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Published in Crop Sci. 44:758-763 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

CROP BREEDING, GENETICS & CYTOLOGY

Pedigree Analysis of a Major QTL Conditioning Soybean Resistance to Southern Root-Knot Nematode

Bo-Keun Haa, J. Brandon Bennetta, Richard S. Husseyb, Steven L. Finnertyb and H. Roger Boerma*,a

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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The southern root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood] (Mi) is a serious pest of soybean [Glycine max (L.) Merr.] in the southern USA. Many soybean cultivars with Mi resistance and high productivity have been developed in the USA during the past few decades. DNA markers have been used to identify a major quantitative trait locus (QTL) near the top of Linkage Group O (LG-O) conferring resistance to Mi. The objectives of this study were to determine the frequency of elite Mi-resistant cultivars that inherited the major Mi resistance QTL on LG-O and determine the ancestral source of the Mi-resistance allele at this QTL. Forty-eight soybean lines, including ancestral, Mi-susceptible, and Mi-resistant genotypes were analyzed at six simple sequence repeat (SSR) loci that flank the major Mi QTL on LG-O. Codescent analysis of markers and phenotypes across six cycles of breeding showed that Mi-resistant cultivars inherited a 200-bp band at Satt358 and a 238-bp band at Sat_132 from ‘Palmetto’. The tight linkage of both Satt358 and Sat_132 to the Mi QTL on LG-O indicates that selection for the Mi-resistant parent's allele at either of these markers should be highly effective in identifying Mi-resistant plants or lines.

Abbreviations: EDTA, ethylenediamine tetraacetic acid • LG-O, Linkage Group O • Mi, Meloidogyne incognita • PCR, polymerase chain reaction • QTL, quantitative trait locus/loci • SSR, simple sequence repeat


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
ROOT-KNOT NEMATODES (Meloidogyne spp.) are economically important plant pathogens that cause extensive crop losses all over the world (Sasser, 1977). They have extensive host ranges, their distribution is worldwide, and they interact with fungi in disease complexes. In the southeastern USA, the southern root-knot nematode (Mi) is the most common root-knot nematode. In a Florida field infested with Mi, soybean yields of susceptible cultivars were reduced by 53 to 90% (Kinloch, 1974). Yield losses in the southern USA due to root-knot nematode were estimated at $80 million in 1996 (Pratt and Wrather, 1998).

The development and use of nematode-resistant soybean cultivars result in reduced yield losses and increased grower profits (Boerma and Hussey, 1992). Thus, many soybean cultivars with root-knot nematode resistance and high productivity have been developed in the USA during the past few decades. The Mi-resistant soybean ‘Bragg’ (Hinson and Hartwig, 1964) originated from the cross ‘Jackson’ x D49-2491 and the Mi-resistant ‘Forrest’ (Hartwig and Epps, 1973) from the cross ‘Dyer’ x Bragg. The Mi-resistant ‘Maxcy’ (Shipe et al., 1994) and ‘Doles’ (Boerma et al., 1994b) were released in the 1990s. Both cultivars share Forrest as a common parent. Forrest was shown to possess a single additive Mi-resistance gene, Rmi1 (Luzzi et al., 1994). In a cross of PI 96354, which possesses a high level of resistance to Mi (Luzzi et al., 1987), and ‘Bossier’ (Mi susceptible), a major QTL was identified which mapped to LG-O of the public soybean genetic linkage map (Tamulonis et al., 1997). With SSR markers, Li et al. (2001) found the major Mi QTL on LG-O was indicated in the Satt492 and Satt358 interval and located 3.1 cM from Satt492. Tamulonis et al. (1997) speculated, based on segregation for gall number in a population of Forrest x PI 96354 and the level of resistance in Forrest, that the Mi-resistance QTL on LG-O was the Rmi1 gene.

The availability of pedigree data and molecular linkage maps provides an opportunity to track genomic regions through the breeding process (Shoemaker et al., 1992). King et al. (1999) tracked Vf for scab (Venturia inaequalis) resistance in apple (Malus) accessions and assessed linkage drag through a genome scan of regions flanking the introgression site. Narvel et al. (2001a)( 2001b) used a molecular pedigree analysis to track the rxp gene for bacterial pustule (Xanthomonas campestris pv. glycines) resistance in elite North American soybean cultivars and to monitor introgression of soybean insect resistance in 15 resistant genotypes.

The initial applications of marker-assisted selection in soybean improvement were for traits conditioned by QTL with relatively large phenotypic effects and in which the positive alleles were rare in the ancestral germplasm (Orf et al., 2004). In these situations, breeders are usually aware of the QTL or genes conditioning the selected trait in their segregating populations and the parental source of the positive alleles at these QTL. In this study, we determine the frequency of elite Mi-resistant cultivars that inherited the major LG-O resistance QTL (Rmi1) and determine the ancestral source of the Mi-resistance allele at this QTL in elite U.S. soybean cultivars.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Forty-eight soybean genotypes, including ancestral, Mi-susceptible, and Mi-resistant genotypes, were chosen on the basis of their potentially informative pedigree and published resistance to Mi. Seed for these cultivars were obtained from the USDA Soybean Germplasm Collection maintained at the Univ. of Illinois (Urbana, IL). The 48 genotypes were evaluated for Mi gall formation in a greenhouse in a randomized complete block experimental design with six replications. Three seed of each genotype were planted into individual 20.6-cm Ray Leach Single Cell Cone-tainers (Stuewe & Sons, Inc., Corvallis, OR) containing approximately 15.6 cm2 of methyl-bromide-fumigated Pacolet sandy loam soil, a member of the fine, kaolinitic, thermic family of Typic Kanhapludults, amended with methyl-bromide-fumigated sand to a texture of 73% sand, 16% silt, and 11% clay (by weight). Cones were placed in every other row of a Ray Leach 98-cone tray. Resistant and susceptible checks were planted in each tray to develop a gall index. The checks included ‘Bryan’ (highly resistant), ‘Perrin’ (resistant), and ‘GaSoy 17’ (susceptible).

Approximately 7 to 10 d after planting, the plants were thinned to one plant per cone and then inoculated with 4000 Mi eggs. The Mi inoculum was propagated on ‘Marion’ tomato (Lycopersicon esculentum Mill.) and egg inoculum was collected according to the procedure described by Hussey and Barker (1973). The number of eggs per milliliter of inoculum was adjusted so that the desired inoculum density was applied in a volume of 3 to 5 mL. The inoculum was placed at a soil depth of 2 to 3 cm with an ARTEK Systems digital dispensing pump (Asteck Systems Corp., Farmingdale, NY). Each plant was fertilized weekly with 6 mg N, 3 mg P, and 5 mg K. Fifteen hours of supplemental light was provided by 400-W Multivapor metal halide lamps (Westinghouse Electric Corp., Lamp Division, Bloomfield, NJ) which were suspended 1.4 m above the greenhouse bench. The greenhouse was maintained at 28 ± 5°C. The plants were irrigated twice a day by a mechanical overhead irrigation system with water that was heated to 36 ± 3°C.

The experiment was terminated 30 d after inoculation when galls had developed on the susceptible checks. Following removal of plants from the cones, the roots were excised, washed free of soil, and evaluated for gall number. The number of galls on the resistant and susceptible standards was used to develop a gall index, where 1 ≤ 10 galls per plant, 2 = 11 to 20, 3 = 21 to 30, 4 = 31 to 40, and 5 > 40 galls. Data for gall number were analyzed by ANOVA with SAS (SAS Institute, 1992).

Soybean DNA was extracted from seeds of each genotype according to modified procedures of Kang et al. (1998), quantified by a FL600 Microplate Fluorescence Reader (Bio-Tek Instruments, Winooski, VT), and diluted to 20 ng µL–1 for the polymerase chain reaction (PCR). Seven seeds from each cultivar were ground with a coffee grinder (Braun KSM2, Boston, MA), and 0.1 g of each homogenate was transferred to a new 1.5-mL tube containing 500 µL of extraction buffer [200 mM Tris pH 8, 200 mM NaCl, 25 mM ethylenediamine tetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate], 10 µL Proteinase K (20 mg mL–1). The samples were incubated in a water bath at 50°C for 1 h and then 500 µL CTAB solution (2% cetyltrimethylammonium bromide, 100 mM Tris-HCl pH 8, 20 mM EDTA pH 8, 1.4 M NaCl) was added. After shaking, the samples were spun at 12000 rpm (Beckman Microfuge E, Beckman Instruments, Carlsbad, CA) for 10 min. The supernatant was transferred to a new 1.5-mL tube, and then one volume chloroform/isoamyl alcohol (24:1, v/v) was added. After shaking for 1 min at room temperature, the samples were spun at 12000 rpm for 10 min. The supernatant was transferred to a new 1.5-mL tube, and then 80% volume of isopropyl alcohol was added to precipitate DNA. The supernatant was decanted and the pellets were washed with 70% (v/v) ethanol. The DNA pellets were then dried and dissolved in 100 µL of tris-EDTA buffer.

On the basis of the integrated genetic linkage map of soybean (Cregan et al., 1999), six SSR markers (Satt358, Satt487, Satt500, Satt492, Satt445, and Sat_132) located near Mi-resistance QTL on LG-O were chosen (Li et al., 2001). These six markers span an approximately 15-cM region of LG-O. The primer sequences for each SSR were obtained from SoyBase, a USDA-sponsored genome database (http://129.186.26.94/ssr.html; verified 22 Jan. 2004). Fluorescent-labeled forward primers and nonlabeled reverse primers were obtained from PE-ABI (Foster City, CA). Polymerase chain reactions were prepared on the basis of the protocol of Diwan and Cregan (1997), with slight modifications. The 10-µL reaction mix contained 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 units of Taq DNA polymerase. The reactions were performed in a dual 384-well GeneAmp PCR System 9700 (Perkin Elmer, Norwalk, CT). The PCR amplicons were analyzed on an ABI-Prism 377 DNA sequencer (PE-ABI, Foster City, CA) with a 4.8% acrylamide to bisacrylamide (19:1) gel at 750 V for 2 h. Marker data were collected with DNA Sequencer Collection software v. 2.5. The marker fragments were analyzed with GeneScan software v. 3.0 and scored with Genotyper software v. 2.1 (PE-ABI, Foster City, CA).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The published pedigrees and Mi reactions of the 48 genotypes included in this study are shown in Table 1. Seed was not available for two parental breeding lines, R66-1517 and N70-2173, and Bossier was included in the experiment as the susceptible check. As reported by Hussey et al. (1991), a gall index of ≤1.5 indicated a high level of Mi resistance, an index of ≥1.6 to ≤2.5 was considered to indicate a moderate level of Mi resistance, and a gall index of ≥2.6 was considered susceptible. After screening with an inoculum density of 4000 Mi eggs per plant, 24 genotypes were identified resistant to Mi (Table 2). Also, Bragg, ‘Wright’, and ‘Cook’ had moderate resistance to Mi. However, ‘Sharkey’, Dyer, and FC 33243 produced a large number of galls, which is inconsistent with their previously reported Mi reactions (Hartwig and Epps, 1968; Caviness et al., 1975; Hartwig et al., 1988). The susceptible reaction of Sharkey is consistent with the result of Hussey et al. (1991). In addition, ‘Pharaoh’ was identified resistant to Mi in this study, but Schmidt et al. (1993) reported Pharaoh as moderately susceptible. We also screened unreported genotypes to Mi reaction. Palmetto was resistance to Mi, but Volstate, Tokyo, and PI 54610 were identified as susceptible to Mi. These results indicate that the source of the Mi resistance in Jackson which originated from the cross Volstate(2) x Palmetto was most likely Palmetto (Table 1).


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  		Table 1. Pedigree and published southern root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood] (Mi) reaction of 48 soybean genotypes.

 

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  		Table 2. Mean southern root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood] (Mi) gall index and band size at six simple sequence repeat (SSR) markers that flank the major Mi quantitative trait loci (QTL) on Linkage Group O (LG-O) for 48 soybean genotypes.

 
The ability to detect the major Mi-resistance QTL on LG-O through SSR marker analysis was tested by genotyping 48 soybean lines, including ancestral, Mi-susceptible, and Mi-resistant cultivars (Table 2). At Satt358, three alleles, 160, 192, and 200 bp, were detected across the 48 genotypes. The 200-bp band of Satt358 was present in all the Mi-resistant genotypes with the exception of ‘Lee 74’, which will be discussed in greater detail later is this section. All the Mi-susceptible genotypes possessed a 192-bp band at this marker except FC 33243. The cultivars Gregg and Maxcy were resistant to Mi, but were heterogeneous for the two bands at Satt358 (Table 2).

Six different allele sizes were found among the 48 genotypes at Sat_132 (Table 2). All of the Mi-resistant genotypes with the exception of Maxcy had a 238-bp band at Sat_132. The Mi-resistant Gregg was heterogeneous for the 238- and 252-bp bands. Maxcy possesses a 236-bp band at Sat_132, which was also present in the Mi-susceptible genotypes ‘Hampton’, ‘Coker 338’, ‘Johnston’, and ‘Coker 488’. This band was not present in any of the other genotypes in our study, and all the other Mi-susceptible genotypes possess a 246-, 248-, 250-, or 252-bp band at this marker. It is interesting that the Mi-resistant Maxcy and the Mi-susceptible Coker 338 and Coker 488 share Hampton as a common ancestor (Table 1). Maxcy appears to possess a crossover between Sat_132 and the Mi QTL. The alleles in the 48 genotypes at markers Satt487, Satt445, and Satt500 were not as strongly associated with Mi resistance as Satt358 or Sat_132 (Table 2). Satt492 had a same allele in all genotypes except Young and Gasoy 17.

In the cases where we found a Mi reaction that was different from that reported in the literature, the Satt358 marker data supported our classification. For example, Pharaoh, which was previously reported as moderately susceptible to Mi but was resistant in our study, had the 200-bp band at Satt358 (Tables 1 and 2). Sharkey and Dyer, which were previously reported as resistant to Mi but were found to be susceptible in our study, possess the 192-bp band at Satt358, which is present in all the other Mi-susceptible genotypes. When Pharaoh, Dyer, and Sharkey were evaluated for their Mi reaction in a common experiment with 45 other genotypes, their Mi reaction was predicted on the basis of the presence or absence of the 200-bp allele for Mi resistance at Satt358.

Lee 74 originated from the cross ‘Lee 68’ x R66-1517. The breeding line R66-1517 was selected from a backcross population of ‘Lee’ (5) x FC 33243. The donor parent of Lee 74's Mi resistance was reported to be FC 33243 (Caviness et al., 1975). The 160-bp band at Satt358 from FC 33243 was present in the Mi-resistant Lee 74. The problem with use of these data to delineate the association of Satt358 and the Mi QTL on LG-O is that FC 33243 was susceptible to Mi in our study (Table 2). Lee and its parents ‘S100’ and ‘CNS’ were also susceptible. During our Mi screening experiment, we noticed that FC 33243 possessed a greater amount of plant-to-plant variation than the other genotypes. To verify this observation, we determined the Mi reaction on an additional 40 individual FC 33243 plants. After counting the number of galls on these 40 plants, we transplanted seven plants with the fewest galls (9 to 19 galls) and the six plants with the most galls (73 to 90 galls). The progeny from these plants produced either a low or high number of galls similar to the gall number of their progenitor. It is assumed that one of the FC 33243 Mi-resistant plants was the source of the Mi resistance in Lee 74. Thus, FC 33243 possesses phenotypic heterogeneity for its Mi reaction and is homogenous for the 160-bp band at Satt358 and homogeneous for the other five SSR markers we evaluated in this region of LG-O. Since the FC 33243 resistant and susceptible selections possessed the 160-bp band at Satt358, it is possible that Lee 74 possesses a Mi-resistance gene other than Rmi1. Another explanation would be a mutation in the Rmi1 gene in the Mi-susceptible FC 33243 plants.

The results of the Mi screening and SSR marker genotyping for the Satt358 and Sat_132 markers were integrated in Fig. 1 . Genotypes in nonshaded boxes are Mi susceptible, genotypes in gray-shaded boxes are Mi resistant and inherited a 200-bp band at Satt358 and a 238-bp band at Sat_132, and genotypes in dark-shaded boxes are Mi resistant and inherited a 160-bp band at Satt358 and a 248-bp band at Sat_132. On the basis of these results, we can conclude that there are two ancestral sources of Mi resistance in elite southern U.S. soybean cultivars. One is Palmetto and the other is FC 33243. Palmetto is likely the source of the Mi-resistance gene (Rmi1) that was found in Forrest (Luzzi et al., 1994), and most elite Maturity Group V, VI, VII, and VIII cultivars with Mi resistance contain the Rmi1 gene.



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  		Fig. 1. A representation of the pedigree relationships and the simple sequence repeat (SSR) bands at two SSR markers near the major southern root-knot nematode [Meloidogyne incognita (Kofoid and White) Chitwood] (Mi) quantitative trait loci (QTL) on Linkage Group O (LG-O) among Mi-resistant and Mi-susceptible cultivars. Note: FC 33243 was found to possess both susceptible and resistant plants. Solid lines indicate a direct parent and progeny relationship and broken lines indicate an indirect parent and progeny relationship. No shading indicates high Mi galls; gray shading indicates low Mi galls, 200-bp band at Satt358, and 238-bp band at Sat_132; and dark shading indicates low Mi galls, 160-bp band at Satt358, and 248-bp band at Sat_132.

 
Our study provides strong evidence of the co-segregation of the 200-bp band at Satt358 and Mi resistance (Rmi1). This co-segregation has been maintained across approximately six cycles of breeding (crossing and inbreeding). The apparent tight linkage of Satt358 and Sat_132 and the major QTL for Mi resistance on LG-O, the major phenotypic effect of this QTL on Mi gall number, and the relatively rare occurrence of the resistance allele at this QTL among the ancestors of southern U.S. elite cultivars suggest that these markers could be effectively employed in breeding for Mi resistance.


   ACKNOWLEDGMENTS
 
This research was supported by funds allocated to the Georgia Agricultural Experiment Stations and by grants from the Georgia Agricultural Commodity Commission for Soybeans, and the Georgia Seed Development Commission.

Received for publication August 2, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 


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