Crop Science 40:700-703 (2000)
© 2000 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Inheritance and Allelism Analysis of Hypernodulating Genes in the NOD3-7 and NOD2-4 Soybean Mutants
T.D. Vuonga and
J.E. Harperb
a Dep. of Crop Sciences, Univ. of Illinois, Urbana, IL 61801 USA
b Soybean/Maize Germplasm, Pathology and Genetics Unit, USDA-ARS and Dep. of Crop Sciences, Univ. of Illinois, 1201 W. Gregory Dr., Urbana, IL 61801 USA
j-harper{at}uiuc.edu
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ABSTRACT
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Hypernodulating soybean [Glycine max (L.) Merr.] mutants derived from cv. Williams showed greater early-season dinitrogen fixation potential and partial tolerance to high levels of NO-3. Understanding genetic control is essential to manipulation of this trait. Genetic analysis of the NOD1-3 and NOD4 mutants, and the En6500 mutant, had previously shown that these three mutants were controlled by the same recessive allele at the rj7 locus. However, genetic control of two additional hypernodulating mutants, NOD3-7 and NOD2-4, remained to be verified. We investigated the inheritance and allelic relationship of these latter mutations in relation to those previously studied. The cultivar Harosoy 63 was crossed to the NOD3-7 and NOD2-4 mutants to produce F1 hybrids, F2 progeny, and F2-derived families. Phenotypic segregation was examined. For allelism tests, hypernodulating progeny exhibiting purple hypocotyl, which were isolated from the F2 segregants of the crosses with Harosoy 63, were used for crossing with the NOD2-4 mutant. Resulting phenotypes were visually evaluated for hypernodulation at 14 d after planting (DAP) in the greenhouse. The results of genetic analysis indicated that a recessive allele was responsible for hypernodulation in the NOD3-7 and NOD2-4 mutants. Allelism analysis revealed that although the hypernodulating mutants were isolated from independent mutational events, the rj7 locus controlled NOD-type hypernodulating mutants and the En6500 mutant.
Abbreviations: DAP, days after planting
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INTRODUCTION
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THE SYMBIOTIC INTERACTION between host plants and rhizobial bacteria results in a multistep process of nodulation in various legumes, including soybean. The nodulation process has been reported to be adversely affected by numerous environmental conditions. Of these, the availability of soil residual NO-3 is considered to be a primary factor inhibiting the symbiotic N2 fixation process (Gibson and Harper, 1985; Harper, 1987). The sensitivity of the nodulation process to the high level of residual soil NO-3 has been the subject of numerous investigations, but a definitive mechanism of the NO-3 inhibition still remains elusive. Many approaches, including selection of insensitive or less sensitive bacterial strains and manipulation of the host plant, have been used in attempts to circumvent the inhibitory effects of NO-3. It has been shown that the latter would be more likely to provide greater potential for overcoming the problem because the host plant has primary control of the level of N2 fixation (Gibson and Harper, 1985).
Soybean researchers (Betts and Herridge, 1987; Herridge and Betts, 1988) attempted to identify soybean genotypes capable of enhanced nodulation and N2 fixation under high levels of soil residual NO-3. However, naturally occurring differences among legume genotypes are not large for this trait. All legume cultivars studied have NO-3 sensitive N2 fixation, and conventional selection approaches seem unlikely to produce NO-3 tolerant legume nodules (Streeter, 1988). Therefore, several researchers have used chemical mutagenesis as an alternative approach to generate greater genetic variability in host nodulation response. Several hypernodulating mutants with enhanced nodulation and partial tolerance to the presence of high levels of NO-3 were isolated (Akao and Kouchi, 1992; Buzzell et al., 1990; Carroll et al., 1985; Gremaud and Harper, 1989; Lee et al., 1997).
The following genetic studies indicated that a single recessive mutant gene was involved in hypernodulating or supernodulating mutations. Delves et al. (1988) concluded that all nodulation mutants derived from the cultivar Bragg were controlled by single recessive mutant genes, with the possible exception of the nts1116 mutant. These genes were allelic and inherited as single Mendelian recessive genes in spite of the differences in the degree of nodulation expression. Buzzell et al. (1990) isolated five supernodulating mutants from the mutagenized cv. Elgin 87. Analyzing phenotypic segregation in F2 generations derived from several crosses, the authors hypothesized that there might be two genes involved in supernodulation expression in these soybean mutants. However, no evidence was provided to confirm the hypothesis. Akao and Kouchi (1992) isolated a supernodulating mutant, En6500, from cv. Enrei. In a followup genetic study, Kokubun and Akao (1994) reported that a recessive mutant gene was responsible for the mutation and that this gene was allelic to that found in nts382, a Bragg-derived mutant. Although these two mutants were derived from parental cultivars with markedly different genetic backgrounds, they were controlled by the same recessive gene.
Analyzing the allelic relationship of three mutants derived from the cultivar Williams (NOD1-3, NOD4, and NOD2-4) and the En6500 mutant, Vuong et al. (1996) reported that the hypernodulation characteristics in the NOD1-3, NOD4, and En6500 mutants were controlled by a recessive mutant gene, previously designated rj7 (Harper and Nickell, 1995). In contrast, the NOD2-4 mutant appeared to be controlled by an additional recessive mutant gene. This gene was tentatively proposed as rj8 (Vuong et al., 1996). However, in a study of genetic interaction between the rj7 and rj8 genes, the results showed that segregation for nodulation phenotype did not fit the expected ratio of 9:7 in the F2 generation. The observed ratio of phenotypes was 13:3 normal/hypernodulating. Based on the data of F2:3 progeny tests, it was speculated that a complex interaction might result in the apparent atypical segregation where one gene played the role of a modifier and the another gene as being modified. Alternatively, we could not rule out seed contamination due to lack of visual markers. Additionally, previous genetic analysis (Vuong, 1996, unpublished data) of the NOD3-7 mutant was suspect because of apparent contamination of the NOD3-7 seed source. Therefore, the approach taken in this study was to develop hypernodulated lines with a visual marker (purple hypocotyl) to facilitate identification of true crosses and rule out seed contamination.
The objectives of our study were (i) to develop hypernodulating progeny with purple hypocotyl from crosses with cv. Harosoy 63, (ii) to investigate the inheritance of mutant gene(s) conferring the hypernodulation of the NOD3-7 and NOD2-4 mutants, and (iii) to analyze allelic relationships between the mutant genes identified.
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Materials and methods
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Plant Materials
For genetic studies, the NOD3-7 and NOD2-4 mutants were purified using single plant selection from the original seed lots. These mutants were then crossed with cv. Harosoy 63 as a male parent. From the previous crosses of hypernodulating mutants and cv. Harosoy 63, several F2 segregants exhibiting hypernodulation and purple hypocotyl were saved and allowed to self for four additional cycles for homozygosity. They were designated as their parental mutants followed by an asterisk. For example, NOD1-3* represents a hypernodulating line with purple hypocotyl that was isolated from the NOD1-3 x Harosoy 63 cross. Several crosses were made between the isolated lines and the corresponding hypernodulating mutants for the confirmation of mutant gene identity. The supernodulating En6500 mutant (Akao and Kouchi, 1992) was also included for allelism analyses. Parental cultivars and lines were grown on the Agronomy and Plant Pathology South Farm, University of Illinois at Urbana-Champaign for crossing in summers of 1996 and 1997.
Progeny Analysis and Visual Evaluation of Nodulation
Crosses between the purified NOD3-7 and NOD2-4 mutants and the normally nodulating Harosoy 63 were made in the field. The F1 progeny were grown and visually evaluated in the greenhouse as previously described by Pracht et al. (1993) and Vuong et al. (1996). Briefly, seeds were inoculated with a commercial peat-based Bradyrhizobium japonicum inoculant (Urbana Laboratory, St. Joseph, MO) and sown directly into the gravel beds.1
An irrigation regime was set up to promote uniform emergence of seeds. Fourteen days after planting, the beds were flooded and seedlings were carefully pulled up. The nodulation characteristics of progeny were visually evaluated. The F1 plants were then transplanted to gravel beds and were hydroponically cultured for F2 seed production. Phenotypic segregation for nodulation of F2s was evaluated following the same procedure as done with F1s. Subsequently, the F2 seedlings were transplanted to the field for F3 seed production. The F2:3 families were also evaluated in the greenhouse as described above.
To analyze allelism of mutant genes, crosses between original hypernodulating mutants and hypernodulating lines with purple hypocotyls were made. The F1 nodulation phenotypes were visually evaluated. The purple vs. green hypocotyl color present in the parents was used as a visible genetic marker for the verification of true hybrid seeds. Observed segregation ratios were tested for goodness of fit to expected ratios by chi-square analysis.
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Results
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Development of Hypernodulating Lines Expressing Purple Hypocotyl
Crosses of NOD1-3, NOD3-7, and NOD4 with Harosoy 63 as the male parent were made to develop hypernodulated lines that carried purple hypocotyls as visual markers. All crosses resulted in F1 plants that were normally nodulated with purple hypocotyls, indicative of single recessive genes controlling hypernodulation and the dominant gene for purple hypocotyl color. Progeny were advanced to F2, selected for hypernodulation and purple hypocotyl, and selfed for four cycles to ensure homozygosity for hypernodulation and purple hypocotyl color to provide progeny used for subsequent allelism analysis
Inheritance of Hypernodulation in the NOD3-7 and NOD2-4 Mutants
Confirmation of the recessive nature of the hypernodulating gene in NOD3-7 and NOD2-4 was made by evaluating progeny in the F2 for segregation into normal and hypernodulating phenotypes. Segregation ratios approached the expected 3:1 normal/hypernod for both mutant lines (Table 1)
. Chi-square tests of heterogeneity were performed to examine the heterogeneity among populations and indicated that the data from individual F2 populations could be pooled. The pooled data analysis revealed high levels of probability of observed to expected values. These results indicated that hypernodulating mutations in the NOD3-7 and NOD2-4 mutants were controlled by single recessive genes.
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Table 1 Nodulation phenotypes in F1 and phenotypic segregation in F2 of soybean crosses between the hypernodulating NOD2-4 and NOD3-7 mutants and cv. Harosoy 63
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Table 2
summarizes F2:3 progeny tests for segregation of nodulation. Approximately one-third of normally nod- ulating F2 plants did not segregate in the F3. The remaining F2 families were segregating for normal and hypernodulation and thus were heterozygous in the F2. Lack of F3 segregation in hypernodulating F2 plants confirmed homozygosity. The observed ratio of phenotypic segregation of normally nodulating F2 plants fit 1:2:1 normal/segregating/hypernodulating. It was confirmed that single recessive mutant genes were responsible for hypernodulating mutation in the NOD3-7 and NOD2-4 mutants, resembling the genetic control of mutant genes in the NOD1-3 and NOD4 mutants.
Allelism Analysis of Mutant Genes
Table 3
(upper half) presents the expression of nodulation and hypocotyl color from crosses of NOD2-4 (green hypocotyl) with the selected NOD* lines and with En6500 (purple hypocotyl). All F1 plants involving NOD2-4 showed hypernodulation and purple hypocotyls, indicating that the recessive mutant gene in NOD2-4 was allelic to those in En6500 and other NOD mutants. Testing the segregation in F2, no phenotypic segregation was found for nodulation type, whereas hypocotyl color character displayed segregation into an expected ratio of 3:1 purple/green.
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Table 3 Allelism analysis of recessive mutant genes in the hypernodulating NOD2-4 and NOD3-7 soybean mutants derived from mutagenized Williams
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The results of F1 and F2 testing of NOD3-7 (Table 3, lower half) were consistent with those in the allelism analysis of the NOD2-4 mutant. In the cross of NOD3-7 and En6500, F1 plants displayed hypernodulation and purple hypocotyl. F2 progeny were not segregating for nodulation, but hypocotyl color was segregating into an expected ratio of 3:1. These results were in agreement with those of previous work and provided strong support for our conclusion that all of these mutant alleles are at the same locus.
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Discussion
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Our study provided conclusive evidence that the hypernodulation trait in NOD2-4 and NOD3-7 is controlled by a single recessive gene that is allelic to the rj7 gene previously reported (Harper and Nickell, 1995) as controlling hypernodulation and confirmed in NOD1-3 and NOD4 (Vuong et al., 1996). Because these results were obtained using the visual marker of purple hypocotyl color to verify true crosses, we are convinced that these results are valid and that the previous conclusion that the NOD2-4 mutant may involve a second gene, tentatively designated rj8 (Vuong et al, 1996), was not correct. This earlier conclusion could have resulted from seed contamination or revertants within the parents used for crosses. It has been noted that both NOD2-4 and NOD3-7 show normally nodulated plants with some frequency in field trials, while the NOD1-3 line has been quite stable since the original report in 1989 (Gremaud and Harper, 1989). Whether this is due to outcrossing, revertants, or seed contamination has not been resolved. We used remnant M5 seed of the initially isolated NOD2-4 hypernodulating line (Gremaud and Harper, 1989), and several cycles of seed purification were undertaken to acquire a genetically pure line of NOD3-7. We also incorporated a visual marker, purple hypocotyl, into the hypernodulated mutants to aid in verification of crosses. We therefore are confident in the present results that all four hypernodulating mutants (NOD) derived from Williams are allelic and controlled by the rj7 recessive gene.
In addition, our results are consistent with a single loci being responsible for most hyper- or supernodulating mutants. Delves et al. (1988) reported that 15 of 16 hyper- and supernodulating lines were allelic, while Buzzell et al (1990) reported that three of five supernodulating lines from their group were allelic and they suggested a possible involvement of a second gene. However, subsequent test crosses did not support a second gene involvement (Buttery and Buzzell, 1998), and these authors suggested that a homozygous lethal effect in the embryo could have impacted segregation ratios. Thus, in both of these cases where a tentative suggestion for a second gene was made, no subsequent work has provided confirmation that a second gene exists in control of hyper- or supernodulation. Furthermore, the observation that supernodulation mutant nts382 from Bragg and En6500 from Enrei are allelic (Kokubun and Akao, 1994) and that En6500 is allelic to the four NOD mutants evaluated here provides good evidence that the Rj7 gene may be a particularly susceptible gene target for chemical mutagenesis.
The fact that it now appears that rj7 is the gene responsible for loss of autoregulatory control in soybean classified as either supernodulated or hypernodulated by various groups does raise the issue about distinction between these two terms. It appears to us that these relative terms may not in fact be due to differential genetic control, but rather due to environmental or growth condition responses that allow more or less expression of nodule number by plants that have lost autoregulatory control. These mutant classes are certainly distinct from normally nodulated lines, but it is less certain that there is a true distinction between lines originally designated supernodulated or hypernodulated. It is well known that normally nodulated plants express a large range in nodule number depending on conditions that the plants are exposed to during the infection and subsequent nodule development stages. Our interpretation of a lack of consistent distinction between hypernodulated and supernodulated lines is consistent with a single gene (rj7) controlling autoregulatory response in soybean, and with observations on a range of nodule number within normally nodulated lines depending on growth condition.
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NOTES
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1 Trade and manufacturers names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. 
Received for publication September 19, 1999.
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REFERENCES
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- Buzzell, R.I., B.R. Buttery, and G. Ablett. 1990. Supernodulation mutants in Elgin 87 soybean. In P.M. Gresshoff et al. (ed.) Nitrogen fixation: Achievements and objectives. Chapman Hall, New York.
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ABSTRACT
INTRODUCTION
