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Published online 30 July 2007
Published in Crop Sci 47:1681-1690 (2007)
© 2007 Crop Science Society of America
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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Single Nucleotide Polymorphism Detection of the Rcs3 Gene for Resistance to Frogeye Leaf Spot in Soybean

A. M. Missaouia, B. K. Haa, D. V. Phillipsb and H.R. Boermaa,*

a The Univ. of Georgia Center for Applied Genetic Technologies, Athens, GA 30602
b Dep. of Plant Pathology, Univ. of Georgia–Griffin Campus, Griffin, GA 30223; M. Missaoui, current address: Monsanto Co., 1551 Hwy. 210, Huxley, IA50124

* Corresponding author (rboerma{at}uga.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Frogeye leaf spot (FLS), caused by Cercospora sojina Hara, has become a more frequent disease in soybean, Glycine max (L.) Merr., across the USA. The Rcs3 gene provides resistance to all known races of C. sojina. To provide resources for efficient marker-assisted selection (MAS) of Rcs3, we developed and evaluated several single nucleotide polymorphisms (SNP) and insertions or deletions (InDels) markers linked to Rcs3. We surveyed 13 bacterial artificial chromosome (BAC) end sequences that were anchored with simple sequence repeat (SSR) markers Satt244 and Satt547 along with the two SSRs containing clones in six soybean cultivars (Davis, Cook, and Young that have the Rcs3 allele and Blackhawk, Bragg, and Lee that have the rcs3 allele) for SNPs. A total of 19 SNPs/InDels were identified and validated, but only 11 mapped near Rcs3 in a F2 population of Davis x Blackhawk. The Rcs3 gene was positioned near Satt244 and 0.50 cM from SNPs AZ573TA150 and AZ573CA393. None of the SNPs or InDels identified appeared to contribute directly to the Rcs3 phenotype, but 11 mapped within a 3-cM interval surrounding Rcs3. These 11 markers were further validated for association with Rcs3 and for their potential in MAS in 64 lines and cultivars. Our results suggest that the two markers AZ573TA150 and AZ573CA393 could be used in MAS for Rcs3. Allele specific oligonucleotide probes specific for MAS were developed for a direct hybridization assay on a Luminex 100 flow cytometry platform.

Abbreviations: ABC, ATP-binding cassette • ASO, allele-specific oligonucleotide • BAC, bacterial artificial chromosome • EST, expressed sequence tag • FLS, frogeye leaf spot • InDel, insertion or deletion • LG, linkage group • MAS, marker-assisted selection • MFI, mean fluorescence intensity • PCR, polymerase chain reaction • SBE, single base extension • SNP, single nucleotide polymorphism • SSR, simple sequence repeat


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
FROGEYE LEAF SPOT (FLS) is a fungal disease of soybean [Glycine max (L.) Merr.] caused by Cercospora sojina Hara (Hara, 1915). The pathogen infects the aboveground parts of the plant and reduces yield by nearly 31% (Mian et al., 1998). Even though the disease is a frequent problem in the soybean production areas of the southern USA, it recently has become more prevalent in north-central states, possibly because of the unusually warmer winters and the lack of resistance in the commonly grown cultivars.

Complete resistance to all known races of C. sojina was found to be conditioned by the Rcs3 gene in the cultivar Davis (Phillips and Boerma, 1982; Boerma and Phillips, 1983; Missaoui et al., 2007). The Rcs3 gene was mapped on linkage group (LG-J), near the simple sequence repeat (SSR) markers Satt244 and Satt547 (Mian et al., 1999). The association between the two SSR markers and Rcs3 was confirmed in 64 cultivars and breeding lines from the ancestors and descendants of Davis (Missaoui et al., 2007). However, since SSR markers are based on repeat length variants and their detection requires electrophoresis, they are not ideal for high throughput genotyping. Simple sequence repeats are also less suitable for association studies because of the occurrence of SSR alleles of identical size but different evolutionary origin (homoplasy) (Viard et al., 1998).

With the decreasing cost of DNA sequencing and the accumulation of large libraries of genomic DNA and expressed sequence tag (EST) sequences, molecular marker technologies are shifting toward sequence variation. Single nucleotide polymorphisms (SNPs) are present at a greater frequency throughout the genome and usually associated with lower genotyping error rates than microsatellite markers (Kennedy et al., 2003). Evans and Cardon (2004) showed that the information content associated with a traditional map of microsatellite markers (1 marker approximately every 10 cM) is lower than the information content associated with a dense map of SNPs, suggesting that linkage studies that were previously based on sparse microsatellite maps could benefit from the generation of highly saturated SNP maps. Declining costs of SNP typing have made whole genome scans feasible even for linkage studies, and the lesser power of a single SNP is compensated for by their much greater density and lower typing costs than multiallelic markers (Morton and Collins, 2002).

Single nucleotide polymorphisms are becoming the marker of choice for genetic studies and due to their abundance they can provide a large supply of markers for crop improvement programs (Gupta et al., 2001, Nasu et al., 2002; Feltus et al., 2004). The advances in SNP-based marker technology have had a great impact on the generation of high-density genetic maps (Snelling et al., 2005), trait mapping (Jin et al., 2003; Hayashi et al., 2004), association studies (Sellick et al., 2004; Syvanen, 2005), and positional cloning of genes underlying complex traits (Monna et al., 2002; Shen et al., 2004).

Single nucleotide polymorphisms were used for candidate gene mapping of barley (Hordeum vulgare L.) mutants involved in plant architecture (Rossini et al., 2006) and genetic mapping and quantitative trait loci identification in sunflower (Helianthus annuus L.) (Lai et al., 2005). In soybean, Jeong and Saghai Maroof (2004) assayed and genotyped SNPs linked to two Soybean mosaic virus resistance genes, Rsv1 and Rsv3, with a modified allele-specific polymerase chain reaction (PCR) procedure. Kim et al. (2005) identified a SNP associated with the soybean GmNARK gene (G. max nodule autoregulation receptor kinase) controlling autoregulation of nodulation between wild-type ‘Sinpaldalkong 2’ and its mutant counterpart, SS2–2. They converted the SNP into a single nucleotide amplified polymorphism marker to allow direct marker-assisted selection (MAS) for supernodulation at an early growth stage without need for inoculation and root phenotyping.

Several approaches have been applied for the SNP discovery including the mining of EST sequence data sets that offer a valuable resource for the SNP detection due to the relatively high redundancy of gene sequences, the diversity of genotypes represented in the databases, and the likelihood that each SNP is associated with an expressed gene (Picoult-Newberg et al., 1999; Batley et al., 2003a; Grivet et al., 2003). This resource has recently been used in large-scale SNP identification in several plant species including wheat (Triticum aestivum L.) (Somers et al., 2003), melon (Cucumis melo L.) (Morales et al., 2004); Arabidopsis (Arabidopsis thaliana) (Schmid et al., 2003), barley (Kota et al., 2003); maize (Zea mays L.) (Batley et al., 2003a), and sugarcane (Saccharum officinarum L.) (Grivet et al., 2003). The major drawback to this approach is the elevated frequency of false positives arising from base calling errors present in the databases.

DNA sequences in noncoding regions, such as introns, 3' untranslated regions, and bacterial artificial chromosome (BAC) end sequences are also considered a useful resource for SNP discovery. These regions provide up to threefold higher frequency of SNPs than coding regions (Rafalski, 2002; Zhu et al., 2003). Another source of SNPs includes regions flanking microsatellites. Several studies have shown that the flanking regions of the repeat sequences are highly variable in maize (Mogg et al., 2002; Batley et al., 2003b) and wheat (Ablett et al., 2006). Identifying nucleotide polymorphisms surrounding SSRs has the advantage that many of these markers have already been mapped. Single nucleotide polymorphism genotyping methods and platforms are also continuously being developed and improved to the extent that it is challenging to keep to date and to decide on the best technology available (Kwok, 2001; Tsuchihashi and Dracopoli, 2002).

The objectives of the present study are the identification of SNP markers associated with the Rcs3 locus conditioning broad resistance to FLS and the development of an efficient SNP genotyping assay for use in MAS of Rcs3.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Materials
Six soybean cultivars were selected for SNP and insertion or deletion (InDel) discovery. Three of these, Davis, ‘Cook’, and ‘Young’, were known to contain the Rcs3 allele conditioning broad resistance to FLS (Missaoui et al., 2007). The other three cultivars, Blackhawk, Bragg, and Lee, possess the rcs3 allele and are susceptible to FLS. A F2 population of 92 plants derived from a cross of Davis x Blackhawk was used for mapping the nucleotide variants (SNPs and InDels) identified. This population was originally used for mapping the Rcs3 gene to LG-J (Mian et al., 1999). In addition to this population, we evaluated 64 cultivars and breeding lines from the ancestors and descendants of Davis for the confirmation of the association between the SNP or InDel markers and Rcs3.

DNA Extraction, PCR, Sequence Analysis, and Genotyping
Genomic DNA was extracted from lyophilized plant tissue using a 2% CTAB extraction buffer as described in (Missaoui et al., 2007). Bacterial artificial chromosomes that were anchored with the SSR markers Satt244 and Satt547, both tightly linked to Rcs3, were identified in Soybase (http://soybase.org/; verified 24 Apr. 2007) and their end sequences were obtained from GenBank (Table 1). Sequences of the clones bearing the SSR marker repeats Satt244 and Satt547 were also obtained from GenBank. Forward and reverse primers were designed with the program primer3_www.cgi v 0.2 (Rozen and Skaletsky, 2000). Oligonucleotide primers were synthesized by Biosource International (Camarillo, CA).


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  		Table 1. Bacterial artificial chromosome (BAC) end and simple sequence repeat (SSR)-containing sequences, primer combinations used for detection of Rcs3 polymorphisms, number of single nucleotide polymorphism (SNP) and insertions or deletions (InDels) identified, and putative function of the sequences analyzed.

 
The primers developed from each BAC end sequence and the SSR clones were used to PCR amplify the corresponding genomic regions from the three resistant cultivars known to have the Rcs3 gene and the three susceptible (rcs3) cultivars (Table 1). Polymerase chain reaction was performed with PTC-225 DNA Engine Tetrad (MJ Research, Waltham, MA) thermal cyclers. Amplification using 2 µL of DNA (~100 ng µL–1), was performed in a total volume of 20 µL, containing 1x GeneAmp PCR Gold Buffer (50 mM KCl and 15 mM Tris-HCl pH 8.0), 2.5 mM MgCl2, 0.5 µM of each primer, 200 µM of each dNTP, and 1 unit of AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA). Thermocycling conditions consisted of an initial denaturation and enzyme activation step of 94°C for 10 min, followed by 40 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 45 s, and a final extension of 72°C for 7 min.

The PCR products were electrophoresed on a 1% low-melting SeaPlaque agarose gel (BMA Bioproducts, Rockland, ME) to check for multiple fragments. Polymerase chain reaction products that amplified a single fragment were digested with shrimp alkaline phosphatase (0.1 U µL–1) and ExoI nuclease (0.02 U µL–1) according to manufacturer protocol and directly sequenced in both directions using the individual primers used for PCR amplification in separate sequencing reactions. Each sequencing reaction consisted of 2 µL of sequencing mix BigDye Terminator Cycle Sequencing Ready Reaction v 3.0 Kit (Applied Biosystems), 1.0 µM of each primer, 1% DMSO, 2 µL of 5x sequencing buffer (supplied with the sequencing kit), and 4 µL of PCR product in a total volume of 10 µL. Cycle sequencing conditions were as recommended by the kit manufacturer except that 90 cycles were used. Sequencing reactions were purified using the MultiScreen Filtration System (Millipore Corporation, Bedford, MA) using Sephadex G50 Superfine (Sigma-Aldrich, St. Louis, MO) per the manufacturer's protocol. Sequence analysis was carried on a PerkinElmer 3730 XL capillary DNA Analyzer (Applied Biosystems).

SNP Identification and Genotyping
Sequences from the six cultivars, together with the consensus sequence were aligned using the multiple sequence alignment program Clustal X (Thompson et al., 1997) and SNP variants (SNPs and InDels) were identified by visual inspection of the alignments. A targeted approach was followed so that polymorphisms were accepted only if the sequence trace was of high quality and the nucleotide variant was polymorphic between the two sets of three resistant and three susceptible cultivars. The SNP and InDel interrogation primers were designed with melting temperatures ranging from 58 to 73°C using the program SBE-primer (Kaderali et al., 2003). The single base extension (SBE) primers were designed to be specific for each locus and to terminate one nucleotide 5'-upstream of the SNP location (Table 2).


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  		Table 2. Single nucleotide polymorphism (SNP) and insertion or deletion (InDel) markers developed from the validated nucleotide variants, interrogation primer sequences used for their genotyping with the single base extension (SBE) assay, and allele genotypes in Davis and Blackhawk.

 
Sequence variants detected by sequence alignment were first validated with the SNaPshot assay on DNA from the six cultivars used in their identification according to the protocol provided with the ABI PRISM SNaPshot Multiplex kit (Applied Biosystems). The validated SNPs were developed into PCR markers and assigned names that include the GenBank sequence ID, the SNP genotype, and the position of the SNP in the sequence (i.e., AZ573TA150). The markers were genotyped in a F2 population of 92 progenies derived from a cross between Davis x Blackhawk and 64 ancestors and descendants of Davis using the minisequencing or SBE assay as described in Lee et al. (2004). Briefly, the procedure consists of a first PCR amplification of the target genomic region using the same primers originally used for sequencing followed by enzymatic removal of excess primers and nucleotides. The resulting amplicons are used as templates for a second PCR reaction based on primers (capture probe) designed to terminate immediately upstream of the SNP to incorporate one of two fluorescent terminators (ddNTP) representing the allelic SNP nucleotides into products which are detected by differential fluorescence polarization of the two terminator dyes. All capture probes for SBE were designed with a fluorescent tag at their 5' end complementary to a ZipCode sequence that was coupled to a specific Luminex xMAP micorsphere.

The Luminex xMAP system, which is a microsphere-based suspension array platform, provided the opportunity to multiplex multiple primers in a single reaction. Detection for each SNP was performed on a Luminex 100 flow cytometer (Luminex Corporation, Austin, TX) equipped with a Luminex XY Platform plate reader and Luminex-compatible analysis software from MiraiBio (Alameda, CA). The fluorescence on the surface of the microspheres was measured and converted to a mean fluorescence intensity (MFI) value based on a minimum of 100 microspheres of each type in a 50-µL sample volume.

SNP Mapping and Confirmation of the Association with Rcs3
The SNPs were mapped in the Davis x Blackhawk F2 population that was used previously in the linkage mapping of the Rcs3 gene (Mian et al., 1999). Linkage analysis of the SNP markers, SSR markers, and Rcs3 were performed using Map Manager QTX version b20 (Manly et al., 2001). The SNPs that showed linkage with Rcs3 were further analyzed for confirmation of their association with Rcs3 in 64 cultivars and breeding lines including ancestors and descendants of Davis (the known source of Rcs3).

Microsphere-Direct Hybridization Assay with Biotinylated Upstream Primer
Microsphere-direct hybridization was performed according to Dunbar and Jacobson (2000) with slight modifications. The upstream primer for amplification of the SNP-containing fragment was labeled at the 5' terminus with biotin (Integrated DNA Technologies, Inc., Coralville, IA). Targets were amplified and used in a total 10-µL reaction mix containing 2 µL of 40 ng template DNA, 1.0x PCR buffer, 2.5 mM MgCl2, 100 µM of each dNTP, 0.5 µM each of 5'-biotinylated primer and an unmodified reverse primer, and 0.5 unit of Taq DNA polymerase. Polymerase chain reaction cycling conditions were 30-s DNA denaturation step at 94°C, a 30-s annealing step at 52°C, and a 30-s extension step at 72°C for 35 cycles on a MJ Tetrad thermocycler (MJ Research, Watertown, MA).

Allele-specific oligonucleotides (ASOs) specific for each genotype sequence (Davis and Blackhawk) were synthesized with a 5' amine Uni-Link modification (Integrated DNA Technologies, Inc.). The ASOs were coupled to carboxylated microspheres using a carbodiimide coupling method (Fulton et al., 1997).

After PCR amplification of the target region, SNP genotyping was performed in a 50-µL hybridization reaction containing 10 µL unpurified PCR product, and 2500 ASO-coupled microspheres from each set in 1 x TMAC buffer (4.5 M tetramethylammonium chloride, 75 mM Tris-HCl [pH 8.0], 6 mM EDTA [pH 8.0], 0.15% Sarkosyl). Reactions were denatured at 95°C for 4 min and incubated at 50°C for 30 min. The beads were pelleted by microcentrifugation, and the supernatant was removed. The beads were labeled with 50 µL of freshly made 6 µg mL–1 streptavidin–phycoerythrin in 1x TMAC at 50°C for 20 min. Reactions were then analyzed using the Luminex 100 instrument (Luminex Corporation). The data collection software was set to analyze 100 beads from each set and the MFI was used for analysis.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
SNP Survey and Identification
The approach adopted was to search for SNPs in a region of LG-J that is known to be closely linked to the Rcs3 gene controlling broad resistance to C. sojina. Two pairs of primers were designed from the sequences of the clones bearing the two SSR repeats of Satt244 and Satt547 (Table 1). Thirteen pairs of primers were designed from end sequences of BAC clones that were identified with the SSR markers Satt244 and Satt547 that previously were shown as tightly linked to Rcs3. These primer pairs were used to amplify genomic DNA from the three resistant and three susceptible cultivars. Ten pairs of primers from the BAC ends amplified single bands and produced high quality sequences. These primers were used for identification of nucleotide polymorphisms between the two groups of cultivars (Table 1). The primers designed from the clone containing the SSR marker Satt244 amplified multiple bands. We redesigned the primers to amplify separately the first 505 bp of the 981-bp sequence and the second part consisting of 476 bp. The first part of the sequence, which contains the ATT repeat, amplified several bands and did not yield a sequence of acceptable quality. Therefore, it was excluded from further analysis. The second part of the clone amplified a single band and resulted in a good quality sequence that was used for polymorphism identification between the two groups of cultivars (Table 1). To increase the chances of success in identifying SNPs associated with Rcs3, we considered only the polymorphisms, whether single nucleotides or InDels, that had a similar allele that characterized the three resistant and a common unique allele for the three susceptible cultivars.

The results of this survey of nucleotide variations between the Rcs3 and rcs3 groups, including SNPs and small nucleotide InDels, are summarized in Table 1. Among the 12 sequences analyzed, five did not have nucleotide variations and seven contained 15 SNPs and four InDels between the two groups of cultivars. The SSR clones contained the most nucleotide variations with three SNPs and two InDels in the clone bearing Satt244 and six SNPs in the one bearing Satt547 (Table 1). Previous studies in maize indicated that SNPs and InDels occur at a relatively high frequency in flanking regions of maize microsatellites, suggesting the possibility of converting SSR markers into SNP-based markers (Mogg et al., 2002; Batley et al., 2003b). The BAC end sequences contained at most two SNPs per sequence with the exception of AZ044573, which contained one SNP and two InDels (Table 1). Transitions (C/T or G/A, and vice versa) were the most common variations between the two groups of cultivars (67%) compared to transversions (A/C, A/T, G/C, or G/T, and vice versa) (Table 2).

BLAST Search of SSR Flanking Regions and BAC End Sequences
The two SSR-containing sequences and each BAC end sequence were analyzed using BLASTN and TBLASTX against the nonredundant and the EST databases to determine if any of the sequences were coding regions. Neither of the SSR-containing sequences was similar to a coding region. Five BAC end sequences were similar to coding regions in Arabidopsis, rice, and Medicago truncatula Gaertn. (Table 1). Two BAC end sequences were similar to known sequences from genes involved in pathogenesis and plant defense mechanisms. AQ989240 was similar to a putative ATP-binding cassette (ABC) transporter in the Oryza sativa japonica cultivar group. The role of ABC transporters in pathogenesis has been demonstrated for the plant pathogens Magnaporthe grisea (Hebert) Barr. (Urban et al., 1999), Botrytis cinerea Pers. (Schoonbeek et al., 2001), and Gibberella pulicaris (Fries) Sacc. (Fleissner et al., 2002). AZ301449, which did not have nucleotide variations between the two groups of cultivars was similar to leucine-rich repeat (LRR) sequence in M. truncatula, a major component of the largest class of disease resistance proteins NBS-LRR, which recognize and transmit pathogen-derived signals.

The coding sequences totaling 1565 nucleotides in size contained one SNP. The noncoding sequences totaled 3.875 kb and contained 14 SNPs and four InDels (1/277 bp). In soybean, at least one SNP was identified on average every 470 bp (Choi et al., 2005). Zhu et al. (2003) reported the presence of 280 SNPs in 143 amplicons totalling about 76.3 kb of DNA sequence averaging one SNP every 273 bp. Ching et al. (2002) reported the frequency of one ncSNP per 31 bp and one cSNP per 124 bp in 18 maize genes assayed in 36 inbred lines.

Mapping and Confirmation of Candidate SNPs
Among the 19 SNPs identified, six could not be genotyped because they were in very close proximity (<20 bp) to other SNPs which hindered the development of a stable capture probe (Table 2). The remaining 13 polymorphisms were mapped in the F2 population of Davis x Blackhawk and allowed the construction of a dense genetic map of the segment of LG-J where Rcs3 was mapped previously. Our LG-J map incorporated six SSR markers, 11 SNPs, and Rcs3 (total of 18 loci) (Fig. 1 ). Two SNPs derived from the end sequence AQ989336 from the BAC clone Gm_ISb001_066_A14R that was supposedly anchored with Satt547 did not map to LG-J indicating that it was possibly incorrectly anchored. The SNPs identified in the clone AF237406 that contained the SSR repeat Satt547 mapped at the same location as Satt547. The four SNPs identified in the clone CC453957 that contains the SSR Satt244 mapped 1.6 cM away from Satt244, a possible indication that this locus is in a region of tandem duplications and the recombinations observed represent a locus from a nearby duplication. These SNPs were identified in the last 476 bp section of the 981-bp sequence since the first 505 bp that contain the ATT repeat were not analyzed because they amplified several bands and did not yield a sequence of acceptable quality. This region of LG-J is known to be highly duplicated. In a population of 64 cultivars and breeding lines, Satt244 amplified 10 marker bands of various sizes with two to five fragments in each genotype, an indication of the duplication of this locus (Missaoui et al., 2007). The Soybean GBrowse Database (SoyGD) (http://soybeangenome.siu.edu [verified 23 Apr. 2007]; Shultz et al., 2006) shows that there are at least four homoeologous regions related to the region of LG-J encompassing Satt244 even though it has not been determined where the homoeologous amplicons and contigs map.


Figure 1
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  		Figure 1. Integrated single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) map of the segment of linkage group (LG-J) containing the Rcs3 gene conferring resistance to frogeye leaf spot in soybean. The map on the left represents the USDA consensus map (http://soybase.org/; verified 24 Apr. 2007).

 
The Rcs3 gene was positioned on the map near Satt244 and within 0.50 cM from InDels AZ573TA150 and AZ573CA393 (Fig. 1). Several other SNPs, such as CC957AG685, CC957TC536, CC957AG730, and CC957TA799, showed a high degree of redundancy and mapped to the same locus. This was expected because SNPs identified from the same source sequence would be expected to map to the same location. It appears that none of the SNPs and InDels identified contributes directly to the Rcs3 phenotype but several are tightly linked to the phenotype (Fig. 1 and Table 3).


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  		Table 3. Single nucleotide polymorphism (SNP) genotypes based on the single base extension assay at five SNP and insertion or deletion (InDel) markers and the direct hybridization assay in 64 ancestors and descendants of the soybean cultivar Davis (original source of Rcs3).

 
Since all the SNP markers mapped within 0.50 to 2.1 cM from the Rcs3 gene, five SNPs (one from each source sequence) were further validated for association with Rcs3 and for their potential for MAS in 64 cultivars and breeding lines including ancestors and descendants of Davis (Table 3). Among the 21 cultivars and breeding lines shown previously to contain the Rcs3 gene, only one, N97–9612, did not have the Davis allele at CC957TA799 and three were heterogeneous at this locus. The Davis allele at the marker locus AF406GA207 was absent in three lines known to have the Rcs3 (Au94–863, N97–9658, and SC97–1075). Five lines were heterogeneous for the Davis allele at this marker locus. The markers AZ573CA150, AQ455GA396, and AQ166AG280 showed the closest association with the presence of Rcs3. The Davis alleles at these three marker loci were present in all the cultivars and lines known to have the Rcs3 gene. N97-9612, which was shown previously to be heterogeneous in the segment of LG-J spanning Satt244 and Satt547, was also heterogeneous at this locus. These three markers appear to be tightly linked and therefore may suffice to be used as a haplotype tag for the Rcs3 gene. Johnson et al. (2001) suggested that the information provided by SNPs is most useful when several (two to four) closely spaced SNPs completely define haplotypes in the region of interest. Because some SNPs are redundant in the presence of linkage disequilibrium, only a small subset defining the haplotype of interest need to be assayed. Taillon-Miller et al. (2004) predicted through power calculations that linkage disequilibrium blocks can be represented by SNP tags in genome-wide association studies to identify alleles associated with common diseases within linkage disequilibrium blocks.

Our results suggest that the marker AZ573TA150 and AZ573CA393, derived from the same GenBank accession sequence, could be used successfully in MAS for Rcs3. AZ573TA150 is an InDel of three nucleotides (ACT) and AZ573CA393 an InDel of four nucleotides (CATA) that are missing in the Rcs3 cultivars and breeding lines.

Direct Hybridization for Marker-Assisted Selection
To provide resources for MAS, we developed a direct hybridization assay to detect Rcs3. The direct hybridization assay was shown to be more cost effective than the SBE or even allele specific primer extension when detected on the Luminex 100 flow cytometry platform (Lee et al., 2004). The InDel of AZ573TA150 and a SNP AZ573AG159 were incorporated into ASO probes corresponding each to the Davis or Blackhawk allele. Each probe consisted of a 19-mer oligonucleotide (Davis, 5'-TAA TGG CCA TTG TAT GAT G-3'; Blackhawk, 5'-TAA TGG TCA TTG TAG TAT G-3'). The 64 ancestors and descendants of Davis were genotyped with these probes using the direct hybridization assay (Table 3). The results were similar to that obtained using the SBE assay for AZ573TA150 (Table 3). All cultivars and breeding lines carrying the Davis allele had positive signal for the Davis ASO probe. G92-2739, N97-9612, G-Gordon-Rcs3, G92-1306, and Motte were heterogeneous for AZ573TA150 with SBE and showed positive signals for both the Davis and Blackhawk ASO probes. In addition, we genotyped the F2 population of Davis x Blackhawk using the ASO probes and the direct hybridization assay (Fig. 2 ). All 92 lines had good signal intensity and a clear clustering pattern for lines that were homozygous for the Davis allele, homozygous for the Blackhawk allele, or were heterozygous. The SNP genotypes obtained from the direct hybridization assay were accurate and 100% (92/92) congruent with the genotypes previously obtained using SBE assay for AZ573TA150 and the SRR markers Satt244 and Satt547. These results suggest that the direct hybridization assay using the ASOs for AZ573TA150 and AZ573TA199 provide a robust assay for MAS of the Rcs3 gene conditioning broad resistance to FLS in soybean.


Figure 2
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  		Figure 2. Results of direct hybridization assay for the F2 population of Davis x Blackhawk. Genotypes are represented by homozygous for the Davis allele ({diamondsuit}), heterozygous ({blacksquare}), and homozygous for the Blackhawk allele ({blacktriangleup}) determined by the single base extension (SBE) assay. Numbers on the x- and y-axis represent mean fluorescent intensity (MFI).

 

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Received for publication November 12, 2006.


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The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome