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a Agronomy Dep., Purdue Univ., West Lafayette, IN 47907
b Entomology Dep., Purdue Univ., West Lafayette, IN 47907
c International Institute of Tropical Agriculture, Sabo Bakin Zuwo Road, PMB 3112, Kano, Nigeria
* Corresponding author (hohm{at}purdue.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: AFLP, amplified fragment length polymorphism • BSA, bulked segregant analysis • CTAB, hexadecyltrimethylammonium bromide • EDTA, ethylenediamine tetra-acetic acid • IITA, International Institute of Tropical Agriculture • MAS, marker-assisted selection • PCR, polymerase chain reaction • SCAR, sequence characterized amplified region • TBE, Tris-borate
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cowpea production is limited by several abiotic and biotic factors, including parasitic weeds, among which Alectra vogelii Benth. and S. gesnerioides are the most important. Alectra vogelii is restricted to Africa while S. gesnerioides is found in Africa, and in parts of Asia and the USA (Musselman et al., 1991; Parker and Riches, 1993). In West Africa, S. gesnerioides is an increasingly serious problem. When there is drought, its impact becomes even more significant (Obilana, 1987). Yield losses due to S. gesnerioides range from 15 to 100%. Aggarwal and Ouédraogo (1989) have recorded 30% yield losses while farmers in northern Nigeria experienced 100% losses because of Striga (Emechebe et al., 1991). Furthermore, its rapid spread to new regions constitutes a severe threat to cowpea production (Agbobli, 1991).
Control measures including cultural practices, chemical control, biological control, and host plant resistance have been reviewed (Dubé and Alain, 2001; Boukar, 2002). No single method seems to provide a complete control of this parasite, although host plant resistance appears to be the most effective and economical approach. Significant progress toward developing Striga-resistant lines has been reported in different breeding programs and by the International Institute for Tropical Agriculture (IITA), but important constraints linger.
To alleviate these constraints and for other reasons (e.g., speeding breeding efforts, possibility of identifying other strains of the parasites, reduction of environmental factors on the parasite's development), MAS has been proposed as an alternative solution for pyramiding resistance genes (Haley et al., 1994; Ouédraogo et al., 2001). Several molecular marker technologies have been exploited for MAS. Amplified fragment length polymorphism (Vos et al., 1995), combined with bulked segregant analysis (BSA) (Michelmore et al., 1991), has been used to discover markers closely associated with economically important traits in many crop species including cowpea. Recent studies conducted by Ouédraogo et al. (2001) using these techniques (AFLP and BSA) identified three markers tightly linked to the resistance gene Rsg2, effective against S. gesnerioides race 1 from Burkina Faso, and present in IT82D-849; and six AFLP markers associated with the resistance gene Rsg4, effective against S. gesnerioides race 3 from Nigeria, and present in Tvu 14676. Two of the markers, E-AAC/M-CAA300 and E-ACA/M-CAT150, were linked to both Rsg2 and Rsg4, respectively.
To improve the efficiency of molecular marker screening, AFLP markers and other molecular markers, such as randomly amplified polymorphic DNA (RAPD), can be converted into SCAR markers (Paran and Michelmore, 1993). In several species such as carrot (Daucus carota L.) (Bradeen and Simon, 1998), mustard [Brassica juncea (L.) Czern.] (Negi et al., 2000), soybean [Glycine max (L.) Merr.] (Meksem et al., 2001), apple (Malus domestica Borkh.) (Xu et al., 2001), barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) (Shan et al., 1999), and rice (Oryza sativa L.) (Jia et al., 2001), the amplified AFLP band was cloned and sequenced and then used to produce extended primers. The use of a pair of specific primers to amplify genomic DNA allows the development of SCAR markers. These markers are useful to plant breeding and genome analysis because of their simplicity, low cost, and efficiency. Depending on crop species, SCAR markers generally generate a single polymorphic band that is more reproducible across labs, is easier to score, and applicable to use with low-quality DNA obtained through rapid DNA extraction procedures than polymorphic bands generated by AFLP or RAPD markers (Kelly and Miklas, 1998). The codominant feature of certain SCARs has a direct application for MAS in plant breeding. As a contribution to the development and implementation of MAS approaches, the objectives of this study were to map Rsg1 and develop an AFLP-derived SCAR marker to facilitate MAS for this gene.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Population 2 was used to validate the linked molecular markers obtained from population 1. The F2 populations derived from both crosses were screened for resistance to the Nigerian strain of S. gesnerioides (prevailing in Nigeria). Thus, the populations were characterized for resistance conditioned by Rsg1 from parent line B301 and not for resistance from IT88D-867-11. Plants were grown with the pot-culture technique (Singh and Emechebe, 1990, Atokple et al., 1995). Two F2 seeds were planted in each plastic pot, 13-cm diam. and 13 cm deep containing about 1 L of unsterilized sieved sand and top soil (sandy loam) mixture (1:1 vol/vol) previously inoculated uniformly with about 800 S. gesnerioides seeds. About 10 wk after planting, the soil was washed off the plant roots after submerging each pot in a 20-L bucket of water for about 5 min. The roots of each plant were gently separated, carefully freed from any remaining soil and examined for Striga attachment. Plants allowing attachment, healthy development, and emergence of Striga were classified as susceptible. Those without any attachment and free of infection were categorized as resistant.
DNA Extraction
Five weeks after planting, one young fresh leaflet from each plant was collected in an Eppendorf tube and put on ice. The samples were taken to IITA headquarters, Ibadan, Nigeria, where they were lyophilized. Dry leaf samples were transferred to Purdue University, where they were stored at –80°C before their use.
Leaf tissues from individual plants were ground to a fine powder under liquid nitrogen before DNA isolation. Total genomic DNA was extracted by the CTAB (hexadecyltrimethylammonium bromide) method (Saghai-Maroof et al., 1984) following the procedure of Hoisington et al. (1994) with minor modifications. Two-percent CTAB extraction buffer [100 mM Tris-HCl buffer pH 8.0, 2% (w/v) CTAB, 100 mM Na2EDTA (ethylenediamine tetra-acetic acid), and 1.4 M NaCl] was used instead of the 1.67% CTAB outlined by Hoisington et al. (1994). The DNA concentration in each sample was measured with the Hoefer DyNA Quant 200 Fluorometer (Hoefer Pharmacia Biotech Inc., Buckinghamshire, UK). A total of 62 and 35 F2 individual plants from the crosses IT93K-693-2 x IAR1696 and IT93K-693-2 x Kamboinse local, respectively, were screened against S. gesnerioides. The first population was used to identify markers linked to the targeted striga resistance gene, and the second population was used to validate linkage between the striga resistance and the SCAR marker.
Bulked Segregant Analysis
For BSA (Michelmore et al., 1991), equivalent amounts of genomic DNA from 10 resistant F2 plants and 10 susceptible F2 plants from the population derived from the cross between IT93K-693-2 and IAR1696 were pooled to form resistant and susceptible bulks. Both bulks were used along with the parents to identify markers showing polymorphisms between the four samples. These polymorphic markers were further used to analyze individual F2 plants to determine linkages between AFLP markers and the S. gesnerioides resistance gene.
AFLP Analysis
Amplified fragment length polymorphism analysis was performed according to the procedure described by Vos et al. (1995), with a commercially available kit (AFLP® Analysis System I, Invitrogen, Life Technologies, Carlsbad, CA) and following the manufacturer's instructions with minor modifications. Approximately 500 ng DNA of each sample was digested with EcoRI/MseI restriction enzyme solution. After the ligation of the digested DNA, the reaction mixture was diluted five-fold rather than 10-fold with TE buffer containing 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA. The number of cycles was increased to 25 cycles for preamplification reaction and 30 cycles for the selective amplification rather than 20 and 23 cycles, respectively. Primer labeling was performed by phosphorylating the 5' end of EcoR I primers with [
–33P]ATP (adenosine 5'-triphosphate disodium salt) and T4 kinase in selective amplification. The preamplification product was also diluted 1:20 rather than 1:50. The amplification products were separated on 6% (w/v) polyacrylamide gels containing 29:1 acrylamide: Bis-acrylamide (Fisher Scientific, Chicago, IL), 7.5 M urea, and 1 x TBE (1,1,2,2-tetrabromoethane) buffer used with 0.4-mm spacers and a sharktooth comb. The gels were preelectrophoresed for about 20 min with 1 x TBE. Electrophoresis was performed at constant temperature and wattage (45–50°C, 100 W) for about 2.5 h. The gels were transferred onto 3-MW gel blot paper (Midwest Scientific, Valley Park, MO) and dried at 80°C for 2 h on a gel-drier (Bio-Rad, Hercules, CA). The dried gels were exposed to x-ray film (X-OMAT AR, Eastman KODAK Corp., Rochester, NY) for 2 to 3 d at –80°C without an intensifying screen. The bands were visualized with a transluminator (Fisher Scientific, Chicago, IL).
Marker Segregation and Linkage Analysis
Data were analyzed by the chi-square (
2) test to ascertain the goodness of fit between the expected ratio for a single dominant gene, and the segregation of the phenotypic data. Linkage analysis between the AFLP markers and the S. gesnerioides resistance loci was performed with the software package MAPMAKER/EXP version 3.0 (Lander et al., 1987). Map units were computed by applying the Kosambi function (Kosambi, 1944). The LOD score of 5 and the maximum distance of 25 cM were used in the determination of linkages.
Cloning and Sequencing of the Target AFLP Band
The gel slice containing the DNA fragment was excised from the dried AFLP polyacrylamide gel with a sharp-edged clean razor blade, and then eluted with 30 µL of 1 x TE (10 mM Tris-HCl, pH 8.0 and 1mM EDTA pH 8.0) for overnight at 4°C. From this, 1.0 µL of supernatant was used as template for polymerase chain reaction (PCR) amplification with the same primer combination and PCR conditions as that of the selective amplification with the only difference of not labeling the EcoRI primer (E-ACT).
The amplified products were electrophoresed at 70 V in a 1.2% low-melting-point agarose gel. The critical fragment was excised from the gel and purified with QIAquick gel-extraction Kit (QIAGEN Inc., Valencia, CA) following the manufacturer's instructions. An aliquot of 3.0 µL of the purified DNA was ligated into a pGEM-T easy vector (Promega, Madison, WI) according to the procedures described by the manufacturer with minor modifications. The host strain DH5
was used as competent cells for transformation. The recombinant plasmids were plated on selective Luria-Bertani media containing ampicillin {(2S,5R,6R)-6-[(R)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo(3.2.0)heptane-2-carboxylic acid} and X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; BioVectra, Charlottetown, PE, Canada).
QIAprep Miniprep Kit (Qiagen, Inc., Valencia, CA) was used for plasmid DNA extraction. To check the presence of the target insert, PCR amplification was performed with 25 µL total volume of 1 x PCR buffer, 1.5 mM MgCl2, 200 mM dNTP, 0.4 µM T7, 0.4 µM SP6, 0.2 µL of Taq, 10 ng of template DNA. The amplification profile consisted of one cycle at 94°C for 2 min, followed by 36 cycles of 45 s at 94°C, 1 min at 62°C and 1.5 min at 72°C, with a final extension of 7 min at 72°C.
All 10 samples showed a single amplification product in a 1.2% agarose gel immersed in 0.5 x TBE buffer (90 mM Tris-Borate, 1 mM EDTA, pH 8.0). The ten corresponding purified plasmid DNAs were sent to the DNA Sequencing Laboratory at the Genomic Center (Purdue University) for sequencing.
Designing SCAR Primers
The following oligonucleotide primers, designed on the basis of the identical sequence of plasmid DNA, led to polymorphisms between the parents and the bulks. Primer-reverse:
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Primer-forward:
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The 5' end of the reverse and forward primers contained two selective bases (AC and CT) of Mse1 and EcoR1 primers, respectively. These primers were synthesized by Integrated DNA Technologies (Coralville, IA) and were used to screen the parents, both resistant and susceptible bulks and individual F2 plants. The optimal PCR amplification was conducted with 25 µL of reactions containing 25 ng of template DNA; 10 mM Tris HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2; 200 µM of each dNTP; 0.3 µM of each primer; and 1 unit of Taq DNA polymerase. After an initial heat denaturation step at 94°C for 2 min, DNA fragment amplification was performed for 43 cycles, comprising 45 s at 94°C, 1 min at 62°C, and 1.5 min at 72°C. Final extension was for 7 min at 72°C. To separate the amplified products, 3.5% Metaphor agarose gels (BMA, Rockland, ME) stained with ethidium bromide were used and the products were visualized by illumination with ultraviolet light.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F2 populations 1 and 2 segregated 43 resistant:19 susceptible and 28 resistant:7 susceptible, respectively. These segregations fit a 3:1 ratio (2 = 1.05, P = 0.30 and 2 = 0.47, P = 0.49, respectively) indicating that resistance to the Nigerian strain of S. gesnerioides in IT93K-693-2 is monogenic and dominant. The gene providing resistance to the Nigerian strain of S. gesnerioides in these populations is Rsg1, which is from B301.
Marker Analysis
The AFLP analysis conducted on the population derived from the cross of IT93K-693-2 x IAR1696 showed that there were about 65-120 clearly readable bands for each EcoRI and MseI primer combination. From the 64 AFLP primer combinations used, 20 showed polymorphism between the parents and the bulks, and were used to screen individual F2 plants. Of these 20, four primer combinations produced bands that were linked to Rsg1. Primer combination EcoRI-ACT/MseI-CTC showed an approximately 115-bp amplification product present in the susceptible parent IAR1696 and the susceptible bulk but absent in the resistant parent and resistant bulk. This marker linked in repulsion phase to the resistance allele was designated E-ACT/M-CTC115 following AFLP marker nomenclature described by Ouédraogo et al. (2001). The three other primer combinations EcoRI-AAG/MseI-CTA, EcoRI-ACT/MseI-CAC (Fig. 1) , and EcoRI-ACA/MseI-CAG revealed polymorphic fragments of 190, 115, and 108 bp, respectively. These primer combinations generated polymorphisms that were linked in coupling phase to the resistance allele with the marker present only in the resistant phenotype. They were designated E-AAG/M-CTA190, E-ACT/M-CAC115, and E-ACA/M-CAG108, respectively.
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These primers were used to screen the parents, both resistant and susceptible bulks and individual F2 plants. An 85-bp DNA fragment was amplified from the susceptible parent, bulk and individual F2 plants while an 83-bp fragment was amplified from the resistant parent, bulk and some individual F2 plants. Other resistant F2 plants showed both bands, representing heterozygous plants (Fig. 3) . Thus, the AFLP marker was converted into a codominant SCAR marker designated as SEACTMCAC83/85, meaning SCAR marker of size 83 to 85 bp derived from AFLP primer combination E-ACT/M-CAC.
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The SCAR marker SEACTMCAC83/85 was also used to screen the F2 population of IT93K-693-2 x Kamboinse local. Similar band patterns to that of the first population were obtained. The genotypes of plants based on the SCAR marker were the same as observed for the original AFLP marker. All the susceptible plants showed the 85-bp amplification product. The resistant plants showed either the 83-bp band (homozygous) or both the 85 and 83 bp (heterozygous), except that two resistant plants, which were assumed to be recombinant plants, revealed only the 85-bp band.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Markers linked at a distance < 5 cM to the target gene, as those obtained in the present study, can be effectively used for indirect selection (Weber and Wrickle, 1994). The efficiency of MAS can be increased by employing markers flanking the gene of interest. This has been demonstrated for bacterial blight resistance genes in rice (Huang et al., 1997) and suggested, for example, for common bean (Kelly and Miklas, 1998) and wheat (Schachermayr et al., 1997).
In the present study, we were able to convert AFLP markers into easy, inexpensive, and reliable PCR-based markers like SCAR. Several studies in the literature revealed the inability to convert AFLP fragments with sizes < 200 bp (De Jong et al., 1997; Negi et al., 2000). According to Horn et al. (2003), the possibility of converting AFLP markers into sequence-specific markers is often restricted because of the very small size of the markers and the fact that most AFLP polymorphisms seem to originate in differences within the restriction sites. Bradeen and Simon (1998) pointed out that the AFLP fragment is too short for designing an appropriate PCR primer to amplify a polymorphic band, while Prins et al. (2001) reported that different AFLP fragments of the same size may migrate together in the gel, and a target polymorphic band may contain contaminating fragments from adjacent bands. Shan et al. (1999) found that the restoration of the original polymorphism remains difficult since the cloning procedure required for the AFLP conversion often contributes to the loss of the original polymorphism (Wei et al., 1999). Several procedures have been proposed to solve these problems. In the case of AFLP markers which are of 150 to 300 bp in size, Negi et al. (2000) pointed out that it is essential to isolate the flanking regions for the conversion to SCAR markers. These authors suggested the use of a PCR walking approach to isolate fragments adjacent to the AFLP markers. Other groups have reported the use of inverse-PCR to isolate the flanking regions for conversion to SCAR (Bradeen and Simon, 1998; De Jong et al., 1997). Recently, Brugmans et al. (2003) describe a general protocol for the conversion of AFLP markers into single-locus PCR assays and state that in principle, there is no minimal size of an AFLP marker as long as the internal sequence of the AFLP band is sufficiently long to allow the design of a highly specific PCR primer. The codominant nature of the SCAR obtained in the present study is an important factor for reliability in the linkage analysis. It has been reported that the use of dominant markers in linkage analysis with an F2 population can lead to errors, as the amount of information created by each data point is decreased in situations where heterozygous genotypes are found (Beaumont et al., 1996). The conversion of the AFLP marker into a SCAR will facilitate the transfer of the S. gesnerioides resistance gene to desirable cowpea lines via MAS. Selection in segregating populations with this SCAR marker will be more efficient and less expensive than with AFLP markers.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication September 25, 2003.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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