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Published online 16 July 2007
Published in Crop Sci 47:S-73-S-82 (2007)
© 2007 Crop Science Society of America
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Development of SNP Assays for Marker-Assisted Selection of Two Southern Root-Knot Nematode Resistance QTL in Soybean

Bo-Keun Haa,*, Richard S. Husseya and H. Roger Boermab

a Dep. of Crop and Soil Sci., Univ. of Georgia, Center for Applied Genetic Technologies, 111 Riverbend Rd., Athens, GA 30602
b Dep. of Plant Pathology, Univ. of Georgia, 2106 Miller Plant Sciences Bldg., Athens, GA 30602

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The identification of single nucleotide polymorphism (SNP) markers tightly linked to soybean [Glycine max (L.) Merr.] quantitative trait loci (QTL) conditioning resistance to southern root-knot nematode (Meloidogyne incognita) (Mi) would enhance the efficiency and cost effectiveness of marker-assisted selection (MAS) for this trait. Bacterial artificial chromosome (BAC) ends and simple sequence repeat (SSR)-containing genomic DNA clones were used to develop SNP markers linked to two soybean Mi resistance QTL on Linkage Group O (LG-O) and LG-G. A total of 14 BAC-end sequences and seven SSR flanking regions were used to design primers to amplify genomic fragments of PI 96354 (Mi resistant) and ‘Bossier’ (Mi susceptible). We discovered three SNPs in Satt358 source-sequences located near a major Mi-resistant QTL on LG-O and three SNPs in Satt199 source-sequences located near a minor Mi-resistant QTL on LG-G. Using a direct hybridization SNP assay detected on a Luminex 100 flow cytometer, the SNP358 genotypes of 94 F2:3 lines from a cross of PI 96354 x Bossier were congruent with the genotypes of the SSR marker Satt358. The genotypes of SNP199 marker which targets a SNP in Satt199 source-sequence also showed 100% congruence with the genotypes of the SSR marker Satt199. SNP genotyping of 24 known Mi-resistant or Mi-susceptible cultivars showed that SNP358 and SNP199 markers should be highly effective in MAS for the Mi-resistance QTL on LG-O and LG-G.

Abbreviations: ASO, allele-specific oligonucleotide • ASPE, allele-specific primer extension • BAC, bacterial artificial chromosome • DH, direct hybridization • LG, linkage group • MAS, marker assisted selection • MFI, mean fluorescence intensity • Mi, Meloidogyne incognita • OL, oligonucleotide ligation • QTL, quantitative trait loci • SBE, single-base extension • SNP, single nucleotide polymorphism • SSR, simple sequence repeat

Received for publication October 15, 2006.

Development of SNP Assays for Marker-Assisted Selection of Two Southern Root-Knot Nematode Resistance QTL in Soybean

Bo-Keun Haa,*, Richard S. Husseya and H. Roger Boermab

a Dep. of Crop and Soil Sci., Univ. of Georgia, Center for Applied Genetic Technologies, 111 Riverbend Rd., Athens, GA 30602
b Dep. of Plant Pathology, Univ. of Georgia, 2106 Miller Plant Sciences Bldg., Athens, GA 30602

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

The identification of single nucleotide polymorphism (SNP) markers tightly linked to soybean [Glycine max (L.) Merr.] quantitative trait loci (QTL) conditioning resistance to southern root-knot nematode (Meloidogyne incognita) (Mi) would enhance the efficiency and cost effectiveness of marker-assisted selection (MAS) for this trait. Bacterial artificial chromosome (BAC) ends and simple sequence repeat (SSR)-containing genomic DNA clones were used to develop SNP markers linked to two soybean Mi resistance QTL on Linkage Group O (LG-O) and LG-G. A total of 14 BAC-end sequences and seven SSR flanking regions were used to design primers to amplify genomic fragments of PI 96354 (Mi resistant) and ‘Bossier’ (Mi susceptible). We discovered three SNPs in Satt358 source-sequences located near a major Mi-resistant QTL on LG-O and three SNPs in Satt199 source-sequences located near a minor Mi-resistant QTL on LG-G. Using a direct hybridization SNP assay detected on a Luminex 100 flow cytometer, the SNP358 genotypes of 94 F2:3 lines from a cross of PI 96354 x Bossier were congruent with the genotypes of the SSR marker Satt358. The genotypes of SNP199 marker which targets a SNP in Satt199 source-sequence also showed 100% congruence with the genotypes of the SSR marker Satt199. SNP genotyping of 24 known Mi-resistant or Mi-susceptible cultivars showed that SNP358 and SNP199 markers should be highly effective in MAS for the Mi-resistance QTL on LG-O and LG-G.

Abbreviations: ASO, allele-specific oligonucleotide • ASPE, allele-specific primer extension • BAC, bacterial artificial chromosome • DH, direct hybridization • LG, linkage group • MAS, marker assisted selection • MFI, mean fluorescence intensity • Mi, Meloidogyne incognita • OL, oligonucleotide ligation • QTL, quantitative trait loci • SBE, single-base extension • SNP, single nucleotide polymorphism • SSR, simple sequence repeat


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
ROOT-KNOT NEMATODES (Meloidogyne spp.) are major soybean pests that cause severe yield losses in the southern USA. Soybean breeders have been intensifying their efforts to develop improved root-knot nematode resistant cultivars. One of the challenges in the past has been that screening for resistant plants was tedious and time-consuming (Boerma and Hussey, 1992). Recently, breeders have explored the use of DNA markers in place of phenotypic screening once the marker/QTL relationship has been established and confirmed. The use of MAS has many advantages when compared to phenotypic screening. Selection can be performed in early generations of segregating populations, at early stages of plant development, and can be used to pyramid genes. With MAS, breeders can conduct multiple cycles of selection in a year, eliminate dependence on the natural occurrence of the pest or pathogen, and reduce the cost and labor associated with inoculum maintenance (Mohan et al., 1997). However, the successful application of MAS in a plant breeding program depends on several factors: (i) the identification of markers that co-segregate or are closely linked with QTL that condition the desired trait, (ii) the availability of an efficient marker assay for screening large breeding populations, and (iii) a marker assay that is economical to use (Gupta et al., 1999; Francia et al., 2005).

Molecular markers have been used to map the genomic location of QTL for Mi resistance. Using RFLP markers, Tamulonis et al. (1997) mapped a major QTL on LG-O that explained 31% of the phenotypic variation in gall number and a minor QTL on LG-G that accounted for 14% of variation. With SSR markers, Li et al. (2001) mapped the major QTL to a 9.1 cM interval between Satt358 and Satt492 on LG-O and the minor QTL to a 8.2 cM interval between Satt505 and Satt012 on LG-G. The use of SSR markers flanking the two QTL was effective in predicting the Mi phenotypes (Li et al., 2001). Also, Ha et al. (2004) identified SSR markers Satt358 and Sat_132 on LG-O that cosegregated with Rmi1, a Mi resistance gene, using a molecular pedigree analysis in southern U.S. elite soybean cultivars.

The use of either RFLP or SSR markers in MAS has some limitations. They both depend on gel-based assays and are therefore time-consuming and expensive. Recently SNP markers have been considered as the marker of choice for MAS because of their high frequency, widespread distribution throughout the genome, and their suitability for high-throughput, automated genotyping (Schork et al., 2000). In humans, Wang et al. (1998) reported the frequency of SNP was 1.4 per kilobase in a survey of 10 individuals. Lindblad-Toh et al. (2000) found that the frequency of SNP was 0.95 per kilobase in seven inbred mouse lines. Also, SNPs are highly abundant and distributed throughout the genome in plants such as Arabidopsis thaliana (Drenkard et al., 2000), Oryza sativa (Nasu et al., 2002), and Zea mays (Batley et al., 2003). In soybean, Grimm et al. (1999) estimated the frequency of SNP as 3.4 per kilobase in approximately 18000 bases of DNA sequence obtained from 18 genotypes that were the ancestors of modern North American soybean cultivars. Recently, Zhu et al. (2003) found a total of 280 SNPs in 76-kb of sequence from 25 different soybean genotypes. This study also reported that nucleotide diversity in random non-coding genomic sequence from BAC clones and SSR flanking regions was higher than that of the genomic DNA associated with coding regions. This suggested that the BAC-end sequence and SSR flanking regions provide a good source of sequence information for SNP discovery.

Several SNP genotyping techniques are available for use in MAS. These SNP assays are based on hybridization methods, allele-specific PCR, primer extension, oligonucleotide ligation, and endonuclease cleavage (Gupta et al., 2001; Syvänen, 2001). For plant improvement applications, SNP genotyping assays containing single-base extension (SBE), allele-specific primer extension (ASPE), oligonucleotide ligation (OL), and allele-specific oligonucleotide (ASO) hybridization, also referred to as direct hybridization (DH), were compared using the Luminex 100 flow cytometer platform (Luminex Corporation, Austin, TX) (Lee et al., 2004). On the basis of cost, simplicity, and speed, the DH assay was considered the most economical for MAS. The DH assay tests for single base mismatch with two short allele specific oligonucleotide (ASO) probes. The probes are allowed to pair with the target DNA that contains the SNP at assay conditions in which only perfectly matched probe-target hybridizations are stable and hybridizations that contain a mismatch are unstable (Syvänen, 2001). Direct hybridization assays with the Luminex 100 flow cytometer were used in several studies to screen for mutations in the cystic fibrosis transmembrane conductance regulator gene (Dunbar and Jacobson, 2000), genotype β–globin variants (Colinas et al., 2000), and haplotype the NAT2 gene (Hurley et al., 2004).

The objectives of this study were to identify SNPs linked to the previously identified and confirmed Mi resistance QTL on LG-O and LG-G and to develop an efficient SNP detection assay for both QTL for use in MAS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Materials
Ninety-four F2 plants from the cross of PI 96354 x Bossier were used in this study. PI 96354 has a high level of resistance to Mi for both gall formation and nematode reproduction and Bossier is highly susceptible (Luzzi et al., 1987). This population was utilized in the RFLP mapping (Tamulonis et al., 1997) and SSR mapping (Li et al., 2001) of the QTL conferring Mi resistance. DNA was extracted from individual parental genotypes and individual field-grown F2 plants using the modified CTAB procedure of Keim et al. (1988) and diluted to 20 ng µL–1. The F2:3 lines were evaluated for Mi galling in the greenhouse (Tamulonis et al., 1997).

SNP Identification
Based on the integrated genetic linkage map of soybean (Song et al., 2004), three SSR markers (Satt358, Sat_132, and Satt487) located near a major Mi-resistance QTL on LG-O and four SSR markers (Satt199, Satt505, Satt400, and Satt012) located near a minor Mi-resistance QTL on LG-G were chosen (Li et al., 2001; Ha et al., 2004). BAC clones that were anchored with the SSR markers were identified in the Soybean Genome Database (http://soybeangenome.siu.edu/; verified 16 Apr. 2007) (Fig. 1 ). SSR-containing sequences and their BAC end sequences were obtained from GenBank and primers were designed with OLIGO primer design software (Molecular Biology Insights, Inc., Cascade, CO).


Figure 1
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  		Figure 1. Soybean composite genetic map in the region of the Mi resistance quantitative trait loci on linkage group (LG)-G (a) and LG-O (b) (http://soybase.org; verified 16 April 2007). The bacterial artificial chromosome (BAC) clones were positive clones for the SSR markers. The BAC clones prefixed with "IS" (Iowa State University) or "UM" (University of Minnesota) were from the ‘Williams 82’ and ‘Faribault’ BAC libraries, respectively.

 
All PCR primers were used to amplify genomic DNA of PI 96354 and Bossier. PCR amplification used a 10-µL reaction mix containing 2 µL of 40 ng template DNA, 1.0 x PCR buffer, 2.5 mM MgCl2, 100 µM of each dNTP, 0.2 µM each of forward and reverse primers, and 0.5 unit of Taq DNA polymerase. PCR cycling conditions were 30 s DNA denaturation step at 94°C, a 30 s annealing step at 50°C, and a 1 min extension step at 72°C for 35 cycles on a MJ Tetrad thermocycler (MJ Research, Watertown, MA). The products were analyzed on a 1.5% agarose gel stained with ethidium bromide.

After the initial determination that a set of PCR primers appeared to produce a single amplicon from genomic DNA, the PCR product was directly sequenced using one of the PCR primers with a BigDye Terminator v3.1 Cycle Sequencing Kit (PE-ABI, Foster City, CA). These amplicons were analyzed on an ABI 377 automated sequencer (PE-ABI, Foster City, CA) with a 36-cm 4.5% polyacrylamide gel (29:1), 6M Urea. The sequence data from amplicons of PI 96354 and Bossier were analyzed with ClustalW 1.8 (Thompson et al., 1994).

Microsphere-direct Hybridization with Biotinylated Upstream Primer
Microsphere-direct hybridization was performed according to Dunbar and Jacobson (2000) with slight modifications. The upstream or downstream primer for amplification of the SNP-containing fragment was labeled at the 5' terminus with biotin (BioSource Int, Camarillo, CA). Targets were amplified and used in a total 10-µL reaction mix containing 2 µL of 40 ng template DNA, 1.0 x PCR buffer, 2.5 mM MgCl2, 100 µM of each dNTP, 0.5 µM each of the 5'-biotinylated primer and an unmodified reverse primer, and 0.5 unit of Taq DNA polymerase. PCR cycling conditions were a 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 specific for each genotype sequence (PI 96354 and Bossier) were synthesized with a 5' amino modification (BioSource Int, Camarillo, CA). The ASOs were coupled to carboxylated microspheres using a carbodiimide coupling method (Fulton et al., 1997). For each ASO and microsphere set combination, 5 x 106 carboxylated microspheres were suspended in 50 µL 0.1M 2-(N-Morpholino) ethanesulfonic acid, pH 4.5 (MES). One nmol of amine-substituted ASO was added, followed by addition of 25 µg N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide [EDC] and incubation in the dark for 30 min. The EDC addition and incubation were repeated and the microspheres were washed once with 0.02% Tween-20 and once with 0.1% SDS. Coupled microspheres were stored in MES at 4°C in the dark.

After PCR amplification, genotyping was performed in a 50 µL hybridization reaction containing 10 µL unpurified PCR product, and 2500 of each ASO-coupled microsphere 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 4 µg mL–1 streptavidin-phycoerythrin in 1 x TMAC at 50°C for 20 min. Reactions were then analyzed using the Luminex 100 instrument. The data collection software was set to analyze 100 beads from each set and the mean fluorescence intensity (MFI) was used for analysis.

For multiplex assays, each PCR reaction that amplified the SNP containing region was pooled (total volume was 17 µL). To the pooled product, 33 µL of 1.5 x TMAC buffer containing 2500 of each ASO-coupled microsphere set was added. The samples were mixed and 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 1 x TMAC at 50°C for 20 min. Reactions were then analyzed on a Luminex 100 instrument. The data collection software was set to analyze 100 beads from each set and the mean fluorescence intensity (MFI) was used for analysis. The genotype score was calculated: log10 [(fraction of PI 96354 allele signal + 0.01)/(fraction of Bossier allele signal + 0.01)], which results in heterozygotes having values close to 0, homozygotes for PI 96354 allele close to +1, and homozygotes for Bossier allele close to –1 (a score of ± 1 corresponds that MFI of the positive allele is 10 x higher than MFI of the negative allele) (Hirschhorn et al., 2000).

Confirmation of SNP Genotyping
Seed for 12 Mi-resistant soybean lines and 12 Mi-susceptible lines were obtained from the USDA Soybean Germplasm Collection maintained at the Univ. of Illinois (Urbana, IL). Soybean DNA was extracted from seeds of each genotype according to modified procedures of Kang et al. (1998) and diluted to 20 ng µL–1. The SSR genotyping assay followed by the protocol of Diwan and Cregan (1997). The PCR amplicons were analyzed on an ABI-Prism 377 DNA sequencer (PE-ABI, Foster City, CA). The SNP genotyping assay followed the multiplex protocol of microsphere-direct hybridization as described earlier.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
SNP Discovery for Meloidogyne incognita (Mi) Resistance
In soybean, it is still relatively difficult to discover SNPs at defined positions in the genome due to the lack of a complete soybean physical map. However, existing DNA sequence data from SSR flanking regions and RFLP probes, both of which are already positioned on the soybean linkage map, provide an excellent resource for SNP discovery throughout the soybean genome (Cregan, 1999). In addition, a genome-wide, BAC-based physical map and the integrated physical and genetic map of soybean in the Soybean Genome Database (SoyGD) could provide additional sequence resources to find SNPs (Shultz et al., 2006; http://soybeangenome.siu.edu/; verified 11 May 2007).

In this study, we used three SSR-containing clones (Satt358, Sat_132, and Satt487) located near the major Mi-resistance QTL (Rmi1) on LG-O and four SSR-containing clones (Satt199, Satt505, Satt400, and Satt012) located near minor Mi-resistance QTL on LG-G to search for SNPs. Furthermore, we found 9 BAC clones that were anchored with these SSR markers in the SoyGD (Fig. 1).

Sequences from SSR clones and end sequences from BAC clones were obtained from GenBank. A total of 14 BAC-end sequences and seven SSR clones were used to design primers to amplify genomic fragments of PI 96354 and Bossier. A total of 6.9 k bp of sequence data were obtained from 21 PCR products of PI 96354 and Bossier. A total of 12 SNPs including seven single-base changes and five InDels were identified from sequence alignment of each PCR fragment (Table 1). Four InDels were derived from different SSR containing genomic DNA clones between PI 96354 and Bossier for Satt358, Satt199, Satt505, and Satt012, respectively. In two genotypes, the SNP frequency was 1.0 SNPs per kbp and nucleotide diversity ({theta}) was 0.001. The nucleotide diversity in our study was numerically lower than the nucleotide diversity ({theta} = 0.00179) in random noncoding genomic sequence from BAC clones and SSR flanking regions previously estimated among 25 soybean genotypes reported by Zhu et al. (2003). Of the seven single-base changes 57% of the SNPs were transversions (A{leftrightarrow}C, A{leftrightarrow}T, G{leftrightarrow}C, or G{leftrightarrow}T), whereas 43% were transitions (C{leftrightarrow}T, or G{leftrightarrow}A).


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  		Table 1. GenBank accessions selected based on the sequence of seven simple sequence repeat (SSR)-containing clones and the single nucleotide polymorphism (SNP) polymorphisms identified between PI 96354 and Bossier.

 
In the Satt358 flanking region, three SNPs were identified at sequence position 83, 91, and 190 (Fig. 2 ). On the basis of nucleotides at the 83-base (A vs. T) and 91-base (G vs. A) positions, haplotype AG characterized the Bossier genotype (susceptible to Mi), and haplotype TA the PI 96354 genotype (resistant to Mi).


Figure 2
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  		Figure 2. Polymorphic sites identified from PI 96354 and Bossier. A) sequences from the Satt358 containing genomic DNA clone, B) sequences from the Satt199 containing genomic DNA clone, C) sequences from the Satt012 containing genomic DNA clone.

 
On LG-G we identified two SNPs, A/T (base 202) and A/G (base 209), in the Satt199 flanking region (Fig. 2). SNP199 haplotype AA characterized the PI 96354 genotype and haplotype TG characterized the Bossier genotype. In the Satt012 flanking region, one SNP was identified at the 180-base position. Previous research has shown that MAS based on SSR marker Satt358 located on LG-O and SSR markers Satt199 or Satt012 located on LG-G enhanced the level of Mi resistance (Li et al., 2001). Therefore, SNPs identified from the source sequence containing the Satt358, Satt199, and Satt012 repeats were evaluated for SNP assay development.

SNP Genotype Analysis of F2:3 Lines
The Luminex 100 flow cytometer has the capability of conducting multiplexed SNP analyses in a single reaction tube by using reaction-specific microspheres that fluoresce at different frequencies (Dunbar, 2006; Lee et al., 2004). On the basis of cost, simplicity, and speed, we choose to develop the direct hybridization (DH) assay for efficient MAS of Mi resistance.

To evaluate a DH assay, we used SNP358 developed from Satt358 source sequence on LG-O (Table 2). Allele-specific oligonucleotide probes corresponding to each of the PI 96354 and Bossier sequence variants were designed in such a way that the two polymorphic bases at the 83- and 91-base positions in the Satt358 source sequence were positioned in the center of either a 19- or 23-mer probe. This allowed the determination of which probe length provided the best clustering pattern of lines homozygous for PI 96354, homozygous for Bossier, or heterozygous at SNP358 (Table 2). The ASO358-TA probe was specific for the PI 96354 allele and the ASO358-AG probe was specific for the Bossier allele. A clustering method was used to score the genotypes, with heterozygous lines having values close to 0 and homozygous lines generally close to ± 1 (Hirschhorn et al., 2000).


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  		Table 2. Sequences of target amplification primers and allele-specific oligonucleotide (ASO) probes for single nucleotide polymorphism (SNP) genotyping assays.

 
To optimize the assay, three hybridization temperatures and two lengths of ASOs were evaluated (Fig. 3 ). Thirty minute hybridization at 50°, 53°, and 55°C temperatures were evaluated and the same temperatures were used to incubate the reaction containing streptavidin for 20 min. As shown in Fig. 3, the 19-mer ASOs provided the most unambiguous separation of F2:3 lines homozygous for the PI 96354 or Bossier haplotypes or lines containing both haplotypes (heterozygous lines) compared to the 23-mer ASOs under all three temperature-hybridization conditions. As the hybridization temperature increased, the value of the mean fluorescence intensity (MFI) decreased, but the clustering pattern of the three groups (homozygous for PI 96354, homozygous for Bossier, and heterozygous) did not appear significantly affected. Based on the clustering pattern and signal intensity, the 50°C hybridization temperature and the 19-mer ASOs for SNP358 marker gave the most robust clustering pattern to score these F2:3 lines. These results were consistent with previously reports that the effect of a base mismatch on hybridization was greater with decreasing probe length (Armstrong et al., 2000; Dunbar, 2006). The SNP genotypes obtained from this DH assay were extremely accurate, being 100% congruent with genotypes previously obtained for these 94 F2:3 lines by SSR analysis using Satt358 (Li et al., 2001).


Figure 3
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  		Figure 3. Optimization of direct hybridization (DH) assay. SNP358 marker was genotyped across the F2:3 population from the cross of PI 96354 x Bossier using different length of probes and different hybridization temperature conditions. Data are presented as the log10 [(fraction of PI 96354 allele signal + 0.01)/(fraction of Bossier allele signal + 0.01)] versus total mean fluorescence intensity (MFI) of two alleles (Hirschhorn et al., 2000). Genotypes are represented by homozygotes for PI 96354 allele (Figure 3), heterozygotes (Figure 3), and homozygotes for Bossier allele (Figure 3) determined by the band sizes of the Satt358 SSR marker. (H.T. = hybridization temperature; ASO = allele-specific oligonucleotide).

 
For the minor Mi-resistance QTL on LG-G we designed a SNP detection assay which targeted two SNPs in the Satt199 source sequence on LG-G (base position 202 and 209). The ASO199-AA probe was specific for the PI 96354 haplotype and the ASO199-TG probe was specific for the Bossier haplotype (Table 2). Our initial ASOs for SNP199 were designed to detect the PI 96354 and Bossier haplotypes with 19-mers, but MFI of the ASO199-AA allele was not large enough to effectively score the PI 96354 haplotype. The SNP199 ASOs were redesigned with a 20-mer to detect the PI 96354 haplotype and a 19-mer for the Bossier haplotype (Table 2).

In addition, we tested genotyping of the SNP012 marker which targets a G/A SNP at position 180 in Satt012 source sequences on LG-G. However, the SNP012 marker did not produce a clear clustering pattern with good signal strength. This may have resulted from the low GC content of the ASOs used in this assay (less than 25%).

After development and optimization of the DH assay for SNP358 and SNP199, we evaluated a multiplex assay containing both SNP358 and SNP199 probe sets in a single reaction. We used the 50°C hybridization temperature and 19-mer ASOs for SNP358 and the ASOs described above for SNP199. Data in Fig. 4 showed clear clustering patterns with approximately a 10:1 separation ratio of the two haplotypes for both SNP markers. Compared with the single-reaction assay of SNP358 (Fig. 3), its clustering pattern when multiplexed with SNP199 was similar, but some lines produced a weaker signal in the multiplexed reaction. This weaker signal did not prevent the correct characterization of these lines for SNP358. The ability to combine SNP358 and SNP199 marker increases the efficiency of MAS for the LG-O and LG-G Mi-resistance QTL.


Figure 4
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  		Figure 4. Multiplex single nucleotide polymorphism (SNP) genotyping across the F2:3 population from the cross of PI 96354 x Bossier. Data are presented as the log10 [(fraction of PI 96354 allele signal + 0.01)/(fraction of Bossier allele signal + 0.01)] versus total mean fluorescence intensity (MFI) of two alleles (Hirschhorn et al., 2000). Genotypes at SNP358 and SNP199 are represented by homozygotes for PI 96354 allele (Figure 4), heterozygotes (Figure 4), and homozygotes for Bossier allele (Figure 4) determined by the band sizes of the SSR marker Satt358 and SSR marker Satt199, respectively.

 
SNP Genotyping of Known Meloidogyne incognita (Mi) Resistant and Susceptible Lines
The effectiveness of SNP358 and SNP199 were compared with their respective SSR markers, Satt358 and Satt199, by assaying 12 Mi-resistant and 12 Mi-susceptible soybean lines (Table 3). To genotype the SNP markers among these lines, the multiplex assay for SNP358 and SNP199 was used. The relationship between Mi reaction and Satt358 genotypes were previously described by Ha et al. (2004). The study reported that Mi-resistant lines inherited a 200-bp allele and Mi-susceptible lines inherited a 192-bp allele at Satt358. Eleven of the 12 Mi-resistant lines carrying the 200-bp allele at Satt358 had approximately 10 x higher positive signal for the ASO358-TA probe specific to the PI 96354 allele than the ASO358-AG probe specific to the Bossier allele. ‘Gregg’, which is heterogeneous at Satt358 (192/200 bp), showed positive signals for both ASO358-TA and ASO358-AG. All Mi-susceptible lines carrying the 192-bp allele at Satt358 had positive signal for the Bossier-specific ASO358-AG probe. However, FC 33243 with a 160-bp allele at Satt358 showed low MFI for both ASO358-TA and ASO358-AG. To gain more perspective on this observation, we sequenced the PCR product of FC 33243. The sequence showed that FC 33243 has a unique AA haplotype at SNP358. Therefore, SNP358 has three SNP haplotypes; TA, AG, and AA in 24 soybean lines. FC 33243 has been previously shown to be a unique source of Mi-resistance and was found to be heterogeneous for its Mi reaction (Ha et al., 2004).


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  		Table 3. Comparison between simple sequence repeat (SSR) markers and single nucleotide polymorphism (SNP) markers on linkage group-O (LG-O) and LG-G in the Mi-resistant and Mi-susceptible cultivars.

 
At the Satt199 SSR marker on LG-G four alleles, 159, 162, 170, and 200 bp, were detected among the 24 lines (Table 3). PI 96354, which contains a minor Mi-resistance QTL linked to Satt199, possessed the 200-bp allele. However, the 200-bp allele of Satt199 was also present in ‘Cobb’, FC 33243, ‘Gasoy17’, and ‘Volstate’ that likely do not possess the minor Mi-resistance QTL on LG-G. In genotyping the SNP199 marker, all lines homozygous for the 200-bp allele at Satt199 had approximately 10 x higher positive signal for the ASO199-AA specific probe which characterizes the PI 96354 allele than for ASO199-TG which is specific for the Bossier allele. The other lines homozygous for the 159, 162, and 170-bp alleles at Satt199 had positive signal for the ASO199-TG probe. These results suggested that the SNP199 marker had two different haplotypes (AA and TG) in 24 soybean lines. Although the AA haplotype of SNP199 marker was not perfectly associated with the minor Mi-resistance QTL on LG-G, this marker could be used for MAS when it is polymorphic in populations known to contain the PI 96354 Mi-resistance allele at the LG-G QTL.

The time required for multiplex detection of SNP358 and SNP199 was 4 h which includes 2 h for PCR, 1.5 h for hybridization, 0.5 h for assaying on the Luminex instrument. In contrast, time for genotyping with the SSR markers requires 5 h including 2 h for PCR, 1 h for gel preparation, and 2 h for gel electrophoresis. SSR marker analysis required gel electrophoresis that uses moderately hazardous reagents, is difficult to automate, and requires a highly skilled staff to run the gels and for data analysis (Meksem et al., 2001). The SNP assay using direct hybridization eliminates the need for gel electrophoresis and manual gel tracking and therefore allows accurate and high speed detection of SNP haplotype across thousands of individuals. Based on a two-plex assay, the cost of SNP genotyping using Luminex 100 was approximately $0.15 per data point, while the cost of SSR genotyping was $0.20 in our study. The cost per data point for SNP genotyping was approximately 75% of that for SSR genotyping. However, the SNP genotyping using Luminex 100 and SSR genotyping using gel systems or capillary systems still require post-PCR steps that limit a streamlined workflow.

Marker assisted selection generally involves the assaying of a few markers across thousands of individuals, and as such, requires low cost/genotype, short assay time, and simplicity. In this study, we have developed the resources for a relatively high-throughput method of selection for the Mi-resistance QTL on LG-O and LG-G by assaying SNP358 and SNP199 using a DH assay detected on the Luminex 100 flow cytometry platform. Our research has also discovered additional SNPs in the Mi-resistance containing regions of LG-O and LG-G. Other assays for detection of these SNPs along with SNP358 and SNP199 could be developed for the Luminex 100 or other platforms.

Received for publication October 15, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 





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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