity and nodulation. Wani et al. [39] stated that the number of nodules or nitrogenase activity were genotypically variable within grain legume species. A legume plant with effective nodules could meet not only its own N requirements, but also enrich soil N content, thus improving soil fertility and sustainability [40] .
In results from the second screening experiment, Yezin-11 (Rj4) was the most efficient cultivar, with the most nodules, and highest nodule, shoot and root dry weights, and the greatest nitrogenase activity. When we compared the N fixing rates, in terms of ARA per plant inoculated with B. japonicum USDA 110, Yezin-9 (Rj3) and Yezin-10 (Rj2Rj3) were most efficient in N fixation, though they did not differ significantly from the N fixation of Yezin-6 (non-Rj), Yezin-8 (non-Rj) and Yezin-3 (Rj4). This might be related to the Rj genes, which can affect both preference and compatibility for nodulation between the host cultivar and soybean Rhizobia [13] [14] [17] . Yamakawa et al. [22] [23] stated that Rj2Rj3Rj4-gene harboring cultivars had improved N fixation compared with the non-Rj, Rj2, Rj2Rj3 and Rj4 soybean cultivars when inoculated with B. japonicum USDA110. In both experiments, the Rj4-gene harboring Yezin-11 cultivars showed higher N fixation capacity, followed by the Rj3-gene harboring Yezin-9 cultivars. The N fixation activity and the ratio of N fixed from the atmosphere to the total N accumulation in plants vary significantly within soybean cultivars [41] [42] .
5. Conclusion
Most Myanmar soybean cultivars were identified as harboring the Rj4 gene. A few cultivars were classified as non-Rj, Rj2Rj3 and Rj3 gene harboring. This was the first report of Rj2Rj3 and Rj3 genotype soybean cultivars in Myanmar. We evaluated the N fixation and nodulation of Myanmar soybean cultivars by using the standard strain B. japonicum USDA 110. In both experiments, Yezin-11 (Rj4) was the most efficient for N fixation and nodulation. It appeared that Yezin-11 (Rj4) was more compatible with B. japonicum USDA110 based on the results from both experiments. Yezin-11 had about two to three times higher symbiotic N fixation capacity than the other soybean cultivars. Our study provides useful information for breeders seeking to produce cultivars efficiently at N fixation. Further study is needed on the effectiveness of different Rj genotypes with indigenous bradyrhizobia to increase soybean productivity via symbiotic N fixation.
Acknowledgements
This work was supported by Ministry of Education, Culture, Sports, Sciences and Technology of Japan. We are grateful to Dr. Htun Shwe (Researcher, Food Legume Section, Department of Agricultural Research) for providing soybean seeds.
Cite this paper
Sadiq, R., Maqbool, N. and Haseeb, M. (2017) Ameliorative Effect of Chelating Agents on Photosynthetic Attributes of Cd Stressed Sunflower. Agricultural Sciences, 8, 149-160. https://doi.org/10.4236/as.2017.82010
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