Brief Communications
A Dominant, Host Plant Mutation Conferring Ineffective Nodulation in the Chickpea-Rhizobium Symbiosis V. G. Paruvangada and T. M. Davis Chickpea (Cicer arietinum L.) mutant PM638, a derivative of wild-type, desi chickpea line ICC 640, forms yellowish, ineffective root nodules and differs phenotypically from previously described chickpea nodulation mutants. To establish the mode of inheritance of the mutant nodule trait, and to eliminate independent chlorophyll and sterility mutations carried by the mutant, PM638 was crossed with ICC 640 and an F5 generation mutant was reciprocally backcrossed to ICC 640. Segregation analysis indicated that the mutant phenotype was conferred by a single, dominant nuclear gene mutation. In keeping with established nomenclature for chickpea nodulation genes, the mutant allele is tentatively designated Rn7. Chickpea (Cicer arietinum L.), also called garbanzo bean, is an economically important food legume. Like its relatives in the family Viciae (Smartt 1990), chickpea forms indeterminate root nodules characterized by the presence of a persistent nodule meristem. The chickpea symbiont is referred to as Rhizobium ciceri (Cadahia et al. 1986; Gaur and Sen 1979; Kingsley and Bohlool 1983). Chickpea is a self-pollinating diploid (2n 5 2x 5 16), and has a relatively small genome size among legumes, with a haploid DNA content of 1C 5 0.95 pg (Patankar and Ranjekar 1984). As such, chickpea is a tractable subject for genetic investigation of host factors involved in the legume-Rhizobium symbiosis. Plant nodulation or symbiotic genes have been identified by genetic study of
naturally occurring variants or induced mutants in several different legume species (Caetano-Anolle´s and Gresshoff 1991; Rolfe and Gresshoff 1988; Vance et al. 1988). The most extensive analysis of nodulation genes has been in pea (Pisum sativum L.). Several pea nodulation mutants referred to as ‘‘sym’’ mutants have been obtained through induced mutation ( Duc and Messager 1989; Kneen et al. 1987; Kneen and LaRue 1988), and characterized to varying extents (e.g., Fearn and LaRue 1991; Kneen et al. 1990). Mutants with altered symbiotic properties, including nonnodulating and ineffectively nodulating (nitrogen fixation poor or lacking) mutants, have also been identified and characterized at the physiological and biochemical levels in soybean (Glycine max) and alfalfa (Medicago sativa) (Carroll et al. 1985; Egli et al. 1989). Six nodulation mutants have been described in chickpea ( Davis 1988; Davis et al. 1985, 1986; Singh et al. 1992). These nonnodulating and ineffectively nodulating mutants are useful tools for studying the host contribution to the nodulation process in the legume-Rhizobium symbiosis. Radiation-induced chickpea mutants PM233B, PM665B, PM679B, PM405B, and PM796B, carrying mutant genes rn1–rn5, respectively, are particularly valuable for comparative studies because they are all essentially isogenic, differing from a common wild-type parent, ICC 640, only with respect to mutations in single nodulation genes ( Davis 1988; Davis et al. 1986). Ineffectively nodulating chickpea mutant PM638 was a product of the induced mutation study of Davis et al. (1985), but has not yet been described. The transmission genetic study of mutant PM638 is reported here.
Materials and Methods The characteristics of wild-type parental line ICC 640 (a desi-type chickpea) and the
mutagenesis experiment that gave rise to ineffectively nodulating mutant PM638 and other chickpea nodulation mutants have been described elsewhere ( Davis et al. 1985). Morphological features of mutant PM638 were examined by visual comparison of shoots and roots of mutant versus wild type plants. An inheritance study of the ineffective nodulation trait in chickpea mutant PM638 was initiated by crossing PM638 with ICC 640. An F5 generation mutant was then used in backcrosses with ICC 640 to produce BCF2 and BCF3 progenies for segregation analysis. Crosses were made by hand pollination and advanced beyond the F1 generation via natural self-pollination. Several BCF1 plants were grown in soil in the greenhouse to produce seeds for BCF2-generation testing. Other BCF1 plants and all BCF2 and BCF3 plants were grown under nitrogen deficient conditions to test their nodulation phenotypes. Seeds were planted in pots filled with vermiculite, inoculated with Rhizobium ciceri strain CC1192, and watered daily with nitrogenfree nutrient solution ( Davis et al. 1985). Plants were grown in a root zone chamber ( Environmental Growth Chambers, Chagrin Falls, Ohio), which provided a constant root temperature of 268C ( Davis et al. 1986). Ten plants each of the mutant and ICC 640 were included in each trial to serve as controls. Phenotypic segregation data from BCF2 and BCF3 populations were collected 25 days after planting.
Results When grown in the absence of mineral nitrogen and inoculated with R. ciceri, ineffectively nodulating mutants were easily recognized by their yellowish, comparatively spherical nodules and by their shoot nitrogen deficiency symptoms including stunted growth and chlorotic leaves indicative of symbiotic ineffective-
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tant-type BCF3 families. Approximately one-third of the 16 ineffectively nodulating BCF2 plants subjected to progeny testing bred true, while the balance produced segregating BCF3 families ( Table 1).
Discussion
Figure 1. Appearance of shoots in 4-week-old wild-type and ineffectively nodulating mutant chickpeas grown in nitrogen-deficient medium with or without Rhizobium inoculation. Left: wild-type ICC 640 (inoculated). Shoot growth is vigorous due to effective nodulation. Middle: wild-type ICC 640 (uninoculated). Stunted, nitrogen-deficient shoot growth is due to lack of root nodules. Right: mutant PM638A (inoculated). Stunted, nitrogen-deficient shoot growth is due to ineffectiveness of root nodules.
selfing and selection, the albino and sterility mutations were eliminated and a true-breeding nodulation mutant line, designated PM638A, was recovered in the F5 generation. PM638A was used as a parent in reciprocal backcrosses to wild-type parent ICC 640. All BCF1 plants from reciprocal crosses between PM638A and ICC 640 had the mutant nodule phenotype. A 3:1 ratio of ineffectively nodulating to normally nodulating plants was seen in the BCF2 populations and within segregating BCF3 families ( Table 1). All 11 normally nodulating BCF2 plants subjected to progeny testing were true breeding, as would be expected for the homozygous recessive class. A homozygous mutant line for use in future investigations, designated as PM638B, was initiated by seed propagation of a plant selected from one of the nonsegregating, mu-
ness ( Figure 1). This was in contrast to wild-type plants which formed normally elongated, reddish brown nodules, grew vigorously ( Figure 1), and had dark green leaves. Similarly, wild-type and mutant plants were easily distinguishable in all segregating populations used in the inheritance study. PM638 and advanced-generation mutant progeny grew normally in soil when provided with adequate mineral nitrogen. In the F1 and subsequent generations of the initial PM638 3 ICC 640 cross, ineffective nodulation was inherited in the manner of a dominant, nuclear-encoded trait. Satisfactory F2 segregation data were unobtainable for the nodulation trait, however, because a recessive, seedling-lethal chlorophyll mutation and a recessive sterility mutation carried by PM638 were also segregating. Through repeated cycles of
Table 1. Analysis of BCF2 and BCF 3 segregation data in reciprocal crosses between PM638A and ICC 640 x2
No. of plants or families Generation: cross
Mutant
F2: ICC 640 3 PM638A F2: PM638A 3 ICC 640
74 59
F3 families: PM638A 3 ICC 640 Among: ( F2 normal nodules) Among: ( F2 ineffective nodules) Within: ( F2 ineffective nodules)
5 66
298 The Journal of Heredity 1999:90(2)
Segregant
0 11
Normal
Value (ratio)
P
19 18
1.04 (3:1) 0.11 (3:1)
.50–.30 .80–.70
11
0.00 (0:1) 0.03 (1:2) 2.51 (3:1)
.90–.70 .20–.10
31
A single dominant nuclear gene mutation carried by original mutant PM638 was shown to account for the ineffective nodulation phenotype in crosses between mutant line PM638A and ICC 640. This is the first dominant mutation known to affect the chickpea-Rhizobium symbiotic process. The other nodulation gene mutations that impair the symbiotic process in chickpea ( Davis 1988; Davis et al. 1986; Singh et al. 1992), as well as in most other legumes (Caetano-Anolle´s and Gresshoff 1991; Vance et al. 1988), are recessive, with exceptions as follows. Strain-specific ineffective nodulation or nonnodulation responses controlled by dominant genes have been reported in soybean and pea. In soybean variety Hardee, ineffective nodulation in response to Bradyrhizobium japonicum strains 122 and 33 was attributed to dominant genes Rj2 and Rj3, respectively ( Vest 1970), whereas in variety Hill, ineffective nodulation in response to B. japonicum strain 61 was attributed to the dominant Rj4 gene ( Vest and Caldwell 1972). In soybean cultivar Kent, ineffective nodulation in response to fast-growing rhizobial strain USDA 205 was also attributed to a single dominant gene ( Devine 1984). The Rj2 and Rj4 genes were shown to be nonallelic to each other and to the recessive, nonnodulation gene rj1 ( Devine and O’Neill 1989). Strain-specific nonnodulation in response to various strains of European Rhizobium leguminosarum bv. viciae (Rlv) has been studied in detail in wild pea varieties Iran and Afganistan ( Kozik et al. 1995; Lie 1984; Lie et al. 1976). Temperature-sensitive, strain-specific nonnodulation in Iran was attributed to the effects of a single dominant gene, Sym1 ( Lie 1984). When inoculated with Rlv strain PRE, no nodules formed at 208C, but normal nodules formed at 268C ( Lie et al. 1976). Strain-specific nonnodulation in Afganistan was attributed to the Sym2 gene, which behaved as dominant in relation to Rlv strain PRE but recessive or semidominant in relation to strain PF2 ( Lie 1984). Subsequent research showed that Sym1 and Sym2 conditioned similar strain and temperature-sensitive phenotypes and are alleles of the same gene ( Kozik et al. 1995).
As indicated above, strain-specific nonnodulation responses in soybean and pea typically have been conditioned by dominant host genes. It would be interesting to determine whether this pattern extends to the dominant Rn7 mutation in chickpea. However, it has not yet been determined whether the ineffective nodulation of chickpea mutant PM638B occurs in response to R. ciceri strains other than CC1192. For use in future experimental work, mutant line PM638B was derived from original mutant PM638 by crossing and backcrossing to wild-type parent ICC 640, as was done for previously described chickpea nodulation mutants ( Davis 1988). Backcrossing to wild type was required to eliminate undesirable chlorophyll, sterility, and possibly other mutations. In its symbiotic function, ineffectively nodulating mutant PM638 (and its backcross derivatives, PM638A and PM638B) differs phenotypically from previously described chickpea nodulation mutants. Mutants PM233 ( Davis et al. 1985, 1986) and ICC 435M (Singh et al. 1992) lack nodules entirely, while PM665 and PM679 have a temperature-dependent, nonnodulation phenotype ( Davis et al. 1986). PM405 forms numerous, small, white nodules that lack detectable acetylene reduction activity (a measure of nitrogenase activity), while PM796 nodules have a narrow band of leghemoglobin pigmentation near the tips, but are otherwise green, and reduce low but detectable levels of acetylene ( Davis 1988). PM638 nodules are larger than those of PM405 and are yellowish rather than white. Although PM638 has a unique phenotype as compared to other chickpea nodulation mutants, it is not known for certain whether the dominant mutant gene it carries is allelic to any of the previously identified mutant derivatives of ICC 640 ( Davis 1988; Davis et al. 1986). Allelic relationships among the rn1–rn5 genes were determined by complementation tests ( Davis 1988; Davis et al. 1986). However, complementation tests cannot be carried out for PM638 because the mutation is dominant and therefore complementation between it and any other mutations cannot be detected in F1 hybrids. One source of insight into the relationship of chickpea nodulation genes will be to determine their genome map positions.
Mapping of the PM638 mutation to a unique locus will demonstrate its distinction from any other mapped nodulation gene, while mapping to the same locus as a previously known nodulation gene will indicate possible allelism. Results of ongoing investigations ( Davis TM, unpublished data) indicate that the PM638 mutation is closely linked to the rn1 gene and is unlinked to the other known chickpea nodulation genes. However, the recessive rn1 allele, which completely eliminates infection thread and nodule formation (Matthews and Davis 1990), interrupts the symbiotic process at a much earlier developmental stage than does the dominant PM638 mutation. Thus the PM638 mutation appears to define a new nodulation gene to which we tentatively assign the gene symbol Rn7. The naming of the Rn7 gene is in accordance with the nomenclature used for other chickpea nodulation gene mutations identified to date ( Davis 1988; Davis et al. 1986; Singh et al. 1992), indicating that it is the seventh gene in the series of known chickpea genes having a role in the symbiotic process. Because of the novelty of dominant nodulation gene mutations, molecular characterization of Rn7 may offer valuable insight into the regulation of root nodule development and function. From the Department of Plant Biology, University of New Hampshire, Durham, NH, 03824. Address correspondence to T. M. Davis at the address above or email:
[email protected]. This article is scientific contribution no. 1876 from the New Hampshire Agricultural Experiment Station.
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q 1999 The American Genetic Association
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Brief Communications 299
