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Angular leaf spot (ALS), caused by the fungus Phaeoisariopsis griseola (Sacc.) Ferr., is one of the most important diseases of common bean (Phaseolus vulgaris L.) in tropical and subtropical bean producing areas. The common bean accession G 10474 has been widely resistant to P. griseola, including pathotype 63-63, one of the most virulent P. griseola pathotypes characterized. The objectives of this study were to determine the inheritance of angular leaf spot resistance and to identify molecular markers linked to the resistance gene in G 10474. Resistance to P. griseola pathotypes 63-63 in G 10474 was investigated in F^sub 2^ populations derived from a cross between G 10474 and Sprite (universally susceptible). Inoculation of parents, F^sub 1^, F^sub 2^, and backcross-derived plants with race 63-63 revealed that a single dominant gene conditioned ALS resistance in G 10474. A combination of the amplified fragment length polymorphism (AFLP) technique and bulked segregant analysis (BSA) was applied to the F^sub 2^ population to identify molecular markers linked to the ALS resistance gene in G 10474. Three AFLP markers (E-ACA/M-CTT^sub 330^, E-AAC/M-CAG^sub 310^, and E-AAC/M-CAT^sub 285^) segregated in coupling phase with the resistance gene in G 10474. The E-ACA/M-CTT^sub 330^ marker was successfully converted to a codominant sequence characterized amplified region (SCAR) marker at 5 cM from the resistance gene. Validation of the SCAR marker outside the mapping population showed that the utility of this marker for marker-assisted selection (MAS) was limited to the Andean gene pool of P. vulgaris. Therefore, the SCAR marker we report can only be used to introgress the ALS resistance of G 10474 into Andean backgrounds. [PUBLICATION ABSTRACT]
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ABSTRACT
Angular leaf spot (ALS), caused by the fungus Phaeoisariopsis griseola (Sacc.) Ferr., is one of the most important diseases of common bean (Phaseolus vulgaris L.) in tropical and subtropical bean producing areas. The common bean accession G 10474 has been widely resistant to P. griseola, including pathotype 63-63, one of the most virulent P. griseola pathotypes characterized. The objectives of this study were to determine the inheritance of angular leaf spot resistance and to identify molecular markers linked to the resistance gene in G 10474. Resistance to P. griseola pathotypes 63-63 in G 10474 was investigated in F^sub 2^ populations derived from a cross between G 10474 and Sprite (universally susceptible). Inoculation of parents, F^sub 1^, F^sub 2^, and backcross-derived plants with race 63-63 revealed that a single dominant gene conditioned ALS resistance in G 10474. A combination of the amplified fragment length polymorphism (AFLP) technique and bulked segregant analysis (BSA) was applied to the F^sub 2^ population to identify molecular markers linked to the ALS resistance gene in G 10474. Three AFLP markers (E-ACA/M-CTT^sub 330^, E-AAC/M-CAG^sub 310^, and E-AAC/M-CAT^sub 285^) segregated in coupling phase with the resistance gene in G 10474. The E-ACA/M-CTT^sub 330^ marker was successfully converted to a codominant sequence characterized amplified region (SCAR) marker at 5 cM from the resistance gene. Validation of the SCAR marker outside the mapping population showed that the utility of this marker for marker-assisted selection (MAS) was limited to the Andean gene pool of P. vulgaris. Therefore, the SCAR marker we report can only be used to introgress the ALS resistance of G 10474 into Andean backgrounds.
COMMON BEAN is one of the most important food legumes in the world because of its commercial value, extensive production, consumer use, and nutrient value (being a source of carbohydrate, protein, minerals, and vitamins) (Liebenberg and Pretorius, 1997; Pastor-Corrales et al., 1998; Singh, 1999). Pests and disease outbreaks are among the main problems reducing bean yields (Liebenberg and Pretorius, 1997; Wortmann et al., 1998). In Africa, pest and disease problems are the second biggest constraint to bean productivity, with an estimated annual yield loss of 2 288 000 Mg, of which 348 000 Mg (17%) is due to the angular leaf spot disease (Wortmann et al., 1998). Angular leaf spot is currently the most economically important disease that reduces dry bean yields by as much as 50 to 80%, when susceptible varieties are planted (Correa et al., 1994; de Jesus Junior et al., 2001).
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.
MATERIALS AND METHODS
G 10474 is a small red seeded climbing bean germplasm from the highlands of Guatemala that was identified as having high levels of resistance to P. griseola under field conditions in Darien and Sanlander de Quilichao (Pastor-Corrales el al., 1998). It pertains to race G of the Middle American gene pool (Beebe et al., 2000). However, it is susceptible to Bean common mosaic virus (BCMV) and has an intermediate reaction to leaf hoppers (Empoasca spp.).
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 patholype 63-63, a highly virulent race that overcomes the resistance in all the ALS differential varieties (Mahuku et al., 2003a).
Genetic Material
For the inheritance studies, G 10474 was crossed with a universally susceptible bean variety, 'Sprite'. Sprite is a snap bean that is susceptible to more than 400 P. griseola isolates of a diverse origin that we have tested under green house conditions. Simple crosses were made between G 10474 X Sprite to generate F^sub 1^, F^sub 2^, and BCF^sub 1^ to Sprite populations. Populations were established in the green house with one plant per pot, and inoculated with pathotype 63-63 (Table 2). An additional 106 F^sub 2^ plants were inoculated with a less virulent pathotype, 7-35. Pathotype 7-35, was chosen to verify the presence of other genes that might not be detected if a more virulent pathotype is used. In each case, a set of differential varieties was established (3 plants/pot, 3 pots per differential variety) to verify the reaction and designation of pathogen pathotype and disease development conditions.
Inoculum Preparation, Inoculations, and Disease Evaluations
Inoculum was obtained from a 2-wk-old culture of each monosporic isolate grown on Petri dishes containing V8-juice agar (Pastor-Corrales et al., 1998). The first trifoliate leaf of each plant was inoculated 17 d after planting by spraying 2 X 10^sup 4^ conidia/mL with a De Vilbiss air compressor at 0.25 kW (1/3 horsepower) until plants were wet. Inoculated plants were placed in a humid chamber at approximately 22°C and relative humidity >95% with a 12 h dark/light cycle. After 4 d in the humid chamber, plants were put on tables in the greenhouse, and the temperature maintained between 24 to 30°C. Disease evaluations were conducted 10, 12, 14, 17, and 21 d after inoculation by a CIAT 1-to-9 visual scale (van Schoonhoven and Pastor-Corrales, 1987), where 1 represents no visible symptoms and 9 = severe symptoms and disease expression.
Table 1. Virulence phenotypes of 43 pathotypes of Phaeoisariopsis griseola of a diverse origin used to characterize the germ-plasm accession G 10474.
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).
Table 2. Response of parental genotypes G 10474, Sprite, F^sub 1^, and F^sub 2^ plants from the G 10474 x Sprite cross, and backcross populations to inoculation with Phaeoisariopsts griseola races 63-63 and 7-35.
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 > or = 8) of the G 10474 X Sprite F^sub 2^ 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 [mu]L TE (10 mM Tris, 1 mM EDTA pH 8.0) overnight at 4°C. The fragments were reamplified in 12.5-[mu]L reaction volumes containing 1.0 [mu]L 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 1 X 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[alpha]. 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 F^sub 2^ 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 F^sub 2^ population. PCR amplifications were performed in 12.5-[mu]L reaction volumes containing 20 ng of genomic DNA, 10 mM of Tris-HCl pH 8.0, 1.5 mM of MgCl^sub 2^, 50 mM of KCl, 0.25 mM of each dNTP, 0.5 [mu]M of forward primer, 0.5 [mu]M 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 < or = 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:
AUDPC = [Sigma]^sub i^.[(D^sub i^ + D^sub i-1^) * (t^sub i^ - t^sub i-1^)]/2
where D is the disease score from the 1-to-9 severity scale, and t corresponds to days after inoculation, with 1 = 10, 12, 14, 17, or 21 d (Shaner and Finney, 1977). AUDPC measures the rate of disease progression and was used to ascertain the relative resistance of each plant to race 63-63 or 7-35 and confirm the classification of individual plants into resistant and susceptible classes based on the rating score taken 17 d after inoculation. Segregation analysis of the disease reaction of 135 F^sub 2^ plants (for pathotype 63-63) and 106 plants (for pathotype 7-35) was performed by the Chi-square ([chi]^sup 2^) test, according to the Mendelian segregation hypothesis of 3:1 (resistant to susceptible). Similarly, the BCF^sub 1^ 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
Validation of the ALS Resistance Gene(s) in G 10474
Of the 43 pathotypes used to validate the effectiveness of the resistance gene in G 10474 to manage P. griseola, only three pathotypes (15-23, 15-55, 31-55 from Haiti) were able to overcome the resistance in this accession (Table 1). All the other races, including race 63-63 had either an immune response (rating 1) or a resistance response (rating >1-3), demonstrating the utility of this gene to manage the ALS disease. However, race 15-23 from Brazil and 31-55 from Colombia had an incompatible interaction with G 10474 (Table 1).
Fig. 1. AFLP banding patterns from the combination E-ACA/M-CTT^sub 330^ for the cross C 10474 X Sprite. The polymorphic fragments are indicated by the arrow. MP = molecular marker, RP is resistant parent (G 10474), RB is resistant bulk, SB is susceptible bulk, Lanes 1 to 5 are resistant plants while Lanes 6 to 10 are susceptible F^sub 2^ plants, SP is the susceptible parent (Sprite).
Nature and Inheritance of ALS Resistance in G 10474
All F^sub 1^ 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 F^sub 2^ population was consistent with a 3 resistant:1 susceptible ratio ([chi]^sup 2^ = 0.003), while that for the backcross to susceptible parent (Sprite) fit a 1:1 ratio ([chi]^sup 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 F^sub 2^ plants to inoculation with race 7-35 revealed a segregation ratio of 3:1 ([chi]^sup 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 F^sub 2^ plants. Three AFLP fragments E-ACA/M-CTT^sub 330^, E-AAC/M-CAG^sub 310^, and E-AAC/M-CAT^sub 285^ 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-CTT^sub 330^ 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 F^sub 2^ population (Fig. 2). Linkage analysis after testing the marker on 241 F^sub 2^ 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.
Fig. 2. PCR products from F^sub 2^ population showing homozygous resistant plants (305 bp), homozygous susceptible plants (280 bp) and heterozygous plants with both fragments. The amplification products were obtained using the SCAR marker derived from the polymorphic AFLP fragment generated by the primer combination E-ACA/M-CTT^sub 330^. Lane 19 is negative control (no plant DNA) while 20 is the 100-bp molecular size marker.
Primers for the other two AFLP fragments (E-AAC/ M-CAG^sub 310^ and E-AAC/M-CAT^sub 285^) amplified a similar sized fragment from susceptible and resistant plants, indicating the presence of highly homologous sequences between G 10474 and the susceptible bulk within these AFLP markers. PCR products from resistant and susceptible parents amplified with these primers were cloned and sequenced. Alignment of resistant and susceptible plant sequences revealed no nuclcotide polymorphisms for the two AFLP markers and thus no SCAR primers could be developed for these AFLP markers.
Validation of the Utility of the Marker
To verify the utility of the SCAR marker for marker assisted selection (MAS), the SCAR primer-pair (PF5^sub 330^L2, 5'-CTTGTTCTGAGTCATTTACCTTGC-S'; and PFS330-Rl, S'-GAATTCACAGTCCAAACTACTCTA ATC-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.
DISCUSSION
In this paper, we present evidence of a single dominant gene in G 10474 conditioning resistance to two P. griseola pathotypes. We also show that this gene is effective against many Mesoamerican P. griseola pathotypes from different geographical regions. Host pathogen cocvolution studies have demonstrated that P. griseola coevolved with common bean gene pools and in our experience, all isolates belonging to the Andean subgroup do not infect Mesoamerican genotypes. All Andean P. griseola pathotypes that were used to challenge G 10474 during this study did not infect this genotype, which is thus consistent with the P. griseola-common bean gene pool coevolution hypothesis (Busogoro et al., 1999a; Mahuku et al., 2002; Pastor-Corrales et al., 1998). Because of the high levels of resistance to both Andean and Mesoamerican P. griseola races, G 10474 should find wide application in managing angular leaf spot of common bean. Breeders should incorporate this genotype as a source of resistance in breeding programs, especially for areas where Andean races of P. griseola predominate, such as Madagascar in Africa, Ecuador, and Peru in Latin America.
Fig. 3. PCR amplification of common bean angular leaf spot differential genotypes using the SCAR marker derived from the polymorphic AFLP fragment generated by tlie primer combination E-ACA/M-CTT^sub 330^. Lanes 1-6 are Andean common bean genotypes, Lane 7 is resistant parent G 10474, Lane 8 = resistant bulk, Lane 9 = susceptible bulk, Lane 10 = susceptible parent, Sprite, while Lanes 11-16 are Mesoamerican ALS differential bean genotypes, including Mexico 54 in Lane 14. Lane 17 is negative control while MP is the 100-bp molecular size marker.Fig. 4. Effect of different DNA extraction methods on detection of SCAR marker linked to the ALS resistance locus of G 10474. DNA was extracted using the alkaline method, ammonium acetate method, and the phenol/chloroform method as described in Materials and Methods. Lane 1 to 3 are Andean common bean genotypes Don Timoteo, G 11796 and Bolon liayo, while Lanes 4 to 6 are Mcsoamerican genotypes (G 10474, Mexico 54 and Flor de mayo, respectively. Lane c = negative control (no plant DNA added) while lane MP is the 100-bp molecular size marker.
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-6^sup 2^) 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-1^sup a^, Phg-2^sup 2^, Phg-3^sup 2^, and Phg-4^sup 2^, 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 cultivais 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 break-down. 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.
ACKNOWLEDGMENTS
We are grateful to Jorge Fory for greenhouse operations, Guillermo Castellanos for maintaining the isolates and inoculum production, Juan Cuasquer for statistical analysis, Maria del Carmen Hernandez for technical assistance and Dr Martin Fregene for running the Mapmaker program, helpful insights and interesting discussion throughout the course of this work. We are also grateful to the entire Mesoamerican common bean breeding team for the help in establishing the respective populations. This project was supported in part by the Rockefeller Foundation, SDC through PROFRIJOL, USAID through Hurricane George project for Haiti and the COLCIENCIAS young investigator fellowship to Carmenza Montoya.
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George Mahuku,* Carmenza Montoya, Maria Antonia Henriquez, Carlos Jara, Henry Teran, and Stephen Beebe
Centro Internacional de Agricultura Tropical, A. A. 6713, Cali, Colombia. Received 2 Sept. 2003. *Corresponding author (g.mahuku@cgiar. org).
Published in Crop Sci. 44:1817-1824 (2004).
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