SNP marker diversity in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Single nucleotide polymorphism (SNP) markers have become a genetic technology of choice because of their automation and high precision of allele calls. In this study, our goal was to develop 94 SNPs and test them across well-chosen common bean (Phaseolus vulgaris L.) germplasm. We validated and accessed SNP diversity at 84 gene-based and 10 non-genic loci using KASPar technology in a panel of 70 genotypes that have been used as parents of mapping populations and have been previously evaluated for SSRs. SNPs exhibited high levels of genetic diversity, an excess of middle frequency polymorphism, and a within-genepool mismatch distribution as expected for populations affected by sudden demographic expansions after domestication bottlenecks. This set of markers was useful for distinguishing Andean and Mesoamerican genotypes but less useful for distinguishing within each gene pool. In summary, slightly greater polymorphism and race structure was found within the Andean gene pool than within the Mesoamerican gene pool but polymorphism rate between genotypes was consistent with genepool and race identity. Our survey results represent a baseline for the choice of SNP markers for future applications because gene-associated SNPs could themselves be causative SNPs for traits. Finally, we discuss that the ideal genetic marker combination with which to carry out diversity, mapping and association studies in common bean should consider a mix of both SNP and SSR markers.     

Microsatellite characterization of Andean races of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

The Andean gene pool of common bean (Phaseolus vulgaris L.) has high levels of morphological diversity in terms of seed color and size, growth habit and agro-ecological adaptation, but previously was characterized by low levels of molecular marker diversity. Three races have been described within the Andean gene pool: Chile, Nueva Granada and Peru. The objective of this study was to characterize a collection of 123 genotypes representing Andean bean diversity with 33 microsatellite markers that have been useful for characterizing race structure in common beans. The genotypes were from both the primary center of origin as well as secondary centers of diversity to which Andean beans spread and represented all three races of the gene pool. In addition we evaluated a collection of landraces from Colombia to determine if the Nueva Granada and Peru races could be distinguished in genotypes from the northern range of the primary center. Multiple correspondence analyses of the Andean race representatives identified two predominant groups corresponding to the Nueva Granada and Peru races. Some of the Chile race representatives formed a separate group but several that had been defined previously as from this race grouped with the other races. Gene flow was more notable between Nueva Granada and Peru races than between these races and the Chile race. Among the Colombian genotypes, the Nueva Granada and Peru races were identified and introgression between these two races was especially notable. The genetic diversity within the Colombian genotypes was high, reaffirming the importance of this region as an important source of germplasm. Results of this study suggest that the morphological classification of all climbing beans as Peru race genotypes and all bush beans as Nueva Granada race genotypes is erroneous and that growth habit traits have been mixed in both races, requiring a re-adjustment in the concept of morphological races in Andean beans.     

Integration of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) linkage and chromosomal maps

Fluorescent in situ hybridisation of pooled, closely linked RFLP markers was used to integrate the genetic linkage map and the mitotic chromosome map of the common bean. Pooled RFLP probes showed clear and reproducible signals and allowed the assignment of all linkage groups to the chromosomes of two Phaseolus vulgaris cultivars, Saxa and Calima. Low extension values for signals originating from clustered RFLPs suggest that these clones are physically close to each other and that clusters in the genetic map are not a result of suppression of recombination due to the occurrence of chromosome rearrangements. For linkage group K, clustering of markers could be associated with proximity to centromeres. High variation in the number of 45S rDNA loci was observed among cultivars, suggesting that these terminal sites are highly recombinogenic in common bean.     

Symbiotic response of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) to iron deficiency

In order to assess symbiotic activity (the nodules integrity and the iron use efficiency) in common bean (Phaseolus vulgaris L.) under low iron availability, the growth of plants and nodules, the concentration of leghaemoglobin and malondialdehyde, and activity of nitrogenase, catalase, peroxidase and superoxide dismutase were analysed in two (contrasting) common bean varieties subjected to iron deficiency. Results show that nitrogen fixation and leghaemoglobin accumulation decreased at limiting iron availability while malondialdehyde concentration increased under these conditions. The tolerant variety to iron deficiency, ARA14, was clearly less affected than the sensitive one, Coco blanc. A significant stimulation of peroxidase (POD) activity was observed in ARA14 under iron deficiency. At the same conditions, SOD and CAT activities in ARA14 plants were maintained at high level. It was also found that the iron use efficiency for leghaemoglobin accumulation, SOD, CAT and POD activities were critical for the protection of symbiotic system against oxidative burst and for the maintaining of an optimal functioning of N₂ fixing system.     

Immunological detection of bean common mosaic virus in French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) leaves

Bean common mosaic potyvirus (BCMV) is an important seed borne pathogen of French bean. Differential inoculation with bean common mosaic virus at cotylodonary trifoliate leaf stage and pre-flowering stage of crop growth revealed that cotyledonary leaf infection favored maximum disease expression. Under immunosorbent electron microscopy (ISEM) the virus particles of filamentous structure having a diameter of 750 nm (l) and 15 nm (w) were observed. These particles gave positive precipitin tests with polyclonal antiserum of Potato virus Y.     

Meta-QTL analysis of seed iron and zinc concentration and content in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Key message

Twelve meta-QTL for seed Fe and Zn concentration and/or content were identified from 87 QTL originating from seven population grown in sixteen field trials. These meta-QTL include 2 specific to iron, 2 specific to zinc and 8 that co-localize for iron and zinc concentrations and/or content.

Abstract

Common bean (Phaseolus vulgaris L.) is the most important legume for human consumption worldwide and it is an important source of microelements, especially iron and zinc. Bean biofortification breeding programs develop new varieties with high levels of Fe and Zn targeted for countries with human micronutrient deficiencies. Biofortification efforts thus far have relied on phenotypic selection of raw seed mineral concentrations in advanced generations. While numerous quantitative trait loci (QTL) studies have been conducted to identify genomic regions associated with increased Fe and Zn concentration in seeds, these results have yet to be employed for marker-assisted breeding. The objective of this study was to conduct a meta-analysis from seven QTL studies in Andean and Middle American intra- and inter-gene pool populations to identify the regions in the genome that control the Fe and Zn levels in seeds. Two meta-QTL specific to Fe and two meta-QTL specific to Zn were identified. Additionally, eight Meta QTL that co-localized for Fe and Zn concentration and/or content were identified across seven chromosomes. The Fe and Zn shared meta-QTL could be useful candidates for marker-assisted breeding to simultaneously increase seed Fe and Zn. The physical positions for 12 individual meta-QTL were identified and within five of the meta-QTL, candidate genes were identified from six gene families that have been associated with transport of iron and zinc in plants.

     

Exploring the quantitative resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">phaseolicola</Emphasis> in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Pseudomonas syringae pv. phaseolicola is an important disease that causes halo blight in common bean. The genetic mechanisms underlying quantitative halo blight resistance are poorly understood in this species, as most disease studies have focused on qualitative resistance. The present work examines the genetic basis of quantitative resistance to the nine halo blight races in different organs (primary and trifoliate leaf, stem and pod) of an Andean recombinant inbred line (RIL) progeny. Using a multi-environment quantitative trait locus (QTL) mapping approach, 76 and 101 main-effect and epistatic QTLs were identified, respectively. Most of the epistatic interactions detected were due to loci without detectable QTL additive main effects. Main and epistatic QTLs detected were mainly consistent across the environment conditions. The homologous genomic regions corresponding to 26 of the 76 main-effect detected QTLs were positive for the presence of resistance-associated gene cluster encoding nucleotide-binding and leucine-rich repeat (NL) proteins and known defence genes. Main-effect QTLs for resistance to races 3, 4 and 5 in leaf, stem and pod were located on chromosome 2 within a 3.01-Mb region, where a cluster of nine NL genes was detected. The NL gene Phvul.002G323300 is located in this region, which can be considered an important putative candidate gene for the non-organ-specific QTL identified here. The present research provides essential information not only for the better understanding of the plant-pathogen interaction but also for the application of genomic assisted breeding for halo blight resistance in common bean.     

Development of a genome-wide anchored microsatellite map for common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

A total of 150 microsatellite markers developed for common bean (Phaseolus vulgaris L.) were tested for parental polymorphism and used to determine the positions of 100 genetic loci on an integrated genetic map of the species. The value of these single-copy markers was evident in their ability to link two existing RFLP-based genetic maps with a base map developed for the Mesoamerican × Andean population, DOR364 × G19833. Two types of microsatellites were mapped, based respectively on gene-coding and anonymous genomic-sequences. Gene-based microsatellites proved to be less polymorphic (46.3%) than anonymous genomic microsatellites (64.3%) between the parents of two inter-genepool crosses. The majority of the microsatellites produced single bands and detected single loci, however four of the gene-based and three of the genomic microsatellites produced consistent double or multiple banding patterns and detected more than one locus. Microsatellite loci were found on each of the 11 chromosomes of common bean, the number per chromosome ranging from 5 to 17 with an average of ten microsatellites each. Total map length for the base map was 1,720 cM and the average chromosome length was 156.4 cM, with an average distance between microsatellite loci of 19.5 cM. The development of new microsatellites from sequences in the Genbank database and the implication of these results for genetic mapping, quantitative trait locus analysis and marker-assisted selection in common bean are described.Communicated by H.F. Linskens     

Regeneration of different cultivars of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) via indirect organogenesis

A protocol for in vitro regeneration via indirect organogenesis for Phaseolus vulgaris cv. Negro Jamapa was established. The explants used were apical meristems and cotyledonary nodes dissected from the embryonic axes of germinating seeds. Several auxin/cytokinin combinations were tested for callus induction. The best callus production was obtained with medium containing 1.5 μM 2,4-dichlorophenoxyacetic acid. After 2 weeks of growth calli were transferred to shooting medium containing 22.2 μM 6-benzylaminopurine. Shoots regenerated with a frequency of approximately 0.5 shoots per callus, and upon transfer to rooting medium these shoots produced roots with 100% efficiency. Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Greenhouse grown regenerated plants showed normal development and were fertile. The protocol was reproducible for other nine P. vulgaris cultivars tested, suggesting a genotype independent procedure.     

Plasmid content of salt stress-tolerant <Emphasis Type="Italic">Rhizobium</Emphasis> strains from Egyptian soils nodulating common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Plasmid profile analysis is useful to characterize Rhizobium strains within the same species. Among the 16 Rhizobium strains examined, 14 had distinct plasmid profiles. The size of plasmids ranged from 40 to 650 kb, and three plasmids of 650, 510 and 390 kb were common to several strains. Plasmid analysis revealed that Rhizobium etli contained a mega-plasmid, similar in size to Rhizobium tropici. All the salt-tolerant strains examined had a plasmid of 250 kb, except for strain EBRI 29. This suggests that this plasmid may play an important adaptive role under salt stress conditions.     

Mapping QTL for climbing ability and component traits in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Common bean (Phaseolus vulgaris L.) varies in growth habit from aggressive climbing types to bush beans. Growth habit is determined by a combination of factors including determinate versus indeterminate growth, total plant height, degree of branching and internode length. Together these factors make up climbing ability. The objective of this research was to determine the quantitative trait loci (QTL) controlling climbing ability in a F_5:8 recombinant inbred line population derived from an inter-gene pool cross of an aggressive indeterminate climbing bean with type IV growth habit (G2333) by an indeterminate bush bean of type IIb growth habit (G19839). The population was planted in four randomized complete block design experiments across environments that varied in altitude (from 1,000 to 1,750 masl) and soil fertility (low versus high phosphorus). QTL were identified for plant height, internode length and number of branches per plant on a genetic map covering all common bean linkage groups with a total length of 1,175 cM. In addition a scale was developed to evaluate overall climbing ability and was also used to identify QTL. A total of 7 QTL were found for plant height, 9 for climbing ability, 6 for internode length and 1 for branch number. The largest number and most significant QTL were found on the lower half of linkage group B04 suggesting a major pleiotropic locus for growth habit traits at this location of the genome that is distinct from previously characterized genes which control plant morphology of the crop.     

Characterisation of structural genes involved in phytic acid biosynthesis in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Phytate (myo-inositol hexakisphosphate), the major form of phosphorous storage in plant seeds, is an inositol phosphate compound poorly digested by humans and monogastric animals. A major goal for grain crop improvement is the reduction of its content in the seed to improve micronutrient bioavailability and phosphorus utilisation by humans and non-ruminant animals, respectively. We are interested in lowering phytic acid in common bean seed and to this goal we have undertaken a two-strategy approach: the isolation of mutants from an EMS mutagenised population (Campion et al. 2009) and the identification of genes coding for candidate enzymes involved in inositol phosphate metabolism for future targeted mutant isolation and/or study. In this paper we report data referred to the second approach and concerning the isolation and genomic organisation of Phaseolus vulgaris genes coding for myo-inositol 1-phosphate synthase (PvMIPSs and PvMIPSv), inositol monophosphatase (PvIMP), myo-inositol kinase (PvMIK), inositol 1,4,5-tris-phosphate kinase (PvIPK2), inositol 1,3,4-triphosphate 5/6-kinase (PvITPKα and PvITPKβ) and inositol 1,3,4,5,6 pentakisphosphate 2-kinase (PvIPK1). All these genes have been mapped on the common bean reference genetic map of McClean (NDSU) 2007 using a virtual mapping strategy. Bean markers, presumably associated to each gene of the phytic acid pathway, have also been identified. In addition, we provide a picture of the expression, during seed development, of the genes involved in phytic acid synthesis, including those such as MIK, IMP and IPK2, for which this information was lacking.     

Nucleotide diversity of a genomic sequence similar to <Emphasis Type="Italic">SHATTERPROOF</Emphasis> (<Emphasis Type="Italic">PvSHP1</Emphasis>) in domesticated and wild common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Evolutionary studies in plant and animal breeding are aimed at understanding the structure and organization of genetic variations of species. We have identified and characterized a genomic sequence in Phaseolus vulgaris of 1,200 bp (PvSHP1) that is homologous to SHATTERPROOF-1 (SHP1), a gene involved in control of fruit shattering in Arabidopsis thaliana. The PvSHP1 fragment was mapped to chromosome Pv06 in P. vulgaris and is linked to the flower and seed color gene V. Amplification of the PvSHP1 sequence from the most agronomically important legume species showed a high degree of interspecies diversity in the introns within the Phaseoleae, while the coding region was conserved across distant taxa. Sequencing of the PvSHP1 sequence in a sample of 91 wild and domesticated genotypes that span the geographic distribution of this species in the centers of origin showed that PvSHP1 is highly polymorphic and, therefore, particularly useful to further investigate the origin and domestication history of P. vulgaris. Our data confirm the gene pool structure seen in P. vulgaris along with independent domestication processes in the Andes and Mesoamerica; they provide additional evidence for a single domestication event in Mesoamerica. Moreover, our results support the Mesoamerican origin of this species. Finally, we have developed three indel-spanning markers that will be very useful for bean germplasm characterization, and particularly to trace the distribution of the domesticated Andean and Mesoamerican gene pools.     

Characterization of AT-rich microsatellites in common bean (<Emphasis Type=Italic>Phaseolus vulgaris</Emphasis> L.)

Polymorphism of microsatellite markers is often associated with the simple sequence repeat motif targeted. AT-rich microsatellites tend to be highly variable and this appears to be notable, especially in legume genomes. To analyze the value of AT-rich microsatellites for common bean (Phaseolus vulgaris L.), we developed a total of 85 new microsatellite markers, 74 of which targeted ATA or other AT-rich motif loci and 11 of which were made for GA, CA or CAC motif loci. We evaluated the loci for the level of allelic diversity in comparison to previously characterized microsatellites using a panel of 18 standard genotypes and genetically mapped any loci polymorphic in the DOR364 × G19833 population. The majority of the microsatellites produced single bands and detected single loci, however, 15 of the AT-rich microsatellites produced multiple or double banding patterns; while only one of the GA or CA-rich microsatellites did. The polymorphism information content (PIC) values averaged 0.892 and 0.600 for the AT and ATA motif microsatellites, respectively, but only 0.140 for the CA-rich microsatellites. GA microsatellites, which had a large average number of repeats, had high to intermediate PIC, averaging 0.706. A total of 45 loci could be genetically mapped and distribution of the loci across the genome was skewed towards non-distal locations with a greater prevalence of loci on linkage groups b02, b09 and b11. AT-rich microsatellites were found to be a useful source of polymorphic markers for mapping and diversity assessment in common bean that appears to uncover higher diversity than other types of simple sequence repeat markers.     

In silico identification and characterization of putative differentially expressed genes involved in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) seed development

Two genotypes of common bean (Phaseolus vulgaris L.) were studied to determine the structural cause of seed abortion in this species. In the non-abortive control (wild-type, cultivar BAT93), the histological analysis revealed a classical pattern of seed development and showed coordinated differentiation of the embryo proper, suspensor, endosperm tissue and seed coat. In contrast, the ethyl methanesulfonate (EMS) mutant (cultivar BAT93) showed disruption in the normal seed development leading to embryo abortion. Aborted embryos from these degenerate seeds showed abnormalities in suspensor and cotyledons at the globular, heart, torpedo and cotyledon stages. Exploring the feasibility of incorporating the available online bioinformatics databases, we identified 22 genes revealing high homology with genes involved in Arabidopsis thaliana embryo development and expressed in common bean immature seeds. The expression patterns of these genes were confirmed by RT–PCR. All genes were highly expressed in seed tissues. To study the expression profiles of isolated genes during Phaseolus embryogenesis, six selected genes were examined by quantitative RT–PCR analysis on the developing embryos of wild-type and EMS mutant plants. All selected genes were expressed differentially at different stages of embryo development. These results could help to improve understanding of the mechanism of common bean embryogenesis.     

Association mapping of days to flowering in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) revealed by DArT markers

In the current study, 173 common bean genotypes from several geographic regions were studied. Days to flowering (DF) was evaluated in two experimental locations in Izmir, Turkey (Bornova and Menemen) in 2 years (2015 and 2016) and was found to range from 30 to 62.7 days with a mean value of 41.5 days. A total of 22,848 SNPs based on diversity array technology were developed, and after filtering, the remaining 20,766 SNP markers were used for calculating linkage disequilibrium. Chromosomes 1–11 contained 1846, 2342, 2184, 1153, 1351, 1520, 1953, 2080, 2065, 1199, and 1511 SNPs, respectively. A total of 1562 SNPs were identified as scaffold markers. The PIC value was 0.25, ranging from 0.005 to 0.500. Common bean accessions were divided into two main subpopulations, namely POP1 (Mesoamerican) and POP2 (Andean). Mixed linear model using the Q + K model showed that three SNPs had a significant association (p?<?0.01) in Bornova in 2015 and seven SNPs had a significant association (p?<?0.01) in the same location in 2016. Five significant associations (p?<?0.01) were identified in 2015 while six (p?<?0.01) were identified in Menemen in 2016. When the data from both locations and both years was combined, six SNPs were significant (p?<?0.01). For DF, 11 putative candidate genes were predicted from the sequences representing homology to linked SNPs. We conclude that the markers, which were significantly associated with the DF of the common bean genotypes in the current study, can be used for marker-assisted selection in plant breeding program of common bean.     

The plant growth promoting bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> is vertically transmitted in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> (common bean)

Bactrocera dorsalis (Diptera: Tephritidae) is a serious menace to agricultural production worldwide. In order to prevent further damage, it is of paramount important that cost-effective strategies should be developed for their management. Gut bacteria has established diverse relationships with their insect hosts, which could be exploited in pest management programs to improve on control efficiency. In this study, gut bacteria isolates identified by culture dependent technique were incorporated into larval diets in an attempt to understand the roles they play in the development and survival of oriental fruit fly. From our results, the isolated bacteria belonged to four different phyla including the Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. The response of the fly to different gut isolates varied greatly. Diets enriched with Enterococcus phoeniculicola had lower larval developmental duration, higher pupal weight, and an increased percentage survival. On the other hand, diets supplemented with Lactobacillus lactis had negative effects on B. dorsalis development. This study provides clues on how symbiotic bacteria could be exploited in mass rearing for an efficient implementation of the Sterile Insect Technique (SIT) in pest management programs.     

Quantitative trait loci for root morphology traits under aluminum stress in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Aluminum (Al) toxicity is a major limiting factor of crop production in acid soils, which are found mostly in developing countries of the tropics and sub-tropics. Common bean (Phaseolus vulgaris L.) is particularly sensitive to Al toxicity; and development of genotypes with better root growth in Al-toxic soils is a priority. The objectives of the present study were to physiologically assess root architectural traits in a recombinant inbred line (RIL) population of common bean that contrasts for Al resistance (DOR364 × G19833) and to identify quantitative trait loci (QTL) controlling root growth under two nutrient solutions, one with 20 μM Al concentration and the other without Al, both at pH 4.5. A total of 24 QTL were found through composite interval mapping analysis, 9 for traits under Al treatment, 8 for traits under control treatment, and 7 for relative traits. Root characteristics expressed under Al treatment were found to be under polygenic control, and some QTL were identified at the same location as QTL for tolerance to low phosphorous stress, thus, suggesting cross-links in genetic control of adaptation of common bean to different abiotic stresses.     

Inheritance of seed condensed tannins and their relationship with seed-coat color and pattern genes in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Condensed tannins are major flavonoid end products that affect the nutritional quality of many legume seeds. They chelate minerals and interact with proteins, thus reducing their bioavailability. Tannins also contribute to seed coat color and pigment distribution or intensity. The objective of this study was to analyze the relationship between quantitative trait loci (QTL) for seed tannin concentration in common bean and Mendelian genes for seed coat color and pattern. Three populations of recombinant inbred lines, derived from crosses between the Andean and Mesoamerican genepools were used for QTL identification and for mapping STS markers associated with seed color loci. Seed coat condensed tannins were determined with a butanol–HCl method and a total of 12 QTL were identified on separate linkage groups (LGs) in each of the populations with individual QTL explaining from 10 to 64% of the phenotypic variation for this trait. Loci on linkage groups B3 and B10 were associated with the Mendelian genes Z and Bip for partly colored seed coat pattern, while a QTL on linkage group B7 was associated with the P gene which is the primary locus for the control of color expression in beans. In conclusion, this study found that the inheritance of tannin concentration fits an oligogenic model and identifies novel putative alleles at seed coat color and pattern genes that control tannin accumulation. The results will be important for the genetic improvement of nutritionally enhanced or biofortified beans that have health promoting effects from higher polyphenolics or better iron bioavailability.