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Centro Internacional de Agricultura Tropical, A. A. 6713, Cali, Colombia
* Corresponding author (g.mahuku{at}cgiar.org).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development and use of resistance cultivars is the most effective, economical, and environmentally sound strategy for disease control. However, the pathogenic variability of P. griseola (Busogoro et al., 1999a; Mahuku et al., 2002; Pastor-Corrales et al., 1998) and the occurrence of newly evolving virulent pathotypes (Mahuku et al., 2002) complicate the development and limits the lifetime of resistant cultivars. New sources of resistance must always be sought, and the usefulness of the identified gene(s) validated before they are deployed. This can be done by exposing the sources of resistance to existing pathogenic variation over different production areas (Milgroom and Fry, 1997) or by sampling the diversity that exists in the pathogen and using representative races to screen sources of resistance (Milgroom and Fry, 1997). The use of a wide diversity of host resistance genes and knowledge of the pathogen's genetic diversity for virulence and other markers are useful tools in developing and deploying bean varieties with durable ALS resistance.
Several potential sources of ALS resistance have been identified among the primary and secondary P. vulgaris gene pools (Busogoro et al., 1999b; Mahuku et al., 2003a; Pastor-Corrales et al., 1998), but the nature and inheritance of resistance in most of these potential sources have not been elucidated, although this information is crucial to breeding programs. Previous inheritance studies have revealed that one, two, or three dominant or recessive genes condition resistance to P. griseola in the primary and secondary P. vulgaris gene pools (Busogoro et al., 1999b; Carvalho et al., 1998; Ferreira et al., 2000; Nietsche et al., 2000; Sartorato et al., 1999). QTL analysis of recombinant inbred lines (RIL) derived from crossing DOR 364 (Mesoamerican) x G 19833 (Andean) revealed that both minor and major genes condition ALS resistance to different P. griseola pathotypes (Jara 2002).
In a pathogen that is highly variable like P. griseola (Busogoro et al., 1999a; Mahuku et al., 2002), pyramiding several resistance genes into the same background is the most effective breeding strategy. However, this requires or is facilitated by (i) a clear understanding of the inheritance of resistance, (ii) knowledge of the effectiveness of the resistance gene(s) in managing P. griseola, and (iii) markers that are tightly linked to each gene (either physical or specific pathotypes with defined avirulence genes). Such markers will allow indirect selection of resistance genes, overcoming problems of epistatic interactions that can mask one or another resistance gene.
Molecular markers linked to some angular leaf spot resistance genes have been identified in P. vulgaris (Carvalho et al., 1998; Ferreira et al., 2000; Nietsche et al., 2000; Sartorato et al., 1999). However, the molecular markers identified to date are insufficient to monitor all the different ALS resistance genes that have been described. Furthermore, the use of molecular markers in a breeding program requires validation of the markers outside the mapping population. Many identified markers have proved to be monomorphic in relation to other parental materials and therefore, of limited use in MAS (Miklas, 2002).
This study was initiated (i) to validate the usefulness of the ALS resistance gene(s) in the germplasm accession ‘G 10474’, (ii) to elucidate the genetics of ALS resistance in G 10474, (iii) to identify and develop markers that are closely linked to the resistance gene(s), (iv) to validate the usefulness of the identified marker(s) for marker assisted selection breeding outside the mapping population and in different common bean backgrounds, and (v) develop a protocol for use of the identified marker in common bean improvement programs involving G 10474 as source of ALS resistance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Validation of Usefulness of ALS Resistance Gene in G 10474
A total of 43 pathotypes of P. griseola, representing 73 isolates of a diverse origin were used to evaluate the germplasm accession G 10474 under green house conditions (Table 1). Under these conditions, G 10474 had an immune reaction to pathotype 63-63, a highly virulent race that overcomes the resistance in all the ALS differential varieties (Mahuku et al., 2003a).
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DNA Extraction
DNA was extracted from leaves by the procedure of Möller et al., (1992) as modified by Mahuku et al. (2002). DNA quality was determined by electrophoresis through 0.7% (w/v) agarose gels and the concentration was measured with a fluorometer (Hoefer DyNA Quant 2000, Pharmacia Biotech, USA).
Bulked Segregant Analysis (BSA)
Screening of the AFLP markers closely linked to the ALS resistance gene(s) in G 10474 was based on the BSA method developed by Michelmore et al. (1991). Two DNA pools of 10 ALS-resistant (rating 1) and 10 ALS-susceptible individuals (rating 
8) of the G 10474 x Sprite F2 population were used to detect AFLPs linked to the resistance gene. Two leaf disks (2 mm) were excised from each resistant (resistant bulk) or susceptible (susceptible bulk) plant that constituted the bulk, combined, and used to extract DNA by the extraction procedure previously described (Mahuku et al., 2002), except that a single chloroform extraction was included after the RNase A digestion step. DNA from individual plants of each bulk was used to confirm the polymorphism of potential markers. Another 20 individuals representing a full range of the greenhouse scores, were selected to check the cosegregation with resistance of promising AFLP markers identified by bulked segregant analysis.
AFLP Analysis
AFLP fingerprints were generated on the basis of the method of Voss et al. (1995) with the AFLP Analysis System I (Life Technologies, Rockville, MD, USA), following the manufacturer's instructions. The reaction products were resolved on 5% (w/v) polyacrylamide gels, and bands were detected by silver nitrate staining.
Cloning the Selected Marker
AFLP bands linked to resistance gene in G 10474 were excised from the dried polyacrylamide gel with a sterile scalpel and incubated in 50 µL TE (10 mM Tris, 1 mM EDTA pH 8.0) overnight at 4°C. The fragments were reamplified in 12.5-µL reaction volumes containing 1.0 µL of template DNA under the conditions described for selective AFLP amplification. The amplified products were separated on a 1.2% (w/v) low-melting point agarose in 1x TAE (Tris-acetate EDTA) buffer. Following staining in ethidium bromide and visualization under UV light, the bands were excised from the gel and the PCR products isolated with the Qiaex II gel-extraction kit (Qiagen Inc., Alameda, CA, USA) following the manufacturer's instructions. The isolated fragments were A-tailed and cloned into the bacterial plasmid pGEM-T Easy Vector (Promega, Madison, WI, USA) following the manufacturer's instructions. The plasmids were then transformed into the bacterial strain Escherichia coli DH5
. To ensure the correct bands had been cloned, the plasmid DNA was amplified with the appropriate AFLP primers and run adjacent to the original AFLP reactions on a polyacrylamide gel. Five white colonies from each transformation event were selected and the respective inserts were sequenced with the ABI PRISM 377 DNA automated sequencer (Perkin Elmer, Applied Biosystems). Sequencing reaction conditions were chosen following the manufacturers recommendations, and the DNA sequences were analyzed with Seqman within the DNAStar program (DNAStar, Madison, WI, USA). If the sequences of the five clones were identical, new primers internal to the AFLP selective primers were designed.
Conversion of AFLP Marker to a SCAR and Screening of F2 Population
Primers were designed using the software Primer 3 (Center for Genome Research, Whitehead Institute, MA, USA, http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi; verified 19 May 2004), and DNAStar Primer Select (DNAStar, Madison, WI, USA), and commercially synthesized (Integrated DNA Technologies, Inc., Coralville, IA, USA). The primers were tested in resistant and susceptible parents and respective bulks. If polymorphism was maintained, the designed primers were tested in 10 resistant and 10 susceptible individuals. Primers that distinguished resistant from susceptible individuals were then tested in the remaining individuals of the F2 population. PCR amplifications were performed in 12.5-µL reaction volumes containing 20 ng of genomic DNA, 10 mM of Tris-HCl pH 8.0, 1.5 mM of MgCl2, 50 mM of KCl, 0.25 mM of each dNTP, 0.5 µM of forward primer, 0.5 µM of reverse primer, and 1 unit of Taq polymerase (Promega, Madison, WI, USA). The following conditions were used: an initial denaturation step at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60°C for 45 s, 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR products were electrophoresed through a 1.5% (w/v) agarose gel in 1x TBE buffer and photographed under UV light following staining with ethidium bromide. SCAR markers that differed in length after amplification were used as indel (insertion–deletion) markers. In the case of an identical sequence length, the fragment from the susceptible individuals was cloned and sequenced according to the method described above. The sequences derived from resistant and susceptible individuals were then aligned by the program Megalign within DNAStar, and where possible the primer pairs were redesigned to exploit differences between the resistant and susceptible sequences. These primers were tested in susceptible and resistant parents and respective bulks.
Validating the Usefulness of SCAR
The SCAR primer pair was used to amplify DNA obtained from 12 common bean differential varieties that include six Andean genotypes and six Mesoamerican genotypes. All common bean differentials are susceptible to P. griseola pathotype 63-63. An additional 20 common bean cultivars (Andean and Mesoamerican) that were used in crosses with G 10474 were also screened using the polymorphic primers described here. In addition, the specificity of the marker was tested with DNA obtained from alkaline DNA extraction method (Klimyuk et al., 1993) and ammonium acetate method (Mahuku et al., 2002). These methods do not involve the use of organic solvents, and therefore yields DNA that is relatively impure, compared with DNA extracted by the phenol–chloroform methods. However, these methods are simple and designed to handle large sample sizes, and therefore, are suitable for marker-assisted selection.
Data Analysis
Individual plants were scored as resistant (rating of 
3) or susceptible (rating score > 3). The area under disease progress curve (AUDPC) was calculated for each inoculated plant from the disease reaction scores at 10, 12, 14, 17, and 21 d after inoculation as:
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2) test, according to the Mendelian segregation hypothesis of 3:1 (resistant to susceptible). Similarly, the BCF1 to Sprite were tested for goodness of fit to a 1:1 (resistant to susceptible) ratio. The estimation of recombination frequencies and of genetic distances between SCAR and resistance gene was detected by the MAPMAKER/EXP (Lander et al., 1987) program, with a minimum LOD score of 3.0. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Nature and Inheritance of ALS Resistance in G 10474
All F1 plants from G 10474 x Sprite cross were resistant to pathotype 63-63, suggesting that a dominant gene conditioned ALS resistance (Table 2). Segregation for resistance in the F2 population was consistent with a 3 resistant:1 susceptible ratio (2 = 0.003), while that for the backcross to susceptible parent (Sprite) fit a 1:1 ratio (2 = 0.059) (Table 2). These results support the hypothesis that a single dominant gene in G 10474 controls resistance to P. griseola pathotype 63-63. Similarly, the response of 106 F2 plants to inoculation with race 7-35 revealed a segregation ratio of 3:1 (2 = 0.109) that was consistent with the hypothesis of a single dominant gene conditioning ALS resistance in G 10474 (Table 2). Therefore, the resistance of G 10474 to P. griseola pathotypes 63-63 and 7-35 is monogenic and dominant.
Identification of a DNA Marker Linked to Resistance Gene
Among the 55 primer pair combinations screened, three revealed polymorphism between the parents and two bulks of DNA extracted from contrasting F2 plants. Three AFLP fragments E-ACA/M-CTT330, E-AAC/M-CAG310, and E-AAC/M-CAT285 segregated in coupling phase with the resistance gene in G 10474 (Fig. 1)
. All 10 resistant individuals tested contained these three fragments, which were absent in the susceptible bulk, susceptible parent (Sprite) and in all 10 individual susceptible plants tested. In addition, the 20 individual plants representing the full range of the greenhouse rating scores showed results that were consistent with the phenotypic evaluations. The fragments were cloned, sequenced, and primers internal to the EcoRI and MseI restriction sites were designed. Only the marker derived from the E-ACA/M-CTT330 primer pair was polymorphic in the resistant and susceptible parents and the respective bulks. When tested on 20 individuals representing the full range of greenhouse scores, the SCAR marker was codominant, amplifying a 305-bp fragment in the resistant parent, bulk and individual plants and a 280 bp in the susceptible parent, bulk and individual plants (Fig. 2)
. This SCAR marker could distinguish between homozygous resistant, heterozygous resistant and homozygous susceptible plants in the F2 population (Fig. 2). Linkage analysis after testing the marker on 241 F2 individual plants showed that the marker was located 5.0 cM from the resistance gene. Alignment of sequences derived from resistant and susceptible plants revealed that 25 bp were missing from the fragment associated with susceptibility, which could be a result of a deletion of this fragment from a resistant parent. To identify any likely candidates for the resistance gene, a BLAST (Altschul et al., 1990) search in the GenBank database, using the sequence of the AFLP marker as queries was conducted. None of the AFLP sequences showed similarity to known gene sequences.
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Validation of the Utility of the Marker
To verify the utility of the SCAR marker for marker assisted selection (MAS), the SCAR primer-pair (PF5330–L2, 5'-CTTGTTCTGAGTCATTTACCTTGC-3'; and PF5330–R1, 5'-GAATTCACAGTCCAAACTACTCTAATC-3') was used to amplify DNA obtained from Andean and Mesoamerican genotypes that are susceptible to P. griseola pathotype 63-63. A susceptible fragment (280 bp) was associated with Andean genotypes and Mesoamerican genotypes had the resistant fragment (305 bp) (Fig. 3)
. Mexico 54 was the only Mesoamerican genotype that had the 280-bp fragment. The same result was obtained when an additional 16 Mesoamerican genotypes that are susceptible to P. griseola were tested with the SCAR marker. To test for the suitability of the SCAR marker for MAS, we used the marker in a MAS workshop held in Uganda in February 2003, using different DNA extraction methods. The fragment associated with resistance (305 bp) was observed in G 10474 and Mesoamerican genotypes, whereas the fragment associated with susceptibility (280 bp) was detected in Andean genotypes and Mexico 54 (Fig. 4)
, irrespective of the DNA extraction method.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several studies have demonstrated that both major and minor genes condition resistance of common bean to P. griseola (Carvalho et al., 1998; Jara 2002; Mahuku et al., 2003b; Singh and Saini, 1980). It has been shown that resistance of cultivars AND 277, MAR 2, Cornell 49-242, (Phg-3), Mexico 54, and BAT 332 (Phg-62) to ALS is due to one dominant gene (Carvalho et al., 1998; Nietsche et al., 2000; Sartorato et al., 1999). Allelism tests done by Caixeta et al. (2002) revealed that the cultivar AND 227 has four angular leaf spot resistance genes designated Phg-1a, Phg-22, Phg-32, and Phg-42, Mexico 54 has three (Phg-2, Phg-5 and Phg-6) resistance genes, and MAR 2 has two genes (Phg-4 and Phg-5). QTL analysis of the population derived from DOR 364 (Mesoamerican) x G 19833 (Andean) revealed that both minor and major genes, condition ALS resistance to different P. griseola pathotypes, and that resistance to Andean races might be located on different linkage groups than genes conditioning resistance to Mesoamerican races (Jara, 2002). Our study provides further insights into the genetics of inheritance of angular leaf spot resistance in common bean. The identification and characterization of the resistance gene in G 10474 is extremely important to bean breeding programs, given the usefulness of this gene against many P. griseola pathotypes of a diverse origin.
It is not known whether the ALS resistance gene in G 10474 occupies a different locus from those that have been described (Caixeta et al., 2002; Carvalho et al., 1998; Nietsche et al., 2000; Sartorato et al., 1999). Allelism tests with genotypes where ALS resistance genes have been well characterized are needed to confirm if, the ALS resistance gene in G 10474 is new and unique. However, judging from the fact that most races that have a compatible interaction with Mexico 54, MAR 1, MAR 2, AND 277, BAT 332, and Cornell 49-242 do not infect G 10474 (CIAT, 2002), it is probable that G 10474 is carrying a different gene or allele from those conditioning resistance in these cultivars.
We identified three AFLP markers linked in coupling phase to the resistance locus in G 10474. Identification of molecular markers closely linked to the ALS resistance genes is an essential step toward both MAS and map-based cloning of these genes. While the AFLP is powerful in identifying markers closely linked to genes of interest, it is poorly adapted to large-scale, locus-specific uses such as MAS (Shan et al., 1999). It is therefore, necessary to convert AFLP markers to PCR-based markers to expand their utility for MAS. Although AFLP markers have successfully been converted into PCR-based markers for several species (Bradeen and Simon, 1998; Negi et al., 2000; Shan et al., 1999), generally, their conversion is more difficult because of the loss of polymorphisms related to EcoRI or MseI restriction site differences during generation of primers from an internal sequence (Shan et al., 1999). Of the three AFLP markers we identified, only one was successfully converted to a codominant SCAR marker. Attempts to convert the other two AFLP markers were not successful, as polymorphism was lost when primers internal to the EcoRI or MseI restriction site were designed. In addition, no significant polymorphisms were observed between sequences derived from resistant and susceptible plants, and therefore, no primers could be designed. We are in the process of PCR walking into regions 5' of the sequenced fragments, in the hopes of designing new primers that capture the EcoRI or MseI restriction site differences to permit us to develop useful primers.
Before a marker can find wide application, it is important to validate its usefulness for MAS breeding outside the mapping population. Several markers identified for different common bean diseases have been of limited use because (i) the linkage is not tight enough, and as a result, the linkage intensity may vary widely across different genetic backgrounds due to recombination suppression, (ii) the gene is not expressed in certain genetic backgrounds, or (iii) the markers are present in both susceptible and resistant lines outside the mapping population (Miklas, 2002). This therefore implies that markers should be tested for their utility in other cultivars and populations outside the mapping population, to validate their usefulness for MAS. Results from validation of the AFLP derived SCAR marker for G 10474 on common parental genotypes revealed that this SCAR marker is gene pool specific, and therefore, is only useful for introgressing the G 10474 derived ALS resistance gene into Andean backgrounds. Since the Andean gene pool is genetically narrower than the Mesoamerican gene pool (Beebe et al., 2001), the marker described here should find wide application in broadening the ALS resistance in Andean beans.
The utility of a marker for MAS depends on how easy it is to assay. The marker should be relatively stable, not sensitive to crude methods of DNA extraction, and expressed under different conditions. To test for all these traits, we used the marker in a MAS workshop held in Uganda in February 2003, using different DNA extraction methods. It was established that the SCAR marker described here could be used with DNA of varying purity and is suitable for large scale assaying. A protocol for using the codominant marker linked to the resistance locus in G 10474 was developed and the marker is ready for use by our partners.
The identification and characterization of the G 10474 resistance gene is extremely important to bean breeding programs aiming to develop cultivars resistant to this pathogen. Given the fact that several different genes encode resistance to P. griseola, the tagging of a major resistance gene in G 10474 is important for gene pyramiding to reduce the probability of resistance breakdown. An AFLP-derived, PCR-based codominant SCAR marker for the P. griseola ALS resistant locus in G 10474 was developed. The suitability of this marker for MAS was established during a workshop held in Africa and a protocol for assaying this marker under different laboratory conditions was developed. The marker will greatly increase the speed and efficiency of introgressing the G 10474 derived resistance gene into commercial Andean bean types.
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ACKNOWLEDGMENTS |
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Received for publication September 2, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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