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(American Journal of Botany. 2002;89:559-565.)
© 2002 Botanical Society of America, Inc.

Genetics and Molecular Biology

Molecular mapping of the fasciation mutation in soybean, Glycine max (Leguminosae)1

H. Caglar Karakaya2, Yuhong Tang2, Perry B. Cregan3 and Halina T. Knap2,4

2The Interdisciplinary Genetics Program and Crop and Soil Environmental Science Department, Clemson University, 276 Poole Agricultural Center, Clemson, South Carolina 29634-0359 USA 3Soybean Genomics and Improvement Laboratory, USDA, ARS, Bldg. 006, Room 100, 10300 Baltimore Ave., Beltsville, Maryland 20705-2350 USA

Received for publication June 26, 2001. Accepted for publication October 9, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The spontaneous fasciation mutation generates novel developmental diversity in cultivated soybean, Glycine max (L.) Merrill. An increased apical dominance in the mutant inhibits axillary buds, causes a branchless phenotype, and restricts reproduction to shoot apices. The fasciation mutation is encoded by a recessive (f) allele at a single locus. The mutation, despite its importance in soybean development, has no locus assignment on previously reported molecular maps of soybean. A population of 70 F2 progeny was derived from a cross between ‘Clark 63’ and the fasciation mutant. More than 700 molecular markers (amplified restriction fragment length polymorphisms [AFLPs], random amplified polymorphic DNAs [RAPDs], restriction fragment length polymorphisms [RFLPs], and simple sequence repeats [SSRs]) were used in mapping of the fasciation phenotype. Twenty linkage groups (LGs) corresponding to the public soybean molecular map are represented on the Clark 63 x fasciation mutant molecular map that spans 3050 centimorgans (cM). The f locus was mapped on LG D1b+W and linked with two AFLPs and four SSR markers (Satt005, Satt141, Satt600, and Satt703). No linkage was found between the f locus and several cDNA polymorphic loci between the wild type and the mutant. The known map position of the f locus and demonstration of the mutant phenotype from early postembryonic throughout reproductive stages provide an excellent resource for investigations of molecular mechanisms affecting soybean ontogeny.

Key Words: development • fasciation • Glycine max • Leguminosae • map • molecular markers • mutant • polymorphism • soybean


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
In soybean, Glycine max (L.) Merr., the fasciation mutation is a single gene trait encoded by a recessive allele (Nagai, 1926 ; Albertsen et al., 1983 ). The mutation has pleiotropic effects on plant development and pattern formation, and it changes phyllotaxy and plastochron of soybean plants (LaMotte et al., 1988 ; Tang and Knap, 1998 ). In early postembryonic development, the mutation causes enlargement of the shoot apex and its shape is altered from a dome to a ridge-like structure. Our previous studies suggest that the fasciation gene may be involved in balancing meristem maintenance and organ differentiation (Tang and Knap, 1998 ). The apical meristem of the mutant generates almost twice the number of leaves as the wild type plant, owing to meristem enlargement and rapid leaf primordia initiation. Strong apical dominance in the mutant suppresses development of axillary buds and causes a branchless phenotype. Floral production occurs at the shoot apices and results in a pod-set pattern resembling cauliflower. The drastic developmental changes caused by the fasciation allele do not result in a substantial reproductive penalty for the mutant plants (Albertsen et al., 1983 ; Wongyai, Tadahiko, and Matsumoto, 1984 ).

Domestication of plants seems to favor an increase in apical dominance during the evolution of cultivated species. In the domestication of maize, Zea mays subsp. mays, there is a profound increase in apical dominance relative to its wild ancestor teosinte, Zea mays subsp. parviglumi (Doebley, Stec, and Hubbard, 1997 ; Lukens and Doebley, 2001 ). The importance of branching may carry evolutionary implications. In this context, contribution of the apical meristem to plant architecture may prove to be informative about the evolution of morphological characters in plants. The strong dominance of the apical meristem is the most characteristic trait of the fasciation mutant in soybean and generates developmental diversity useful for investigation of genetic and hormonal factors important for plant architecture.

The frequent occurrence of the fasciation mutations in many different species (spontaneous fasciation mutations have been reported in more than 100 vascular plant families; Kiesselbach, 1926 ; White, 1945 ; Zielinski, 1945 ) suggests that the regulatory factors, which are fundamental to plant development, must be affected in individual mutants. Characterization of Arabidopsis thaliana fasciation mutants implicated several genes including CLAVATA1 (Clark, Running, and Meyerowitz, 1993 ; Clark, Williams, and Meyerowitz, 1997 ); CLAVATA2 (Kayes and Clark, 1998 ); CLAVATA3 (Clark, Running, and Meyerowitz, 1995 ); FASCIATA1 and FASCIATA2 (Leyser and Furner, 1992 ; Kaya et al., 2001 ). Changes in the function of these genes lead to developmental alterations resulting in a similar fasciation phenotype (Wilkinson and Haughn, 1995 ; Meyerowitz, 1997 ; Laufs et al., 1998 ). In our previous studies, two GmCLV1 genes were isolated from soybean (Yamamoto, Karakaya, and Knap, 2000 ). Expression and sequencing analysis showed that GmCLV1A and GmCLV1B were not responsible for the fasciation mutation. The GmCLV1A locus maps to linkage group (LG) H of the soybean molecular map (Yamamoto, Karakaya, and Knap, 2000 ). Also, sequencing and expression analysis of a soybean ortholog of the Arabidopsis FASCIATA2 gene excluded that gene as a candidate for the fasciation mutation in soybean (Karakaya, 2001 ).

The genetic approach can provide a valuable avenue to elucidate factors involved in the establishment of the fasciation phenotype. The f locus was localized on LG 11 of the soybean conventional genetic map (Hedges et al., 1990 ). In soybean, several molecular maps have been generated using restriction fragment length polymorphism (RFLP), amplified restriction fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD) markers (Apuya et al., 1988 ; Lark et al., 1993 ; Akkaya, Bhagwat, and Cregan, 1995 ; Shoemaker and Specht, 1995 ; Keim et al., 1997 ; Cregan et al., 1999 ). However, none of these molecular maps contains the f locus.

We developed a population segregating for the fasciation phenotype. More than 700 markers (RFLPs with genomic or cDNA clones, AFLPs, and RAPDs) were used in mapping of the fasciation (f) locus. However, efforts to assign the f locus to the public soybean molecular map were not successful until simple sequence repeat (SSR) markers were used. Simple sequence repeat markers are single locus markers with multiple alleles. The SSR marker density exceeds 600 loci on the soybean molecular map (Cregan et al., 1999 ), thus facilitating information on recombination frequencies in the soybean genome.

In this study, we assigned the fasciation locus on LG D1b+W of the soybean molecular map. Positioning of the f locus is the first step toward a detailed genetic map of the region and physical isolation of this important developmental gene in soybean.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant genotypes
The fasciation mutant line was a generous gift from a plant breeding company, Ring Around Research (Hale Center, Texas, USA). F1 and F2 progeny of crosses between the mutant line and three independent fasciation mutation sources, PI 83945-4, PI 243541, and T17, exhibited the fasciation phenotype, confirming that the same alleles were present in all lines (Tang and Skorupska, 1997 ). A mapping population consisting of 70 F2 progeny was derived from a cross between cultivar Clark 63 and the fasciation mutant. The parental lines were polymorphic for flower color (W1) and seed coat pigmentation (R). The genotype of wild type Clark 63 was W1W1RRFF and the mutant was w1w1rrff. The F2 genotypes of wild type individuals (homozygous or heterozygous) were determined by scoring the phenotype of the F2:3 progeny lines.

DNA isolation and analysis
DNA was extracted from 4 g of young leaf tissue using the method described by Keim, Olson, and Shoemaker (1988) . For bulk segregant analysis, 10 µg of DNA from each of ten homozygous FF F2 progeny were pooled together for the FF bulk. Similarly, equal amounts of DNA from F2 recessive genotypes were pooled to make the ff bulk. Restriction enzymes, EcoRI, EcoRV, DraI, HindIII, TaqI, XhoI, and XbaI, were used individually to digest total genomic DNA of mutant and wild type and F2 progeny DNA. Southern analysis with genomic and cDNA clones was performed as described previously (Skorupska et al., 1993 ).

Restriction fragment length polymorphism marker analysis
Almost 400 clones were used in RFLP analysis. Two hundred forty-nine soybean genomic clones were obtained from Biogenetic Services (Brookings, South Dakota, USA). The selected RFLP probes spanned the linkage groups of the public soybean molecular map at intervals of approximately 20 centimorgans (cM) (Cregan et al., 1999 ). In Southern analysis, the RFLP patterns of genomic clones obtained for Clark 63 and the fasciation mutant were compared with the patterns in SoyBase (http://soybase.agron.iastate.edu) for assignment of loci according to the public soybean molecular map.

One hundred thirty-one clones were isolated from a {lambda}ZapII (Stratagene, LaJolla, California, USA) cDNA library constructed from shoot apices of the fasciation mutant at stages V3–V4 and differentially screened with cDNA from wild type and mutant epicotyls (Tang, 1999 ). For probe synthesis, inserts of {lambda}ZapII cDNA clones were amplified by polymerase chain reaction (PCR) using T3 and T7 primers. The amplification conditions were: 1 min denaturation at 93°C; 30 cycles of 45 s at 93°C, 2 min at 60°C, and 3 min at 72°C; and 5 min at 72°C. The clones were labeled with 15 µCi [{alpha}-32P] dCTP by random oligonucleotide-primed synthesis (Feinberg and Vogelstein, 1983 ).

Two cDNAs, UnP2 and UnP5, were obtained using differential display analysis (GenHunter, Nashville, Tennessee, USA) of cotyledonary tissue of 5-d-old seedlings of mutant and wild type cDNA (Karakaya, 2001 ). The cDNA fragments were cloned into pGEM vector (Promega, Madison, Wisconsin, USA) and amplified with gene-specific primers: UnP1 with forward primer 5'-GCTAACGCAAGGGCATCAGAGG-3' and reverse primer 5'-TGTCCCTTCAGCGGGATCTTGTT-3'; UnP5 with forward primer 5'-GATGAGGATGAGGACGATGATT-3' and reverse primer 5'-CGAAGTAGCAAGACCATTCTCTCT-3'. A cytokinin oxidase cDNA, GmCKOX, was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) using 5'-GAGCAAGGCCCCATAAATAATTG-3' and 5'-CAGGGATATAGGCTAGTCCTTGGAG-3', forward and reverse primers, respectively (Karakaya, 2001 ). The cDNAs were labeled with [{alpha}-32P] dCTP by PCR. The PCR labeling mixture contained 40 ng of template, 1 x PCR buffer (Perkin-Elmer, Norwalk, Connecticut, USA), 2 mmol/L MgCl2, 0.4 mmol/L dATP, 0.4 mmol/L dGTP, 0.4 mmol dTTP, 2 pmol gene specific primers, 50 µCi [{alpha}-32P] dCTP, and 1 unit Taq polymerase (Perkin-Elmer) in a 20-µL volume. The PCR conditions were denaturation at 94°C for 2 min; 30 cycles of 94°C for 1 min; annealing at 60°C for UnP1, 55°C for UnP5, or 57°C for GmCKOX for 40 s; 72°C for 1 min; and extension at 72°C for 5 min.

Sequencing
Clones were sequenced using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction DNA Sequencing Kit (Perkin-Elmer, Foster City, California, USA). Sequencing was performed using an ABI 373 Automated Sequencer (Model Version 2.1.1. Perkin-Elmer). Sequence analysis was conducted using BLASTX (http://www.ncbi.nlm.nih.gov/BLAST/). Additional sequence searches were conducted using expressed sequence tag sequences generated by The Soybean Expressed Sequence Tag (EST) Project, which is an excellent resource for functional and comparative analyses (R. C. Shoemaker, Project Leader; http://soybase.agron.iastate.edu). A MEGABLASTN program with the Est Others database field http://www.ncbi.nlm.nih.gov/BLAST/ was used for sequence comparisons using default parameters.

Amplified restriction fragment length polymorphism marker analysis
The AFLP analysis was performed as described by Vos et al. (1995) using an AFLP Analysis System I, AFLP Starter Primer Kit (Life Technologies, Gaithersburg, Maryland, USA). For DNA digestion, two restriction enzymes, EcoRI and PstI, were used in combination with MseI. A total of 64 EcoRI/MseI primer combinations and 81 PstI/MseI primer combinations were tested in bulk segregant analysis with DNA samples of Clark 63, fasciation mutant, the F2 FF bulk, and the F2 ff bulk. Twenty-five primer combinations that either generated a high number of polymorphic fragments or fragments corresponding to the wild type pattern (Clark 63 and the F2 FF bulk) vs. the mutant pattern (the fasciation mutant and the F2 ff bulk) were tested in the F2 progeny.

Random amplified polymorphic DNA marker analysis
The RAPD analysis was conducted using decamer primers (Operon Technologies, Alameda, California, USA). Amplification conditions used were as described in Skorupska et al. (1994) . A total of 250 primers were tested on parental and the F2 FF and F2 ff DNA bulks. Forty-eight primers that produced distinct polymorphisms were tested in the F2 population.

Simple sequence repeat marker analysis
LG 11 of the classical soybean map containing the f locus was integrated with LG D1b+W of the soybean molecular map (Cregan et al., 1999 ). Twenty-five SSR primer pairs (Satt005, Satt041, Satt069, Satt089, Satt135, Satt141, Satt172, Satt189, Satt198, Satt202, Satt271, Satt274, Satt282, Satt290, Satt350, Satt412, Satt428, Satt459, Satt506, Satt537, Satt546, Satt579, Satt600, Satt604, and Satt703) from LG D1b+W were obtained from the USDA-ARS, Soybean Genomics and Improvement Laboratory, Beltsville, Maryland, USA. The amplification of SSR was conducted as described in Cregan and Quigley (1997) . Amplification products were fractionated on a 3% NuSieve 3 : 1 agarose gel (Bio Whittaker Molecular Applications, Rockland, Maryland, USA) and stained with ethidium bromide for observation of SSR fragments.

Linkage analysis
Linkage analysis was conducted with Mapmaker/Exp 3.0 (Lander et al., 1987 ) using a logarithm of odds (LOD) score of 3.0. Recombination frequencies were converted to map distance in centimorgans by the Kosambi function (Kosambi, 1944 ).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Molecular polymorphism between Clark 63 and the fasciation mutant was investigated with more than 700 markers, including 249 genomic and 134 cDNA clones in RFLP analysis, 250 RAPDs, and 145 AFLP primer combinations. Genomic differences estimated by the number of polymorphic fragments out of the total detected fragments in Southern analysis with genomic probes was 6.6% and with cDNA clones was 8.5% (Table 1). In RAPD analysis, 4.3% of amplified fragments were polymorphic between Clark 63 and the fasciation mutant (Table 1).


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  		Table 1. Molecular marker polymorphism between Clark 63 and the fasciation mutant in soybean

 
In AFLP analysis, the number of DNA fragments generated by each primer combination ranged from 40 to 70, with an average of 55 distinct fragments per reaction. There were no obvious differences between the EcoRI/MseI and the PstI/MseI primer combinations in the total number of fragments generated. The polymorphism frequency between wild type and mutant detected by EcoRI/MseI was 7.2% compared with 4.9% for the PstI/MseI primer combination (data not shown). The difference may be due to the frequency of EcoRI sites in noncoding regions, which may have a higher level of genomic polymorphism.

In bulk segregant analysis, 14 AFLP markers had contrasting patterns between the FF genotypes (wild type and the FF F2 bulk) and the ff genotypes (fasciation mutant and the ff F2 bulk). Two of these markers, EaggMacc245 and EaacMcta250, were linked to the f locus based upon analysis of the F2 population (Fig. 1). The association of these two AFLP markers with the f locus was further confirmed by the AFLP pattern of 26 ff F2 segregants isolated from the independent F2 population generated from Clark 63 and fasciation mutant cross (one recombinant occurred for marker EaacMcta250, and three recombinants were observed for marker EaggMacc245; data not shown). None of the polymorphic RAPD markers observed in bulk segregant analysis linked with the fasciation phenotype.



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  		Fig. 1. Map position of the f locus and 17 cDNA marker loci on the linkage molecular map constructed from the Clark 63 x the fasciation mutant F2 population. Linkage groups A to Y correspond to the USDA-ARS soybean molecular linkage map. Map distances are shown in centimorgans. The fasciation locus is boxed. Only the segments of the linkage groups containing the mapped cDNA loci are shown. AFLP loci: restriction enzymes are indicated by capital letter followed by anchor sequence in small letters and the size in base pairs; RAPD markers: primer is in capital letters followed by DNA fragment size in base pairs. Designation of cDNA marker loci: Vsp27 = vegetative storage protein, BA1 = beta-amylase, FIP1 = putative FH protein interacting protein FIP1, PRC1 = proteasome IOTA subunit, UBPC = putative ubiquitin carboxyl terminal hydrolase, CKOX = cytokinin oxidase, Rps8 = 40S ribosomal protein S8, PrP = putative pre-mRNA splicing factor, THTM = thioredoxin m-type chloroplast precursor, RpsL31 = 60S ribosomal protein L31, AGO1 = Argonaute1 protein, and UnP = unknown protein

 
At an LOD score of 3.0, 241 loci were assigned to 31 LGs, spanning 3050 cM. All 20 LGs of the public molecular map were represented on the map constructed from the Clark 63 x the fasciation mutant F2 population (Fig. 1). Eleven small linkage groups, spanning 509 cM, did not link with the public soybean molecular map (data not shown). The W1 locus (flower color) mapped on LG F. The R locus (pigmentation of seed coat) mapped on LG K. The map assignments of these phenotypic markers are in agreement with a previous report (Shoemaker and Specht, 1995 ). Segregation of the fasciation phenotype gave a good fit to the expected 1 : 2 : 1 ratio based on F2:3 genotypes (wild type : heterozygote : mutant = 19 : 35 : 16, {chi}2 = 0.26, P > 0.75). When using RFLP, AFLP, and RAPD markers, the fasciation phenotype was not assigned a locus on a linkage map. The successful positioning of the f locus was accomplished using SSR markers that facilitated detection of recombination events on LG D1b+W. Three SSR markers, Satt005, Satt141, and Satt600 linked with the f locus within 15 cM (Figs. 1 and 2). The order and map distances of the SSR loci differed from those reported by Cregan et al. (1999) . A new locus, Satt703, mapped close to the terminal region of LG D1b+W permitting orientation of the linked AFLP and SSR markers and the f locus toward the proximal region of LG D1b+W (Fig. 1).

Seventeen RFLP markers obtained with cDNA clones mapped to 11 LGs of the public molecular map. Only cDNAs with similarity to known or unknown proteins were given locus assignments (Fig. 1). The GenBank accession numbers are presented in Table 2. We also used the soybean expressed sequence tag database (http://www.ncbi.nlm.nih.gov/blast/index.html) as a Glycine max species reference for clones isolated in our laboratory. The mapped clones encode housekeeping proteins such as: ribosomal protein subunits S8 (Rps8) and L31 (RpsL31) (mapped on LG G and M, respectively); histone protein (H2B) (mapped on LG B1); and several important development related proteins (Table 2). Five clones (UnP1, UnP2, UnP3, UnP4, UnP5) with similarities to A. thaliana unknown proteins mapped on LG A2, C2, K, or L (Table 2). Expressed sequence tag soybean database searches with the UnP1, UnP2, UnP3, UnP4, or UnP5 sequences identified ESTs, confirming that the mapped clones function during vegetative or reproductive stages in soybean.


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  		Table 2. Soybean cDNA clones with assigned loci on the linkage map constructed from the F2 population of Clark 63 x the fasciation mutant

 
A vegetative storage protein (VSP27), a beta-amylase (BA, an exo-type starch hydrolyzing enzyme), and two unknown proteins (UnP1, UnP2) were assigned loci to LG A2 (Fig. 1). Positioning of the VSP27 confirmed the previous locus assignment to LG A2 (Shoemaker and Specht, 1995 ). The formin homology (FH) protein locus was mapped on LG C1. The FH proteins are implicated in cell polarization or cytokinesis through cytoskeletal function organization (Banno and Chua, 2000 ; Ozaki-Kuroda et al., 2001 ). EcoRI, DraI, HindIII, and TaqI RFLP polymorphisms detected with the clone encoding PRC1, an IOTA subunit of the proteasome, mapped on LG C2, 0.7 cM from RFLP marker A397-1. A full-length PRC1 cDNA (994 bp) was isolated, and the sequence was submitted to GenBank (GBAN-AF034572). In plants, proteasomal subunits, including IOTA subunit, were shown to be related to cell division, suggesting that their role in the regulation of developmental events occurs by controlling the levels of regulatory proteins in proliferating tissues rather than protein degradation in tissue senescence (Ito et al., 1997 ; Bahrami and Gray, 1999 ).

Two cDNA clones, which were mapped on the LG F, encode for a highly conserved ubiquitin carboxyl-terminal hydrolase, UBPC, which is involved in ATP-dependent protein degradation (Viestra, Langan, and Haas, 1985 ) and putative cytokinin oxidase, GmCKOX. In the soybean EST database, which contains more than 189 000 sequences, several CKOX sequences are reported. GmCKOX in this study had sequence homology with GBAN-AW830743 (Table 2). The soybean CKOX EST (GBAN-BG651837) was identified in a cDNA library constructed from mRNA isolated from reproductive shoot apices of the fasciation mutant, which was provided by our laboratory for the Soybean EST sequencing project. Isolation and characterization of the full-length cDNA of this new CKOX gene is in progress.

Two clones were mapped on LG H. These encode a pre-mRNA splicing factor (PrP) associated with the G0/G1 and G1/G2 transitions in the cell cycle and a chloroplast m-type thioredoxin precursor protein (THTM) involved in various redox reactions through the reversible oxidation (Lopez et al., 1994 ).

Argonautel (AGO1) belongs to a novel class of proteins controlling leaf, floral organ, and axillary meristem development in Arabidopsis (Bohmert et al., 1998 ; Lynn et al., 1999 ). In RFLP analysis, the soybean clone GBAN-B1740284 with similarity to Argonautel showed independent assortment from the fasciation locus and mapped on LG Y of the soybean molecular map.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
In this study, different marker systems, including RFLP, RAPD, AFLP, and SSR, were used for mapping the fasciation locus in soybean. Due to the limited germplasm sources used in the development of soybean cultivars, polymorphism in Glycine max is generally low (Shoemaker and Specht, 1995 ). The estimation of the genome diversity between Clark 63 and the fasciation mutant by RFLP and AFLP markers was less than 10%. The mechanisms of RFLP and AFLP are based on the variation of nucleotides at restriction sites of homologous sequences between two genomes, and it is not surprising to find a similar estimation of genomic diversity using these two methods. The lower polymorphism (4.3%) between Clark 63 and the fasciation mutant estimated with RAPD markers might be due to low annealing temperature used in RAPD analysis that allows some mismatching and thereby can mask some of the nucleotide variation between the two genomes.

We observed an overall expansion of genetic distances on the Clark 63 x the fasciation mutant map relative to those on the soybean molecular map of Cregan et al. (1999) . Progeny number or different recombination frequencies in a particular cross might affect overall scores of genetic distances. Genomic similarities between Clark 63 and the fasciation mutant could contribute to higher recombination frequencies and thus map expansion, including LG D1b+W on which the fasciation locus was mapped. Jin et al. (1998) and Hegstad et al. (2000) also reported expansion of genetic distances on soybean LG D1b+W in mapping of a male-sterile gene and the wp flower color locus, respectively. Hegstad et al. (2000) speculate that increased recombination rates in the LG D1b+W region may be caused potentially by rearrangements or insertions generated by a defective wp-m transposable element. Linkage of the f locus and the wp locus with Satt141 and Satt600 on the LG D1b+W suggests that further investigations of the region might elucidate the origin of gene mutation and structural rearrangements of that region of the soybean genome.

An advantage of using cDNA clones for mapping is the identification of actively transcribed regions of the genome. In combination with sequence data, mapping of cDNAs enhances the characterization of gene distribution on each chromosome and within the whole genome. Seventeen cDNAs were mapped to 11 different linkage groups of the public soybean molecular map, adding to previously mapped cDNAs: histone H3 genes (Kanazin, Blake, and Shoemaker, 1996 ), disease resistance gene analogs (Kanazin, Fredrick, and Shoemaker, 1996 ; Yu, Buss, and Maroof, 1996 ), chalcone synthase multigene family (Todd and Vodkin, 1996 ), ß-1,3-glucanases genes (Jin et al., 1999 ), cDNAs from various studies (Matthews et al., 2001 ), and genes associated with cyst nematode infection of soybean (Vaghchhipawala et al., 2001 ).

Mapping of cDNA markers may allow identification of genes corresponding to classical loci and association with phenotype. However, mutations caused by single-base changes or frame shifts are difficult to detect by RFLP analysis unless the restriction sites used are altered by the mutation. Several of the mapped clones were differentially expressed between the mutant and Clark 63 (Tang, 1999 ; Karakaya, 2001 ). None of the tested cDNA markers mapped at the fasciation locus suggesting that several genomic regions might be involved in the establishment of the fasciation mutant phenotype. GmCLV1 and GmFAS2, orthologs of Arabidopsis genes that cause a fasciation phenotype, were excluded as candidate genes for fasciation mutation in soybean based on expression and mapping analyses (Yamamoto, Karakaya, and Knap, 2000 ; Karakaya, 2001 ). Another important soybean clone with similarity to Argonaute1 (mutation in Argonaute1 pleiotropically affects development of apical shoot meristem, axillary meristems, and leaves) mapped to LG Y. Also, GmCKOX showed independent assortment from the fasciation phenotype and mapped to LG F. Cytokinin oxidase inactivates cytokinins by irreversible degradation (Kaminek, Motyka, and Vankova, 1997 ), which could be one of the possible mechanisms for differences in cytokinin levels observed between the mutant and the wild type (Karakaya, 2001 ). Characterization of the second GmCKOX identified by the EST sequencing of a cDNA library from reproductive shoot apices of the fasciation mutant might aid information on association of cytokinin oxidase genes with the fasciation mutant phenotype.

Assignment of the f locus to linkage group D1b+W and identification of flanking markers can be used as an initial step for precise mapping of the f gene. It is likely that the f locus product acts upstream of other genes in soybean development and intervenes directly or indirectly in several signal transduction pathways throughout vegetative and reproductive development. Hence, the mutant can be a valuable resource for investigation of genomic organization, regulation of soybean developmental traits, and evolution of key morphological characters.



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  		Fig. 2. Simple sequence repeat marker polymorphisms detected (A) with Satt141 and (B) Satt005 and linked with the fasciation (f) locus on linkage group D1b+W of the molecular map constructed from the Clark 63 x the fasciation mutant F2 population. Order of genotypes: Clark 63 (F), fasciation mutant (f), 1–17 are F2 progeny expressing the fasciation phenotype. Progeny 11 and 17 are heterozygotes for Satt141 and Satt005 markers. Polymerase chain reaction products were separated in 3% NuSieve 3 : 1 agarose gel. Lane 18 is 100 bp DNA ladder (Promega, Madison, Wisconsin, USA)

 

   FOOTNOTES
 
1 The authors thank Drs. V. Baird and A. Abbott for helpful comments on the manuscript. This paper is technical contribution No. 4729 of the SC Agricultural Experimental Station. Back

4 Author for reprint requests (current address: 272 Poole Agricultural Center, Dept. of Crop & Soil, Clemson University, Clemson, South Carolina 29634-0359 USA; mail: hskrpsk{at}clemson.edu ) Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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