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Crop Science 40:534-537 (2000)
© 2000 Crop Science Society of America

CELL BIOLOGY & MOLECULAR GENETICS

Positioning the wp Flower Color Locus on the Soybean Genome Map

J.M. Hegstada, J.A. Tartera, L.O. Vodkina and C.D. Nickella

a Univ. of Illinois at Urbana-Champaign, Dep. of Crop Sciences, 1102 S. Goodwin, Urbana, IL 61801 USA

cnickell{at}uiuc.edu


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 
The wp allele generates a pink flowered phenotype in soybean [Glycine max (L.) Merr.]. Genotypes with W1_wp wp are associated with larger seed size and higher seed protein content. The influence of a single locus on the anthocyanin pathway and seed protein accumulation has not been documented in other species. It is of interest to clone wp by initially identifying linked molecular markers. The objective of this research was to use restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) molecular markers to position the wp locus on the soybean public genetic map. Of the 74 markers tested, two RFLP and 14 SSR markers were identified as linked to wp. The two RFLP markers linked to wp in this study were previously reported to be associated with quantitative trait loci (QTL) controlling seed protein in a G. soja Siebold & Zucc. x G. max population. It is proposed that wp is located between RFLP locus K011 and SSR locus Satt600. The molecular markers linked to wp were located on linkage group D1b+W of the public soybean genetic map. The knowledge of linked molecular markers will assist in cloning wp, potentially leading to further characterization of how wp influences the anthocyanin pathway and seed protein accumulation.

Abbreviations: AFLP, amplified fragment length polymorphism • cM, centimorgan • PCR, polymerase chain reaction • QTL, quantitative trait loci • RAPD, random amplified polymorphic DNA • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results and discussion
 REFERENCES
 
PINK FLOWERED SOYBEAN plants were discovered by Stephens and Nickell (1991). Genetic analysis revealed the unique color was due to homozygous recessive wp alleles in the presence of W1 (Stephens and Nickell, 1992). Pink flowered lines averaged 22% higher in seed weight, and 4% higher in protein content when compared with purple flowered lines from the same background (Stephens et al., 1993). In a different study, stable purple flowered revertant lines derived from a pink flowered source were significantly lower in protein content when compared with pink flowered sister lines (Hegstad et al., 2000). In addition, stable pink flowered lines derived from a purple flowered source were significantly higher in protein content compared with purple flowered sister lines (Hegstad et al., 2000). There is evidence that the wp allele exerts an influence on both the anthocyanin pathway and seed protein accumulation. It is therefore of interest to clone wp to characterize further its genetic action. The initial step in cloning is to position wp on the soybean genetic map by identifying linked molecular markers.

Molecular marker linkage maps have greatly accelerated recent advancements in plant genomics. RFLP markers are codominant markers that are well suited for the construction of linkage maps and synteny studies between species (Ahn and Tanksley, 1993; Kurata et al., 1994). SSR markers are codominant markers that can be automated using the polymerase chain reaction (PCR). The level of information content and potential for automation has resulted in SSR markers being a preferred marker system for quickly generating saturated linkage maps (Rafalski and Tingey, 1993).

In soybean, the public molecular marker linkage map was initially constructed with RFLP markers on a diverse G. max x G. soja population (Shoemaker and Olson, 1993). A linkage map containing 40 SSR markers was created from a population from a cross of `Clark' x 'Harosoy' (Akkaya et al., 1995). The initial SSR map of Akkaya et al. (1995) was expanded and integrated with the linkage map of Shoemaker and Specht (1995) into the current genetic map. The current soybean genetic map contains 606 SSR, 689 RFLP, 79 random amplified polymorphic DNA (RAPD), 11 amplified fragment length polymorphic (AFLP), 10 isozyme, and 26 classical markers mapped to 20 linkage groups (Cregan et al., 1999). The objective of this research was to position wp onto the genetic map using RFLP and SSR molecular markers.


   Materials and methods
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results and discussion
 REFERENCES
 
Flower color isolines in a Clark genetic background that differ for alleles at the W1, W3, W4, or Wm loci (Bernard, 1974) were obtained from the USDA soybean germplasm collection, Urbana, IL. A molecular marker mapping population was generated by crossing RM55 x LN89-5322-2. RM55 (Chandlee and Vodkin, 1989) is a Clark isoline that is stable for purple flowers and has the genotype W1W1 WpWp. LN89-5322-2 (Stephens et al., 1993) originated from the cross [(Sherman (McBlain et al., 1987) x Asgrow A2943) x Elgin 87 (Fehr et al., 1988)], has the genotype W1W1 wpwp, and has been stable for pink flowers over eight generations of observation. Approximately 160 individual F2 plants of the cross RM55 x LN89-5322-2 were grown in 1994 and tagged for flower color. For each tagged plant, leaf samples were collected and plants were harvested separately. The pink flower phenotype is due to the presence of homozygous wp alleles. Therefore, in order to identify molecular markers linked to wp, emphasis was placed upon analyzing all pink flowered F2 plants (n = 40).

RFLP probes A023, A144, A242, A245, A407, A688, and K011, previously reported to be associated with protein content (Diers et al., 1992), were obtained as DNA agar stabs from Biogenetic Services, (Brookings, SD) and PCR amplified using M13 universal primers. Genomic DNA of the Clark isolines, parental genotypes, and mapping population was extracted from leaf tissue following the protocols described by Dellaporta (1994), with slight modifications. Ten millimoles of 1,10-phenanthroline was added in the extraction buffer as a nuclease inhibitor and the hexadecyltrimethylammonium bromide step was omitted. Genomic DNA (5 µg) was digested with restriction enzymes DdeI, DraI, EcoRI, EcoRV, HindIII, and TaqI for approximately 2 h at 37°C. RFLP procedures of gel electrophoresis, Southern blotting, probe purification, prehybridization, probe hybridization, stringency washing, and autoradiography were performed as described in Hegstad et al. (1998).

The soybean genetic linkage map of Cregan et al. (1999) was examined for linkage groups containing the RFLP markers selected from Diers et al. (1992). SSR primers that were linked to the selected RFLP markers were acquired from Research Genetics (Huntsville, AL). DNA of the parental genotypes and F2 individuals in the mapping population was amplified for presence of SSR alleles using 60 ng sample DNA, 1x PCR buffer (50 mM KCl, 10 mM Tris HCl pH9.0, 0.1% [v/v] Triton X-100), 1.5 mM MgCl, 0.15 µM 3' and 5' primers, 1.25 mM dNTPs, and 1 unit Taq DNA polymerase (Gibco BRL Life Technologies, Gaithersburg, MD). SSR reactions were internally labeled with 0.1 µL (10 µCi/µL) [{alpha}-32P] dATP. Reactions were amplified by PCR under the following conditions: 94°C for 1 min, 52°C for 1 min, 70°C for 45 s (times 34 cycles), with the final cycle having a 70°C extension for 5 min. Reactions were stopped with 2.5 µL SSR loading dye (90% [v/v] formamide, 20 mM EDTA [ethylenediaminetetraacetate], 0.1% [v/v] bromophenol blue, 0.1% [v/v] xylene cyanol), denatured for approximately 2 min at 95°C, and placed immediately into ice.

Sequencing gel solution Gel mix 6 (5.7% [w/v] acrylamide 0.3% [w/v] bis, 7 M urea, 100 mM [pH 8.3] Tris-borate, 1 mM EDTA, 3 mM TEMED [N',N',N',N'-tetramethylethylenediamine]), was obtained from Gibco BRL Life Technologies (Gaithersburg, MD). 0.45 mL of 10% (w/v) ammonium persulfate was added to the premix, swirled for 30 s, and approximately 70 mL of the mix was poured onto a horizontally level DNA sequencing glass plate with 0.4-mm spacers. The top electrophoresis plate was then lowered over the premix liquid in similar fashion to placing a coverslip over a microscope slide. This technique was rapid and usually resulted in complete gels with no bubbles. Gels were allowed to polymerize overnight.

Gels were pre-run with 35 mA, 60 W, 1500 V for approximately 40 min, and urea was purged from the sharkstooth comb area just prior to sample loading. Four microliters of denatured SSR reaction samples were loaded into each lane and gels were electrophoresed at 35 mA, 60 W, 1500 V for approximately 2.5 h, or until the xylene cyanol dye was 2/3 down the gel. Gels were lifted from the electrophoresis plate with Whatman filter paper, wrapped in plastic wrap, vacuum dried at 30°C for approximately 40 min, and exposed to x-ray film overnight.

Forty pink flowered individuals of the F2 mapping population were scored for the size of the SSR allele product in comparison to the parental genotypes. Data were subjected to chi-square analysis against expected ratios to determine absence or presence of linkage between the marker and wp. The computer program Mapmaker 3.0 (Lander et al., 1987) was used to determine linkage distance of RFLP and SSR markers linked to the wp locus based upon maximum likelihood scores at LOD = 3.0.


   Results and discussion
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
REFERENCES
 
Approximately 689 RFLP markers have been mapped on the soybean public genetic linkage map (Cregan et al., 1999). Since the RFLP procedure is labor intensive, the process to map the wp locus began by testing markers that had previously been reported to be significantly associated with protein content. Seven different markers significant for protein content in a G. max x G. soja population were reported by Diers et al. (1992). Markers A023, and A242 were located on linkage group E, A245 was located on linkage group F, and A144, A407, A688, and K011 were mapped to linkage group I (Cregan et al., 1999). When labeled as RFLP probes, A023, A144, A242, A245, and A688 did not have polymorphic bands between the pink flowered parent and the purple flowered parent or Clark flower color isolines using six different restriction enzymes. These markers were therefore considered to be uninformative. These results are not unexpected as the mapping population in this study was created by crossing elite lines of a narrow genetic base. The RFLP markers originally were mapped from a genetically diverse population from a G. max x G. soja cross (Diers et al., 1992).

A RFLP between the pink flowered parent and the purple flowered parent as well as the Clark flower color isolines was detected with A407 and K011. For both A407 and K011, data from the F2 mapping population were significant for linkage to wp when tested by chi-square analysis (Table 1) . K011 and A407 mapped at distances of 4.2 and 10.9 centimorgans (cM), respectively, from the wp locus (Table 2) .


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  		Table 1 Chi-square analysis of selected RFLP and SSR markers to determine absence or presence of linkage with wp

 

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  		Table 2 Linkage map for wp determined by RFLP and SSR molecular markers

 
Simple sequence repeat markers were used to identify additional variation around the wp locus. To initiate this evaluation, RFLP markers A407 and K011 were located on the public linkage map of Cregan et al. (1999). Marker A407 mapped to linkage groups A1 and I, and K011 mapped to B1, D1b+W, D2, I, and Y. Of these regions, no polymorphism was evident between the parental lines of the mapping population for 14 selected SSR markers of linkage groups A1, B1, and I (Hegstad, 1999). Polymorphism between the parental lines was detected for 28 SSR markers selected from linkage groups D1b+W, D2, and O (Hegstad, 1999). The markers that were polymorphic for the parental lines were used on the 40 pink flowered F2 individuals of the mapping population to determine linkage of the marker with wp. Data analysis using Mapmaker 3.0 (Lander et al., 1987) revealed that markers on linkage groups D2 and O were unlinked to wp, as the map distance between these markers and wp was greater than 50 cM.

Several SSR markers selected from linkage group D1b+W were found to be polymorphic between the parental lines (Hegstad, 1999). Chi-square analysis of the F2 mapping population revealed 14 markers were significantly linked (P = 0.001) to wp (Table 1). A linkage map for the wp locus was constructed using data analysis with Mapmaker 3.0 (Table 2). The wp locus is proposed to be located on linkage group D1b+W between RFLP marker K011 and SSR marker Satt600 (Table 2).

The markers in the linkage group for wp are in similar order to those located on linkage group D1b+W of the integrated linkage map (Cregan et al., 1999) (Table 3) . The distances between the mapped markers in this study are greater than previously described by Cregan et al. (1999). It should be noted that in Cregan et al. (1999), a consensus map was generated on the basis of data collected from three independent groups (USDA/Iowa State University, University of Utah, and University of Nebraska). On linkage group D1b+W, the three groups do not have all markers in common on their respective maps. In addition, the relative order of the markers and linkage distances between markers is different between the maps of the three groups.


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  		Table 3 Positioning the soybean pink flower color locus wp on the public genetic map

 
The discrepancy between the data reported in this study and the maps of Cregan et al. (1999) may be due to experimental error associated with the small population size in this analysis. It was of interest to identify markers linked to the homozygous wp genotypes, of which 40 of the 160 individuals in the mapping population were pink flowered. It is evident in the mapping population of this study the particular region of DNA around the wp locus has more recombination events than were observed in the mapping populations of Cregan et al. (1999). The pink flower phenotype is speculated to occur due to the presence of a defective wp-m transposable element located in the Wp allele responsible for purple pigmented flowers (Hegstad et al., 2000). Though it has not been proven at the molecular level, the rearrangements or insertions generated by wp-m potentially may have increased the recombination rates in the region surrounding the wp locus. Although the distances for the map presented in this report are greater than those previously identified, ten molecular markers linked to wp are approximately in the same order when integrated with linkage group D1b+W (Table 3).

In summary, two RFLP and 14 SSR molecular markers were identified as significantly linked to wp. The two RFLP markers linked to wp in this study were identified as linked to QTLs for high protein content contributed by the G. soja accession PI 486916 (Diers et al., 1992). Therefore, the wp locus may potentially be linked or pleiotropically influence protein QTL that are highly conserved among members of the Glycine subgenus soja (Moench) F.J. Herm. Although more recombination and thus greater marker distances were identified in the data presented, the relative position of the linked markers is similar to linkage group D1b+W of the soybean genetic map (Cregan et al., 1999). The assignment of wp to a known linkage group will assist in the cloning of this gene. This could assist future studies to determine how wp influences the biosynthetic pathways of anthocyanin production and seed protein accumulation.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 
Contribution from the Illinois Agric. Exp. Stn., Urbana, IL. Research supported by the Illinois Soybean Program Operating Board.

Received for publication May 11, 1999.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results and discussion
 REFERENCES
 




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