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a Dep. of Crop and Soil Science, Univ. of Georgia, Center for Applied Genetic Technologies, 111 Riverbend Rd., Athens, GA 30602
b Dep. of Plant Pathology, Univ. of Arkansas, Fayetteville, AR 72701
c Pioneer Hi-Bred International, Inc., Johnston, IA 50131
d Dep. of Plant Pathology, Univ. of Georgia, Athens, GA 30602
* Corresponding author (ha{at}uga.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: LG, linkage group • MAS, marker-assisted selection • NIL, near isogenic line • PCR, polymerase chain reaction • Pf, final population • Pi, initial inoculum level • QTL, quantitative trait loci • RFLP, restriction fragment length polymorphism • RI, reproductive index • RIL, recombinant inbred line • SCN, soybean cyst nematode • SSR, simple sequence repeat
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Since nematicides and/or rotation to a nonhost crop are usually not economical for soybean producers, genetic resistance is the most desirable control method (Harville et al., 1985). Rebois et al. (1968) found that the soybean cultivars Pickett and Dyer were resistant to the reniform nematode. Also Rebois et al. (1970) reported that resistance to reniform nematode could be expected in genotypes with resistance to the soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) due to the similarities in the feeding sites. However, Caviness and Riggs (1976) reported that ‘Mack’ was resistant to SCN and susceptible to reniform nematode. Recent studies have shown that SCN-resistant soybean cultivars that derive resistance from Peking (e.g., ‘Forrest’ and ‘Centennial’), PI 437654 (e.g., ‘Hartwig’), or PI 90763 (e.g., ‘Cordell’) are potentially resistant to R. reniformis, whereas those that derive SCN resistance from PI 88788 are not resistant to R. reniformis (Robbins et al., 1994a, 1994b; Davis et al., 1996; Robbins and Rakes, 1996). The phenotypic relationship suggests that common genes and/or linked genes are responsible for resistance to both SCN and reniform nematode in soybean.
The genetics of resistance to R. reniformis has been studied in soybean (Williams et al., 1981; Harville et al., 1985). Williams et al. (1981) reported that in a cross of Forrest (resistant) and ‘Ransom’ (susceptible), reniform nematode resistance was recessive and controlled by alleles at one locus. In other soybean populations, resistance in soybean was controlled by two loci with unequal effects (Harville et al., 1985). The proposed genotypes of the four parental cultivars were (i) susceptible soybean cultivar Davis Rn1 Rn1 Rn2 Rn2, (ii) moderately susceptible soybean cultivar Bragg Rn1 Rn1 rn2 rn2, (iii) moderately resistant soybean cultivar Dare rn1 rn1 Rn2 Rn2, and (iv) resistant soybean cultivar Pickett 71 rn1 rn1 rn2 rn2. In an unpublished study (2000), Pioneer Hi-Bred International, Inc. identified two quantitative trait loci (QTL) for reniform nematode resistance in a cross between ‘BSR101’ and PI 437654 with restriction fragment length polymorphism (RFLP) markers covering whole linkage groups. This study basically employed the same 298 recombinant inbred lines (RILs) and 355 RFLP markers reported by Webb et al. (1995). One QTL on LG-L explained 20% of the total variation in reniform reproductive index (RI), and another QTL on LG-B1 accounted for 11% of variation.
Many studies using DNA markers have concentrated on QTL that control parasitic nematodes in soybean. Southern root-knot nematode [M. incognita (Kofoid and White) Chitwood] (Tamulonis et al., 1997c; Li et al., 2001), peanut root-knot nematode [M. arenaria (Neal) Chitwood] (Tamulonis et al., 1997b), and javanese root-knot nematode [M. javanica (Treub) Chitwood] resistance QTL (Tamulonis et al., 1997a) have been identified. In addition, DNA markers have been used to dissect quantitatively inherited resistance to the SCN (Concibido et al., 1994, 2004; Webb et al., 1995; Cregan et al., 1999). Webb et al. (1995) mapped three major QTL to LG-A2, -G, and -M, respectively, that confer resistance to SCN race 3 in a cross between PI 437654, resistant to SCN and reniform nematode (Robbins and Rakes, 1996), and BSR101. Cregan et al. (1999) reported that marker-assisted selection (MAS) using Satt309, located 1 to 2 cM from rhg1 on LG-G, should be effective to select for SCN resistance at rhg1 derived from Peking, PI 90763, and PI 437654 in crosses with most SCN-susceptible soybean genotypes. In a direct comparison, genotypic selection with Satt309 was 99% accurate in predicting lines that were susceptible in subsequent greenhouse assays (Mudge et al., 1997; Cregan et al., 1999).
The objectives of this study were to use simple sequence repeat (SSR) markers to: (i) refine the location of existing, but unpublished QTL and identify additional QTL conferring resistance to reniform nematode in the population of BSR101 x PI 437654 previously used in reniform and SCN mapping study, (ii) determine the magnitude of resistance of each QTL, and (iii) evaluate the potential interaction of identified QTL.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Reniform Nematode Screening
Reniform nematode resistance screening was conducted in a greenhouse at the University of Arkansas. Seeds of each genotype were germinated in vermiculite and as the cotyledons where starting to open the seedlings were transplanted into 10-cm-diam. clay pots (one seedling per pot) containing 500 cm3 of pasteurized fine sandy loam soil (ca. 91% sand, 5% silt, 4% clay, <1% organic matter). One day after transplanting each pot was inoculated with 1700 vermiform reniform nematodes (i.e., initial population; Pi). The reniform population used was from a culture originating in Jefferson County, Arkansas, and maintained on ‘Braxton’ soybean. The experimental design was a randomized complete block with five replications. All pots were watered at least twice daily and other times, if needed, and fertilized each week with 12 mg N, 6 mg P, and 10 mg K.
Thirteen weeks after inoculation, the number of reniform nematode eggs and vermiform nematodes contained in egg masses on the roots and the number of vermiform nematodes in the soil of each pot were determined. The eggs and vermiform nematodes in the egg masses on roots were collected with 0.525% NaOCl (Hussey and Barker, 1973) and counted. To calculate the final reniform nematode soil population, a 100-cm3 aliquot of well-mixed soil from each pot was suspended in water and poured through nested 850- and 38-µm-pore sieves to remove plant debris and collect the nematodes. Nematodes caught on the 38-µm-pore sieve were separated from soil by sucrose centrifugal-flotation (Jenkins, 1964), counted, and multiplied by 5 to give the total soil number per pot. The total number of reniform nematode eggs and vermiform nematodes per pot was calculated by adding the number from the soil to the number from the roots. A reproductive index (RI), defined as the number of eggs + vermiform nematodes at test termination (Pf)/initial infestation level (Pi), was calculated for each pot. Because of an association between the mean and variance of a genotype, the RI of each pot was transformed to log ratio [log10 (RI + 1)] values for statistical analysis and presented as antilogs.
SSR Marker Data Collection
Soybean DNA was extracted from seeds of the 228 RILs according to modified procedures of Kang et al. (1998), quantified using a PerkinElmer Lambda 2 UV/VIS Spectrometer (PerkinElmer Life and Analytical Sciences, Shelton, CT), and diluted to 20 ng µL–1 for polymerase chain reaction (PCR). Ten seeds from each line were ground with 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 extraction buffer (200 mM Tris pH 8, 200 mM NaCl, 25 mM ethylenediamine tetraacetic acid [EDTA], 0.5% sodium dodecyl sulfate) and 10 µL proteinase K (20 mg m L–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 10000 g (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 10000 g 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 relationship with SCN and reniform nematode resistance, 96 SSR markers were chosen. These markers were near previously reported QTL for SCN and reniform resistance and were located on five linkage groups (A2, B1, G, L, and M) (Concibido et al., 2004). The primer sequences for each SSR were obtained from SoyBase, a USDA-sponsored genome database (http://soybase.org/). Fluorescent-labeled forward primers and nonlabeled reverse primers were obtained from PE-ABI (Foster City, CA). Polymerase chain reactions were prepared based on the protocol of Diwan and Cregan (1997) with slight modifications. The 10-µL reaction mix contained 2 µL of 40 ng template DNA, 1.0x 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. The reactions were performed in a MJ Research PTC-225 thermal cycler (MJ Research, Inc., Reno, NV). The PCR amplicons were analyzed on an ABI-Prism 377 DNA sequencer (PE-ABI) with a 4.8% acrylamide to bisacrylamide (19:1) gel at 750 V for 2 h. The marker fragments were analyzed with GeneScan software v.3.0 and scored with Genotyper software v.2.1 (PE-ABI).
Linkage Map and QTL Analysis
Initially, 134 randomly selected RILs were evaluated for the association of SSR marker alleles and reniform nematode RI. To further increase precision, 94 additional RILs were included. A broad sense heritability estimate based on variance component was calculated on entry-mean basis with the following formula: h2=
RILs2/[RILs2+(e2/r)] (Fehr, 1987), where RILs2 is the genotypic variance component for reniform RI per plant among RILs, e2 is the error variance, and r is the number of replications.
Single factor analysis of variance (SF-ANOVA) was used to determine the significance (P = 0.05) among SSR genotypic class means by use of General Linear Model (GLM) (SAS Institute, 2001). A multiple regression model with a FORWARD option was used for identifying the independent markers linked to the QTL within and among linkage groups at the 5% significance level.
Genetic linkage maps were constructed in MAP MANAGER QTX Version b16 with the Kosambi map function (Manly et al., 2001). Quantitative trait loci analyses of RI were conducted using interval mapping implemented in the program MAP MANAGER QTX (Manly et al., 2001). Genome-wide threshold values for declaring the presence of QTL were determined by 1000 permutations for reniform RI, as implemented by MAP MANAGER QTX. The additive effects (
) for individual QTL were estimated by MAP MANAGER QTX.
Multilocus QTL analyses were performed with three genetic marker loci in mixed linear models (Littel et al., 1996; Tang et al., 2006). Statistical analyses were performed by SAS PROC MIXED, where genotype (G) effects were fixed and RIL nested within genotype (RIL/G), and replications (R) were random. Variance components were estimated by the REML method. Type III sums of squares and F statistics were estimated for genotype effects, and least square means were estimated for genotypes. Additive (), additive x additive ( x ), and additive x additive x additive ( x x ) effects were estimated by ESTIMATE statements.
QTL Confirmation
To evaluate the effect of identified QTL in a different genetic background, F7–derived RILs from the cross of Prichard x Anand were evaluated for reniform RI. Anand originated from the cross ‘Holladay’ x Hartwig and was reported as resistant to reniform nematode (Anand et al., 2001; Robbins et al., 1999). Hartwig was developed from the cross of Forrest (3) x PI 437654 (Anand, 1992). Prichard originated from the cross of Co82–622 x ‘Howard’ and was susceptible to reniform nematode (Boerma et al., 2001). Both Howard and Co82–622 include Forrest in their pedigree. A total of 353 RILs were genotyped using the SSR markers Satt513 on LG-L, Satt359 on LG-B1, and Sat_168 on LG-G. Our goal was to identify eight RILs for each of the eight possible homozygous marker classes. However, one genotypic class contained only two RILs and another class had only one RIL out of 353 RILs. Therefore, a total of 51 RILs were evaluated for reniform nematode resistance in a greenhouse as described earlier. The screening was conducted in a randomized complete block experimental design with five replications. Each plant was inoculated with 1644 vermiform reniform nematode (Pi). Final nematode population (Pf) for each pot was determined only from the soil.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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To further increase the precision for QTL mapping, 94 additional RILs were genotyped with 28 SSR markers on LG-B1, -L, and -G. Data from these 94 RILs were combined with the 134 original RILs. When the marker and phenotypic data from the 228 RILs were analyzed by interval mapping with Map Manager QTX, a QTL mapped to a 25.3-cM interval between Sat_184 and Satt513 on LG-L and explained 21% of the variation in reniform RI (Fig. 2 ). The QTL peak was 8 cM from Satt513 (LOD = 11.8). Another QTL mapped to a 14.5-cM interval between Satt359 and Satt484 on LG-B1 and explained 16% of the phenotypic variation (LOD = 8.8). The QTL peak was 5 cM from Satt359. A third QTL, accounting for 8% of the phenotypic variation in reniform RI (LOD = 4.1), was linked to marker Sat_168 on LG-G (Fig. 2).
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QTL Effects on Reniform Nematode Resistance
Three QTL conferring reniform RI showed a significant (P < 0.05) additive main effect (Table 1). Also, significant (P < 0.01) additive x additive epistasis was detected between Satt359 and Sat_168 (Table 2). The LG-B1 QTL effect was greater than the LG-G QTL effect (Table 1 and 2). However, RILs homozygous for PI 437654 alleles at Satt359 and Sat_168 had a reniform RI of 0.79 which was significantly lower than the mean RI (RI = 4.5) for the other three genotypes (Table 2).
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Of the 228 RILs in this population, 206 RILs were homozygous and without missing data for the SSR markers Satt513 on LG-L, Satt359 on LG-B1, and Sat_168 on LG-G. For the eight possible homozygous classes of the three markers, we expected an equal distribution of lines in each class (approx. 26 lines per class). The actual number of lines in these marker classes was significantly different (
2 = 28.25, P < 0.01) from expectations (Table 3). One genotypic class that had the BSR101 (B) allele at Satt513 and PI 437654 (P) alleles at the Satt359 and Sat_168 included only seven lines. Also, the B/B/P and B/P/B classes for Satt513, Satt359, and Sat_168, respectively, contained fewer than the 26 expected lines. Apparently, the combination of alleles from BSR101 in the region of Satt513 on LG-L with alleles from PI 437654 in the region of Satt359 on LG-B1 and the combination of alleles from BSR101 in the region of Satt513 on LG-L with alleles from PI 437654 in the region of Sat_168 on LG-G were deleterious to survival or were unintentionally selected against during population development.
A distortion in marker classes also had been reported in another study that mapped SCN resistance using the same BSR101 x PI 437654 population that was also maintained and increased in Iowa by Pioneer Hi-Bred International, Inc. (Webb et al., 1995). A recent study with three near isogenic line (NIL) populations segregating at the SCN resistance locus rhg1 on LG-G reported that SCN-resistant NILs had significantly less field emergence than susceptible NILs (Kopisch-Obuch and Diers, 2006). Results of that study suggested that the segregation distortion was caused by pleiotropic effects of rhg1 or genetic linkage with unfavorable genes causing reduced field emergence.
Confirmation of the QTL in a Different Genetic Background
Before the application of MAS it is desirable to confirm QTL in the same population with a different sample of lines or in a different genetic background (Fasoula et al., 2004). A population of 353 RILs derived from the cross of Prichard x Anand was evaluated to confirm the previously described QTL for reniform RI.
Anand and its ancestors (Holladay, Hartwig, Forrest, and PI 437654) were screened with SSR markers flanking QTL on LG-L, -B1, and -G (Table 4). Anand inherited the allele at the reniform QTL on LG-B1 from Hartwig (via Forrest), the allele at the reniform QTL on LG-G from Hartwig (via PI 437654 or Forrest), and the allele at the QTL on LG-L from Holladay. Putatively, Anand appeared to possess resistance alleles at the LG-B1 and LG-G QTL, but not at the LG-L QTL.
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This study was initiated based on the observation that there was a genetic linkage between SCN and reniform nematode resistance. Many studies reported that SCN-resistant soybean cultivars that derive resistance from Peking, PI 437654, or PI 90763 are also resistant to reniform nematode (Robbins et al., 1994a, 1994b; Davis et al., 1996; Robbins and Rakes, 1996). The SCN-resistant genotype PI 437654 has major SCN QTL on LG-G (rhg1) and LG-A2 (Rhg4). The SSR marker Satt309, located 1 to 2cM from rhg1 has been shown to be useful for MAS for SCN resistance (Cregan et al., 1999). Concibido et al. (2004) reported that QTL on LG-A1, -B1, -C1, -J, and -M conditioned SCN resistance in PI 437654. Interestingly, the QTL for SCN resistance linked to RFLP marker A006 on LG-B1 was reported by Vierling et al. (1996) to explain more than 90% of the phenotypic variation in a cross between ‘Williams 82’ and Hartwig. Hartwig was derived from backcrossing SCN resistance from PI 437654 into Forrest (Anand, 1992). Also, a SCN-resistant QTL linked to an RFLP marker (A567a) on LG-B1 was found in a cross from BSR101 x PI 437654, according to a patent claim by Webb (2003).
Harville et al. (1985) suggested that reniform nematode resistance was quantitatively controlled by two genes with unequal effects. By contrast, we found a QTL on LG-L and two epistatically interacting QTL on LG-B1 and LG-G. The reniform resistance QTL on LG-G with its resistance allele derived from PI 437654 was located near SSR marker Sat_168 which is closely linked to SCN resistance gene rhg1. Also, the reniform QTL on LG-B1 located between Satt359 and Satt484 is positioned between RFLP markers A006 and A567a, which were reported to be linked to SCN QTL (Vierling et al., 1996; Webb, 2003). Therefore, our results support linkage between certain SCN and reniform resistance QTL, provide confirmation of the QTL on LG-G, and evidence of an interaction of QTL for reniform RI on LG-G and LG-B1.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication October 9, 2006.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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