Journal of Heredity 2003:94(5)
© 2003 The American Genetic Association 94:425-428
Brief Communication |
Molecular Mapping of the Male-Sterile, Female-Sterile Mutant Gene (st8) in Soybean
From the Department of Crop Science, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido, Japan (Kato), and from the USDA-ARS CICGR and the Departments of Agronomy and Zoology/Genetics, Iowa State University, Ames, IA 50011-1010 (Palmer).
Address correspondence to Dr. Palmer at the above address, or e-mail: rpalmer{at}iastate.edu.
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Abstract |
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Soybean male-sterile, female-sterile mutant genes have been identified by genetic and cytological studies. The St8 gene has been identified as an asynaptic mutation resulting in male and female sterility. This mutant gene was derived from a gene-tagging study using the soybean w4-mutable line. In this report we identified the genetic map position of st8 via restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers. The St8 gene mutation was located between RFLP marker E107 and SSR markers Satt132, Sct_065, and Satt414 on molecular linkage group J and linked to each by 7.8 cM and 3.4 cM, respectively.
Meiosis comprises a complex series of events including chromosome pairing, synaptonemal complex formation and crossing over, chromosome segregation, and cytokinesis. The complexity of events suggests that many genes are tightly regulated to ensure each successful meiotic division. In plants, mutants exhibiting alterations in meiosis have been identified as sterile or partially fertile mutants: maize (Golubovskaya 1989; Staiger and Cande 1993), rice (Kitada et al. 1983), tomato (Moens 1969), and Arabidopsis thaliana (Chaudhury et al. 1994; Dawson et al. 1993; He et al. 1996; Peirson et al. 1996; Ross et al. 1997). In meiosis, synapsis of homologous chromosomes is a key event because it is essential for normal chromosome segregation and is implicated in the regulation of crossover frequency. Synaptic mutants represent an important class of meiotic mutants (reviewed by Riley and Law 1965) and are subdivided into two groups: (1) asynaptic mutation, in which asynaptic mutants are defective in homologue synapsis, and (2) desynaptic mutation, in which desynaptic mutants are defective in maintenance of synapsis after a normal synaptic process.
In soybean a total of five recessive mutations, st2, st3, st4, st5, and st8, and one duplicate-factor inheritance gene mutation, st6 and st7, affecting chromosome synapsis have been identified by genetic and cytological analyses (Ilarslan et al. 1997; Palmer and Horner 2000; Palmer and Kilen 1987; Skorupska and Palmer 1990). In accordance with Riley and Law (1965), st2, st3 (Palmer and Kilen 1987), and st8 (Palmer and Horner 2000) are classified as an asynaptic mutation, whereas st4, st5, st6, and st7 are classified as desynaptic mutations. These mutants are male-sterile, female-sterile plants. In addition, a new male-sterile, female-fertile desynaptic mutant in soybean has been identified (Bione et al. 2002). The St8 mutation has been identified among progenies of germinal revertants at the w4-mutable locus (Palmer et al. 1989). Cytological analyses demonstrated that chromosome pairing was completely absent in homozygous recessive st8st8 sterile plants (Palmer and Horner 2000).
The objective of the present study was identification of the genetic map position of the st8 gene via molecular markers restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR).
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Materials and Methods |
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Plant Materials
Cv. Minsoy (PI 27890; female parent) was crossed with T352H (St8st8) plants with use of standard soybean crossing techniques at the Bruner Farm near Ames in summer 2000. The F1 seeds were advanced to the F2 at the University of Puerto Rico–Iowa State University Soybean Nursery near Isabela, Puerto Rico. All F1 plants were fertile. F2 seeds were planted at the Bruner Farm, in May 2001. Segregation for fertile or sterile plants, or for both, was recorded at maturity. Seventy-nine F2 plants were used for the mapping study. F3 seeds from fertile F2 plants were harvested individually. These F3 seeds were planted at the University of Puerto Rico–Iowa State University Soybean Nursery in September 2001. Segregation of fertile or sterile plants, or for both, in each F3 line was recorded at maturity to classify F2-plant genotype.
SSR Analysis
Soybean DNA was isolated from freeze-dried leaf tissue of parental and 79 F2 plants of a cross between Minsoy and T352H, in accordance with Keim et al. (1988). SSR markers (Akkaya et al. 1992) were evaluated as follows. Polymerase chain reaction (PCR) mixture contained 50 ng of soybean genomic DNA, 1.75 mM Mg2+, 0.15 mM of sense and antisense primers, 150 mM of each nucleotide, 1 x PCR buffer, and 0.5 U Taq DNA polymerase (Promega) in a total volume of 30 µl. Cycling consisted of 45 s at 94°C, 45 s at 47°C, and 45 s at 68°C for 32 cycles on a PTC-100TM Programmable Thermal Controller (MJ Research, Inc.). PCR products were run on 2.0 % (w/v) Agarose 3:1TM E776 (AMRESCO) gel in 0.5 x TBE (0.089 M Tris-borate, 0.089 M boric acid, 0.002 M EDTA) buffer with ethidium bromide incorporated in the gel or on sequencing gel; 8% (w/v) acrylamide to bis-acrylamide (29:1); 5.6 M urea; and 30% (v/v) formamide in Tris-acetate EDTA (TAE) buffer.
RFLP Analysis
For Southern blotting experimentation, extracted DNA, was purified with phenol and chloroform, according to the modified CTAB method (Murray and Thompson 1980). Parental and F2 DNA (7 µg) was digested overnight with restriction enzymes, HindIII, EcoRI, EcoRV, DraI, TaqI, XhoI, BamHI, SspI, and HaeIII. Digested DNA was electrophoresed in a 0.8% agarose gel in 0.5 x TBE buffer, and transferred onto Hybond-N+ nylon membranes. Transferred membranes were baked for 2 h at 80°C. Labeling of probes, prehybridization, hybridization, and detection were conducted with ECL direct nucleic acid labeling and detection systems (Amerham Pharmacia Biotech, Inc.). The probes for the present study were kindly provided by R. C. Shoemaker (USDA-ARS, Iowa State University, Ames). The following labeling and detection procedure was modified to be suitable for Southern hybridization in soybean, in accordance with original protocol (Amersham Pharmacia Biotech, Inc.). Probes were labeled directly with enzyme peroxidase for 1 h at 37°C. Membranes were prehybridized at 38°C for 1 h and subsequently hybridized overnight at the same temperature. Membranes were washed with primary wash buffer at 55°C for 10 min and 5 min. Subsequently, membranes were washed with 2 x 0.3 M NaCl, 0.03 M Na3 citrate·2H2), pH to 7.0 at room temperature for 10 min and 5 min. Washed membranes were soaked with mixed detection reagents for 1 min at room temperature. Membranes were exposed to X-ray film at room temperature for 1 h.
Linkage Analysis
The MapMaker program (Lander et al. 1987) was used to construct a linkage map. A LOD score of 3 was used as the lower limit for accepting linkage between two markers. Recombination frequencies were converted to map distances in centiMorgans (cM) by the Kosambi (Kosambi 1944) function. On the basis of two-point analysis, MapMaker generated log-likelihood values for the most probable order.
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Results |
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Segregation Patterns
In F2 generation we observed 59 fertile and 20 sterile plants. Sterile F2 plants were completely male and female sterile and provided no progeny. Fertile F2 plants were tested in F3 generation to determine genotype. On the basis of the F3 progeny test, F2 fertile plants were divided into two classes: 21 F2:3 families were true breeding fertile, and 38 F2:3 families segregated approximately 3:1, fertile to sterile plants. As a result, three genotype classes in F2 generation were segregated 21:38:20 and followed a 1:2:1 ratio (
2 = 0.14, P =.93).
Identification of SSR Markers Linked to st8
Initial screening of the F2 population was conducted by selecting several SSR markers from each linkage group (Cregan et al. 1999). The markers were chosen to divide each linkage group into segments of less than 30 cM. SSR marker Satt414 on molecular linkage group J was identified as linked to the st8 locus (LOD = 26.83). An additional 23 markers, Satt405, Satt249, Satt287, Sct_046, Satt285, Sct_065, Satt596, Scaa003, Satt280, Satt456, Satt406, Satt380, Sat 093, Satt183, Satt529, Sct_001, Satt244, Satt431, Satt132, Satt215, Satt547, Satt686, and Sat 259 from this linkage group (Cregan et al. 1999; Cregan PB, unpublished data) were screened between parental lines. We detected polymorphisms for SSR markers: Satt285, Satt132, Sct_065, Satt414, Satt686, Sat_259, Satt596, Satt456, Satt406, Satt380, Satt183, Satt280, Satt215, Satt547, and Satt431. We tried to locate RFLP markers for the gap between two SSR markers, Satt285 and Satt132. Five RFLP markers, A060, B046, A204, B074, and E107, from this linkage group (Cregan et al. 1999) were screened between parental lines. We detected polymorphism for E107 between parental lines after digestion with HaeIII. Among these polymorphic marker loci, Satt132, Sct_065, and Satt414; Satt686 and Sat_259; Satt596 and Satt456; and Satt406, Satt380, and Satt183 cosegregated, respectively. On the basis of LOD scores generated from the MapMaker program, the st8 locus was linked to markers Satt285; Satt132 and Sct_065; Satt686 and Sat_259; Satt596 and Satt456; Satt406, Satt380, and Satt183; and Satt280, Satt215, and E107, with LOD scores of 3.71, 26.83, 24.05, 25.40, 24.35, 10.01, 19.05, and 18.53, respectively. The most likely order of markers is shown in Figure 1. The St8 locus was between locus E107 and loci Satt132, Sct_065 and Satt414 and linked to each by 7.8 cM and 3.4 cM, respectively. Segregation ratios of all tested SSR and RFLP markers on molecular linkage group J provided good fits to 1:2:1 in codominant marker loci, or 1:3 in dominant marker locus (Table 1).
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Discussion |
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Mutant studies on meiosis are commonly used to dissect complex cellular processes. Many genes causing various meiotic mutations have been reported in diverse organisms, including yeast (Roeder 1995), Drosophila (Orr-Weaver 1995), Caenorhabditis elegans (Zetka and Rose 1995), humans (Bickel and Orr-Weaver 1996), and many higher plants (Kaul and Murthy 1985). Plants have been used for decades to study meiosis because of accessibility of the male gametophyte, microsporocytes, and pollen grains, and synchronous development among sprogenous cells within an anther. Mutations exhibiting alterations in meiosis have been identified in numerous plant species, including maize (Golubovskaya 1989; Staiger and Cande 1993), rice (Kitada et al. 1983), tomato (Moens 1969), and Arabidopsis thaliana (Chaudhury et al. 1994; Dawson et al. 1993; He et al. 1996; Peirson et al. 1996; Ross et al. 1997). Most of these plants provided good models for meiotic studies because of the large size of their anthers and chromosomes. In soybean, in spite of the small size of the anthers and chromosomes, a total of five single recessive gene mutations, st2, st3, st4, st5, and st8, and one duplicate-factor inheritance gene mutation, st6 and st7, affecting chromosome synapsis have been genetically and cytologically identified (Ilarslan et al. 1997; Palmer and Horner 2000; Palmer and Kilen 1987; Skorupska and Palmer 1990). In Arabidopsis mutant, asy1, with defects in both male and female meiosis, encodes a polypeptide exhibiting similarity to the HOP1 gene of Saccharomyces for synaptonemal complex assembly and normal synapsis (Caryl et al. 2000). The asy1 protein is required for meiotic chromosome synapsis, localized to axis-associated chromatin in Arabidopsis and Brassica (Armstrong et al. 2002). In addition, much effort has been expended in identifying and characterizing the meiotic proteins that are involved in synapsis and the genes that encode them or regulate their expression (Roeder 1997). Although cytological studies of higher plant meiosis, especially male meiosis, have provided a wealth of information regarding the physical changes associated with meiotic division (Bass et al. 2003; Brown and Lemmon 1996; Rhoades 1950; Tanaka 1991; Wolniak 1976), very little is known about the genes and proteins involved in this complex process. Map position is essential for map-based gene isolation. In soybean, st5 has been identified on classical linkage group 8 (Mahama et al. 2002), which has been integrated into molecular linkage group F (Cregan et al. 1999). This is the first report showing the molecular genetic map position of a soybean male-sterile, female-sterile mutant gene. We are just at the starting point in clarifying the complex genetic mechanism of synapsis in soybean.
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Acknowledgments |
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We are grateful to Dr. P. B. Cregan, USDA-ARS, Soybean and Alfalfa Research Lab, Beltsville, MD, and to Monsanto for kindly providing DNA sequence information of unpublished primers. We are grateful to Dr. R. C. Shoemaker, USDA-ARS, Iowa State University, Ames, for kindly providing RFLP probes. This is a joint contribution of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 3769 and the USDA-ARS, Corn Insects and Crop Genetics Research Unit, and is supported by the Hatch Act and the State of Iowa. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by Iowa State University or the USDA, and the use of the name by Iowa State University or the USDA implies no approval of the product to the exclusion of others that may also be suitable.
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Footnotes |
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Corresponding Editor: Halina T. Knap
Received December 5, 2002
Accepted June 17, 2003
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References |
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