Phylogenetic study on wild allies of Lima bean,Phaseolus lunatus (Fabaceae), and implications on its origin

An investigation was made of the phylogenetic relationships among wild accessions of Lima bean (Phaseolus lunatus) and wild allies of Mesoamerican and Andean origins, using electrophoresis of seed storage proteins and isozymes. Mesoamerican wild species are phylogenetically more distant fromP. lunatus than Andean species, and apparently belong to the tertiary gene pool of Lima bean. The Andean wild species, which are investigated for the first time, reveal a high similarity to the Lima bean, and particularly with its Mesoamerican gene pool. These Andean species probably constitute a secondary gene pool of Lima bean, and are thus of considerable interest in the context of genetic improvement of the crop. Based on these observations, an Andean origin is suggested for the Andean wild species and forP. lunatus. These results point out the importance of collecting and conserving AndeanPhaseolus germplasm.     

Use of chloroplast DNA polymorphisms for the phylogenetic study of seven Phaseolus taxa including P. vulgaris and P. coccineus

The genetic variability of seven Phaseolus taxa has been evaluated on the basis of molecular data and the results have used to clarify the phyletic relationships between several taxa of the P. coccineus L. complex. Chloroplast DNA (cpDNA) from 33 populations was digested with six restriction endonucleases, revealing some polymorphisms that made it possible to divide most of the taxa into two main groups: the subspecies of P. coccineus on the one hand, and P. vulgaris L., P. polyanthus Greenman and P. costaricensis (Freytag and Debouck) on the other hand. P. polyanthus is closer to P. vulgaris than the other taxa of the second group and should be considered as a separate species. The position of the wild species P. costaricensis is intermediate between P. coccineus and P. polyanthus. P. glabellus shows sufficient polymorphisms at the cpDNA level to be recognized as a separate species, as previously suggested from total seed-protein electrophoretic studies. These results favour the hypothesis of a common phylogeny for P. vulgaris, P. polyanthus, P. costaricensis and P. coccineus from a single wild ancestor. Although cpDNA is generally known to be uniform at the intraspecific level, some additional polymorphisms were also detected within P. vulgaris, P. polyanthus and P. coccineus. Further studies are required to understand the significance of the latter.     

Biochemical evidence bearing on the domestication of<Emphasis Type="Italic">Phaseolus</Emphasis> (Fabaceae) beans

The genusPhaseolus (Fabaceae) consists of some 50 species, all of which are distributed in the Americas. Four of these contain cultigens.P. vulgaris (common bean),P. lunatus (lima bean),P. acutifolius (tepary bean),P. coccineus subsp.coccineus (runner bean); andP. coccineus subsp.polyanthus (no English vernacular name). Biochemical markers—phaseolin seed storage protein and isozymes—have provided new evidence on the organization of the first three species. Domestication has possibly caused a strong reduction in genetic diversity inP. vulgaris andP. acutifolius. BothP. vulgaris andP. lunatus cultivars result from at least two independent domestications, in Mesoamerica and in the Andes. These two species consist of two gene pools, each of which includes wild ancestors and their respective cultivated descendants. Our findings suggest the need for additional emphasis on genetic conservation of wild ancestors and their use in breeding programs and for a comparison of inter-gene pool vs. intra-gene pool crosses in breeding programs.     

Regeneration of Fertile Plants from Callus in Phaseolus polyanthus Greenman (Year Bean)

For the first time, plant regeneration of several domesticatedgenotypes of Phaseolus polyanthus Greenman (year bean) has beenachieved. Thidiazuron in combination with indole-3-acetic acidwas used to induce morphogenic, green nodular callus from explantsthat had been obtained either from greenhouse-grown plants orfrom in vitro -germinated seeds. Of the six genotypes of P.polyanthus tested, five produced shoots in vitro. Regeneratedshoots that formed roots in vitro were established in the greenhouse,whereas non-rooted shoots could be established in vitro by grafting.Morphologically normal progeny plants were obtained from thegreenhouse-established regenerants. However, by using the sameprocedure, no regeneration response was observed in two domesticatedand two wild genotypes of P. coccineus L. (runner bean). Thisprotocol should help achieve Agrobacterium - or particle bombardment-mediatedgenetic transformation to improve this important food legume.Copyright 2001 Annals of Botany Company Callus, genetic transformation, grafting, legumes, organogenesis, Phaseolus coccineus L., Phaseolus polyanthus Greenman, Phaseolus vulgaris L     

Genetic structure of a Lima bean base collection using allozyme markers

 Genetic diversity and structure within a Lima bean (Phaseolus lunatus L.) base collection have been evaluated using allozyme markers. The results obtained from the analysis of wild and cultivated accessions confirm the existence of Andean and Mesoamerican gene pools characterised by specific alleles. Wild and cultivated accessions of the same gene pool are grouped. The Andean natural populations have a very limited geographic distribution between Ecuador and northern Peru. The Mesoamerican wild form extends from Mexico up to Argentina through the eastern side of the Andes. Andean and Mesoamerican cultivated accessions of pantropical distribution contribute substantially to the genetic diversity of the Lima bean base collection. Population genetic parameters, estimated from allozymes, confirmed the predominant selfing mating system of the Lima bean. The selfing mating system, the occurrence of small populations, and low gene flow lead to an interpopulation gene diversity (D_ST=0.235) higher than the intrapopulation gene diversity (H_S=0.032). On the basis of the results, guidelines are given to preserve and exploit the genetic diversity of this threatened species. The results also confirm the independent domestication of the Lima bean in at least two centres, one of which is located at medium elevation in the western valleys of Ecuador and northern Peru. Received: 3 June 1997 / Accepted: 17 June 1997     

Chloroplast DNA as an evolutionary marker in thePhaseolus vulgaris complex

We have analyzed the changes occurring in the chloroplast DNA (cpDNA) of taxa belonging to thePhaseolus vulgaris complex to help clarify relationships among species of this complex. Two restriction maps for 11 restriction enzymes comprising the whole chloroplast genome from a wildP. vulgaris and a wildP. coccineus accession were constructed. These maps allowed us to compare a total of 330 restriction sites between the two genomes in order to identify polymorphisms, assess the type of mutations detected, and identify regions of high variability. A region, located in the large single-copy region near the borders with the inverted repeats, accounted for a large portion of the variation. Most of the mutations detected were due to restriction sites gains or losses. Variable and conserved regions were then evaluated in 30 accessions belonging to taxa of theP. vulgaris complex. Phylogenetic analyses were made using parsimony methods. Conclusions obtained from such analyses were the following: (1) there was high cpDNA variability withinP. coccineus but not inP. vulgaris. (2)P. coccineus subsp.glabellus showed a very distinct cpDNA type that strongly suggests that it actually belongs to a different but as yet undetermined section of the genus. Our cpDNA observations are supported by distinctive morphological traits and reproductive biology of this taxon. (3) InP. coccineus subsp.darwinianus (also classified asP. polyanthus), the cpDNA lineage was in disagreement with data obtained from nuclear markers and suggested a reticulated origin by hybridization betweenP. coccineus as the male parent and an ancestralP. polyanthus type, closely allied toP. vulgaris, as the seed parent. This initial cross was presumably followed by repeated backcrossing toP. coccineus. Our cpDNA studies illustrate the importance of molecular markers in elucidating phylogenetic relationships. They also indicate that accurate phylogenies will require analyses of both nuclear and cytoplasmic genomes.     

The bean polygalacturonase-inhibiting protein 2 (PvPGIP2) is highly conserved in common bean (Phaseolus vulgaris L.) germplasm and related species

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant protein inhibitors of endo-polygalacturonases (PGs) that belong to the leucine-rich repeat (LRR) protein family. In bean, PGIP is encoded by a small gene family of four members among which Pvpgip2 encodes the most wide-spectrum and efficient inhibitor of fungal PGs. In order to evaluate the sequence polymorphism of Pvpgip2 and its functional significance, we have analyzed a number of wild and cultivated bean (P. vulgaris) accessions of Andean and Mesoamerican origin, and some genotypes from the related species P. coccineus, P. acutifolius, and P. lunatus. Our analyses indicate that the protein encoded by Pvpgip2 is highly conserved in the bean germplasm. The few detected polymorphic sites correspond to synonymous substitutions and only two wild genotypes contain a Pvpgip2 with a single non-synonymous replacement. Sequence comparison showed a slightly larger variation in the related bean species P. coccineus, P. acutifolius, and P. lunatus and confirmed the known phylogenetic relationships with P. vulgaris. The majority of the replacements were within the xxLxLxx region of the leucine rich repeat (LRR) domain and none of them affected residues contributing to structural features. The variant PGIPs were expressed in Nicotiana benthamiana using PVX as vector and their inhibitory activity compared to that of PvPPGIP2. All the variants were able to fully inhibit the four fungal PGs tested with minor differences. Taken together these results support the hypothesis that the overall sequence conservation of PGIP2 and minor variation at specific sites is necessary for high-affinity recognition of different fungal PGs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lectin and lectin-related proteins in Lima bean (Phaseolus lunatus L.) seeds: biochemical and evolutionary studies

Lectin-related polypeptides are a class of defence proteins found in seeds of Phaseolus species. In Lima bean (P. lunatus), these proteins and their genes have been well characterized in the Andean morphotype, which represents one of the two gene pools of this species. To study the molecular evolution of the lectin family in Lima bean we characterized the polypeptides belonging to this multigene family and cloned the genes belonging to the Mesoamerican gene pool. The latter gene pool contains components similar to those of the Andean pool, namely: an amylase inhibitor-like (AIL), an arcelin-like (ARL) lectin and the less abundant Lima bean lectin (LBL). These proteins originate from an ancestor gene of the lectin type which duplicated to yield the lectin gene and the progenitor of ARL and AIL. In this species. ARL represents an evolutionary intermediate form that precedes AIL. Phylogenetic analysis supports an Andean origin for Lima bean. The molecular evolutionary studies were extended to the genes of common bean and demonstrated that true lectin genes and the ancestor of lectin-related genes are the result of a duplication event that occurred before speciation. Lima and common bean followed different evolutionary pathways and in the latter species a second duplication event occurred that gave rise, in Mesoamerican wild genotypes, to arcelin genes.     

Distribution of the protein “phaseolin“ in some representatives ofViciaceae

Using immunochemical methods the authors investigated the evolutionary taxonomic distribution of the reserve seed protein “phaseolin” in cultivars ofPhaseolus vulgaris, in a series of species ofPhaseolus, and in representatives of some additional genera ofViciaceae. “Phaseolin” is typical of the seed ofPhaseolus vulgaris L.: it was detected in all 658 investigated cultivars — and also in species related toPhaseolus vulgaris L.(Ph. vulgaris L. ssp.aborigineus Burk.,Ph. polyanthus Green,Ph. dumosus Macf.,Ph. coccineus L., and in an undescribed species from the group ofPh. vulgaris L. -Ph. coccineus L.). A protein immunochemically somewhat similar to ?phaseolin“ occurs inPh. acufifolius A. Gray. In all other taxa“phaseolin” is absent.     

Genetic diversity and structure of a worldwide collection of Phaseolus coccineus L

Phaseolus coccineus L. is closely related to P. vulgaris and is the third most important cultivated Phaseolus species. Little is known about the patterns of its diversity. In this work, a representative collection of its worldwide diversity was initially developed. The collection includes 28 wild forms (WFs) and 52 landraces (LRs) from Mesoamerica (the crop domestication area), and 148 LRs from Europe (where the crop was introduced in the sixteenth century). The collection was studied by using 12 SSR molecular markers that were developed for the P. vulgaris genome. They were proved to be effective and reliable in P. coccineus in this work. Fourteen LRs of P. dumosus (previously identified as a subspecies of P. coccineus) were also studied. The genetic diversity, population structure and phylogenetic relationships were investigated. The results indicate that: (a) the European and Mesoamerican gene pools are clearly differentiated, (b) a certain reduction of diversity occurred with introduction into Europe, and (c) the Mesoamerican LRs (P. dumosus included) and WFs are closely related and are connected by a high gene flow. Inferences on the domestication process of P. coccineus are also presented. This study provides a picture of the genetic diversity distribution and outcomes with introduction into the Old World, which was not available before. It also underlines that the genetic diversity of both WFs and LRs is an important source for Phaseolus spp. breeding programs and deserves to be preserved in situ and ex situ.     

Hybrid Weakness in Phaseolus vulgaris L. II. Disruption of Root-Shoot Integration

We have been examining the importance of the root system on shoot growth and development using a developmentally disabled hybrid of the common bean Phaseolus vulgaris L. Parental cultivars (P. Vulgaris cv. Redkloud of Mesoamerican origin, and P. vulgaris cv. Batt of Andean origin) grow normally, but crosses produce F1 hybrids exhibiting hybrid weakness associated with reduced root and shoot growth. In this study, applications of benzylaminopurine (BAP) to roots of F1 hybrids increased the number of root tips and leaves. Reciprocal grafting was used to study the effects of the root system on shoots. Grafting of roots of the Mesoamerican cultivar onto shoots of F1 hybrids increased the cytokinin concentrations in leaves of F1 hybrids and removed the characteristics associated with hybrid weakness. To determine whether factors in the xylem sap enhanced leaf growth, leaf discs were incubated on sap collected from Mesoamerican and Andean cultivars. Sap from Mesoamerican plants enhanced the growth of leaf discs excised from F1 hybrids more than sap collected from Andean cultivars. Estimates of the transport of zeatin riboside (ZR)–type cytokinins from roots of F1 hybrids indicated that transport out of hybrid roots was reduced compared with those transported out of Mesoamerican or Andean roots. Results suggest that ZR-type cytokinins are involved in hormonal integration between roots and shoots of P. vulgaris and that one of the barriers to hybridization between Andean and Mesoamerican landraces is related to hormone transport. Received October 15, 1998; accepted May 12, 1999     

Isozyme diversity, RFLP of the rDNA and phylogenetic affinities among cultivated Lima beans,Phaseolus lunatus (Fabaceae)

Genetic variation inPhaseolus lunatus (Lima bean) was investigated at isozyme and DNA levels. Sixty cultivated accessions, including representatives of the Mesoamerican and Andean gene pools and intermediate types, were analyzed for variability at 17 isozyme loci. Some accessions were also examined for restriction fragment length polymorphism (RFLP) at the rDNA level. These data were used to construct two dendrograms showing clear separation in two distinct groups corresponding to each of the gene pools and an intermediate one probably representing a transitional group.     

A unique cytoplasmic male sterility (CMS) determinant is present in three Phaseolus species characterized by different mitochondrial genomes

Previous results have shown that cytoplasmic male sterility (CMS) in lines from Phaseolus coccineus and Phaseolus vulgaris contain the same CMS-specific sequence, raising the question of whether this sequence rearrangement arose before divergence of the two species or afterward with subsequent transfer by introgression. Hybridization patterns of total DNA from eight P. vulgaris lines with cytoplasm from P. coccineus and three P. vulgaris lines were examined in order to analyze the mitochondrial DNA (mtDNA) diversity within each species and to determine differences between CMS lines derived from the two species. Three restriction enzymes and 17 heterologous mtDNA sequences were used. The analysis of the different hybridization patterns revealed a considerable diversity in mtDNA organization particularly within P. coccineus. We obtained distinctive hybridization patterns for the five CMS lines tested. The resulting classification showed that mitochondrial genomes from P. coccineus CMS lines group with those of fertile P. coccineus but not with CMS lines from P. vulgaris. The groupings concur with the taxonomic classification of these lines. The results support the hypothesis of a single ancient origin of the CMS determinant and exclude the transfer of cytoplasm by introgression from P. vulgaris to P. coccineus and P. coccineus ssp polyanthus.     

Hybrid Weakness in Phaseolus vulgaris L. I. Disruption of Development and Hormonal Allocation

A reduced concentration of cytokinins may cause the abnormal growth and development found in F1 hybrids between Andean and Mesoamerican races of Phaseolus vulgaris L. In this study, concentrations of the transportable cytokinin zeatin riboside (ZR) were measured by ELISA for ZR (cross reactivities dihydrozeatin, 14%, zeatin 7.6%) in roots, stems, and leaves of a Phaseolus Mesoamerican landrace (P. vulgaris L. cv. Redkloud), an Andean landrace (P. vulgaris L. cv. Batt), and their F1 hybrids. Concentrations of ZR in roots and leaves of F1 hybrids were significantly less than that found in roots and leaves of parental cultivars. Approximately 90% of the ZR found in F1 hybrids was found sequestered in the stems, whereas cytokinins of the parental cultivars were distributed throughout the plant (roots: Batt 37%, Redkloud, 44%; stems: Batt 35%, Redkloud 42%; leaves: Batt 28%, Redkloud 14%). These results suggest that abnormal growth and development of F1 hybrids may involve interruption of the regulation of cytokinin allocation, thereby disrupting the root-shoot feedback loop between root-sourced cytokinins and putative shoot-produced factors. Received October 15, 1998; accepted May 12, 1999     

Isolation and characterization of a new repetitive DNA family recently amplified in the Mesoamerican gene pool of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L., Fabaceae)

The common bean (Phaseolus vulgaris) is one of the most important crop plants. About 50% of its genome is composed of repetitive sequences, but only a little fraction was isolated and characterized so far. In this paper, a new repetitive DNA family from the species, named PvMeso, was isolated and characterized in both gene pools of P. vulgaris (Andean and Mesoamerican) and related species. Two fragments, 1.7 and 2.3 kb long, were cloned from BAC 255F18, which has previously shown a repetitive pattern. The subclone PvMeso-31 showed a terminal block in chromosome 7. This subclone contains a 1,705 bp long, AT-rich repeat with small internal repeats and shares a 1.2 kb region with PvMeso-47, derived from the 2.3 kb fragment. The presence of this repetitive block was restricted to Mesoamerican accessions of the common bean. In P. acutifolius, P. leptostachyus and Andean P. vulgaris, only a faint, 2.3 kb fragment was visualized in Southern experiments. Moreover, in Mesoamerican accessions, two other fragments (1.7 kb and 3.4 kb) were strongly labelled as well. Taken together, our results indicate that PvMeso is a recently emerged, repeat family initially duplicated in chromosome 11, on ancestral Mesoamerican accession, and later amplified in chromosome 7, after the split of the two major gene pools of the common bean.     

Analyses of Phaseolus vulgaris L. and P. coccineus Lam. hybrids by RFLP: preferential transmission of P. vulgaris alleles

Summary Restriction fragment length polymorphism (RFLP) was determined among P. vulgaris genotypes and Phaseolus species using 19 probes. The incidence of polymorphism was high (70–86%) between species, but relatively low (22–26%) between genotypes of P. vulgaris. Suitable probes were identified for the analysis of P. vulgaris and P. coccineus hybrids. The segregation pattern in F₂ populations was Mendelian for two probes (LHB and VEE20) and non-Mendelian for GS-g, CHS, and CHI. Statistical analyses indicated gametic selection with preferential transmission of the P. vulgaris alleles, which may account for the selective recovery of P. vulgaris progeny types observed earlier. The available hybrids of P. vulgaris and P. coccineus and the high degree of interspecific RFLP will facilitate the construction of a linkage map for Phaseolus.     

Species relationships inPhaseolus: Electrophoretic analysis of seed storage proteins

Relationships among storage proteins in seeds from cultivars and primitive accessions of the four economically most important species ofPhaseolus — P. vulgaris, P. coccineus, P. acutifolius andP. lunatus — were studied. Analysis of SDS-polyacrylamide gel electrophoretic patterns of storage seed proteins revealed common characteristics in the major groups of polypeptides forP. vulgaris, P. coccineus andP. acutifolius, while clear differences existed between thesePhaseolus species and P.lunatus.     

Gene pools in wild Lima bean (Phaseolus lunatus L.) from the Americas: Evidences for an Andean origin and past migrations

The aims of this research were to assess the genetic structure of wild Phaseolus lunatus L. in the Americas and the hypothesis of a relatively recent Andean origin of the species. For this purpose, nuclear and non-coding chloroplast DNA markers were analyzed in a collection of 59 wild Lima bean accessions and six allied species. Twenty-three chloroplast and 28 nuclear DNA haplotypes were identified and shown to be geographically structured. Three highly divergent wild Lima bean gene pools, AI, MI, and MII, with mostly non-overlapping geographic ranges, are proposed. The results support an Andean origin of wild Lima beans during Pleistocene times and an early divergence of the three gene pools at an age that is posterior to completion of the Isthmus of Panama and major Andean orogeny. Gene pools would have evolved and reached their current geographic distribution mainly in isolation and therefore are of high priority for conservation and breeding programs.     

Metabolism of C-zeatin in phaseolus embryos : occurrence of o-xylosyldihydrozeatin and its ribonucleoside

The metabolism of trans-[8-¹⁴C]zeatin was examined in embryos of Phaseolus acutifolius A. Gray P.I. 321637 and Phaseolus coccineus Lam. cvs Scarlet Runner and Desiree. In both species zeatin was converted to ribosylzeatin, ribosylzeatin 5′-monophosphate, O-glucosyl-9-ribosylzeatin and the recently discovered O-xylosyl derivatives of zeatin and ribosylzeatin (Turner, JE, DWS Mok, MC Mok, G Shaw 1987 Proc Natl Acad Sci USA. In press). Two new metabolites, identified by enzyme degradation and gas chromatography-mass spectrography analyses as O-xylosyldihydrozeatin and its ribonucleoside, were recovered from P. coccineus embryos. From this and previous studies it may be concluded that the potential to form O-xylosyl derivatives of zeatin is present only in embryos of three Phaseolus species (P. vulgaris L., P. coccineus, and P. acutifolius), but not in P. lunatus L., while the reduction of the side chain is most prominent in P. coccineus.