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a Dep. of Agronomy, Iowa State Univ., Ames, IA 50011-1010, USA
b Crop Research Institute, Anhui Academy of Agric. Sciences, Hefei, Anhui, P.R. China
c Dep. of Crop Science, Obihiro Univ. of Agric. and Veterinary Medicine, Inada, Obihiro, Hokkaido, Japan
d Dep. of Genetics, Development and Cell Biology, and Bessey Microscopy Facility, Iowa State Univ., Ames, IA 50011-1020, USA
e USDA-ARS-CICGR and Dep. of Agronomy, Iowa State Univ., Ames, IA 50011-1010, USA; present address: G301 Department of Agronomy, Iowa State University, Ames, Iowa 50011-1010 USA
* Corresponding author (rpalmer{at}iastate.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: BSA, bulk segregant analysis • CLG, classical linkage group • cM, centiMorgans • MAS, marker-assisted selection • MLG, molecular linkage group • SSR, simple sequence repeats
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Manual cross-pollination for the production of large quantities of hybrid seeds can be difficult and time consuming. Male-sterile mutants are considered a powerful tool in hybrid breeding programs. A cms system is the ideal system for hybrid seed production (Kaul, 1988). Two cms systems have been reported in soybean (Davis, 1985; Sun et al., 1997). One is being studied for its potential in hybrid seed production (Sun et al., 1997).
Alternatively, several methods have been proposed to use genic male-sterile plants to produce hybrid seed (Rao et al., 1990; Horner and Palmer, 1995). The use of CMS and genic nuclear male-sterile plants to produce hybrid soybean seed would require insect pollinators to transfer pollen from the male plants to the female plants. Genic male sterility in soybean is used to improve efficiency of hybridization by eliminating tedious hand emasculations, to enhance random mating for population development (Specht et al., 1985; St. Martin and Ehounou, 1989; Specht and Graef, 1992) and recurrent selection (Lewers and Palmer, 1997), and to facilitate the development of testers for inbred line evaluation and development (St. Martin et al., 1996; Ortiz-Perez, 2005). So far in soybean, nine nuclear male-sterile mutations have been reported (Palmer et al., 2004).
The male-sterile, female-fertile T259 mutant (ms2ms2) was found at Eldorado, Illinois (Bernard and Cremeens, 1975; Graybosch et al., 1984). The ms2 mutation causes abortion of young microspores at the tetrad stage of development, probably due to abnormalities of the tapetal layer (Graybosch and Palmer, 1985). The T259H (Ms2ms2) mutant line was used to develop the ms2 (‘Beeson’), ms2 (‘Clark’), ms2 (‘Wells’), and ms2 (‘Williams’) near iso-genic lines (Bernard et al., 1991). The ms2 (A00–63) mutant is a backcross-derived near-isogenic line of cultivar Beeson (Bernard et al., 1991). The T360 (ms2ms2) mutant was found at Ames, Iowa (Palmer, 2000). The ms? (A00–39) mutant was found in 1999 among progeny of a germinal revertant of T322 (w4–m), identified as ARS-10–483. Based on anther phenotype, male-sterile plants in family A00–39 were suspected to carry an allele at the ms2 locus.
Isozyme screening of near-isogenic male-sterile soybean lines, showed that the ms2 locus was linked with the phosphoglucomutase (Pgm1) locus with an estimated map distance of 18.73 ± 2.4 cM (Sneller et al., 1992). These two loci and the E2 locus comprise classical linkage group (CLG 15) (Palmer et al., 2004). Pgm1 is located on molecular linkage group (MLG) O. The position of the ms2 locus on MLG O has not been determined.
Another male-sterile, female-fertile mutant, T359 (ms9ms9), has pollen grains that stain very lightly without any evidence of colpi, although pollen grains are similar in size to mature pollen grains of fertile plants (Palmer, 2000). The T359 mutant has been used as a parent to produce large quantities of F1 hybrid seed for heterosis tests (Ortiz-Perez, 2005). The ms9 locus has not been located on the classical or the molecular soybean map.
The objectives were to verify the chromosome location of the ms2 (A00–63) mutation, to determine the allelism and the chromosome location of the suspected ms2 (A00–39) mutation, and to determine the chromosome location of the ms9 (T359) mutation. These results should facilitate the transfer of the ms2 and ms9 alleles in soybean breeding programs by using marker-assisted selection (MAS).
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In the allelism test, if the F1 plant segregation was in the ratio of 1 fertile:1 sterile (Ms?ms?: ms?ms?) plant and if the fertile (Ms?ms?) 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 1 (3:1, fertile:sterile plants): 1 (9:7, fertile:sterile plants), the unknown was considered nonallelic to the known tester. This suggests two separate and independent male-sterile, female-fertile mutant genes. The 
2 test was performed to determine whether the observed data fit the expected ratio.
Progeny Testing
F3 seeds from individual F2 fertile plants were harvested, and planted in summer 2004 for Pop-1, and summer 2005 for Pop-2 and Pop-3 at the Bruner Farm, near Ames, Iowa (Table 1). Segregation for fertility/sterility in the progeny rows was used to determine the genotype of each fertile F2 plant. The expected phenotypic ratio in the F2 generation was 3:1 (male-fertile:male-sterile plants), and the expected genotypic ratio of the F2:3 population was 1:2:1, [(Ms?Ms?:Ms?ms?:ms?ms?), (Ms2Ms2:Ms2ms2:ms2ms2), and (Ms9Ms9:Ms9ms9:ms9ms9)].
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Segregating F2 populations were classified at stage R1, at the beginning of flowering (Fehr and Caviness, 1979). Anthers from 5 to 10 plants per population were collected to identify presence/absence of pollen. On that basis only one segregating male-sterile F2 population tracing to just one F1 plant was selected as the mapping population. Individual F2 plants were phenotypically scored as fertile or sterile at maturity. The genotype of each F2 plant was determined in the F2:3 generation.
Pop-1 and Pop-2 were grown during fall 2003 and fall 2004, respectively, in the USDA greenhouse at Iowa State University (Table 1). Seeds were placed on germination paper, and each seedling was transplanted to individual pots, having 116 F2 plants for Pop-1 and 114 F2 plants for Pop-2 (Table 1). Plants were subjected to artificial photoperiod after transplanting, i.e., 3 wk with 18-h photoperiod, 2 wk with a photoperiod of 16-h, 2 wk with 14-h photoperiod, and for the remainder of the growing period until harvest, plants were subjected to 13-h photoperiod. During summer 2004, 148 F2 plants for Pop-3 were grown at the Bruner Farm near Ames, IA.
DNA Extraction
Two to three grams of young trifoliolates were collected from each F2 plant of the three mapping populations, Pop-1, Pop-2, and Pop-3, stored in individual labeled plastic bags, and freeze-dried for 48 to 72 h. Dry leaves were transferred to 15 mL sterile propylene tubes, ground to powder using glass beads, and kept at –80°C until DNA extraction. The protocol for DNA extractions used in previous research was followed (Kato and Palmer, 2003a; 2003b; 2004).
Also during summer 2004, 50 seeds of each of the parent lines used to develop the populations were planted in the USDA greenhouse at Iowa State University. Young trifoliolates of each parental line were collected in bulk and prepared for DNA extraction using the same procedure used for F2 plants.
SSR Analysis
The ms2 gene was mapped to CLG 15 (Sneller et al., 1992), which corresponded to MLG O (Cregan et al., 1999). Therefore, for Pop-1 and Pop-2 only 20 markers from MLG O were selected to test for polymorphism, using as criterion for selection an approximate genetic distance of less than 25 cM between two adjacent markers. For Pop-3, the parents were screened with 229 markers from all 20 MLGs to test for polymorphisms. The markers were selected using the same criterion as for Pop-1 and Pop-2.
At maturity, each F2 plant was phenotypically scored as either sterile or fertile. The initial SSR screening was done as separate experiments for each of the populations using bulk segregant analysis (BSA) method (Michelmore et al., 1991). The contrasted bulks were DNA of 10 F2 plants with sterile phenotype, and DNA of 10 F2 plants with fertile phenotype. The initial molecular screening was made using the two parents and the two contrasted bulks for each SSR marker to find polymorphisms between parents of each cross, and to identify the SSR markers that could be linked to each of the ms?, ms2, and ms9 loci.
Each PCR reaction mixture had a volume of 15 µL, including 1x PCR buffer, 1.8 µM of MgCl2, 0.17 mM of each of dNTPs, 0.25 units Taq polymerase (Bioline, Inc., Randolph, MA), 0.3 µM of forward and reverse primers, and 50 ng of template DNA (10 ng/µL). PCR was done at a PTC-100TM Programmable Thermal Controller (MJ Research, Inc., Waltham, MA), PCR condition was 45 s at 94°C, 45 s at 47°C, and 45 s at 68°C for 32 cycles.
PCR products were separated on 2–5% agarose gels (Agarose 3:1, Amresco Inc., Solon, OH), containing 0.50 µg/mL ethidium bromide, in 1x TBE buffer (0.089M Tris base, 0.089M boric acid, and 0.002M EDTA). The percentage of agarose gels depended on the difficulty of band separation. The band patterns were checked and photographed under UV light.
Data Analysis
Each plant in the population was scored according to the alleles at the locus, i.e., a score of A was assigned if the plant was homozygous for alleles from Minsoy, B if it was homozygous for alleles from male-sterile mutants, and H if it was heterozygous. After scoring the population, recombination values were calculated to determine if a specific primer was linked to the gene of interest, using the Linkage-1 program (Suiter et al., 1983).
Mapmaker V3.0 (Whitehead Institute, Cambridge, MA) (Lander et al., 1987) program was used to make the initial map, using a minimum logarithm of the odd ratio (LOD score) of 3, for accepting linkage between two markers as suggested by Kato and Palmer (2004). Recombination frequencies were converted to map distances in centiMorgans (cM) using Kosambi map function (Kosambi, 1944). JoinMap 3.0 (Kyazma B.V., Wageningen, Netherlands) (Van Ooijen and Voorrips, 2001) was used to make an integrated map, and (or) to make a final map.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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2(1:1) = 0.36, P = 0.55. This 1:1 ratio is expected if the mutants are allelic. Progeny from 20 fertile F1 plants segregated 3 fertile: 1 sterile in the F2 generation. For the combined F2 population, segregation was 1971 fertile: 683 sterile plants, 2(3:1) = 0.76, P = 0.38. Furthermore, a 9 fertile: 7 sterile plants class was not found in the F2 generation. Thus two separate and independent male-sterile, female-sterile mutant genes were not evident. The conclusion is that the ms? allele in the ms?ms? (A00–39) mutant line is an allele at the ms2 locus. The A00–39 mutant line was assigned Genetic Type Collection number T375H and the gene symbol Ms2ms2 (Ames 2). Then Genetic Type T360H becomes Ms2ms2 (Ames 1).
Molecular Mapping of the ms2 (Pop-1) and ms2 (Pop-2) Locus in Soybean
The molecular screening of the two parents and the two contrasted bulks for each SSR marker of MLG O of Pop-1, indicated linkage between the ms2 locus and the SSR marker Sat_190 for MLG O. In Pop-2, linkage between the ms2 locus and SSR markers Sat_190 and Scaa001 from MLG O was shown.
To construct a map for ms2 (A00–39) and ms2 (A00–63), additional polymorphic markers on MLG O were tested. The polymorphic SSR markers were Satt477, Satt592, Satt581, Satt153, Sat_231, Sat_109, and Sat_190 for Pop-1 (Table 2), and SSR markers Sat_274, Satt153, Sat_231, Sat_109, Sat_190 and Scaa001 for Pop-2 (Table 3).
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2 calculations from the F2:3 populations' progeny tests and the F2 populations molecular screenings of Pop-1 and of Pop-2 with 116 and 114 individuals, respectively, showed a good fit to the expected genotypic ratio of 1 male-fertile, female-fertile homozygote: 2 heterozygotes: 1 male-sterile, female-fertile homozygote with 2(1:2:1) = 2.34 and P = 0.31 for Pop-1, and 2(1:2:1) = 1.00 and P = 0.61 for Pop-2 (Tables 2 and 3). Mapping data for Pop-1 and Pop-2 were combined and integrated using JoinMap 3.0 (Van Ooijen and Voorrips, 2001), resulting in a 2(1:2:1) value of 1.07 with P = 0.59 (Table 4).
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Molecular Mapping of the ms9 Locus (Pop-3)
The molecular screening of the two parents and the two contrasted bulks for Pop-3 using 229 SSR markers, indicated linkage between ms9 and Satt 521 on MLG N. To construct a map for ms9, eight additional polymorphic markers on MLG N were tested. Linkage values were calculated between the ms9 locus and polymorphic SSR markers Sat_033, Sat_266, Sat_236, Satt521, Satt237, Sat_091, Sat_239, Sat_241, and Sat_306.
The F2:3 progeny test and the F2 population molecular data from 148 plants gave 2 calculations that showed a good fit to the genotypic ratio of 1:2:1. This indicated that the F2:3 segregation for the ms9 gene fit a 1 male-fertile, female-fertile homozygote: 2 heterozygotes: 1 male-sterile, female-fertile homozygote ratio with 2(1:2:1) = 4.75 and P = 0.09 (Table 5). A map was constructed using Mapmaker V3.0 (Lander et al., 1987). The ms9 locus was located between markers Satt521 and Satt237 and linked to each by 8.5 cM and 16.2 cM, respectively (Fig. 2
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The present study identified molecular markers closely linked to the ms2 and ms9 loci, respectively. Among nine soybean male-sterile mutant genes, only two mutant genes (MWO ms, MLG D1b and ms3, MLG D1b) had been mapped using molecular markers (Jin et al., 1998; Cervantes-Martinez, 2005). Molecular markers are now available to introgress these alleles into breeding lines by the backcross procedure.
The allelism tests confirmed that the ms? mutation of A00–39 was allelic to the ms2 allele of the A00–63 ms2 mutant line. Both male-sterile, female-fertile mutants were mapped, as expected, to MLG O. A total length of 16.9 cM between Sat_190 and Scaa001 in the present study was similar to the 16.6 cM in the saturated molecular linkage map (Song et al., 2004). In this chromosomal region, two RFLP markers, A644_1 and A955_2, had been reported (Song et al., 2004). Thus, these four markers are potential molecular markers for MAS to determine if Ms2Ms2 or Ms2ms2 is used in the backcross-breeding programs and to narrow the genetic map position for the map based cloning of ms2.
The ms9 locus was located between Satt521 and Satt237 on molecular linkage group N, and linked to each by 8.5 cM and 16.2 cM, respectively (Fig. 2). In a total length of 24.7 cM between Satt521 and Satt237 in the present study, an RFLP marker of BLT015_1 and two SSR markers, Satt660 and GMABAB, had been reported (Song et al., 2004). These five markers are also potential molecular markers for MAS to determine if Ms9Ms9 or Ms9ms9 is used in the backcross breeding program and to narrow the genetic map position for the map based cloning of ms9.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication March 2, 2006.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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