Microscopical Analysis of Autofluorescence as a Complementary and Useful Method to Assess Differences in Anatomy and Structural Distribution Underlying Evolutive Variation in Loss of Seed Dispersal in Common Bean.

The common bean has received attention as a model plant for legume studies, but little information is available about the morphology of its pods and the relation of this morphology to the loss of seed dispersal and/or the pod string, which are key agronomic traits of legume domestication. Dehiscence is related to the pod morphology and anatomy of pod tissues because of the weakening of the dorsal and ventral dehiscence zones and the tensions of the pod walls. These tensions are produced by the differential mechanical properties of lignified and non-lignified tissues and changes in turgor associated with fruit maturation. In this research, we histologically studied the dehiscence zone of the ventral and dorsal sutures of the pod in two contrasting genotypes for the dehiscence and string, by comparing different histochemical methods with autofluorescence. We found that the secondary cell wall modifications of the ventral suture of the pod were clearly different between the dehiscence-susceptible and stringy PHA1037 and the dehiscence-resistant and stringless PHA0595 genotypes. The susceptible genotype had cells of bundle caps arranged in a more easily breakable bowtie knot shape. The resistant genotype had a larger vascular bundle area and larger fibre cap cells (FCCs), and due to their thickness, the external valve margin cells were significantly stronger than those from PHA1037. Our findings suggest that the FCC area, and the cell arrangement in the bundle cap, might be partial structures involved in the pod dehiscence of the common bean. The autofluorescence pattern at the ventral suture allowed us to quickly identify the dehiscent phenotype and gain a better understanding of cell wall tissue modifications that took place along the bean's evolution, which had an impact on crop improvement. We report a simple autofluorescence protocol to reliably identify secondary cell wall organization and its relationship to the dehiscence and string in the common bean.

Decoding Gene Expression Signatures Underlying Vegetative to Inflorescence Meristem Transition in the Common Bean.

The tropical common bean (Phaseolus vulgaris L.) is an obligatory short-day plant that requires relaxation of the photoperiod to induce flowering. Similar to other crops, photoperiod-induced floral initiation depends on the differentiation and maintenance of meristems. In this study, the global changes in transcript expression profiles were analyzed in two meristematic tissues corresponding to the vegetative and inflorescence meristems of two genotypes with different sensitivities to photoperiods. A total of 3396 differentially expressed genes (DEGs) were identified, and 1271 and 1533 were found to be up-regulated and down-regulated, respectively, whereas 592 genes showed discordant expression patterns between both genotypes. Arabidopsis homologues of DEGs were identified, and most of them were not previously involved in Arabidopsis floral transition, suggesting an evolutionary divergence of the transcriptional regulatory networks of the flowering process of both species. However, some genes belonging to the photoperiod and flower development pathways with evolutionarily conserved transcriptional profiles have been found. In addition, the flower meristem identity genes APETALA1 and LEAFY, as well as CONSTANS-LIKE 5, were identified as markers to distinguish between the vegetative and reproductive stages. Our data also indicated that the down-regulation of the photoperiodic genes seems to be directly associated with promoting floral transition under inductive short-day lengths. These findings provide valuable insight into the molecular factors that underlie meristematic development and contribute to understanding the photoperiod adaptation in the common bean.

Ancient relaxation of an obligate short-day requirement in common bean through loss of CONSTANS-like gene function.

Common bean (Phaseolus vulgaris L.) is a major global food staple and source of dietary protein that was domesticated independently in Mexico and Andean South America. Its subsequent development as a crop of importance worldwide has been enabled by genetic relaxation of the strict short-day requirement typical of wild forms, but the genetic basis for this change is not well understood. Recently, a loss of photoperiod sensitivity was shown to result from mutations in the phytochrome photoreceptor gene Ppd/PHYA3 that arose independently within the two major domesticated lineages. Here, we define a second major photoperiod sensitivity locus, at which recessive alleles associate with deleterious mutations affecting the CONSTANS-like gene COL2. A wider survey of sequence variation in over 800 diverse lines, including wild, landrace, and domesticated accessions, show that distinct col2 haplotypes are associated with early flowering in Andean and Mesoamerican germplasm. The relative frequencies and distributions of COL2 and PHYA3 haplotypes imply that photoperiod adaptation developed in two phases within each gene pool: an initial reduction in sensitivity through impairment of COL2 function and subsequent complete loss through PHYA3. Gene expression analyses indicate that COL2 functions downstream of PHYA3 to repress expression of FT genes and may function in parallel with PvE1, the bean ortholog of a key legume-specific flowering repressor. Collectively, these results define the molecular basis for a key phenological adaptation, reveal a striking convergence in the naturally replicated evolution of this major crop, and further emphasize the wider evolutionary lability of CONSTANS effects on flowering time control.

Transcriptional Dynamics and Candidate Genes Involved in Pod Maturation of Common Bean (Phaseolus vulgaris L.).

Pod maturation of common bean relies upon complex gene expression changes, which in turn are crucial for seed formation and dispersal. Hence, dissecting the transcriptional regulation of pod maturation would be of great significance for breeding programs. In this study, a comprehensive characterization of expression changes has been performed in two common bean cultivars (ancient and modern) by analyzing the transcriptomes of five developmental pod stages, from fruit setting to maturation. RNA-seq analysis allowed for the identification of key genes shared by both accessions, which in turn were homologous to known Arabidopsis maturation genes and furthermore showed a similar expression pattern along the maturation process. Gene- expression changes suggested a role in promoting an accelerated breakdown of photosynthetic and ribosomal machinery associated with chlorophyll degradation and early activation of alpha-linolenic acid metabolism. A further study of transcription factors and their DNA binding sites revealed three candidate genes whose functions may play a dominant role in regulating pod maturation. Altogether, this research identifies the first maturation gene set reported in common bean so far and contributes to a better understanding of the dynamic mechanisms of pod maturation, providing potentially useful information for genomic-assisted breeding of common bean yield and pod quality attributes.

Characterization of QTL and Environmental Interactions Controlling Flowering Time in Andean Common Bean (Phaseolus vulgaris L.).

Genetic variation for response of flowering time to photoperiod plays an important role in adaptation to environments with different photoperiods, and as consequence is an important contributor to plant productivity and yield. To elucidate the genetic control of flowering time [days to flowering (DTF); growing degree days (GDD)] in common bean, a facultative short-day plant, a quantitative trait loci (QTL) analysis was performed in a recombinant inbred mapping population derived from a cultivated accession and a photoperiod sensitive landrace, grown in different long-day (LD) and short-day (SD) environments by using a multiple-environment QTL model approach. A total of 37 QTL across 17 chromosome regions and 36 QTL-by-QTL interactions were identified for six traits associated with time to flowering and response to photoperiod. The DTF QTL accounted for 28 and 11% on average of the phenotypic variation in the population across LD and SD environments, respectively. Of these, a genomic region on chromosome 4 harboring the major DTF QTL was associated with both flowering time in LD and photoperiod response traits, controlling more than 60% of phenotypic variance, whereas a major QTL on chromosome 9 explained up to 32% of flowering time phenotypic variation in SD. Different epistatic interactions were found in LD and SD environments, and the presence of significant QTL × environment (QE) and epistasis × environment interactions implies that flowering time control may rely on different genes and genetic pathways under inductive and non-inductive conditions. Here, we report the identification of a novel major locus controlling photoperiod sensitivity on chromosome 4, which might interact with other loci for controlling common bean flowering time and photoperiod response. Our results have also demonstrated the importance of these interactions for flowering time control in common bean, and point to the likely complexity of flowering time pathways. This knowledge will help to identify and develop opportunities for adaptation and breeding of this legume crop.

Parallel origins of photoperiod adaptation following dual domestications of common bean.

Common bean (Phaseolus vulgaris L.) is an important grain legume domesticated independently in Mexico and Andean South America approximately 8000 years ago. Wild forms are obligate short-day plants, and relaxation of photoperiod sensitivity was important for expansion to higher latitudes and subsequent global spread. To better understand the nature and origin of this key adaptation, we examined its genetic control in progeny of a wide cross between a wild accession and a photoperiod-insensitive cultivar. We found that photoperiod sensitivity is under oligogenic control, and confirm a major effect of the Ppd locus on chromosome 1. The red/far-red photoreceptor gene PHYTOCHROME A3 (PHYA3) was identified as a strong positional candidate for Ppd, and sequencing revealed distinct deleterious PHYA3 mutations in photoperiod-insensitive Andean and Mesoamerican accessions. These results reveal the independent origins of photoperiod insensitivity within the two major common bean gene pools and demonstrate the conserved importance of PHYA genes in photoperiod adaptation of short-day legume species.

Dissection of Resistance Genes to Pseudomonas syringae pv. phaseolicola in UI3 Common Bean Cultivar.

Few quantitative trait loci have been mapped for resistance to Pseudomonas syringae pv. phaseolicola in common bean. Two F2 populations were developed from the host differential UI3 cultivar. The objective of this study was to further characterize the resistance to races 1, 5, 7 and 9 of Psp included in UI3. Using a QTL mapping approach, 16 and 11 main-effect QTLs for pod and primary leaf resistance were located on LG10, explaining up to 90% and 26% of the phenotypic variation, respectively. The homologous genomic region corresponding to primary leaf resistance QTLs detected tested positive for the presence of resistance-associated gene cluster encoding nucleotide-binding and leucine-rich repeat (NL), Natural Resistance Associated Macrophage (NRAMP) and Pentatricopeptide Repeat family (PPR) proteins. It is worth noting that the main effect QTLs for resistance in pod were located inside a 3.5 Mb genomic region that included the Phvul.010G021200 gene, which encodes a protein that has the highest sequence similarity to the RIN4 gene of Arabidopsis, and can be considered an important candidate gene for the organ-specific QTLs identified here. These results support that resistance to Psp from UI3 might result from the immune response activated by combinations of R proteins, and suggest the guard model as an important mechanism in pod resistance to halo blight. The candidate genes identified here warrant functional studies that will help in characterizing the actual defense gene(s) in UI3 genotype.

Genomic history of the origin and domestication of common bean unveils its closest sister species.

BACKGROUND: Modern civilization depends on only a few plant species for its nourishment. These crops were derived via several thousands of years of human selection that transformed wild ancestors into high-yielding domesticated descendants. Among cultivated plants, common bean (Phaseolus vulgaris L.) is the most important grain legume. Yet, our understanding of the origins and concurrent shaping of the genome of this crop plant is limited. RESULTS: We sequenced the genomes of 29 accessions representing 12 Phaseolus species. Single nucleotide polymorphism-based phylogenomic analyses, using both the nuclear and chloroplast genomes, allowed us to detect a speciation event, a finding further supported by metabolite profiling. In addition, we identified ~1200 protein coding genes (PCGs) and ~100 long non-coding RNAs with domestication-associated haplotypes. Finally, we describe asymmetric introgression events occurring among common bean subpopulations in Mesoamerica and across hemispheres. CONCLUSIONS: We uncover an unpredicted speciation event in the tropical Andes that gave rise to a sibling species, formerly considered the "wild ancestor" of P. vulgaris, which diverged before the split of the Mesoamerican and Andean P. vulgaris gene pools. Further, we identify haplotypes strongly associated with genes underlying the emergence of domestication traits. Our findings also reveal the capacity of a predominantly autogamous plant to outcross and fix loci from different populations, even from distant species, which led to the acquisition by domesticated beans of adaptive traits from wild relatives. The occurrence of such adaptive introgressions should be exploited to accelerate breeding programs in the near future.

Natural rhizobial diversity helps to reveal genes and QTLs associated with biological nitrogen fixation in common bean.

Common bean is one of the most important crops for human feed, and the most important legume for direct consumption by millions of people, especially in developing countries. It is a promiscuous host legume in terms of nodulation, able to associate with a broad and diverse range of rhizobia, although the competitiveness for nodulation and the nitrogen fixation capacity of most of these strains is generally low. As a result, common bean is very inefficient for symbiotic nitrogen fixation, and nitrogen has to be supplied with chemical fertilizers. In the last years, symbiotic nitrogen fixation has received increasing attention as a sustainable alternative to nitrogen fertilizers, and also as a more economic and available one in poor countries. Therefore, optimization of nitrogen fixation of bean-rhizobia symbioses and selection of efficient rhizobial strains should be a priority, which begins with the study of the natural diversity of the symbioses and the rhizobial populations associated. Natural rhizobia biodiversity that nodulates common bean may be a source of adaptive alleles acting through phenotypic plasticity. Crosses between accessions differing for nitrogen fixation may combine alleles that never meet in nature. Another way to discover adaptive genes is to use association genetics to identify loci that common bean plants use for enhanced biological nitrogen fixation and, in consequence, for marker assisted selection for genetic improvement of symbiotic nitrogen fixation. In this review, rhizobial biodiversity resources will be discussed, together with what is known about the loci that underlie such genetic variation, and the potential candidate genes that may influence the symbiosis' fitness benefits, thus achieving an optimal nitrogen fixation capacity in order to help reduce reliance on nitrogen fertilizers in common bean.

Seedling Emergence and Phenotypic Response of Common Bean Germplasm to Different Temperatures under Controlled Conditions and in Open Field.

Rapid and uniform seed germination and seedling emergence under diverse environmental conditions is a desirable characteristic for crops. Common bean genotypes (Phaseolus vulgaris L.) differ in their low temperature tolerance regarding growth and yield. Cultivars tolerant to low temperature during the germination and emergence stages and carriers of the grain quality standards demanded by consumers are needed for the success of the bean crop. The objectives of this study were (i) to screen the seedling emergence and the phenotypic response of bean germplasm under a range of temperatures in controlled chamber and field conditions to display stress-tolerant genotypes with good agronomic performances and yield potential, and (ii) to compare the emergence of bean seedlings under controlled environment and in open field conditions to assess the efficiency of genebanks standard germination tests for predicting the performance of the seeds in the field. Three trials were conducted with 28 dry bean genotypes in open field and in growth chamber under low, moderate, and warm temperature. Morpho-agronomic data were used to evaluate the phenotypic performance of the different genotypes. Cool temperatures resulted in a reduction of the rate of emergence in the bean genotypes, however, emergence and early growth of bean could be under different genetic control and these processes need further research to be suitably modeled. Nine groups arose from the Principal Component Analysis (PCA) representing variation in emergence time and proportion of emergence in the controlled chamber and in the open field indicating a trend to lower emergence in large and extra-large seeded genotypes. Screening of seedling emergence and phenotypic response of the bean germplasm under a range of temperatures in controlled growth chambers and under field conditions showed several genotypes, as landraces 272, 501, 593, and the cultivar Borlotto, with stress-tolerance at emergence, and high yield potential that could be valuable genetic material for breeding programs. Additionally, the potential genetic erosion in genebanks was assessed. Regarding bean commercial traits, under low temperature at sowing time seed reached larger size, and crop yield was higher compared to warmer temperatures at the sowing time. Therefore, early sowing of bean is strongly recommended.

Genome and transcriptome analysis of the Mesoamerican common bean and the role of gene duplications in establishing tissue and temporal specialization of genes.

BACKGROUND: Legumes are the third largest family of angiosperms and the second most important crop class. Legume genomes have been shaped by extensive large-scale gene duplications, including an approximately 58 million year old whole genome duplication shared by most crop legumes. RESULTS: We report the genome and the transcription atlas of coding and non-coding genes of a Mesoamerican genotype of common bean (Phaseolus vulgaris L., BAT93). Using a comprehensive phylogenomics analysis, we assessed the past and recent evolution of common bean, and traced the diversification of patterns of gene expression following duplication. We find that successive rounds of gene duplications in legumes have shaped tissue and developmental expression, leading to increased levels of specialization in larger gene families. We also find that many long non-coding RNAs are preferentially expressed in germ-line-related tissues (pods and seeds), suggesting that they play a significant role in fruit development. Our results also suggest that most bean-specific gene family expansions, including resistance gene clusters, predate the split of the Mesoamerican and Andean gene pools. CONCLUSIONS: The genome and transcriptome data herein generated for a Mesoamerican genotype represent a counterpart to the genomic resources already available for the Andean gene pool. Altogether, this information will allow the genetic dissection of the characters involved in the domestication and adaptation of the crop, and their further implementation in breeding strategies for this important crop.

Major Contribution of Flowering Time and Vegetative Growth to Plant Production in Common Bean As Deduced from a Comparative Genetic Mapping.

Determinacy growth habit and accelerated flowering traits were selected during or after domestication in common bean. Both processes affect several presumed adaptive traits such as the rate of plant production. There is a close association between flowering initiation and vegetative growth; however, interactions among these two crucial developmental processes and their genetic bases remain unexplored. In this study, with the aim to establish the genetic relationships between these complex processes, a multi-environment quantitative trait locus (QTL) mapping approach was performed in two recombinant inbred line populations derived from inter-gene pool crosses between determinate and indeterminate genotypes. Additive and epistatic QTLs were found to regulate flowering time, vegetative growth, and rate of plant production. Moreover, the pleiotropic patterns of the identified QTLs evidenced that regions controlling time to flowering traits, directly or indirectly, are also involved in the regulation of plant production traits. Further QTL analysis highlighted one QTL, on the lower arm of the linkage group Pv01, harboring the Phvul.001G189200 gene, homologous to the Arabidopsis thaliana TERMINAL FLOWER1 (TFL1) gene, which explained up to 32% of phenotypic variation for time to flowering, 66% for vegetative growth, and 19% for rate of plant production. This finding was consistent with previous results, which have also suggested Phvul.001G189200 (PvTFL1y) as a candidate gene for determinacy locus. The information here reported can also be applied in breeding programs seeking to optimize key agronomic traits, such as time to flowering, plant height and an improved reproductive biomass, pods, and seed size, as well as yield.

Uncovering the genetic architecture of Colletotrichum lindemuthianum resistance through QTL mapping and epistatic interaction analysis in common bean.

Colletotrichum lindemuthianum is a hemibiotrophic fungal pathogen that causes anthracnose disease in common bean. Despite the genetics of anthracnose resistance has been studied for a long time, few quantitative trait loci (QTLs) studies have been conducted on this species. The present work examines the genetic basis of quantitative resistance to races 23 and 1545 of C. lindemuthianum in different organs (stem, leaf and petiole). A population of 185 recombinant inbred lines (RIL) derived from the cross PMB0225 × PHA1037 was evaluated for anthracnose resistance under natural and artificial photoperiod growth conditions. Using multi-environment QTL mapping approach, 10 and 16 main effect QTLs were identified for resistance to anthracnose races 23 and 1545, respectively. The homologous genomic regions corresponding to 17 of the 26 main effect QTLs detected were positive for the presence of resistance-associated gene cluster encoding nucleotide-binding and leucine-rich repeat (NL) proteins. Among them, it is worth noting that the main effect QTLs detected on linkage group 05 for resistance to race 1545 in stem, petiole and leaf were located within a 1.2 Mb region. The NL gene Phvul.005G117900 is located in this region, which can be considered an important candidate gene for the non-organ-specific QTL identified here. Furthermore, a total of 39 epistatic QTL (E-QTLs) (21 for resistance to race 23 and 18 for resistance to race 1545) involved in 20 epistatic interactions (eleven and nine interactions for resistance to races 23 and 1545, respectively) were identified. None of the main and epistatic QTLs detected displayed significant environment interaction effects. 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 anthracnose resistance improvement in common bean through application of marker-assisted selection (MAS).

Genetic analysis of single-locus and epistatic QTLs for seed traits in an adapted × nuña RIL population of common bean (Phaseolus vulgaris L.).

KEY MESSAGE: The QTLs analyses here reported demonstrate the significant role of both individual additive and epistatic effects in the genetic control of seed quality traits in the Andean common bean. Common bean shows considerable variability in seed size and coat color, which are important agronomic traits determining farmer and consumer acceptability. Therefore, strategies must be devised to improve the genetic base of cultivated germplasm with new alleles that would contribute positively to breeding programs. For that purpose, a population of 185 recombinant inbred lines derived from an Andean intra-gene pool cross, involving an adapted common bean (PMB0225 parent) and an exotic nuña bean (PHA1037 parent), was evaluated under six different--short and long-day--environmental conditions for seed dimension, weight, color, and brightness traits, as well as the number of seed per pod. A multi-environment Quantitative Trait Loci (QTL) analysis was carried out and 59 QTLs were mapped on all linkage groups, 18 of which had only individual additive effects, while 27 showed only epistatic effects and 14 had both individual additive and epistatic effects. Multivariate models that included significant QTL explained from 8 to 68 % and 2 to 15 % of the additive and epistatic effects, respectively. Most of these QTLs were consistent over environment, though interactions between QTLs and environments were also detected. Despite this, QTLs with differential effect on long-day and short-day environments were not found. QTLs identified were positioned in cluster, suggesting that either pleiotropic QTLs control several traits or tightly linked QTLs for different traits map together in the same genomic regions. Overall, our results show that digenic epistatic interactions clearly play an important role in the genetic control of seed quality traits in the Andean common bean.

Marker-based linkage map of Andean common bean (Phaseolus vulgaris L.) and mapping of QTLs underlying popping ability traits.

BACKGROUND: Nuña bean is a type of ancient common bean (Phaseolus vulgaris L.) native to the Andean region of South America, whose seeds possess the unusual property of popping. The nutritional features of popped seeds make them a healthy low fat and high protein snack. However, flowering of nuña bean only takes place under short-day photoperiod conditions, which means a difficulty to extend production to areas where such conditions do not prevail. Therefore, breeding programs of adaptation traits will facilitate the diversification of the bean crops and the development of new varieties with enhanced healthy properties. Although the popping trait has been profusely studied in maize (popcorn), little is known about the biology and genetic basis of the popping ability in common bean. To obtain insights into the genetics of popping ability related traits of nuña bean, a comprehensive quantitative trait loci (QTL) analysis was performed to detect single-locus and epistatic QTLs responsible for the phenotypic variance observed in these traits. RESULTS: A mapping population of 185 recombinant inbred lines (RILs) derived from a cross between two Andean common bean genotypes was evaluated for three popping related traits, popping dimension index (PDI), expansion coefficient (EC), and percentage of unpopped seeds (PUS), in five different environmental conditions. The genetic map constructed included 193 loci across 12 linkage groups (LGs), covering a genetic distance of 822.1 cM, with an average of 4.3 cM per marker. Individual and multi-environment QTL analyses detected a total of nineteen single-locus QTLs, highlighting among them the co-localized QTLs for the three popping ability traits placed on LGs 3, 5, 6, and 7, which together explained 24.9, 14.5, and 25.3% of the phenotypic variance for PDI, EC, and PUS, respectively. Interestingly, epistatic interactions among QTLs have been detected, which could have a key role in the genetic control of popping. CONCLUSIONS: The QTLs here reported constitute useful tools for marker assisted selection breeding programs aimed at improving nuña bean cultivars, as well as for extending our knowledge of the genetic determinants and genotype x environment interaction involved in the popping ability traits of this bean crop.