Monitoring Stimulated Darkening from UV-C Light on Different Bean Genotypes by NMR Spectroscopy.

The use of UV-C cool white light on bean (Phaseolus vulgaris L.) seeds significantly increases the biochemical seed coat post-harvest darkening process, whilst preserving seed germination. The aim of this work consists in monitoring the effect caused by the incidence of UV-C light on different bean genotypes using NMR spectroscopy. The genotype samples named IAC Alvorada; TAA Dama; BRS Estilo and BRS Pérola from the Agronomic Institute (IAC; Campinas; SP; Brazil) were evaluated. The following two methodologies were used: a prolonged darkening, in which the grain is placed in a room at a controlled temperature (298 K) and humidity for 90 days, simulating the supermarket shelf; an accelerated darkening, where the grains are exposed to UV-C light (254 nm) for 96 h. The experiments were performed using the following innovative time-domain (TD) NMR approaches: the RK-ROSE pulse sequence; one- and two-dimensional high resolution (HR) NMR experiments (1H; 1H-1H COSY and 1H-13C HSQC); chemometrics tools, such as PLS-DA and heat plots. The results suggest that the observed darkening occurs on the tegument after prolonged (90 days) and accelerated (96 h) conditions. In addition, the results indicate that phenylalanine is the relevant metabolite within this context, being able to participate in the chemical reactions accounted for by the darkening processes. Additionally, it is possible to confirm that a UV-C lamp accelerates oxidative enzymatic reactions and that the NMR methods used were a trustworthy approach to monitor and understand the darkening in bean seeds at metabolite level.

Genetic mapping reveals complex architecture and candidate genes involved in common bean response to Meloidogyne incognita infection.

Root-knot nematodes (RKNs), particularly Meloidogyne incognita, are among the most damaging and prevalent agricultural pathogens due to their ability to infect roots of almost all crops. The best strategy for their control is through the use of resistant cultivars. However, laborious phenotyping procedures make it difficult to assess nematode resistance in breeding programs. For common bean, this task is especially challenging because little has been done to discover resistance genes or markers to assist selection. We performed genome-wide association studies and quantitative trait loci mapping to explore the genetic architecture and genomic regions underlying the resistance to M. incognita and to identify candidate resistance genes. Phenotypic data were collected by a high-throughput assay, and the number of egg masses and the root-galling index were evaluated. Complex genetic architecture and independent genomic regions were associated with each trait. Single nucleotide polymorphisms on chromosomes Pv06, Pv07, Pv08, and Pv11 were associated with the number of egg masses, and SNPs on Pv01, Pv02, Pv05, and Pv10 were associated with root-galling. A total of 216 candidate genes were identified, including 14 resistance gene analogs and five differentially expressed in a previous RNA sequencing analysis. Histochemical analysis indicated that reactive oxygen species might play a role in the resistance response. Our findings open new perspectives to improve selection efficiency for RKN resistance, and the candidate genes are valuable targets for functional investigation and gene editing approaches.

Genetic diversity and inter-gene pool introgression of Mesoamerican Diversity Panel in common beans.

Brazil is among the largest producers and consumers of common bean (Phaseolus vulgaris L.) and can be considered a secondary center of diversity for the species. The aim of this study was to estimate the genetic diversity, population structure, and relationships among 288 common bean accessions in an American Diversity Panel (ADP) genotyped with 4,042 high-quality single nucleotide polymorphisms (SNPs). The results showed inter-gene pool hybridization (hybrids) between the two main gene pools (i.e., Mesoamerican and Andean), based on principal component analysis (PCA), discriminant analysis of principal components (DAPC), and STRUCTURE analysis. The genetic diversity parameters showed that the Mesoamerican group has higher values of diversity and allelic richness in comparison with the Andean group. Considering the optimal clusters (K), clustering was performed according to the type of grain (i.e., market group), the institution of origin, the period of release, and agronomic traits. A new subset was selected and named the Mesoamerican Diversity Panel (MDP), with 205 Mesoamerican accessions. Analysis of molecular variance (AMOVA) showed low genetic variance between the two panels (i.e., ADP and MDP) with the highest percentage of the limited variance among accessions in each group. The ADP showed occurrence of high genetic differentiation between populations (i.e., Mesoamerican and Andean) and introgression between gene pools in hybrids based on a set of diagnostic SNPs. The MDP showed better linkage disequilibrium (LD) decay. The availability of genetic variation from inter-gene pool hybridizations presents a potential opportunity for breeders towards the development of superior common bean cultivars.

Genome-wide association mapping reveals new loci associated with light-colored seed coat at harvest and slow darkening in carioca beans.

BACKGROUND: Common bean (Phaseolus vulgaris L.) is a legume whose grain can be stored for months, a common practice among Brazilian growers. Over time, seed coats become darker and harder to cook, traits that are undesirable to consumers, who associate darker-colored beans with greater age. Like commercial pinto and cranberry bean varieties, carioca beans that have darker seeds at harvest time and after storage are subject to decreased market values. RESULTS: The goal of our study was to identify the genetic control associated with lightness of seed coat color at harvest (HL) and with tolerance to post-harvest seed coat darkening (PHD) by a genome-wide association study. For that purpose, a carioca diversity panel previously validated for association mapping studies was used with 138 genotypes and 1,516 high-quality SNPs. The panel was evaluated in two environments using a colorimeter and the CIELAB scale. Shelf storage for 30 days had the most expressive results and the L* (luminosity) parameter led to the greatest discrimination of genotypes. Three QTL were identified for HL, two on chromosome Pv04 and one on Pv10. Regarding PHD, results showed that genetic control differs for L* after 30 days and for the ΔL* (final L*-initial L*); only ΔL* was able to properly express the PHD trait. Four phenotypic classes were proposed, and five QTL were identified through six significant SNPs. CONCLUSIONS: Lightness of seed coat color at harvest showed an oligogenic inheritance corroborated by moderate broad-sense heritability and high genotypic correlation among the experiments. Only three QTL were significant for this trait - two were mapped on Pv04 and one on Pv10. Considering the ΔL, six QTL were mapped on four different chromosomes for PHD. The same HL QTL at the beginning of Pv10 was also associated with ΔL* and could be used as a tool in marker-assisted selection. Several candidate genes were identified and may be useful to accelerate the genetic breeding process.

Genome-Wide Association Study Reveals Genomic Regions Associated with Fusarium Wilt Resistance in Common Bean.

Fusarium wilt (Fusarium oxysporum f. sp. phaseoli, Fop) is one of the main fungal soil diseases in common bean. The aim of the present study was to identify genomic regions associated with Fop resistance through genome-wide association studies (GWAS) in a Mesoamerican Diversity Panel (MDP) and to identify potential common bean sources of Fop's resistance. The MDP was genotyped with BARCBean6K_3BeadChip and evaluated for Fop resistance with two different monosporic strains using the root-dip method. Disease severity rating (DSR) and the area under the disease progress curve (AUDPC), at 21 days after inoculation (DAI), were used for GWAS performed with FarmCPU model. The p-value of each SNP was determined by resampling method and Bonferroni test. For UFV01 strain, two significant single nucleotide polymorphisms (SNPs) were mapped on the Pv05 and Pv11 for AUDPC, and the same SNP (ss715648096) on Pv11 was associated with AUDPC and DSR. Another SNP, mapped on Pv03, showed significance for DSR. Regarding IAC18001 strain, significant SNPs on Pv03, Pv04, Pv05, Pv07 and on Pv01, Pv05, and Pv10 were observed. Putative candidate genes related to nucleotide-binding sites and carboxy-terminal leucine-rich repeats were identified. The markers may be important future tools for genomic selection to Fop disease resistance in beans.

Genome-wide association mapping reveals race-specific SNP markers associated with anthracnose resistance in carioca common beans.

Brazil is the largest consumer of dry edible beans (Phaseolus vulgaris L.) in the world, 70% of consumption is of the carioca variety. Although the variety has high yield, it is susceptible to several diseases, among them, anthracnose (ANT) can lead to losses of up to 100% of production. The most effective strategy to overcome ANT, a disease caused by the fungus Colletotrichum lindemuthianum, is the development of resistant cultivars. For that reason, the selection of carioca genotypes resistant to multiple ANT races and the identification of loci/markers associated with genetic resistance are extremely important for the genetic breeding process. Using a carioca diversity panel (CDP) with 125 genotypes and genotyped by BeadChip BARCBean6K_3 and a carioca segregating population AM (AND-277 × IAC-Milênio) genotyped by sequencing (GBS). Multiple interval mapping (MIM) and genome-wide association studies (GWAS) were used as mapping tools for the resistance genes to the major ANT physiological races present in the country. In general, 14 single nucleotide polymorphisms (SNPs) showed high significance for resistance by GWAS, and loci associated with multiple races were also identified, as the Co-3 locus. The SNPs ss715642306 and ss715649427 in linkage disequilibrium (LD) at the beginning of chromosome Pv04 were associated with all the races used, and 16 genes known to be related to plant immunity were identified in this region. Using the resistant cultivars and the markers associated with significant quantitative resistance loci (QRL), discriminant analysis of principal components (DAPC) was performed considering the allelic contribution to resistance. Through the DAPC clustering, cultivar sources with high potential for durable anthracnose resistance were recommended. The MIM confirmed the presence of the Co-14 locus in the AND-277 cultivar which revealed that it was the only one associated with resistance to ANT race 81. Three other loci were associated with race 81 on chromosomes Pv03, Pv10, and Pv11. This is the first study to identify new resistance loci in the AND-277 cultivar. Finally, the same Co-14 locus was also significant for the CDP at the end of Pv01. The new SNPs identified, especially those associated with more than one race, present great potential for use in marker-assisted and early selection of inbred lines.

Angular Leaf Spot Resistance Loci Associated With Different Plant Growth Stages in Common Bean.

Angular leaf spot (ALS) is a disease that causes major yield losses in the common bean crop. Studies based on different isolates and populations have already been carried out to elucidate the genetic mechanisms of resistance to ALS. However, understanding of the interaction of this resistance with the reproductive stages of common bean is lacking. The aim of the present study was to identify ALS resistance loci at different plant growth stages (PGS) by association and linkage mapping approaches. An BC2F3 inter-gene pool cross population (AND 277 × IAC-Milênio - AM population) profiled with 1,091 SNPs from genotyping by sequencing (GBS) was used for linkage mapping, and a carioca diversity panel (CDP) genotyped by 5,398 SNPs from BeadChip assay technology was used for association mapping. Both populations were evaluated for ALS resistance at the V2 and V3 PGSs (controlled conditions) and R8 PGS (field conditions). Different QTL (quantitative trait loci) were detected for the three PGSs and both populations, showing a different quantitative profile of the disease at different plant growth stages. For the three PGS, multiple interval mapping (MIM) identified seven significant QTL, and the Genome-wide association study (GWAS) identified fourteen associate SNPs. Several loci validated regions of previous studies, and Phg-1, Phg-2, Phg-4, and Phg-5, among the 5 loci of greatest effects reported in the literature, were detected in the CDP. The AND 277 cultivar contained both the Phg-1 and the Phg-5 QTL, which is reported for the first time in the descendant cultivar CAL143 as ALS10.1UC. The novel QTL named ALS11.1AM was located at the beginning of chromosome Pv11. Gene annotation revealed several putative resistance genes involved in the ALS response at the three PGSs, and with the markers and loci identified, new specific molecular markers can be developed, representing a powerful tool for common bean crop improvement and for gain in ALS resistance.

Genetic Diversity, Population Structure, and Andean Introgression in Brazilian Common Bean Cultivars after Half a Century of Genetic Breeding.

Brazil is the largest consumer and third highest producer of common beans (Phaseolus vulgaris L.) worldwide. Since the 1980s, the commercial Carioca variety has been the most consumed in Brazil, followed by Black and Special beans. The present study evaluates genetic diversity and population structure of 185 Brazilian common bean cultivars using 2827 high-quality single-nucleotide polymorphisms (SNPs). The Andean allelic introgression in the Mesoamerican accessions was investigated, and a Carioca panel was tested using an association mapping approach. The results distinguish the Mesoamerican from the Andean accessions, with a prevalence of Mesoamerican accessions (94.6%). When considering the commercial classes, low levels of genetic differentiation were seen, and the Carioca group showed the lowest genetic diversity. However, gain in gene diversity and allelic richness was seen for the modern Carioca cultivars. A set of 1060 'diagnostic SNPs' that show alternative alleles between the pure Mesoamerican and Andean accessions were identified, which allowed the identification of Andean allelic introgression events and shows that there are putative introgression segments in regions enriched with resistance genes. Finally, genome-wide association studies revealed SNPs significantly associated with flowering time, pod maturation, and growth habit, showing that the Carioca Association Panel represents a powerful tool for crop improvements.

Mapping QTLs for drought tolerance in a SEA 5 x AND 277 common bean cross with SSRs and SNP markers

Abstract The common bean is characterized by high sensitivity to drought and low productivity. Breeding for drought resistance in this species involves genes of different genetic groups. In this work, we used a SEA 5 x AND 277 cross to map quantitative trait loci associated with drought tolerance in order to assess the factors that determine the magnitude of drought response in common beans. A total of 438 polymorphic markers were used to genotype the F8 mapping population. Phenotyping was done in two greenhouses, one used to simulate drought and the other to simulate irrigated conditions. Fourteen traits associated with drought tolerance were measured to identify the quantitative trait loci (QTLs). The map was constructed with 331 markers that covered all 11 chromosomes and had a total length of 1515 cM. Twenty-two QTLs were discovered for chlorophyll, leaf and stem fresh biomass, leaf biomass dry weight, leaf temperature, number of pods per plant, number of seeds per plant, seed weight, days to flowering, dry pod weight and total yield under well-watered and drought (stress) conditions. All the QTLs detected under drought conditions showed positive effects of the SEA 5 allele. This study provides a better understanding of the genetic inheritance of drought tolerance in common bean.

Mapping QTLs for drought tolerance in a SEA 5 x AND 277 common bean cross with SSRs and SNP markers.

The common bean is characterized by high sensitivity to drought and low productivity. Breeding for drought resistance in this species involves genes of different genetic groups. In this work, we used a SEA 5 x AND 277 cross to map quantitative trait loci associated with drought tolerance in order to assess the factors that determine the magnitude of drought response in common beans. A total of 438 polymorphic markers were used to genotype the F8 mapping population. Phenotyping was done in two greenhouses, one used to simulate drought and the other to simulate irrigated conditions. Fourteen traits associated with drought tolerance were measured to identify the quantitative trait loci (QTLs). The map was constructed with 331 markers that covered all 11 chromosomes and had a total length of 1515 cM. Twenty-two QTLs were discovered for chlorophyll, leaf and stem fresh biomass, leaf biomass dry weight, leaf temperature, number of pods per plant, number of seeds per plant, seed weight, days to flowering, dry pod weight and total yield under well-watered and drought (stress) conditions. All the QTLs detected under drought conditions showed positive effects of the SEA 5 allele. This study provides a better understanding of the genetic inheritance of drought tolerance in common bean.

Linkage and mapping of quantitative trait loci associated with angular leaf spot and powdery mildew resistance in common beans

Abstract Angular leaf spot (ALS) and powdery mildew (PWM) are two important fungi diseases causing significant yield losses in common beans. In this study, a new genetic linkage map was constructed using single sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), in a segregating population derived from the AND 277 x SEA 5 cross, with 105 recombinant inbred lines. Phenotypic evaluations were performed in the greenhouse to identify quantitative trait loci (QTLs) associated with resistance by means of the composite interval mapping analysis. Four QTLs were identified for ALS resistance. The QTL ALS11AS, linked on the SNP BAR 5054, mapped on chromosome Pv11, showed the greatest effect (R2 = 26.5%) on ALS phenotypic variance. For PWM resistance, two QTLs were detected, PWM2AS and PWM11AS, on Pv2 and Pv11, explaining 7% and 66% of the phenotypic variation, respectively. Both QTLs on Pv11 were mapped on the same genomic region, suggesting that it is a pleiotropic region. The present study resulted in the identification of new markers closely linked to ALS and PWM QTLs, which can be used for marker-assisted selection, fine mapping and positional cloning.

Linkage and mapping of quantitative trait loci associated with angular leaf spot and powdery mildew resistance in common beans.

Angular leaf spot (ALS) and powdery mildew (PWM) are two important fungi diseases causing significant yield losses in common beans. In this study, a new genetic linkage map was constructed using single sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs), in a segregating population derived from the AND 277 x SEA 5 cross, with 105 recombinant inbred lines. Phenotypic evaluations were performed in the greenhouse to identify quantitative trait loci (QTLs) associated with resistance by means of the composite interval mapping analysis. Four QTLs were identified for ALS resistance. The QTL ALS11AS, linked on the SNP BAR 5054, mapped on chromosome Pv11, showed the greatest effect (R2 = 26.5%) on ALS phenotypic variance. For PWM resistance, two QTLs were detected, PWM2AS and PWM11AS, on Pv2 and Pv11, explaining 7% and 66% of the phenotypic variation, respectively. Both QTLs on Pv11 were mapped on the same genomic region, suggesting that it is a pleiotropic region. The present study resulted in the identification of new markers closely linked to ALS and PWM QTLs, which can be used for marker-assisted selection, fine mapping and positional cloning.

Genome-Wide Association Studies of Anthracnose and Angular Leaf Spot Resistance in Common Bean (Phaseolus vulgaris L.).

The common bean (Phaseolus vulgaris L.) is the world's most important legume for human consumption. Anthracnose (ANT; Colletotrichum lindemuthianum) and angular leaf spot (ALS; Pseudocercospora griseola) are complex diseases that cause major yield losses in common bean. Depending on the cultivar and environmental conditions, anthracnose and angular leaf spot infections can reduce crop yield drastically. This study aimed to estimate linkage disequilibrium levels and identify quantitative resistance loci (QRL) controlling resistance to both ANT and ALS diseases of 180 accessions of common bean using genome-wide association analysis. A randomized complete block design with four replicates was performed for the ANT and ALS experiments, with four plants per genotype in each replicate. Association mapping analyses were performed for ANT and ALS using a mixed linear model approach implemented in TASSEL. A total of 17 and 11 significant statistically associations involving SSRs were detected for ANT and ALS resistance loci, respectively. Using SNPs, 21 and 17 significant statistically associations were obtained for ANT and angular ALS, respectively, providing more associations with this marker. The SSR-IAC167 and PvM95 markers, both located on chromosome Pv03, and the SNP scaffold00021_89379, were associated with both diseases. The other markers were distributed across the entire common bean genome, with chromosomes Pv03 and Pv08 showing the greatest number of loci associated with ANT resistance. The chromosome Pv04 was the most saturated one, with six markers associated with ALS resistance. The telomeric region of this chromosome showed four markers located between approximately 2.5 Mb and 4.4 Mb. Our results demonstrate the great potential of genome-wide association studies to identify QRLs related to ANT and ALS in common bean. The results indicate a quantitative and complex inheritance pattern for both diseases in common bean. Our findings will contribute to more effective screening of elite germplasm to find resistance alleles for marker-assisted selection in breeding programs.

Developing a common bean core collection suitable for association mapping studies.

Because of the continuous introduction of germplasm from abroad, some collections have a high number of accessions, making it difficult to explore the genetic variability present in a germplasm bank for conservation and breeding purposes. Therefore, the aim of this study was to quantify and analyze the structure of genetic variability among 500 common bean accessions to construct a core collection. A total of 58 SSRs were used for this purpose. The polymorphism information content (PIC) in the 180 common bean accessions selected to compose the core collection ranged from 0.17 to 0.86, and the discriminatory power (DP) ranged from 0.21 to 0.90. The 500 accessions were clustered into 15 distinct groups and the 180 accessions into four distinct groups in the Structure analysis. According to analysis of molecular variance, the most divergent accessions comprised 97.2% of the observed genetic variability present within the base collection, confirming the efficiency of the selection criterion. The 180 selected accessions will be used for association mapping in future studies and could be potentially used by breeders to direct new crosses and generate elite cultivars that meet current and future global market needs.

Increasing the density of markers around a major QTL controlling resistance to angular leaf spot in common bean.

Angular leaf spot (ALS) causes major yield losses in the common bean (Phaseolus vulgaris L.), an important protein source in the human diet. This study describes the saturation around a major quantitative trait locus (QTL) region, ALS10.1, controlling resistance to ALS located on linkage group Pv10 and explores the genomic context of this region using available data from the P. vulgaris genome sequence. DArT-derived markers (STS-DArT) selected by bulk segregant analysis and SCAR and SSR markers were used to increase the resolution of the QTL, reducing the confidence interval of ALS10.1 from 13.4 to 3.0 cM. The position of the SSR ATA220 coincided with the maximum LOD score of the QTL. Moreover, a new QTL (ALS10.2(UC)) was identified at the end of the same linkage group. Sequence analysis using the P. vulgaris genome located ten SSRs and seven STS-DArT on chromosome 10 (Pv10). Coincident linkage and genome positions of five markers enabled the definition of a core region for ALS10.1 spanning 5.3 Mb. These markers are linked to putative genes related to disease resistance such as glycosyl transferase, ankyrin repeat-containing, phospholipase, and squamosa-promoter binding protein. Synteny analysis between ALS10.1 markers and the genome of soybean suggested a dynamic evolution of this locus in the common bean. The present study resulted in the identification of new candidate genes and markers closely linked to a major ALS disease resistance QTL, which can be used in marker-assisted selection, fine mapping and positional QTL cloning.