Fine-mapping of a major QTL controlling angular leaf spot resistance in common bean (Phaseolus vulgaris L.)

Key message A major QTL for angular leaf spot resistance in the common bean accession G5686 was fine-mapped to a region containing 36 candidate genes. Markers have been developed for marker-assisted selection. Abstract Common bean (Phaseolus vulgaris L.) is an important grain legume and an essential protein source for human nutrition in developing countries. Angular leaf spot (ALS) caused by the pathogen Pseudocercospora griseola (Sacc.) Crous and U. Braun is responsible for severe yield losses of up to 80 %. Breeding for resistant cultivars is the most ecological and economical means to control ALS and is particularly important for yield stability in low-input agriculture. Here, we report on a fine-mapping approach of a major quantitative trait locus (QTL) ALS4.1GS, UC for ALS resistance in a mapping population derived from the resistant genotype G5686 and the susceptible cultivar Sprite. 180 F3 individuals of the mapping population were evaluated for ALS resistance and genotyped with 22 markers distributed over 11 genome regions colocating with previously reported QTL for ALS resistance. Multiple QTL analysis identified three QTL regions, including one major QTL on chromosome Pv04 at 43.7 Mbp explaining over 75 % of the observed variation for ALS resistance. Additional evaluation of 153 F4, 89 BC1F2 and 139 F4/F5/BC1F3 descendants with markers in the region of the major QTL delimited the region to 418 kbp harboring 36 candidate genes. Among these, 11 serine/threonine protein kinases arranged in a repetitive array constitute promising candidate genes for controlling ALS resistance. Single nucleotide polymorphism markers cosegregating with the major QTL for ALS resistance have been developed and constitute the basis for marker-assisted introgression of ALS resistance into advanced breeding germplasm of common bean. Electronic supplementary material The online version of this article (doi:10.1007/s00122-015-2472-6) contains supplementary material, which is available to authorized users.

Angular leaf spot (ALS) caused by the hemibiotrophic fungus Pseudocercospora griseola (Sacc.) Crous and U. Braun (Bassanezi et al. 2002;Crous et al. 2006) was reported to be responsible for up to 60 and 80 % yield losses in Brazil (de Jesus et al. 2001) and Colombia (Schwartz et al. 1981), respectively. P. griseola spores germinate on the leaf surface after 3 days of moist conditions, enter the leaf through the stomata and grow intercellularly, limited by the leaf veins resulting in an angular lesion shape (Monda et al. 2001;Willocquet et al. 2004). Infection and sporulation occurs in a broad temperature range, from 10 to 33 °C (reviewed in Allorent and Savary 2005). Following the two gene pools of common bean, Mesoamerican and Andean, the pathogen evolved in each gene pool separately (Guzman et al. 1995;Crous et al. 2006). Whereas Andean pathogens are mainly virulent on Andean beans, Mesoamerican and some Andean isolates found in Africa attack both Andeans and Mesoamerican beans (Pastor-Corrales et al. 1998;Mahuku et al. 2002). Since P. griseola is highly variable (Abadio et al. 2012), breeding for a broad ALS resistance involves genes of Andean and Mesoamerican origin or introduction of resistance genes from the secondary gene pool (Mahuku et al. 2003). Genetic resistance is the most efficient strategy to prevent yield loss considering ecological and economic factors (Miklas et al. 2006). However, only few resistance genes have been tagged with closely linked markers.
In Mesoamerican germplasm, the ALS resistance gene Phg-2 was identified in the cultivar Mexico 54 on chromosome Pv08 (Sartorato et al. 2000;Mahuku et al. 2011). Localization of reported markers revealed ALS resistance loci at the same region in Mesoamerican germplasm MAR 2 (Ferreira et al. 2000), Cornell 49-242 , Ouro Negro (Corrêa et al. 2001;Faleiro et al. 2003), G10474 (Mahuku et al. 2004) and G10909 (Mahuku et al. 2011). A genetic test for allelism with one pathotype indicated that the Mesoamerican line BAT 322 contained resistance locus Phg-2 as Mexico 54 (Namayanja et al. 2006), whereas in MAR 2, Mexico 54 and Ouro Negro, five additional independent dominant genes (Phg-3 to Phg-7) were identified using different pathotypes (Caixeta et al. 2003(Caixeta et al. , 2005Sanglard et al. 2013). As for the Andean lines, several markers linked to ALS resistance have been reported (Table 1). Phg-1 of the Andean cultivar AND 277 (Carvalho et al. 1998) was mapped to chromosome 1 (Goncalves-Vidigal et al. 2011). In the Andean accession G5686, Mahuku et al. (2009) identified a major resistance locus on Pv04 later confirmed by Oblessuc et al. (2012) and named ALS4.1 GS, UC . In addition, Mahuku et al. (2009) reported two complementary resistance genes in G5686 on Pv09 (ALS9.1 GS ) and Pv04 (ALS4.2 GS ). Further QTL studies support a more quantitative nature of ALS resistance (Lopez et al. 2003;Teixeira et al. 2005;Mahuku et al. 2011;Oblessuc et al. 2012). Wang et al. (2005) reported a genotyping method based on shifting melting temperatures (T m ) of PCR amplicons introduced by allele-specific primers differing in T m . However, the primer design is rather inflexible due to the necessity for the primers to end exactly on the SNP. High-resolution melting curve analysis (HRM) developed by Gundry et al. (2003) and Wittwer et al. (2003) can overcome this disadvantage: Any sequence polymorphism between amplicons can be detected by fluorescence in much larger PCR amplicons (Reed and Wittwer 2004;Montgomery et al. 2007). HRM was used to fine-map resistance loci based on a well-defined DNA sequence polymorphism (Lehmensiek et al. 2008) or even when type, number and composition of the DNA sequence polymorphism in a particular PCR amplicon were unknown (Studer et al. 2009).
In this study, we aimed at (1) validating effective ALS resistance regions in common bean, (2) fine-mapping the source of resistance to identify candidate genes for ALS resistance and (3) developing closely linked markers for breeding applications.

Plant material
A cross between the resistant Andean common bean genotype G5686 and the susceptible cultivar Sprite had been used for the development of an F2 population to characterize the genetics of ALS resistance (Mahuku et al. 2009). This population was now advanced to an F 3 and F 4 population with 180 and 153 individuals, respectively. Additionally, a resistant F 1 plant backcrossed to the susceptible parental genotype was used to generate a BC1F 2 mapping population (89 individuals). G5686 is a highly ALS-resistant Andean dry bean accession from Ecuador (Mahuku et al. 2009). Sprite is an Andean snap bean cultivar (Cunha et al. 2004;Gepts et al. 2008) and was susceptible to over 400 of 503 tested ALS isolates (Mahuku et al. 2009 Mahuku et al. 2009). The inoculation with P. griseola was done according to the CIAT practical guide (Castellanos et al. 2011). The standard visual scale in which 1-3 means resistant, 4-6 intermediate and 7-9 susceptible was used for phenotypic scoring (Schoonhoven and Pastor-Corrales 1987). The plants were evaluated 11, 15 and 20 days after inoculation in the first cycle. Additionally, at the first evaluation, visible hypersensitive reaction (HR) was recorded.
In a second infection cycle with 139 selected recombinant descendants, faster disease development was noted. Plants were visually scored after 9, 13 and 15 days post-inoculation. The calculated area under the disease progress curve (AUDPC) was used for classification into susceptible and resistant plants (Mahuku et al. 2009).

Genotyping
Genotyping was conducted according to the strategy of Peleman et al. (2005): The 180 F 3 plants were genotyped using two SSR and 17 T m markers which were found to be polymorphic out of a set of 64 tested T m markers (Table 1, Supplemental Table S1). The 153 F 4 , 89 BC 1 F 2 and 139 F 4 / F 5 /BCF 3 descendants were genotyped using three selected SNPs (Marker63, Marker50 or Marker9) in order to identify recombination events in a particular region of interest on chromosome Pv04. Finally, 47 recombinant plants were selected and genotyped using three HRM markers, which were found to be polymorphic out of 27 tested amplicons. Primers were designed by the Primer3web version 4.0.0 (Koressaar and Remm 2007;Untergasser et al. 2012). DNA was extracted according to Xin et al. (2003) from approximately 30 mm 2 young trifoliate leaf sample tissue or young unopened trifoliate leaves, using 100 µl buffer A (50 mM NaOH, 2 % Tween 20) and 75 µm buffer B (100 mM Tris-HCl, 1.7 mM EDTA, pH 7.3). The extract was diluted 1:10 in distilled water for PCR.
Pv-ag004 and Pv-ctt001 were amplified under the following PCR conditions: initial denaturation at 94 °C for 3 min followed by 35 cycles of denaturation at 94 °C for 30 s, 48 °C annealing temperature (Yu et al. 2000) for 30 s For the SNP markers genotyped by melting temperature (T m ) shifts, the Tm shift was enhanced by a gcgggc or gcgggcagggcgg-tail added to the forward primer sequences for each SNP allele [in brackets] as described in Wang et al. (2005). Previously reported markers linked to ALS resistance and their physical positions are given along with the respective source of resistance and the corresponding reference a SSR marker derived from Yu et al. (2000) evaluated by Mahuku et al. (2009) Oblessuc et al. (2013) and extension at 72 °C for 8 min. The SSR marker visualization was described in Mahuku et al. (2009) with a separation at 80 V for approximately 1 h using an Owl T-Rex™ vertical S3S camera (ThermoFisher Scientific Inc, USA).

Melting temperature (T m ) shift genotyping
The physical positions of reported markers linked with ALS resistance were identified using sequences from the PhaseolusGenes Toolbox (http://phaseolusgenes.bioinformatics.ucdavis.edu) (2013) and positional information from the Phytozome platform (Goodstein et al. 2012). In those regions (Table 1), SNPs were selected out of a SNP collection provided by The Common Bean Coordinated Agricultural Project (BeanCAP) available on NCBI (2013) and Blair et al. (2013) presenting over 3,300 SNPs. SNP assays were designed following Wang et al. (2005). DNA was amplified by PCR in a total volume of 20 μl containing 4 μl of genomic DNA, 1X Taq buffer, 1.5 mM MgCl2, 0.2 mM dNTPs mix, 0.15 μM each primer (two allele-specific forward primers and the common reverse primer), 1X EvaGreen ® (Biotium) and 0.1 μl of homemade Taq polymerase. Amplification was carried out with the following program: initial denaturation at 94 °C for 3 min followed by 35 cycles of denaturation at 92 °C for 15 s, annealing of 15 s (temperature specific to each primer trio, Table 1) and extension at 72 °C for 15 s, finally followed by 10 min extension at 72 °C and 5 min at 10 °C. T m shifts of the amplicons were measured by melting point analysis in a fluorescence-detecting thermocycler (Mx3000P Stratagene) and used to classify the samples into GG (homozygous DNA sequence of G5686), GS (heterozygous) or SS (homozygous DNA sequence of Sprite) genotypes.
Genotyping using high-resolution melting curve analysis (HRM) HRM genotyping was used to further delimit the position of the QTL ALS4.1 GS, UC . Genes of common bean within this region were selected using the Phytozome genome browser (Phytozome.net 2014), and primer pairs spanning introns were designed with the Primer3web version 4.0.0 (Rozen and Skaletsky 2000). For genes without introns, EST sequences of different common bean genotypes were extracted from the database of Ramirez et al. (2005) and CleanEST (Lee and Shin 2009) and aligned to the common bean genome in order to identify SNPs. Primer pairs were designed to flank the SNPs in 150-300 bp distance. Primer sequences were compared against other closely related species such as soybean (Glycine max) or barrel medic (Medicago truncatula) using the Phytozome platform (Goodstein et al. 2012) to select most conserved primer sequences.
Thirteen EST-derived and fourteen intron-flanking primer pairs were designed between 43.5 and 44.5 Mbp on Pv04. The amplification was carried out in a PCR volume of 7.3 μl containing 1X LightScanner high sensitivity master mix (BioFire Diagnostics Inc., UT, USA) including LCGreen ® PLUS, 0.10 mM of each forward and reverse primer and 1.3 μl of DNA. Additionally, 14 μl of mineral oil was added to each sample, covering the mix to prevent evaporation during PCR and melting analysis. PCR conditions were set as following: denaturation for 2 min at 95 °C, 40 cycles of 30 s at 94 °C, 30 s annealing at the optimal temperature for each primer pair (Table 1) and 30 s for elongation at 72 °C followed by a final cycle of 2 min at 72 °C, 30 s at 94 °C and 30 s at 25 °C. Using a LightScanner Instrument (BioFire Diagnostics Inc.; 96-well plate format), the amplification product was melted ramping from 60 to 95 °C in 0.05 °C steps per second under continuous fluorescence measurement. The melting curves were related to genotype GG, SS and GS using the LightScanner ® and Call-IT ® software modules (BioFire Diagnostics Inc.).
Identification of candidate genes within the region of QTL ALS4.1 GS, UC In order to identify homologies to previously reported candidate resistance genes, an NCBI megaBLAST query (Zhang et al. 2000) was conducted using the sequence in the region cosegregating with ALS4.1 of the common bean reference genome (Andean accession G19833, Schmutz et al. 2014). Function of genes analogous to Arabidopsis was studied using The Arabidopsis Information Resource (TAIR) (Swarbreck et al. 2008). Phylogenetic analysis of candidate genes and related common bean genes was carried out using the amino acid sequences of the candidate protein kinases. First, the sequences of the candidate kinases were used for a protein BLAST against the reference genome, collecting the first 100 hits of each candidate sequence. All sequences were then aligned using Clustal Omega (Sievers et al. 2011) and the results analyzed with the software package Mega5 for a comparative analysis and the construction of a maximum-likelihood tree (Tamura et al. 2011).

Statistical analysis
Single and multiple QTL analyses were carried out using the R package qtl developed by Broman et al. (2003). Single QTL analysis and LOD score calculation were done by marker regression and standard interval mapping (200,000 bp steps, 1,000 permutations, 0.01 assumed genotyping error rate) in order to analyze the major QTL. p values were derived by the analysis of variance (ANOVA) if the residues showed normal distribution and using lmPerm R package of Wheeler (2010) if this was not the case. A multiple QTL model was built using the function stepwise with 200,000 bp steps, 1,000 permutations, 0.01 assumed genotyping error rate and 256 imputations in order to calculate a penalized LOD score, followed by analyses omitting one QTL at a time to obtain an ANOVA table.

Phenotypic evaluation for angular leaf spot (ALS) resistance
In order to validate QTL reported in various Andean germplasm, 180 F 3 (65 families) of the Sprite × G5686 population were evaluated with the P. griseola pathotype 31-0. Plants were classified into 119 resistant and 61 susceptible plants, showing AUDPC resistance values of <20 and ≥20, respectively. The ratio between the resistant and the susceptible plants is in accordance with one dominant resistance gene segregating in an expected 5:3 ratio, assuming independent F 3 individuals (observed Chisquared = 1.001, p = 0.317). HR was observed in 18 out of the 180 F 3 plants. Hypersensitive plants were healthy and vigorous, showing only bright green dots of few millimeters of diameter on the leaf surface without damage to the leaf tissue.

Multiple QTL analysis
Genotyping of these 180 F 3 plants with 2 SSR and 20 SNP markers (Table 1) and subsequent multiple QTL analyses identified one major and two minor QTL explaining in total 80.1 % of the phenotypic variation for ALS resistance ( Table 2). The major QTL (ALS4.1 GS, UC ), closely linked to Marker50 localized on chromosome Pv04 at around 43.7 Mbp, explained 75.3 % of the ALS resistance. Two smaller QTL (ALS10.1 DG, UC, GS and ALS9.1 GS ) explained 4.9 and 1.7 % of the ALS resistance, respectively. Out of three markers genotyped on Pv10, Marker17, localized at around 38 Mbp, was best linked to Table 2 Multiple QTL analyses resulted in three significant QTL for angular leaf spot (ALS) resistance on chromosomes Pv04, Pv09 and Pv010. QTL models were designed using the stepwise function of R package qtl (Broman et al. 2003) LOD score, percentage of explained variance and p value were derived from F statistics by dropping one QTL at a time resulting in the ANOVA table. An additional QTL (ALS5.2 UC, GS ) was identified by marker regression on Pv05 Significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001 a Ferreira da Silva et al.(2003), b Lopez et al. (2003), c Oblessuc et al. (2012) and d Mahuku et al. (2009)    observations, Fig. 2c). However, QTL analysis resulted in a LOD score (1.5) below the calculated significance threshold requiring a LOD score above 14.5 according to a 5 % significance level. It was the only marker on Pv05 and could therefore not be included in multiple QTL analysis.
Fine-mapping of the major QTL for ALS resistance Fine-mapping of the ALS4.1 region was conducted on 47 recombinants out of 561 screened plants (selected from 180 F 3 , 153 F 4 , 89 BC 1 F 2 and 139 F 4 /F 5 /BC 1 F 3 descendants that were investigated for recombination events in the genomic region of ALS4.1) using seven polymorphic markers: four SNP markers (Marker8, Marker63, Marker50 and Marker9) and three HRM markers (4M437, 4M439 and 4M442). Homozygous plants at 4M437 showed no recombination between the marker and the resistance gene, whereas the flanking Marker63 identified four and 4M439 two recombinant plants (Fig. 3). Hence, the region of ALS4.1 was delimited by the markers Marker63 and 4M439, extending from 43,497,706 to 43,915,434 bp.

Toward marker-assisted selection
To make this resistance locus available for markerassisted selection (MAS) to breeding programs, which do not have SNP genotyping facilities, we set up two assays at this locus for outsourcing at the commercial provider LGC genomics (Hertfordshire, UK, http://www.lgcgenomics.com). Assay MAS_ALS4a is based on the polymorphism from Marker50 (Chr04, pos_43,773,443_T/G) and assay MAS_ALS4b (Chr04, pos_43,794,830_T/C) may have a higher specificity, as it was identified based on the recent whole-genome resequencing of genotype G5686 as a unique polymorphism compared with eight other resequenced genotypes (AFR298, G10474, SEA5, MDE23-24, AND696, G40001, G35346 and VAX1, unpublished data). Genotypic data in Table 3 suggest using MAS_ALS4b for marker-assisted selection.
Identification of putative resistance genes within ALS4.1 GS, UC According to the reference sequence of the Andean common bean accession G19833, 36 genes are predicted in the region of the ALS4.1. Resequencing data did not reveal any polymorphisms in the coding sequences of the candidate genes (unpublished data), pointing toward undetected insertions. Among the 36 genes, 11 very closely related to serine/threonine protein kinases (STPK) are annotated (Fig. 3). BLAST searches revealed two Arabidopsis thaliana (At)-like STPKs described as putative resistance genes: Probable LRR receptor-like At1g07560 and At1g18390 involved in response to chitin and  (Schmutz et al. 2014) respiratory burst defense, best matching common bean genes Phvul.004G154600 and Phvul.004G154800, respectively (Phytozome.net 2014). To further characterize the candidate kinases, the amino acid sequence of Phvul.004G155000 was used for a BLAST search to identify related genes in the bean genome. The amino acid sequences of the first 100 hits were aligned with the candidate STPKs, and a maximum-likelihood tree was built. Eight of the 11 kinases are part of a subfamily, together with other two kinases from Pv02 and 10, and are most related to each other, suggesting that they originate from recent local duplications. The other three kinases were grouped apart and are more closely related to kinases present in Pv07, Pv03 and Pv02. (Supplemental Figure S1). Predicted functions of homologs in plant defense and the repetitive nature make these kinases prime candidates to cause the observed resistance ALS.

Characterization of a major resistance locus
Phenotypic evaluation of an F 3 mapping population derived from a resistant Andean common bean genotype G5686 and a susceptible cultivar Sprite for ALS resistance demonstrated the presence of one dominant major resistance locus on chromosome Pv04 and three minor loci. Molecular characterization and multiple QTL analyses confirmed a major resistance source (ALS4.1 GS, UC ) explaining 75.3 % of the phenotypic variation for ALS resistance, which was previously tagged with the SSR marker Pv-ag004 (Mahuku et al. 2009). In the present study, ALS4.1 was delimited to a region between 43,497,706 and 43,915,434 bp on Pv04 and tagged with two markers linked to ALS resistance. The two markers, HRM marker 4M437 and T m shift Marker50, were developed based on EST sequence alignments (Ramirez et al. 2005;Shin 2009) or SNP collections (NCBI 2013;Blair et al. 2013), respectively. This approach enabled the development of markers at any genomic region, independent of polymorphic microsatellite motifs as necessary for SSR markers. In combination with the HRM technology, allowing for fast and accurate closed-tube genotyping in any genetic background, the markers presented here enable efficient marker-assisted introgression of ALS4.1 into advanced breeding germplasm of common bean. The value of G5686 as an Andean source of resistance to withstand both Andean and Mesoamerican pathotypes of P. griseola has been recognized in previous studies (Pastor-Corrales et al. 1998;Mahuku et al. 2009). Ferreira da Silva et al. (2003) reported marker PV-atct001 linked to a resistant allele in a cross of ESAL 550 and Carioca MG. Lopez et al. (2003) reported a QTL near RGA14 in DOR364 × G19833. Now available physical marker positions suggest these may be the same QTL ALS4.1 EC, DG, GS, UC which remains to be confirmed. The ALS resistance gene in ALS4.1 is likely to be one of the genes Phg-2, Phg-3 or Phg-4, reported in AND277 by Caixeta et al. (2005). But as these are not mapped and resistance evaluations used other isolates, further allelism tests are required to identify which gene exactly is underlying ALS4.1 ALS4.1 GS, UC embeds a repetitive genome region including potential resistance genes According to the common bean reference sequence, the ALS4.1 region harbors among its 36 genes repetitive homologs of putative disease resistance genes (Schmutz et al. 2014). Similar observations were reported for other major resistance loci, as, for example, the Mla locus conferring resistance to powdery mildew in barley (Hordeum   (Wei et al. 2002). In the present study, 11 annotated genes containing STPK domains were identified in the region of ALS4.1, eight and three genes, respectively, in two phylogenetically closely related groups. This is interesting since plant resistance genes are often clustered in the genome and evolve rapidly via diverse mechanisms (Smith et al. 2004;David et al. 2009). For example, resistance against soybean cyst nematode is mediated by copy number variation of a 31-kbp genome segment fortifying the expression of several dissimilar genes in a repetitive multigene region (Cook et al. 2012). STPKs phosphorylate hydroxyl groups of serine or threonine residues which is essential for various signaling pathways in eukaryotes including pathogentriggered immunity (Park et al. 2011;Zhang et al. 2013). For example, the Pto protein in tomato containing a STPK domain (Martin et al. 1993;Loh et al. 1998) induces HR by recognizing two specific Pseudomonas syringae pathogen effectors (Dong et al. 2009). Similar STPK-based defenses were reported in Arabidopsis (Warren et al. 1999;Swiderski and Innes 2001) and wheat against powdery mildew (Cao et al. 2011). The involvement of STPKs in pathogen response and the repetitive arrangement also found at other previously described resistance loci (e.g., Vallejos et al. 2006) make this STPK cluster a likely candidate to cause the observed ALS resistance. Resequencing data of G5686 did not reveal any polymorphisms to the reference G19833 that may cause the resistance. While this may be explained with the difficult assembly of this region due to its various local duplications, we hypothesize the G5686 likely contains additional duplications. To detect new genes, a de novo assembly necessitating deeper sequencing data is required.
Hypersensitive reaction is probably linked with ALS4.1 GS, UC and ALS10.1 DG, UC, GS Plants usually respond to biotrophic fungi such as P. griseola at early infection stages with HR (Glazebrook 2005 showed both HR and GG genotype at Marker17, possibly explaining the small but significant resistance improvement when both QTL present. This is in line with Zhou et al. (1995), who observed enhanced HR in the presence of additional genes besides resistance genes Pto and Prf. The fact that HR was observed only in 18 out of 180 plants of the F 3 population indicates that HR may require several genes (Zhou et al. 1995;Salmeron et al. 1996), a low phenotypic penetrance, or that activation of systemic acquired resistance (Oh and Martin 2011) prevented the plants from visible symptoms.
ALS4.1 GS, UC is a major QTL suitable for marker-assisted introgression of ALS resistance In a previous study, three dominant and complementary genes linked with SSR markers Pv-ag004 (ALS4.1 region), Pv-at007 (ALS10.1 region) and Pv-ctt001 (beginning of Pv04), respectively, were reported to confer ALS resistance in an F 2 population of the same cross infected with the same P. griseola pathotype (Mahuku et al. 2009 Mahuku et al. (2009) and confounded the effect of Pv-ag004. Moreover, environmental conditions might have affected the otherwise identical phenotyping procedure. In the current study, ALS4.1 was masking the minor effect of Pv-at007 linked to ALS9.1 GS and an effect of Pv-ctt001 could not be detected (permutation p > 0.5 including 78 observations). Consistent detection of ALS4.1 across repeated experiments explaining the major proportion of the observed resistance rather indicates that ALS resistance in this population is controlled by one independent major locus modified by few minor QTL. We conclude that ALS4.1 has a high value as a source for marker-assisted introgression of ALS resistance, with polymorphism T/C on Chr04 at 43,794,830 bp (marker MAS_ ALS4b) showing highest specificity for G5686.
Various QTL control ALS resistance ALS resistance loci in the region of ALS4.1 were reported in different germplasm screened with several pathotypes (Faleiro et al. 2003;Lopez et al. 2003;Caixeta et al. 2005;Mahuku et al. 2011) and in different environments (Oblessuc et al. 2012). For example, Ferreira da Silva et al. (2003) identified a major ALS resistance locus in the same region as ALS4.1 evaluating the ESAL 550 cultivar in a field experiment. Lopez et al. (2003) and Oblessuc et al. (2012) reported QTL in the same region explaining a smaller part of the variation using various Andean genotypes and experimental conditions. Oblessuc et al. (2012) named two closely adjacent QTL on Pv04, ALS4.1 and ALS4.2, which encase the QTL reported here. Hence, we hypothesize that these are actually one QTL named ALS4.1 GS, UC . AND277, the only genotype to share the G5686 allele for the MAS_ALS4b marker in the evaluated set (Table 1), is similar but not identical to G5686 (correlation 0.88 based on 650 markers, unpublished data). Also available pedigree information does not indicate G5686 in the ancestry of AND277; hence, there is no clear evidence that G5686 and AND277 carry the same allele. On Pv10, Oblessuc et al. (2012) identified the major QTL ALS10.1 DG, UC, GS (Marker17) explaining over 20 % of ALS resistance in the field, in both dry and wet season using CAL 143 as a source of resistance (Oblessuc et al. 2012). ALS9.1 GS was previously described by Mahuku et al. (2009) but not named. Taken together, the significant QTL in this study was also found in other experiments and genotypes, supporting the findings in this work. ALS resistance initially reported to be monogenetic evaluating only one pathotype (Ferreira et al. 2000;Corrêa et al. 2001) was shown to be quantitatively inherited evaluating different pathotypes (Faleiro et al. 2003;Caixeta et al. 2005). Pathotype-specific resistance in common bean was also reported considering different pathotypes of rust (Park et al. 1999;Faleiro et al. 2003) and anthracnose (Faleiro et al. 2003;Rodriguez-Suarez et al. 2007). Since G5686 proved resistance against a wide range of pathotypes (Mahuku et al. 2009), it appears likely that minor QTL or additional QTL will add specific resistance to other pathotypes.

Conclusion
A major QTL explaining 75.3 % of ALS resistance in the G5686 × Sprite population was validated, mapped to 418 kbp on chromosome Pv04 and tagged with two closely linked SNP markers (Marker50 and 4M437) allowing efficient MAS. ALS4.1 GS, UC defines a region of 36 genes including 11 STPKs, which are likely candidates for the resistance gene. Additionally, three minor QTLs were identified. The Andean resistance loci ALS4.1 and Phg-1 as well as the Mesoamerican Phg-2 can now be combined and tested in elite cultivars in order to pyramid resistance genes.
Author contribution statement BR, CJ, CM, BS and BK conceived and designed the study, and BK performed the experiments. BR, CJ and BS contributed with material, reagents and analysis tools, while BK, BR, JDL and CM helped analyzing the data. The manuscript was drafted by BK and further improved by CM, BR and BS. All authors read and approved the manuscript for publication.