Genetic structure of Lima bean (<Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L.) landraces grown in the Mayan area

Lima bean (Phaseolus lunatus L.) is an important crop in the Mayan culture. The Mayan area, considered as a main center of Mesoamerican diversity, has been divided into two subareas: the Mayan lowlands and the Mayan highlands. The Yucatan Peninsula is part of the Mayan lowlands and holds the highest number of Lima bean landraces of Mexico, but Lima beans are in high risk of genetic erosion due to intensification of the traditional Mayan agriculture. However, information on genetic diversity of Lima beans of the Mayan highlands is lacking. By using 46 landraces collected in the Mayan area (23 from each subarea) and 73 ISSR loci (inter-simple sequence repeats), we analyzed the structure, diversity and genetic relationships of Lima beans of this part of Mesoamerica. High levels of diversity (H _BAY = 0.45) and genetic structure (F _ST = 0.66) were found for the whole Mayan area. Genetic diversity in the Mayan lowlands was apparently higher than the Mayan highlands (H _BAY = 0.44 and 0.36, respectively); but differences were not statistically significant. Genetic structure between the subareas was high (AMOVA = 30% of total variation), most landraces grouping according to their geographic origin. This study shows the importance of the Mayan culture in the diversification and conservation of Lima beans. The results provide important information that should be considered when implementing strategies to collect Lima bean landraces and planning in situ and ex situ programs to conserve these landraces in the Mayan region.     

Genetic diversity analysis in Phaseolus vulgaris L. using morphological traits

Common bean (Phaseolus vulgaris) is an important export crop in Kyrgyzstan since the end of the twentieth century. Genetic diversity analysis of common bean populations is useful for breeding programs, as it helps to select genetic material to be used for further crossing. Twenty-seven common bean accessions were analyzed using 13 qualitative morphological traits. In some cases, obtained morphological data were combined with previously published results based on microsatellite markers. The similarity matrices generated from the molecular and morphological data were significantly correlated (r = 0.49, P < 0.01). Cluster analyses based on Dice’s similarity coefficient were constructed based on morphological data and the combined data set of morphology and microsatellite, and both grouped the 27 accessions according to their origin: 15 belonged to the Andean and 12 to the Mesoamerican gene pool. On average, the Andean accessions were less diverse than the Mesoamerican accessions. The average diversity based on the Shannon diversity index for the 13 qualitative morphological traits was 0.05. Overall, this study revealed that qualitative morphological markers are efficient in assigning modern cultivars to their gene pools of origin.     

Italian common bean landraces: diversity and population structure

Phaseolus vulgaris L. is an important species that originated in Mesoamerica. A Mesoamerican and an Andean gene pool are usually distinguished in the domesticated forms. Many bean landraces are still cultivated in Italy and the Department of Applied Biology maintains an ex situ collection of 146 landraces. Although protection schemes are being developed in Italy, most landraces are extinct or at risk of extinction. To facilitate their conservation and use, geographical, morphological, biochemical and molecular (SSR) data were collected and analysed to estimate the diversity and the genetic structure of the collection. Data confirmed that both the Mesoamerican and the Andean gene pools were introduced in Italy and, although a distinction between the two gene pools exists, the Italian landrace diversity is clearly structured in three clusters that are not simply ascribable to the original gene pools. The observed structure appears also to be due to adaptation to the different environmental conditions determined by altitude. This was confirmed by assessing the presence of selective effects for some of the SSR used in this study. Finally, a certain extent of admixture in Italian landrace diversity suggests past (or recurring) hybridisation events among gene pools. The combined use of morphological, biochemical and molecular data clearly distinguished almost all the landraces. The data gathered here can assist landrace in situ protection schemes that are being developed in Italy, be used to register landraces in the European common catalogue of ‘conservation varieties’ for seed commercialisation and contribute to a better use of Italian common bean diversity in breeding for organic and conventional production systems.     

Molecular characterisation of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) accessions from Southwestern Uganda reveal high levels of genetic diversity

Common bean (Phaseolus vulgaris L.) was introduced to East Africa over 400 years ago and is today a fundamental part of food and income security of many smallholder farmers in the region. East Africa is among the top three bean producing and consuming regions of Africa with Uganda, Rwanda and Kenya being leaders in production. The leading common bean producing region in Uganda is the Southwestern (SW) highlands. Production is subsistent and farmers grow mixed varieties (accessions) to minimize losses. The aim of this study was to investigate the impact of farmer selection and production practices on genetic diversity of common bean in SW Uganda. 100 accessions were assembled from SW Uganda and assayed with 6 DNA simple sequence repeat (SSR) markers. A total of 41 alleles were detected giving an overall average gene diversity of 0.299 (30%) in both districts. The accessions clustered into two major gene pools i.e., Mesoamerican and Andean. Within each gene pool there was evidence of clonal populations suggesting wide distribution of certain accessions. Kabale district had a higher average gene diversity (38%) compared to Kisoro district (22%). Detection of Andean phaseolin in a clone set of Mesoamerican gene pool suggests introgession between the two groups.     

The Sierra Norte of Madrid: an agrobiodiversity refuge for common bean landraces

At the mountain area close to the city of Madrid, common beans were one of the main food crops present in everyday diet until 1960. This paper describes the morphological diversity, for forty-six phenological and morphological traits, found in forty-three traditional varieties of common beans collected in this area and seven commercial varieties used as reference. That comparison suggests that common bean breeding programs have led to later varieties with a higher production of straight pods. Quantitative traits showed also similar values when compared to the Iberian common bean collection, except for the phenological and seed size characters. The phenological differences could suggest an ecological adaptation of the studied landraces to the regional environmental conditions. The different seed size averages of both collections might correspond to the scarce presence of smaller seed-type common beans in Madrid collection. All the accessions collected in the Sierra Norte of Madrid belong to any of the groups included in the Spanish core collection. Madrilenian collection is also composed by indeterminate growth habit varieties, while the presence of bush accessions is relatively uncommon. Most of the Madrilenian landraces have a remarkable fitness for green-pod consumption (42 %) and their seeds are mainly white (30 %), ovate-shape (49 %) and medium-large (40 %). The study of seed storage protein allowed to classify the landraces according to their domesticated gene pools. Most of them (72 %), with T and C phaseolin type, seem to belong to Andean germplasm, while the remaining (28 %), with S and B phaseolin type, to Mesoamerican one. The remarkable morphological diversity of common beans found in this small area is a symptom of a broad genetic base despite genetic erosion, what indicates a widespread crop in the past. Therefore, it is advisable to design agro-environmental policies to promote the production and commercialization of common bean landraces in Sierra Norte of Madrid.     

Assessment of genetic variation in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) from Nebrodi mountains (Sicily,Italy)

This study was carried out to estimate the level of diversity existing within some common bean landraces still cultivated in Nebrodi mountains, North-western area of Sicily. The multidisciplinary approach adopted to reach this goal involved the characterisation of collected material through morphological, biochemical and molecular marker analyses. The nutritional quality of seeds was also investigated in view of the proposition of the best landraces as niche products. Results showed that those bean landraces retain a considerable level of heterogeneity. The use of both biochemical and molecular markers showed that all landraces clustered into two main groups, corresponding to the Andean and Mesoamerican gene pools. Our results suggest that the best strategy for preserving the diversity of common bean from a restricted area such as Nebrodi mountains, necessitates of a deep knowledge of germplasm to avoid the loss of precious genetic resources or, on the contrary, the safeguard of populations genetically redundant.     

Multiple domestications of the Mesoamerican gene pool of lima bean (Phaseolus lunatus L.): evidence from chloroplast DNA sequences

The Mesoamerican (MA) gene pool of lima bean (Phaseolus lunatus L.) is widely distributed from northern Mexico to northern Argentina in its wild forms and from the southern United States to the east coast of Brazil in its domesticated forms. This broad distribution and lack of wild accessions of the MA gene pool in many areas of its natural distribution has impeded determining its center of domestication and establishing whether it has a single or multiple centers. To answer these questions, we evaluated 262 accessions of P. lunatus using two intergenic spacers of chloroplast DNA: atpB-rbcL and trnL-trnF. The data were analyzed using a maximum likelihood tree (ML), a haplotype network and two estimators of genetic differentiation (N _ST and G _ST). Nucleotide diversity (π) and haplotype (Hd) were quantified to estimate the percentage of reduction in genetic diversity (%r) as a founder effect. The ML tree and haplotype network indicated the existence of three groups (AI, MI and MII), which was supported by the high values of N _ST (0.61–0.80). Values for %r were high (58.67–60.83 %). Existence of Mesoamerican and Andean gene pools was confirmed, with two genetically and geographically distinct groups (MI and MII) within the MA gene pool. We present the first evidence for multiple origins of domestication for the MA gene pool. For MI, we propose western central Mexico as the domestication area and between Guatemala and Costa Rica for MII. We observed a founder effect in the MA gene pool as a result of domestication.     

Genetic, morphological and cyanogen content evaluation of a new collection of Caribbean Lima bean (Phaseolus lunatus L.) landraces

Lima bean is a species cultivated broadly in the Americas and has been cultivated in the Caribbean for at least 500 years. In order to determine the genetic structure and diversity of Lima bean from the Caribbean, 50 landraces from Haiti, the Dominican Republic and Puerto Rico were collected and analyzed using 24 SSR markers. All landraces in this Caribbean collection were found to be of Middle-American descent. The genetic diversity (H_E) was highest in the landraces from Puerto Rico and lowest in Haitian landraces. The observed proportion of heterozygotes (H_O) was higher in the Haitian landraces and lowest in Puerto Rican landraces. Unweighted pair-group method with arithmetic averaging analysis showed that the landraces clustered into 3 clusters with all Haitian landraces grouping in one cluster. We also assessed the agro-morphological characteristics of the collection as well as the content of cyanogenic glucoside, linamarin, in leaves and dry seeds. Lima bean, which is a model crop for indirect plant defenses against herbivory, also possess linamarin as a source of direct plant defense. Upon tissue damage, linamarin is converted to toxic hydrogen cyanide. In our collection 44.6 % of the landraces had average seed HCN content ≤200 ppm which is the permitted level for Lima bean seed in the US. Our results also identified the landraces in this collection which have high linamarin levels in the leaves while having low levels in the seeds. Such landraces have the desirable combination of traits and will be the focus of our future plans for agronomic trait improvement though breeding.     

Population structure and genetic differentiation among the USDA common bean (Phaseolus vulgaris L.) core collection

Genetic diversity data were collected from a large population of common bean (Phaseolus vulgaris L.) landraces representing the United States Department of Agriculture core collection. The data were based on microsatellite data from all linkage groups. A procedure was developed to determine if we collected sufficient marker data to adequately estimated pairwise diversity. The diversity data were used to define populations using distance and model-based approaches. Genetic differentiation and genetic isolation by distance data were collected. Diversity was also compared for markers linked and unlinked to domestication loci. Using a model-based approach, the landraces were divided into the traditional Middle American and Andean gene pools. Diversity was greater for the Middle American gene pool. Six Middle American and three Andean subpopulations were defined, and the Middle American subpopulations exhibited strong geographic identity. Unlike other studies, seed size varied considerably with subpopulations, and a number of the subpopulations contained landraces from multiple common bean races. All of the subpopulations were highly differentiated, with the Middle American subpopulations showing the greatest differentiation. Genetic isolation by distance was observed among the Middle American and Andean subpopulations but not among subpopulations within a gene pool. Within each gene pool, diversity was lower for markers linked to domestication loci.     

Estimation of natural outcrossing rate and genetic diversity in Lima bean (<Emphasis Type="Italic">Phaseolus lunatus</Emphasis> L. var. <Emphasis Type="Italic">lunatus</Emphasis>) from Brazil using SSR markers: implications for conservation and breeding

Lima bean (Phaseolus lunatus L.) is an important food source in Brazil, especially in the northeast region, where its production and consumption are high. The goals of the present study were to estimate natural outcrossing rates and genetic diversity levels of Lima bean from Brazil, using ten microsatellite loci to obtain information for their conservation and breeding. Fourteen accessions were selected from an experiment in field with open-pollinated and with the presence of pollinating insects. Twelve seeds of each of the 14 selected accessions were grown in screenhouse for tissue harvest and DNA extraction. The multilocus model was used to determine the reproductive system. The outcrossing rate was 38.1 % (t_m = 0.381; t_s = 0.078), and the results indicated a mixed mating system with a predominance of selfing (1 ? t_m = 61.9 %). The biparental inbreeding rate was high (t _m  ? t _s  = 0.303) and the multilocus correlated paternity was quite high (r _p(m) = 0.889), indicating that the progeny was mostly composed of full sibs. The average effective number of pollen donors per maternal plant (N _ep ) was low (1.12), and the fixation index for maternal genotypes (F _m ) was 0.945, indicating that most genitors resulted from inbreeding. The studied families presented considerable genetic variability: A = 6.10;  %P = 30; H _e  = 0.60 and H _o  = 0.077. Total diversity was high (H _T = 0.596), and a portion was distributed within families (H _S = 0.058). In addition, diversity was higher between families (D _ST = 0.538), and genetic differentiation was high (G _ST = 0.902). The results presented here can be used in the implementation of Lima bean conservation and breeding programs in Brazil.     

Sustainability of the traditional management of Agave genetic resources in the elaboration of mezcal and tequila spirits in western Mexico

Over the last 30 years, cultivation of Agave tequilana Weber var blue for industrial production of tequila, has generated soil erosion, chemical pollution, displacement of traditional food crops and traditional Agave landraces used for preparing “mezcals” in the Appellation of Origin Tequila area. It is also associated with harmful cycles 8–10 years long of surplus-shortfall availability of raw material. Mezcal is the common traditional name given in Mexico to spirits like tequila, prepared with Agave tissue. We examined the traditional strategies of management of the Agave genetic resources for elaborating mezcal and tequila spirits by people of southern Jalisco, Mexico, analyzing the bases of their sustainable management, compared with management of the industrial tequila system. We found that mezcal spirits are prepared with different landraces of A. angustifolia Haw. and A. rhodacantha Trel. which are cultivated as living fences within the “milpa”, the traditional Mesoamerican multi-crop system. The whole system allows simultaneous production of agricultural, livestock and forest resources, permitting the adjustment of mezcal production to demand. Agave borders and terraces are laid out perpendicular to slopes to increase rainfall capture and filtration, and decrease soil erosion. The high species richness creates niche heterogeneity, substantially reducing pest and disease incidence. Genetic and morphological analyses indicated that traditional management and selection of A. angustifolia landraces have produced high genetic diversity (H_BT = 0.438 ± 0.003) and structure (θ^B = 0.408) when compared with wild populations (H_BT = 0.428 ± 0.015; θ^B = 0.212). Morphological differentiation is associated to artificial selection pressures. Differential precocity of Agave landraces and scaled planting favors continuous, year round spirit production. Growers directly market their mezcals, and a portion of the profits is reinvested in the parcel to ensure system continuity. The technological advantages of the traditional mezcal system could attenuate some challenges caused by the tequila industrial agriculture.     

A note on the variation of phaseolin among common bean landraces collected in Eastern Asia

Summary Phaseolin seed protein was used as a marker to reveal the origin, Mesoamerican or Andean, of common bean (Phaseolus vulgaris) accessions cultivated in some countries (Korea, China, and Japan) of eastern Asia. Andean cultivars (T and C phaseolin patterns) are predominant in this geographical area. Introductions from Middle America, represented by cultivars with S phaseolin type, also occurred at lower rate. In all cases, genotypes with larger seeds have been favoured.     

Variation and genomic polymorphism of lectin-related proteins in Lima bean (Phaseolus lunatus L.) seeds

Variation of the lectin and the two lectin-related proteins, AIL (-amylase inhibitor-like) and ARL (arcelin-like) was examined in wild and cultivated accessions of Lima bean (Phaseolus lunatus L.) using electrophoresis of total seed proteins, immunoblot and RFLP analysis of lectin-related genes. Results confirm that divergence of the two major Lima bean gene pools, Andean and Mesoamerican, also apply to this protein family. All three members of the family are present in both gene pools, with differences in size, abundance and composition between gene pools, giving the possibility to distinguish Andean from Mesoamerican lectin pattern types. Both patterns show some variants, such as lack of lectin or its presence as an abundant protein. The observed variation reflects, at least in part, into genomic polymorphism. The presence of arcelin- and -amylase-related proteins in Lima bean could represent a tool to increase our knowledge in the evolution of the lectin family in Phaseolus species.     

Oligonucleotide DNA fingerprinting revealing polymorphism in Phaseolus lunatus L.

Digoxigenated synthetic oligonucleotides complementary tosimple repetitive DNA sequences were used to detect polymorphisms inPhaseolus lunatus L. (Lima bean)genotypes. Twenty two accessions, including wild and cultivatedrepresentatives of the Mesoamerican and Andean gene pools andintermediate forms were included in this study. It could be shownthat five repetitive motifs, namely (AAT)₅,(ATG)₅,(GACA)₄,(GATA)₄,(GGAT)₄, were able to give clear-cutbands when used as probes and to reveal high levels of variation. Thehighest resolving power was revealed by the(AAT)₅ probe, which was able to detect eveninter-individual polymorphisms. Therefore, oligonucleotidefingerprinting has proved to be a useful tool to analyse geneticdiversity in Lima bean. Phylogenetic reconstruction based onJACCARD's similarity index confirmed the existence of two majorgene pools and the presence of a minor group of genetically separatedintermediate genotypes.     

Diversity of common bean (Phaseolus vulgaris L.) germplasm from Portugal

Common bean (Phaseolus vulgarisL.) is a traditional crop in Portugal, where farmers growvarieties selected and maintained by themselves. A collection of 88landraces of common bean was evaluated for 17 quantitative andqualitative traits and the biochemical marker phaseolin to displaythe degree of variation of this germplasm. Agronomic data weresubjected to cluster analysis and several groups were identified,with three groups clustering most of the landraces. Regardingphaseolin variation the C and T banding patterns are the mostfrequent ones, so the origin of the Portuguese beans is thus probablythe Andean region of South America. These results give informationabout the origin, diversity and breeding value of the Portuguesegermplasm, that could be useful to widen the genetic base ofcurrently cultivated bean varieties in Europe.     

New insights into the origins and evolution of rhizobia that nodulate common bean (Phaseolus vulgaris) in Brazil

It is generally accepted that there are two major centers of genetic diversification of common beans (Phaseolus vulgaris L.): the Mesoamerican (Mexico, Colombia, Ecuador and north of Peru, probably the primary center), and the Andean (southern Peru to north of Argentina) centers. Wild common bean is not found in Brazil, but it has been grown in the country throughout recorded history. Common bean establishes symbiotic associations with a wide range of rhizobial strains and Rhizobium etli is the dominant microsymbiont at both centers of genetic diversification. In contrast, R. tropici, originally recovered from common bean in Colombia, has been found to be the dominant species nodulating field-grown common-bean plants in Brazil. However, a recent study using soil dilutions as inocula has shown surprisingly high counts of R. etli in two Brazilian ecosystems. In the present study, RFLP-PCR analyses of nodABC and nifH genes of 43 of those Brazilian R. etli strains revealed unexpected homogeneity in their banding patterns. The Brazilian R. etli strains were closely similar in 16S rRNA sequences and in nodABC and nifH RFLP-PCR profiles to the Mexican strain CFN 42^T, and were quite distinct from R. etli and R. leguminosarum strains of European origin, supporting the hypothesis that Brazilian common bean and their rhizobia are of Mesoamerican origin, and could have arrived in Brazil in pre-colonial times. R. tropici may have been introduced to Brazilian soils later, or it may be a symbiont of other indigenous legume species and, due to its tolerance to acidic soils and high temperature conditions became the predominant microsymbiont of common bean.     

Variation in primitive landraces of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) from Argentina

The phenotypic variation found in four common bean (Phaseolus vulgaris L.) complex primitive landraces, among a group of accessions collected in Northwestern Argentina in several missions is described, with particular attention to the wide diversity found in some small areas. It is presented a hypothesis about the maintenance of such diversity in bean mixtures or complex primitive landraces that grow close to their wild relative. Wide diversity regarding to seed type and plant characteristics was displayed by the landraces MCM-SV (composed of 11 lines), MCM-292 (14 lines), MCM-298 (5 lines) and VAV-3716 (14 lines). Food uses of dry seed and fresh pod seemed to be more relevant than the aesthetic use although all of them were presumably considered by humans for centuries resulting in the current phenotypes of these complex primitive landraces. Additionally, some weedy types (intermediate between wild and domesticated types) were detected in the landracesMCM-292 and MCM-298. The four complex landraces described consisted of highly diverse mixtures and they could play a role in breeding to enlarge the genetic basis of domesticated bean varieties belonging to the Andean gene pool.     

Insight into the origin of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) grown in the state of Jammu and Kashmir of north-western Himalayas

Common bean (Phaseolus vulgaris L.) is considered as one of the principal grain legume crops grown in north-western Himalayan state of Jammu and Kashmir (J&K), India. Huge diversity has been observed for this crop in state Jammu and Kashmir. The hilly regions of the state J&K are famous for producing high quality, tasty and highly flavoured beans. In order to characterise this huge diversity and trace the origin of common bean, systematic efforts have been made for the first time in collection, evaluation and characterization of bean collection from Jammu & Kashmir. A set of 428 common bean lines were initially collected/procured and based on cluster analysis using few qualitative traits/site of collection, a diverse set of 96 lines was selected. The PCR assay for phaseolin locus led to the characterization of 96 lines into Meso-american and Andean types. Out of 96 lines tested, 45 possessed “S” type phaseolin and 51 possess “T” type phaseolin. The ITS region of selected local and exotic lines was Sanger sequenced and the sequence analysis of ~ 800 bp long region revealed the presence of 12 SNPs including one promising SNPs showing significant association with phaseolin patterns. The clustering based on ITS sequence data led to the clear cut separation of common bean lines into two distinct clusters based on their phaseolin types. The results of the present study helped to gain insights into the origin of common bean landraces grown in state of J&K, India.     

Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N2 with common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N₂ fixation.     

Performance of Common Bean (Phaseolus vulgaris L.) Landraces from Spain in the Atlantic and Mediterranean Environments

Ninety-five common bean (Phaseolus vulgaris L.) landraces from Spain were evaluated in three different environments in northern Spain for their agronomic performance and seed quality. Significant differences among landraces were found for 14 quantitative traits related to phenology, yield and its components, and seed quality traits. Environmental effects were significant for all traits evaluated except for seeds pod^?1, seed width/thickness, seed weight, and seed water absorption. Landrace by environment interactions were significant for all traits except for seeds pod^?1 and seed water absorption. Selection of new breeding lines for agronomic performance and seed quality within landraces should be reliable because many of them are mixture of lines. Some heirloom varieties belonging to the types faba, caparrón,riñón,ganxet and tolosana had the best performances regarding to seed quality and yield. Principal component analysis revealed differences among environments affecting the performance of the bean landraces. Variation in the landraces seems to be organized in a different way in each one of the test environments, therefore, low plasticity and specific adaptation of Spanish bean landraces to different environments is derived from this study. Some landraces, especially those large and white seeded should be an useful resource for sustainable farming systems in different biogeographical areas and a worthy germplasm for the genetic improvement of agronomic value and seed quality.