Microsatellite marker diversity in common bean (Phaseolus vulgaris L.)

A diversity survey was used to estimate allelic diversity and heterozygosity of 129 microsatellite markers in a panel of 44 common bean (Phaseolus vulgaris L.) genotypes that have been used as parents of mapping populations. Two types of microsatellites were evaluated, based respectively on gene coding and genomic sequences. Genetic diversity was evaluated by estimating the polymorphism information content (PIC), as well as the distribution and range of alleles sizes. Gene-based microsatellites proved to be less polymorphic than genomic microsatellites in terms of both number of alleles (6.0 vs. 9.2) and PIC values (0.446 vs. 0.594) while greater size differences between the largest and the smallest allele were observed for the genomic microsatellites than for the gene-based microsatellites (31.4 vs. 19.1 bp). Markers that showed a high number of alleles were identified with a maximum of 28 alleles for the marker BMd1. The microsatellites were useful for distinguishing Andean and Mesoamerican genotypes, for uncovering the races within each genepool and for separating wild accessions from cultivars. Greater polymorphism and race structure was found within the Andean gene pool than within the Mesoamerican gene pool and polymorphism rate between genotypes was consistent with genepool and race identity. Comparisons between Andean genotypes had higher polymorphism (53.0%) on average than comparisons among Mesoamerican genotypes (33.4%). Within the Mesoamerican parental combinations, the intra-racial combinations between Mesoamerica and Durango or Jalisco race genotypes showed higher average rates of polymorphism (37.5%) than the within-race combinations between Mesoamerica race genotypes (31.7%). In multiple correspondance analysis we found two principal clusters of genotypes corresponding to the Mesoamerican and Andean gene pools and subgroups representing specific races especially for the Nueva Granada and Peru races of the Andean gene pool. Intra population diversity was higher within the Andean genepool than within the Mesoamerican genepool and this pattern was observed for both gene-based and genomic microsatellites. Furthermore, intra-population diversity within the Andean races (0.356 on average) was higher than within the Mesoamerican races (0.302). Within the Andean gene pool, race Peru had higher diversity compared to race Nueva Granada, while within the Mesoamerican gene pool, the races Durango, Guatemala and Jalisco had comparable levels of diversity which were below that of race Mesoamerica.     

Novel Phaseolin types in wild and cultivated common bean (Phaseolus vulgaris,Fabaceae)

Forty-one wild types and 41 cultivars of common bean (Phaseolus vulgaris) from Meso-and South America were screened for variability of phaseolin seed protein using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) and two-dimensional isoelectric focusing SDS/PAGE. Wild accessions from the Andean region showed phaseolin types which had not been previously identified in wild material from that region. Other wild accessions from Argentina exhibited novel phaseolin patterns collectively designated as ‘J’ (‘Jujuy’) phaseolin types, and one accession from northern Peru exhibited a novel phaseolin type, the ‘I’ (‘Inca’) type. The ‘H’ and ‘C’ phaseolins, previously identified only in cultivars, were observed in several wild accessions from Argentina. Among cultivars, two minor variants of the ‘S’ phaseolin type were identified. The ‘Sb’ (‘S Brazil’) was characteristic of a limited number of cultivars from Brazil whereas the ‘Sd’ (‘S Durango 222’) predominated in cultivars of the Mexican central highlands. The distribution of the previously described ‘B’ phaseolin appeared to be larger than formerly known as it extended not only in Colombia but also in Central America. It is possible to correlate the ‘Sb’, ‘Sd’, and ‘B’ phaseolin types with certain agronomic traits.     

RFLP diversity of common bean (Phaseolus vulgaris) in its centres of origin.

Eighty-five wild and cultivated accessions of common bean (Phaseolus vulgaris L.), representing a wide geographic area in the centres of domestication were tested for restriction fragment length polymorphisms (RFLPs). Genomic DNA was digested with one of three restriction enzymes (EcoRI, EcoRV, and HindIII) and hybridized to 12 probes distributed throughout the common bean genome. Accessions could be classified into two major groups with a distinct geographical distribution in Middle America and the Andes. Within each gene pool, cultivated accessions clustered together with wild forms from the same geographical area supporting the multiple domestications hypothesis for this crop. Estimates of Nei's genetic distances among the cultivated races from the two different gene pools varied from 0.12 to 0.56 and among races from the same gene pool from 0.04 to 0.12, suggesting that the divergence in Phaseolus vulgaris has reached the subspecies level. The level of genetic diversity (Ht = 0.38) was twice the value obtained with isozyme analysis. Genetic diversity within races (Hs = 0.27) was four to five times higher compared with isozymes, but genetic diversity between races (Dst = 0.11) was similar for both categories of markers. These results corroborate previous studies on the characterization of genetic diversity in common bean that clearly showed two distinct gene pools, Middle American and Andean. Moreover, RFLP markers are superior to isozymes because they provide better coverage of the genome and reveal higher level of polymorphisms.     

Genetic diversity of Chinese common bean (Phaseolus vulgaris L.) landraces assessed with simple sequence repeat markers

Common beans were introduced from the Americas to China over 400 years ago and presently constitute an important export crop in many areas of the country. Evaluation of the genetic diversity present in Chinese accessions of common beans is essential for conservation, management and utilization of these genetic resources. The objective of this research was to evaluate a collection of 229 Chinese landraces with 30 microsatellite markers to evaluate the genetic variability, genepool identity and relationships within and between the groups identified among the genotypes. A total of 166 alleles were detected with an average of 5.5 alleles per locus for all microsatellites. The landraces were clustered into two genepools with two subgroups each. The level of diversity for Chinese landraces of Andean origin was higher than for the Chinese landraces of Mesoamerican origin due to the presence of more infrequent alleles in this first group. The range of marker prevalence indices was from 0.288 to 0.676 within the Andean group and from 0.426 to 0.754 within the Mesoamerican group. Two subgroups were identified in each genepool group with one of the Mesoamerican subgroups arising from introgression. Gene flow (N ( m )) was 0.86 or below between subgroups from different gene pools and 2.6 or above between subgroups within the genepools. We discuss the existence of a secondary center of diversity for common beans in China and the importance of inter genepool introgression.     

Demographic factors shaped diversity in the two gene pools of wild common bean Phaseolus vulgaris L.

Wild common bean (Phaseolus vulgaris L.) is distributed throughout the Americas from Mexico to northern Argentina. Within this range, the species is divided into two gene pools (Andean and Middle American) along a latitudinal gradient. The diversity of 24 wild common bean genotypes from throughout the geographic range of the species was described by using sequence data from 13 loci. An isolation–migration model was evaluated using a coalescent analysis to estimate multiple demographic parameters. Using a Bayesian approach, Andean and Middle American subpopulations with high percentage of parentages were observed. Over all loci, the Middle American gene pool was more diverse than the Andean gene pool (π_sil=0.0089 vs 0.0068). The two subpopulations were strongly genetically differentiated over all loci (F_st=0.29). It is estimated that the two current wild gene pools diverged from a common ancestor ∼111 000 years ago. Subsequently, each gene pool underwent a bottleneck immediately after divergence and lasted ∼40 000 years. The Middle American bottleneck population size was ∼46% of the ancestral population size, whereas the Andean was 26%. Continuous asymmetric gene flow was detected between the two gene pools with a larger number of migrants entering Middle American gene pool from the Andean gene pool. These results suggest that because of the complex population structure associated with the ancestral divergence, subsequent bottlenecks in each gene pool, gene pool-specific domestication and intense selection within each gene pool by breeders; association mapping would best be practised within each common bean gene pool.     

Races of common bean (Phaseolus vulgaris,Fabaceae)

Evidence for genetic diversity in cultivated common bean (Phaseolus vulgaris) is reviewed. Multivariate statistical analyses of morphological, agronomic, and molecular data, as well as other available information on Latin American landraces representing various geographical and ecological regions of their primary centers of domestications in the Americas, reveal the existence of two major groups of germplasm: Middle American and Andean South American, which could be further divided into six races. Three races originated in Middle America (races Durango, Jalisco, and Mesoamerica) and three in Andean South America (races Chile, Nueva Granada, and Peru). Their distinctive characteristics and their relationships with previously reported gene pools are discussed.     

The amino acid sequence of plastocyanin from French bean (Phaseolus vulgaris)

The amino acid sequence of the plastocyanin from French bean (Phaseolus vulgaris) was determined. The protein consists of a single polypeptide chain of 99 residues, and the sequence was determined by characterization of CNBr, tryptic, chymotryptic and thermolysin peptides. When the sequence is compared with that from the plastocyanin of the unicellular green alga Chlorella fusca, the French-bean protein shows the deletion of the N-terminal residue, a two residue insertion and 53 identical residues. Detailed evidence for the sequence of the protein has been deposited as Supplementary Publication SUP 50037 (16pp., 1 microfiche) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Molecular diversity in Ligurian local races of common bean (Phaseolus vulgaris L.)

Abstract

Eight varieties of Ligurian common beans (Phaseolus vulgaris L.) were analysed using molecular approaches. Results were compared with two commercial cultivars (‘Cannellino’ and ‘Borlotto’). Data suggest that all Ligurian bean varieties have a low genetic variability and are very close to the commercial varieties. In particular, the three ‘Bianco’ varieties showed a molecular affinity, probably due to their common genomic origin.     

Genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in Nepal

Background and Aims

This study was conducted to reveal the genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in various agroecological regions in Nepal.

Method

A total of 63 strains were isolated from common bean grown in the soils collected from seven bean fields in Nepal and characterized based on the partial sequences of 16S–23S internal transcribed spacer (ITS) regions, 16S rDNA, nodC, and nifH. Symbiotic properties of some representative strains with host plants were examined to elucidate their characteristics in relation to genotype and their origin.

Results

The isolated strains belonged to Rhizobium leguminosarum, Rhizobium etli, Rhizobium phaseoli, and one unknown Rhizobium lineage, all belonging to a common symbiovar (sv.) phaseoli. Nine ITS genotypes were detected mainly corresponding to a single site, including a dominant group at three sites harboring highly diverse multiple ITS sequences. Three symbiotic genotypes corresponded to a geographical region, not to the ribosomal DNA group, suggesting horizontal transfer of symbiotic genes separately in each region. Great differences in nitrogenase activity and nodule forming ability among the strains irrespective of their species and origin were observed.

Conclusions

Nepalese Himalaya harbor phylogenetically highly diverse and site-specific strains of common bean rhizobia, some of which could have high potential of symbiotic nitrogen fixation.     

Genetic diversity of common bean (Phaseolus vulgaris L.) ecotypes from Pakistan using Simple Sequence Repeats

Common bean (Phaseolus vulgaris L.) is a legume crop grown all over the world and is a very important food of mountain population of Pakistan for protein intake. The Western Himalayan Mountains are rich in biodiversity including unexplored landraces of the common bean crop. Unfortunately, very little attention has been given to this valuable crop in Pakistan, and it is being exported, majorly from Ethiopia, to meet the country’s requirements. The exploitation, utilization, preservation and multiplication of existing germplasm within the area are very important for sustainable production of the crop and enhancing the nutrition value for the local community in mountain regions. A research study was conducted for evaluation of biological diversity of common bean landraces from Azad Kashmir and Northern areas of Pakistan using morpho-physiological and molecular markers. Thirty-five common bean ecotypes along with one check variety were collected from different altitudes of Azad Kashmir and Northern Pakistan and screened for biological diversity. Morphological characterization revealed high genetic diversity in parameters including stem anthocyanin, growth type, days to flowering, pods/plant and 100 seeds weight. Genomic characterization using SSR markers, for allelic diversity evaluation among germplasm, also provided diverse profile with 83.3% polymorphism in banding pattern. The bulk of gene pool diversity evaluated within bean landraces may help to initiate breeding program for common bean improvement.     

Purification and characterisation of a novel papain inhibitor from common bean (Phaseolus vulgaris) seed

A proteinaceous inhibitor of papain was purified to apparent homogeneity from mature seeds of common bean ( Phaseolus vulgaris L.). After four chromatographic steps, the papain inhibitor was purified 219‐fold with 12% recovery. On the basis of papain inhibitory activity, cystatins have been estimated to account for about 0.1% of the total protein content of mature common bean seeds. The purified protein, as other plant cystatins, is an acidic protein, heat stable and insensitive to reducing agents. Its molecular mass is about 37 kDa as judged by size exclusion chromatography and SDS‐PAGE. Moreover it is immunologically related to oryzacystatins, since it is recognised by a specific oryzacystatin I antiserum. Based on its biochemical properties the papain inhibitor described here belongs to the phytocystatin family. Papain inhibitory assays carried out during seed development showed that bean cystatin is active since early maturation stages. Our results suggest that, in common bean seed, cysteine proteinase inhibitors are important during seed development with a putative role in the control and regulation of endogenous thiol protease activity.     

Development of mapped simple sequence repeat markers from common bean (Phaseolus vulgaris L.) based on genome sequences of a Chinese landrace and diversity evaluation

Microsatellite or single sequence repeat (SSR) markers have been commonly used in genetic research in many crop species, including common bean (Phaseolus vulgaris L.). A limited number of existing SSR markers have been designed from high-throughput sequencing of the genome, warranting the exploitation of new SSR markers from genomic regions. In this paper, we sequenced total DNA from the genotype Hong Yundou with a 454-FLX pyrosequencer and found numerous SSR loci. Based on these, a large number of SSR markers were developed and 90 genomic-SSR markers with clear bands were tested for mapping and diversity detection. The new SSR markers proved to be highly polymorphic for molecular polymorphism, with an average polymorphism information content value of 0.44 in 131 Chinese genotypes and breeding lines, effective for distinguishing Andean and Mesoamerican genotypes. In addition, we integrated 85 primers of the 90 polymorphism markers into the bean map using an F2 segregating population derived from Hong Yundou crossed with Jingdou. The distribution of SSR markers among 11 chromosomes was not random and tended to cluster on the linkage map, with 14 new markers mapped on chromosome Pv01, whereas only four loci were located on chromosome Pv04. Overall, these new markers have potential for genetic mapping, genetic diversity studies and map-based cloning in common bean.     

Genetic diversity and population structure of common bean (Phaseolus vulgaris) landraces from China revealed by a new set of EST-SSR markers

A new set of EST-SSR markers were developed and employed to analyze the genetic diversity and population structure of Phaseolus vulgaris in China. A total of 2452 microsatellites were identified in 2144 unigenes assembled from P. vulgaris ESTs, indicating that merely 6.9% of the 30,952 unigene sequences contained SSRs. Seventeen of 153 randomly designed EST-SSR primer pairs successfully amplified polymorphic products in 31 landraces from six major production provinces of China, with the mean number of alleles per locus of 2.700 and polymorphism information content of 0.378. The observed and expected heterozygosity ranged from 0.100 to 0.954 and 0.081 to 0.558, respectively. Using these markers, both an unrooted neighbor-joining tree and principal coordinates analysis showed that almost all of the landraces were separated according with their regional distribution. Moreover, population structure analysis revealed that all genotypes formed into three distinct clusters (k = 3), suggesting that geographic and climatic factors could provide diverse degrees of selection pressure. Accordingly, germplasm collection and cross breeding among different regions are suggested to accelerate the process of diverse germplasm creation and broaden germplasm resources of Chinese common bean.     

New gene-derived simple sequence repeat markers for common bean (Phaseolus vulgaris L.)

Common bean is an important and diverse crop legume with several wild relatives that are all part of the Phaseoleae tribe of tropical crop legumes. Sequence databases have been a good source of sequences to mine for simple sequence repeats (SSRs). The objective of this research was to evaluate 14 sequence collections from common bean for SSRs and to evaluate the diversity of the polymorphic microsatellites derived from these collections. SSRs were found in 10 of the GenBank sequence collections with an average of 11.3% of sequences containing microsatellite motifs. The most common motifs were based on tri- and dinucleotides. In a marker development programme, primers were designed for 125 microsatellites which were tested on a panel of 18 common bean genotypes. The markers were named as part of the bean microsatellite-database (BMd) series, and the average polymorphism information content was 0.404 for polymorphic markers and predicted well the genepool structure of common beans and the status of the wild and cultivated accessions that were included in the study. Therefore, the BMd series of microsatellites is useful for multiple studies of genetic relatedness and as anchor markers in future mapping of wide crosses in the species.     

Cloning and genetic diversity analysis of a new P5CS gene from common bean (Phaseolus vulgaris L.)

Δ¹-pyrroline-5-carboxylate synthetase (P5CS) is the rate-limiting enzyme involved in the biosynthesis of proline in plants. By the 3′ rapid amplification of cDNA ends (3′-RACE) approach, a 2,246-bp cDNA sequence was obtained from common bean (Phaseolus vulgaris L.), denominated PvP5CS2 differing from another P5CS gene that we cloned previously from common bean (PvP5CS). The predicted amino acid sequence of PvP5CS2 has an overall 93.2% identity GmP5CS (Glycine max L. P5CS). However, PvP5CS2 shows only 83.7% identity in amino acid sequence to PvP5CS, suggesting PvP5CS2 represents a homolog of the soybean P5CS gene. Abundant indel (insertion and deletion events) and SNP (single nucleotide polymorphisms) were found in the cloned PvP5CS2 genome sequence when comparing 24 cultivated and 3 wild common bean accessions and these in turn reflected aspects of common bean evolution. Sequence alignment showed that genotypes from the same gene pool had similar nucleotide variation, while genotypes from different gene pools had distinctly different nucleotide variation for PvP5CS2. Furthermore, diversity along the gene sequence was not evenly distributed, being low in the glutamic-g-semialdehyde dehydrogenase catalyzing region, moderate in the Glu-5-kinase catalyzing region and high in the intervening region. Neutrality tests showed that PvP5CS2 was a conserved gene undergoing negative selection. A new marker (Pv97) was developed for genetic mapping of PvP5CS2 based on an indel between DOR364 and G19833 sequences and the gene was located between markers Bng126 and BMd045 on chromosome b01. The relationship of PvP5CS2 and a previously cloned pyrroline-5-carboxylate synthetase gene as well as the implications of this work on selecting for drought tolerance in common bean are discussed.     

Cloning, quantification and characterization of a minisatellite DNA sequence from common bean Phaseolus vulgaris L.

 We describe the cloning and the characterization of a 130-bp DNA fragment, called OPG9-130, amplified from bean (Phaseolus vulgaris L.) genomic DNA. This fragment corresponds to a minisatellite DNA sequence containing seven repeats of 15 bp which differ slightly from each other in their sequence. Southern analysis showed that the core sequence of 15 bp is repeated in clusters dispersed throughout the genome. The use of this fragment as a probe allowed us to identify common bean lines by their DNA fingerprints. We suggest that OPG9-130 will be useful for line identification as well as for the analysis of genetic relatedness between bean species and lines. Received: 14 February 1998 / Accepted: 10 February 1998     

Integration of simple sequence repeat (SSR) markers into a molecular linkage map of common bean (Phaseolus vulgaris L.)

Microsatellite or simple sequence repeat (SSR) markers have been successfully used for genomic mapping, DNA fingerprinting, and marker-assisted selection in many plant species. Here we report the first successful assignment of 15 SSR markers to the Phaseolus vulgaris molecular linkage map. A total of 37 SSR primer pairs were developed and tested for amplification and product-length polymorphism with BAT93 and Jalo EEP558, the parental lines of an F7 recombinant inbred (RI) population previously used for the construction of a common bean molecular linkage map. Sixteen of the SSRs polymorphic to the parental lines were analyzed for segregation and 15 of them were assigned to seven different linkage groups, indicating a widespread distribution throughout the bean genome. Map positions for genes coding for DNAJ-like protein, pathogenesis-related protein 3, plastid-located glutamine synthetase, endochitinase, sn-glycerol-3 phosphate acyltransferase, NADP-dependent malic enzyme, and protein kinase were determined for the first time. Addition of three SSR loci to linkage group B4 brought two separated smaller linkage groups together to form a larger linkage group. Analysis of allele segregation in the F7 RI population revealed that all 16 SSRs segregated in the expected 1:1 ratio. These SSR markers were stable and easy to assay by polymerase chain reaction (PCR). They should be useful markers for genetic mapping, genotype identification, and marker-assisted selection of common beans.     

Anatomical root variations in response to water deficit: wild and domesticated common bean (Phaseolus vulgaris L)

Root anatomical responses to water deficit are diverse and regulation of water uptake strongly depends on plant anatomy. The ancestors of common bean (Phaseolus vulgaris L.) cultivars are the wild common beans. Because wild beans adapt and survive well in the natural environment, it is hypothesized that wild common bean roots are less affected than those of domesticated beans at low substrate water potential (ψW). A wild common bean accession from Chihuahua Mexico and cv. Bayomex were studied. Seedlings with a mean root length between 3 and 4 cm were maintained for 24 h in vermiculite at ψW of -0.03 (well hydrated), -0.65, -1.48 and -2.35 MPa (partially dry). Ten anatomical characteristics of differentiation and cell division in root regions were evaluated. Thickness of epidermis and protoderm diminished similarly in wild and domesticated beans growing at low substrate ψW (between -0.65 and -2.35 MPa). At the same time, parenchymatic cell area diminished by 71 % in the domesticated variety, but by only 32 % in the wild bean at -2.35 MPa. The number of cells in the cortex and the thickness of the xylem wall increased in both wild and domesticated beans at low substrate ψW; nevertheless, the effect was significantly lower in the wild bean. The number of xylem vessels increased in the cultivar (up to 40 %) while in the wild bean it decreased (up to 33 %). The diameter of xylem vessels and transverse root area diminished (15 and 57 %, respectively) in the cultivar, but in the wild common bean were not affected. Anatomical root characteristics and their modifications in both differentiation and cell division in root regions demonstrated that the wild bean reacted quite differently to substrate ψW than the domesticated common bean.