Crop Science 40:78-83 (2000)
© 2000 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Genetics of Four Male-Sterile, Female-Fertile Soybean Mutants
R.G. Palmera
a USDA-ARS, CICG Research Unit and Dep. of Agronomy and Dep. of Zoology/Genetics, Iowa State Univ., Ames, IA 50011 USA
rpalmer{at}iastate.edu
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ABSTRACT
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Mutations affecting male cell and organ development may produce male-sterile, female-fertile plants that have application in plant breeding. In studies designed to test recombination frequencies and allelism, four such mutants were identified independently in soybean [Glycine max (L.) Merr.] populations that were characterized by chromosomal instability. The objectives of this study were to determine the inheritance of these four mutants and to test allelism with the seven known male-sterile, female-fertile soybean mutants. Allelism tests were done with male-sterile, female-fertile plants as female parents and known heterozygotes as male parents. All four mutants were inherited as single recessive nuclear genes. Male-sterile mutants (MSM)-1, MSM-2, MSM-3, and MSM-4 were nonallelic to each other. Mutants MSM-1, MSM-2, and MSM-3 were nonallelic to known mutants. They were assigned Genetic Type Collection numbers T357H, T358H, and T359H and the gene loci were assigned the symbols Ms7 ms7, Ms8 ms8, and Ms9 ms9, respectively. Mutant MSM-4 was allelic to T259H (Ms2 ms2) and was designated T360H Ms2 ms2 (Ames). The T259H Ms2 ms2 mutant becomes T259H Ms2 ms2 (Eldorado). Dehisced anthers from MSM-1 and MSM-4 were similar in phenotype. Aborted pollen grains of MSM-2 and MSM-3 were different in phenotype from each other and from the seven known male-sterile, female-fertile soybean mutants. These four independently derived male-sterile, female-fertile mutants could be used in plant breeding to produce hybrid seed.
Abbreviations: CMS, cytoplasmic male sterility • MSM, male-sterile mutant
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INTRODUCTION
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MUTATIONS affecting male function but leaving female reproductive capacity unaffected are useful in developmental studies of microsporogenesis and microgametogenesis, plant breeding, genetics, and molecular biology. Male sterility in higher plants has been reviewed by Kaul (1988).
In soybean, cytoplasmic male sterility (CMS) systems with restorer genes have been reported. Davis (1985) described a CMS line that resulted from cross-pollinations between cultivated soybeans (G. max) from widely separated maturity groups. Three reports from China announced different cytoplasmic male-sterility systems. Gai et al. (1995) and Zhang and Dai (1997) noted differences in male fertility among F1 plants from reciprocal cross-pollinations. Ding et al. (1998) reported that male sterility was complete and female fertility was excellent in the CMS line described by Gai et al. (1995). Their preliminary evidence strongly supported two cytoplasmic male steriles in soybean of independent origin. The best-characterized CMS soybean line was described by Sun et al. (1997), which resulted from a cultivated soybean crossed by the wild annual soybean [G. soja (Zucc. and Sieb.)].
Seven genic male-sterile, female-fertile mutations (ms1 to ms6 and the MWO mutant) have been described in soybean (Rubaihayo and Gumisiriza, 1978; Mariani et al., 1991; Palmer et al., 1992; Jin et al., 1997, 1998; Ilarslan et al., 1999). Several methods have been proposed to use genic steriles to produce hybrid seed (Rao et al., 1990; Horner and Palmer, 1995). In soybean, the traditional, dilution, and cosegregation methods were evaluated for hybrid seed production, and the cosegregation method was the most efficient (Lewers et al., 1996). The cosegregation method was used to determine the utility of per se and testcross evaluation for selecting germplasm to be included in cultivar development programs (Lewers et al., 1998a, 1998b), and in marker-assisted recurrent selection in soybean (Lewers and Palmer, 1997).
Four progeny rows, from experiments designed to test for recombination or allelism at the k2 (tan saddle) locus, y20 (yellow foliage) locus, or the Mdh1-n (mitochondrial malate dehydrogenase one null) locus, had both fertile and sterile plants (Chen and Palmer, 1996b, 1998b). The sterile plants had several one-, two-, and three-seeded pods and were suspected to be male-sterile, female-fertile mutants. Fertile plants in each of the four progeny rows were threshed individually. The objectives of this study were to determine inheritance of the four mutants and to test allelism with the seven known genic male-sterile, female-fertile soybean mutants.
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Materials and methods
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Inheritance
Genetic Types T239, T261, Clark isoline L67-3483, and PI 567.630A were obtained from R.L. Nelson (USDA ARS, Urbana, IL). Genetic Types T317 (Amberger et al., 1992), T323, and T325 (Hedges and Palmer, 1992) were isolated previously. The latter three genetic type mutants are yellow foliage (y20) and mitochondrial malate dehydrogenase one null (Mdh1-n) (Palmer et al., 1989). The four new male-sterile, female-fertile mutants were from different crosses (Table 1)
. Fertile plants in segregating families of the four original populations were threshed individually. In the next generation, the number of nonsegregating/segregating families and the number of fertile/sterile plants within segregating families were recorded.
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Table 1 Gene symbol and Genetic Type Collection number of new male-sterile, female-fertile soybean mutants with information on parents of the original cross combinations
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Allelism
Within segregating families of the four new genic male steriles and within segregating families of the seven known genic male steriles, sibling pollinations were made. Male-sterile, female-fertile plants were used as female parents and fertile plants as male parents. The resulting fertile F1 plants were confirmed heterozygotes (e.g., Ms ms) because upon self-pollination the F2 generation segregated the expected 3:1 fertile/sterile plants. These fertile F1 plants were used as male parents in the allelism tests. Tests of allelism were conducted to determine whether the new mutations arose at new loci or represented independent mutations at previously described loci. A recessive sterile homozygote was used as a female parent, and a known heterozygote, at the other tested locus, was used as the male parent.
F1 seed was planted either in the USDA-ARS glasshouse at Iowa State University in Ames or at the USDA-ARS Tropical Crops and Germplasm Research station near Isabela, Puerto Rico. The soil is a very fine, kaolinitic, isohyperthermic Typic Hapludox. F1 plants were classified at maturity as either fertile or sterile. The fertile F1 plants were threshed individually. The F2 seed was planted at the Bruner Farm near Ames, which has a Clarion–Nicollet loam soil type (fine-loamy, mixed, superactive, mesic Typic Hapludoll and fine-loamy, mixed, superactive, mesic Aquic Hapludoll). At maturity the total numbers of fertile and sterile plants within each progeny were recorded.
If the F1 plant segregation was in the ratio of 1:1 fertile/sterile plant and if the fertile F1 plants always segregated in the F2 about 3:1 fertile/sterile plants, the unknown was considered allelic to the known tester. If all the F1 plants were fertile and if the F2 family classification was one (3:1 fertile/sterile plants) : one (9:7 fertile/sterile plants), the unknown was considered nonallelic to the known tester. The 
2 test was used to determine whether the observed data fit the expected ratio.
Malate dehydrogenase (EC 1.1.1.37) isozyme pattern determination followed the procedure of starch gel electrophoresis described by Cardy and Beversdorf (1984a, 1984b).
Pollen
Male-fertile and male-sterile plants were identified at anthesis by squashing anthers in an aqueous solution of I2KI (Jensen, 1962). Anthers from male-fertile plants displayed densely stained pollen grains, whereas anthers from male-sterile plants were devoid of pollen grains or contained lightly stained pollen grains. Anther squashes were viewed on a Leitz orthoplan light microscope (Leica, Inc., Deerfield, IL) and photographed with Kodak Techpan film (Eastman Kodak, Co., Rochester, NY).1
Gene Symbols and Genetic Type Collection Numbers
The Soybean Genetics Committee approved the gene symbols; the curator of the USDA soybean germplasm collection, Dr. R.L. Nelson, assigned Genetic Type Collection numbers (Table 1). In addition, the Soybean Genetics Committee approved the change of T259H Ms2 ms2 to T259H Ms2 ms2 (Eldorado) (Table 1).
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Results and discussion
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All four male-sterile, female-fertile mutants, MSM-1, MSM-2, MSM-3, and MSM-4, were homozygous recessive Mdh1-n Mdh1-n, mitochondrial malate dehydrogenase one null. This was expected for the MSM-1 and MSM-4 mutants because both parents were Mdh1-n. However, for MSM-2 and MSM-3, only one of the parents was Mdh1-n (and y20), and the other parents were homozygous recessive k2. Chen and Palmer (1998a) observed that all mutants that they tested generated by instability at the k2 Mdh1-n y20 chromosome region were Mdh1-n. From the experiments, it is not known whether or not this instability was responsible for generating the four male-sterile, female-fertile mutants.
Progenies that resulted from crosses with L67-3483 (MSM-3; Table 1) gave fertile plants, sterile plants, and a new phenotype: partial-female-sterile plants (Tables 2, 3, and 4)
. This latter class was counted with the fertile plants because this phenotype is not related to the male-sterile, female-fertile phenotype. These partial-female-sterile plants have a phenotype similar to the PS-1 to PS-4 partial-female-sterile mutants (Pereira et al., 1997a, 1997b), but the inheritance is different (Palmer, 1997). This partial-female-sterile trait is not linked to the k2 or the t (pubescence color) locus (Chen and Palmer, 1997).
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Table 2 Segregation of fertile and sterile plants from F2, F3, and F4 generations and number of families from original soybean cross combinations
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Table 3 Allelism tests of new male-sterile, female-fertile mutants with known heterozygous genotypes of new male-sterile, female-fertile mutants in soybean, F2 data
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Table 4 Allelism tests of new male-sterile, female-fertile mutant MSM-3 (heterozygous genotype) with known mutations at the ms1, ms2, ms3, ms4, ms5, ms6, and Midwest Oilseed (MWO) sterile loci in soybean, F2 data
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Progenies that resulted from crosses with PI 567.630A (MSM-4; Table 1) also gave fertile plants, sterile plants, and a new phenotype: partial-sterile plants (Tables 2, 3, and 5)
. This latter class was counted with the fertile plants. This class is partial sterile (semisterile) for both male and female gametes because of the presence of a heterozygous translocation (Chen and Palmer, 1996a).
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Table 5 Allelism tests of new male-sterile, female-fertile mutant MSM-4 (heterozygous genotype) with known mutations at the ms1, ms2, ms3, ms4, ms5, ms6, and Midwest Oilseed (MWO) sterile loci in soybean, F2 data
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Genetics
The four independently-derived male-sterile, female-fertile lines each behaved as a single recessive nuclear gene (Chen and Palmer, 1996b; Table 2). This was confirmed in the F2, F3, and F4 generations. Allelism tests among the four independently derived lines indicated nonallelism (Table 3). All F1 plants were fertile, and the F2 families were approximately equally represented by families segregating 3:1 fertile/sterile plants and families segregating 9:7 fertile/sterile plants.
Male-sterile, female-fertile Mutant 1 (MSM-1) gave all fertile F1 plants and F2 families of approximately equal frequency segregating 3:1 fertile/sterile plants and segregating 9:7 fertile/sterile plants for all seven known male-sterile, female-fertile soybean mutants (Table 6)
. Similar results with the seven known mutants and the other three unknown mutants (MSM-2, MSM-3, and MSM-4) were recorded (Tables 4, 5, and 7)
. The only exception was with the cross of MSM-4 and the ms2 mutant. The F1 plant segregation was 6:9 fertile/sterile plants (2 = 0.60; P = 0.44). This 1:1 ratio is expected if the mutants are allelic. Progeny from the six fertile F1 plants segregated 3:1 fertile/sterile plants (718:272; 2 = 3.23, P = 0.07) in the F2 generation. A 9:7 fertile/sterile plants class was not found in the F2 generation. Additional cross-pollinations of this genetic combination using male-sterile, female-fertile plants by fertile plants in segregating families, gave 47:20 fertile/sterile F1 plants, a good fit to the expected 2:1 ratio (2 = 0.37, P = 0.55), based on the hypothesis of allelism. The 47 fertile F1 plants segregated 3:1 fertile/sterile plants (3886:1331; 2 = 0.73, P = 0.39) in the F2 generation. The 9:7 fertile/sterile plants class was not found in the F2 generation.
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Table 6 Allelism tests of new male-sterile, female-fertile mutant MSM-1 (heterozygous genotype) with known mutations at the ms1, ms2, ms3, ms4, ms5, ms6, and Midwest Oilseed (MWO) sterile loci in soybean, F2 data
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Table 7 Allelism tests of new male-sterile, female-fertile mutant MSM-2 (heterozygous genotype) with known mutations at the ms1, ms2, ms3, ms4, ms5, ms6, and Midwest Oilseed (MWO) sterile loci in soybean, F2 data
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The conclusions are that the four male-sterile, female-fertile mutants are conditioned by single recessive genes, and that MSM-4 is a new allele at the ms2 locus. Gene symbol ms7 was assigned to MSM-1, ms8 to MSM-2, ms9 to MSM-3, and MSM-4 became ms2 (Ames). The corresponding Genetic Type Collection numbers are T357H, T358H, T359H, and T360H.
Pollen
In anthers from fertile plants, the pollen grains stained a dark red-brown with I2KI. These engorged pollen grains filled the anther and were released by gently squashing the anther. Three colpi were evident for each pollen grain (Fig. 1A)
. Anthers from MSM-1 male-sterile plants were devoid of pollen grains (Fig. 1B). Dehisced anthers failed to release any aborted pollen grains or remnants of pollen grains. Pollen grains from MSM-2 male-sterile plants were not similar in appearance to any of the known ms soybean mutants. Dark-staining regions seemed to be in clusters (Fig. 1C). Pollen grains from MSM-3 male-sterile plants were similar in size to mature pollen grains from fertile plants. However, pollen grains were very lightly stained and colpi were not evident (Fig. 1D, 1E). Anthers from MSM-4 male-sterile plants, at anthesis, appeared shrunken and collapsed and were devoid of pollen grains (Fig. 1F). The MSM-4 and T295 ms2 (Graybosch et al., 1984) mutants are in different genetic backgrounds, but the anther phenotype was similar.
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Fig. 1 Pollen grains or anthers of fertile or male-sterile, female-fertile soybean plants. (A) Well-stained robust pollen grains with three colpi from fertile plant. (B) Whole anther devoid of pollen grains from MSM-1 plant. (C) Pollen grains with dark-staining clusters from MSM-2 plant. (D) and (E) Aborted pollen grains with shrunken cytoplasm and lightly stained cell wall from MSM-3 plant. (F) Whole anther devoid of pollen grains from MSM-4 plant. Bars for Fig. 1A, 1C, and 1E = 25 µm, and for Fig. 1B, 1D, and 1F = 100 µm
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Summary
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The k2 Mdh1-n y20 chromosomal region in soybean shows instability when introduced into certain genetic backgrounds. Three k2, 17 Mdh1-n, and 16 y20 mutational events have been genetically analyzed (Chen and Palmer, 1998a and references cited therein). In control populations, no mutations were evident in 883 F2 families. It cannot be determined from these results whether these four male-sterile, female-fertile mutants were due to instability at the k2 Mdh1-n y20 chromosomal region, the result of this region influencing instability at some other chromosome region of the soybean genome, or were due to an unrelated phenomenon. All four mutants are mitochondrial malate dehydrogenase one null (Mdh1-n), and MSM-1, MSM-2, and MSM-4 have tan saddle seedcoat (k2). Seven independent ms1 mutations, three independent ms3 mutations, and two independent mutations of ms4 and ms6 are known (Skorupska and Palmer, 1990; Palmer et al., 1992; Ilarslan et al., 1999). This report identifies the second independent event at the ms2 locus.Pereira Ilarslan Palmer 1997; Pereira Lersten Palmer 1997
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ACKNOWLEDGMENTS
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The author acknowledges Dr. X.F. Chen for making the initial cross-pollinations and Dr. H. Ilarslan for taking the photomicrographs. Ivy Andersen helped with the field studies and the author thanks her. The microscopic aspects of this study were carried out in the Bessey Microscopy Facility at Iowa State University.
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NOTES
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Joint contribution of the USDA-ARS, CICG Research Unit and Journal Paper no. J-18251 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011; Project 3352, and supported by Hatch Act and State of Iowa.
1 The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture or Iowa State University and does not imply its approval to the exclusion of other products that may be suitable. 
Received for publication March 8, 1999.
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REFERENCES
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- Amberger L.A., Shoemaker R.C., Palmer R.G. Inheritance of two independent isozyme variants in soybean plants derived from tissue culture. Theor. Appl. Genet. 1992;84:600-607.
- Cardy B.J., Beversdorf W.D. Identification of soybean cultivars using isoenzyme electrophoresis. Seed Sci. Technol. 1984;12:943-954 a.
- Cardy B.J., Beversdorf W.D. A procedure for the starch gel electrophoretic detection of isozymes in soybean [Glycine max (L.) Merr.]. Department of Crop Science Technology Bull. 119/8401. ON, Canada: University of Guelph, 1984 b.
- Chen X.F., Palmer R.G. Inheritance and linkage with the k2 and Mdh1-n loci in soybean. J. Hered. 1996;87:433-437 a.[Abstract/Free Full Text]
- Chen X.F., Palmer R.G. Isolation of four independently monogenically inherited male-sterile mutants in soybean. Soybean Genet. Newsl. 1996;23:134-139 b.
- Chen X.F., Palmer R.G. Isolation of a heterozygous female semisterile factor and its linkage relationship with three morphological markers in soybean. Soybean Genet. Newsl. 1997;24:170-174.
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- Davis, W.H. 1985. Route to hybrid soybean production. U.S. Patent 4595146. Date issued: 8 Oct. 1985.
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- Hedges B.R., Palmer R.G. Inheritance of malate dehydrogenase nulls in soybean. Biochem. Genet. 1992;30:491-502.[Web of Science][Medline]
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- Ilarslan H., Horner H.T., Palmer R.G. Genetics and cytology of a new male-sterile, female-fertile soybean mutant. Crop Sci. 1999;39:58-64.[Abstract/Free Full Text]
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- Jin W., Horner H.T., Palmer R.G. Genetics and cytology of a new genic male-sterile soybean [Glycine max (L.) Merr.] Sex. Plant Reprod. 1997;10:13-21.
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- Mariani P., Lucchin M., Guzzo F., Varotto S., Parrini P. Cytological evidence of a new male-sterile mutant in soybean [Glycine max (L.) Merr.] Sex. Plant Reprod. 1991;4:197-202.
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- Palmer R.G., Hedges B.R., Benavente R.S., Groose R.W. w4-mutable line in soybean. Dev. Genet. 1989;10:542-551.
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ABSTRACT
INTRODUCTION
