The effect of arcelin-1 on the structure of the midgut of bruchid larvae and immunolocalization of the arcelin protein

Some wild accessions of the common bean (Phaseolus vulgaris) contain a family of proteins called arcelins, that are toxic to the larvae of certain bruchid species. Among the six allelic variants of arcelin tested so far, arcelin-5 and arcelin-1 confer the highest level of resistance against the Mexican bean weevil, Zabrotes subfasciatus. The same proteins are not toxic to the bean weevil, Acanthoscelides obtectus, which is also a serious pest of cultivated beans. Arcelins belong to the bean lectin family that includes phytohemaggutinins and alpha-amylase inhibitors. Although homologous to lectins, arcelins are themselves only very weak lectins, and their binding properties have not been clearly established. The toxic properties of arcelins may be related to their recognition of and interaction with the glycoproteins and other constituents of the membranes along the digestive tract of insects. Since arcelin-1 was shown to have growth inhibitory effects for the larvae of Z. subfasciatus but not of A. obtectus, we examined the effect of an arcelin-1 containing diet on the structure of the cells that line the intestinal tract of the larvae of these two bruchid species, and used antibodies against arcelin to examine the distribution of arcelin within the cells and tissues. Here we show that dietary arcelin-1 caused an alteration of the gut structure and the penetration of arcelin into the haemolymph in Z. subfasciatus but not in A. obtectus. These results lead us to suggest that arcelins exert their toxic effect by severely damaging the epithelial cells.     

Bruchid resistance of common bean lines having an altered seed protein composition

 Arcelin seed proteins of common bean (Phaseolus vulgaris L.) are toxic to one of the most damaging pests of bean seeds, Zabrotes subfasciatus (Boheman), but they appear to have little effect on another important bean pest, Acanthoscelides obtectus (Say), when introduced into standard cultivars by backcrossing. With the goal of increasing arcelin concentration to improve resistance, we modified seed-protein composition by introducing a null allele for the major seed protein, phaseolin, into lines (SMARC1, 2 and 4) or three phytohemagglutinin types (SMPHA lines). These lines were tested for resistance to both insects by measuring percentage insect emergence (%E) and days-to-adult emergence (DAE). For SMARC lines, arcelin type was the most important factor in resistance levels, with SMARC1 lines being most resistant, SMARC2 lines intermediate, and SMARC4 lines the least resistant to both bruchids. Additionally, the absence of phaseolin was a significant factor in the resistance of SMARC lines to A. obtectus. SMARC1 lines without phaseolin had half the percentage insect emergence of lines with phaseolin. SMARC1 lines with an altered seed composition had the highest levels of resistance to both bruchids of any large-seeded line reported to-date. Received: 2 April 1997 / Accepted: 20 May 1997     

Protective mechanism of the Mexican bean weevil against high levels of alpha-amylase inhibitor in the common bean.

Alpha-amylase inhibitor (alpha AI) protects seeds of the common bean (Phaseolus vulgaris) against predation by certain species of bruchids such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (Callosobruchus chinensis), but not against predation by the bean weevil (Acanthoscelides obtectus) or the Mexican bean weevil (Zabrotes subfasciatus), insects that are common in the Americas. We characterized the interaction of alpha AI-1 present in seeds of the common bean, of a different isoform, alpha AI-2, present in seeds of wild common bean accessions, and of two homologs, alpha AI-Pa present in seeds of the tepary bean (Phaseolus acutifolius) and alpha AI-Pc in seeds of the scarlet runner bean (Phaseolus coccineus), with the midgut extracts of several bruchids. The extract of the Z. subfasciatus larvae rapidly digests and inactivates alpha AI-1 and alpha AI-Pc, but not alpha AI-2 or alpha AI-Pa. The digestion is caused by a serine protease. A single proteolytic cleavage in the beta subunit of alpha AI-1 occurs at the active site of the protein. When degradation is prevented, alpha AI-1 and alpha AI-Pc do not inhibit the alpha-amylase of Z. subfasciatus, although they are effective against the alpha-amylase of C. chinensis. Alpha AI-2 and alpha AI-Pa, on the other hand, do inhibit the alpha-amylase of Z. subfasciatus, suggesting that they are good candidates for genetic engineering to achieve resistance to Z. subfasciatus.     

Assessment of the importance of α-amylase inhibitor-2 in bruchid resistance of wild common bean

Both α-amylase inhibitor-2 (αAI-2) and arcelin have been implicated in resistance of wild common bean (Phaseolus vulgaris L.) to the Mexican bean weevil (Zabrotes subfasciatus Boheman). Near isogenic lines (NILs) for arcelin 1–5 were generated by backcrossing wild common bean accessions with a cultivated variety. Whereas seeds of a wild accession (G12953) containing both αAI-2 and arcelin 4 were completely resistant to Z. subfasciatus, those of the corresponding NIL were susceptible to infestation, suggesting that the principal determinant of resistance was lost during backcrossing. Three independent lines of transgenic azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi] expressing αAI-2 accumulated high levels of this protein in seeds. The expression of αAI-2 in these lines conferred protection against the azuki bean weevil (Callosobruchus chinensis L.), likely through inhibition of larval digestive α-amylase. However, although the seed content of αAI-2 in these transgenic lines was similar to that in a wild accession of common bean (G12953), it did not confer a level of resistance to Z. subfasciatus similar to that of the wild accession. These results suggest that αAI-2 alone does not provide a high level of resistance to Z. subfasciatus. However, αAI-2 is an effective insecticidal protein with a spectrum of activity distinct from that of αAI-1, and it may prove beneficial in genetic engineering of insect resistance in legumes.     

QUES, a new Phaseolus vulgaris genotype resistant to common bean weevils, contains the Arcelin-8 allele coding for new lectin-related variants

In common bean (Phaseolus vulgaris L.), the most abundant seed proteins are the storage protein phaseolin and the family of closely related APA proteins (arcelin, phytohemagglutinin and α-amylase inhibitor). High variation in APA protein composition has been described and the presence of arcelin (Arc) has been associated with bean resistance against two bruchid beetles, the bean weevil (Acanthoscelides obtectus Say) and the Mexican bean weevil (Zabrotes subfasciatus Bohemian). So far, seven Arc variants have been identified, all in wild accessions, however, only those containing Arc-4 were reported to be resistant to both species. Although many efforts have been made, a successful breeding of this genetic trait into cultivated genotypes has not yet been achieved. Here, we describe a newly collected wild accession (named QUES) and demonstrate its resistance to both A. obtectus and Z. subfasciatus. Immunological and proteomic analyses of QUES seed protein composition indicated the presence of new Arc and arcelin-like (ARL) polypeptides of about 30 and 27 kDa, respectively. Sequencing of cDNAs coding for QUES APA proteins confirmed that this accession contains new APA variants, here referred to as Arc-8 and ARL-8. Moreover, bioinformatic analysis showed the two proteins are closely related to APA components present in the G12949 wild bean accession, which contains the Arc-4 variant. The presence of these new APA components, combined with the observations that they are poorly digested and remain very abundant in A. obtectus feces, so-called frass, suggest that the QUES APA locus is involved in the bruchid resistance. Moreover, molecular analysis indicated a lower complexity of the locus compared to that of G12949, suggesting that QUES should be considered a valuable source of resistance for further breeding purposes.     

Analysis of bruchid resistance in the wild common bean accession G02771: no evidence for insecticidal activity of arcelin 5

Arcelins are abundant seed storage proteins thought to be implicated in the resistance of wild Phaseolus vulgaris (L.) genotypes against Zabrotes subfasciatus (Boheman), an important storage insect pest of common bean. Here, the insecticidal activity of the arcelin-5 variant that is present in the highly resistant P. vulgaris accession G02771 was investigated. No correlation could be established between the presence of arcelin 5 and the insecticidal effects observed in G02771 seeds. Insect feeding assays with artificial seeds into which purified arcelin-5 protein was incorporated and with transgenic P. acutifolius (A. Gray) seeds in which the arcelin-5 genes were expressed, showed that the presence of arcelin-5 proteins, even at elevated levels, was not sufficient to achieve adequate resistance against Z. subfasciatus. The same might apply to other arcelin variants. Nevertheless, as resistance is clearly closely linked to the presence of the arcelin-1 or arcelin-5 locus, arcelins remain useful markers in breeding programmes aimed at introgressing high levels of resistance to Z. subfasciatus in P. vulgaris cultivars.     

Bean arcelin

Summary SDS-PAGE of seed proteins from the seeds of a nondomesticated bean of Mexican origin (Phaseolus vulgaris L., PI 325690) revealed the presence of a novel 38 kd protein which appeared to be neither an altered phaseolin nor a lectin fraction. The protein was named arcelin, after Arcelia, the town in the state of Guerrero near which PI 325690 had been collected. The pure line, UW 325, was derived by self fertilization of the plant from a single arcelin-containing seed of PI 325690. Despite a low percentage seed phaseolin (14.6%), seed phenotype, seed germination, plant growth, pollen fertility, and percentage seed protein of UW 325 were normal. Analyses of F₂ and F₃ seeds from a single F₁ plant of the cross SanilacXPI 325690-3 revealed that arcelin expression was inherited as a single gene and that presence was dominant to absence of arcelin. The mean percentage phaseolin in the seeds of homozygous dominant Arc/Arc F₃ families (14.0%) was significantly lower than that of the homozygous recessive arc/arc seeds (44.7%). The distribution of percentage phaseolin values for seeds within segregating families was bimodal and nonoverlapping. Without exception, seeds containing arcelin (Arc+phenotype) contained a lower percentage phaseolin than seeds lacking arcelin (Arc-phenotype). Although arcelin presence was associated with low percentage phaseolin, the Arc/Arc and Arc/arc genotypes were similar for seed weight and percentage total seed protein.     

Lectin-related resistance factors against bruchids evolved through a number of duplication events

Abundant lectin-related proteins found in common beans (Phaseolus vulgaris L.) have been shown to confer resistance against the larvae of a number of bruchid species. Genes encoding for these proteins are members of the lectin multigene family, the most representative components being arcelins, phytohemagglutinins and -amylase inhibitors. Arcelins have been described in seven variants, some of which are resistance factors against the Mexican bean weevil (Zabrotes subfasciatus), a major bean predator. In this study the isolation and sequencing of arcelin genes from wild P. vulgaris genotypes, containing Arc3 and Arc7 variants, is reported, and similarities and evolutionary relationships among the seven known arcelins are described. The evolutionary analysis shows that arcelins 3 and 4 cluster together and are the most-ancient variants. A duplication event gave rise to two additional clusters, one comprising arcelins 1, 2 and 6 and separated from the cluster of arcelins 5 and 7. A multiple number of arcelin genes were found in arcelin 3 and 4 genotypes indicating that more than one type of arcelin gene may be present in the same locus. Some of these sequences are reminiscent of ancient duplication events in arcelin evolution demonstrating that arcelins have evolved through multiple duplications. A further aim of this paper was to better understand and describe the evolution of the entire lectin multigene family. Beside arcelins, a number of other types of sequences, such as putative lectins and sequences not easily classifiable, were found in genotypes containing Arc3 and Arc4. These results, together with the evolutionary analysis, indicate that lectin loci are quite complex and confirm their origin by multiple duplication events.Communicated by J.S. Heslop-HarrisonL. Lioi and F. Sparvoli contributed equally to the work     

Development of Zabrotes subfasciatus (Boh.) (Coleoptera: Chrysomelidae, Bruchinae) in genotypes of Phaseolus vulgaris L. (Fabaceae) cultivated in the State of Parana and containing arcelin

This research intended to evaluate the development of Zabrotes subfasciatus (Boh.), a stored-grain pest, on bean genotypes (Phaseolus vulgaris L.) commonly cultivated in the State of Parana and containing arcelin, and the possible resistance of these genotypes to the bruchine. Tests were performed under laboratory conditions (27 masculineC, fotophase 12h, 50 +/- 10 % RH) with the genotypes TPS-Bionobre, IAC-Una, IPR-Uirapuru, IAPAR 44, IPR Juriti, IAPAR 81, Pérola, Carioca, Bolinha, and two others containing arcelin, Arc 1 and Arc 2. The genotypes with Arc 1 and 2 alleles caused higher mortality of immature stages; in Arc 1 developmental period was prolonged and the male and female dry weights were the lowest, suggesting an antibiosis mechanism of resistance. Non-preference for oviposition was not observed for these two genotypes. Among varieties without arcelin, IAPAR 44 was the most resistant to the bruchid, being the least preferred for oviposition, and promoting low percentage of viable eggs, long developmental period and reduced male and female adult dry weight. Perola, IPR Juriti and Bolinha with high number of eggs and viable eggs, low mortality of immature stages, were the most susceptible.     

Characterization of resistance to the bean weevil <Emphasis Type="Italic">Acanthoscelides obtectus</Emphasis> Say, 1831 (Coleoptera: Bruchidae) in common bean genotypes

The bean weevil Acanthoscelides obtectus (Say, 1831) (Coleoptera: Bruchidae) is one of the most serious pests of stored beans worldwide because of the damage it causes to grains within warehouses. The use of resistant genotypes may offer a control strategy for this pest. In the current study, we screened common bean genotypes of Andean American and Mesoamerican origin in laboratory and greenhouse bioassays to select the most promising beans for resistance to the bean weevil. In the laboratory, we evaluated number of eggs, period of development (egg-adult), number of emerged adults, dry weight of adults, and weight of consumed grains. In the greenhouse, number of pods per plant and number of grains per pod were evaluated. We also assessed the percentages of damaged pods per plant and damaged grains per pod. Combining the results obtained in the laboratory and greenhouse assays, the common bean genotypes Arc.1, Arc.2, Arc.1S, Arc.5S, and Arc.3S were identified as resistance expressing antibiosis against A. obtectus. The lowest percentages of damaged pods were found in the Arc.1 and Arc.1S genotypes, and their resistance to damage was apparently morphological (antixenotic) because they possessed structures that prevented contact between larvae and grains. The use of resistant genotypes in combination with other techniques may improve management of the weevil. Additionally, the resistant genotypes identified here can be used in breeding programs to develop common bean lines with resistance to A. obtectus.     

Bean arcelin

Summary Crude proteins from seeds of wild bean accessions of Mexican origin were analyzed by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE). Several accessions had electrophoretic patterns showing unique protein bands. When analyzed by two-dimensional isoelectric focusing (IEF)-SDS/PAGE, four protein variants which had electrophoretic mobilities similar to each other but different from the other major seed proteins, phaseolin and lectin, were observed. All four variants, which have not been described in cultivated beans, were tentatively named arcelin proteins and designated as arcelin 1, 2, 3 and 4. Arcelins 3 and 4 had polypeptides that comigrated on two-dimensional gels and these variants occurred in accessions that were collected in the same location. Analysis of single F₂ seeds from crosses among arcelin-containing lines and from crosses between cultivated beans lines without arcelin and arcelin-containing lines revealed that differences in arcelin polypeptide expression were inherited monogenically. The alleles for different arcelin variants were codominant to each other and dominant to the absence of arcelin. The gene(s) controlling arcelin proteins were unlinked to those controlling phaseolin expression and tightly linked to genes controlling the presence of lectin proteins (< 0.30% recombination). The possible origins of arcelin genes and their potential role in bruchid resistance are discussed.     

Toxicity assessment of wild bean seed protein--arcelin on Asian armyworm, Spodoptera litura (Fabricius)

Arcelin, an anti-metabolic protein was purified from the seeds of wild bean, Lablab purpureus. The feeding assay containing arcelin at 5, 10 and 15 microg concentrations revealed no antifeedant effect against fifth instar larvae of S. litura. However, the enhanced activity of alpha- and beta-naphthyl esterases in the mid-gut samples of S. litura treated with arcelin suggests countermeasure against the toxic effect of arcelin.     

The arcelin-5 Gene of Phaseolus vulgaris Directs High Seed-Specific Expression in Transgenic Phaseolus acutifolius and Arabidopsis Plants

The regulatory sequences of many genes encoding seed storage proteins have been used to drive seed-specific expression of a variety of proteins in transgenic plants. Because the levels at which these transgene-derived proteins accumulate are generally quite low, we investigated the utility of the arcelin-5 regulatory sequences in obtaining high seed-specific expression in transgenic plants. Arcelin-5 is an abundant seed protein found in some wild common bean (Phaseolus vulgaris L.) genotypes. Seeds of Arabidopsis and Tepary bean (Phaseolus acutifolius A. Gray) plants transformed with arcelin-5 gene constructs synthesized arcelin-5 to levels of 15% and 25% of the total protein content, respectively. To our knowledge, such high expression levels directed by a transgene have not been reported before. The transgenic plants also showed low plant-to-plant variation in arcelin expression. Complex transgene integration patterns, which often result in gene silencing effects, were not associated with reduced arcelin-5 expression. High transgene expression was the result of high mRNA steady-state levels and was restricted to seeds. This indicates that all requirements for high seed-specific expression are cis elements present in the cloned genomic arcelin-5 sequence and trans-acting factors that are available in Arabidopsis and Phaseolus spp., and thus probably in most dicotyledonous plants.     

Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences

Over the past decade, several high value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds. Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops. In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large scale production.     

Talisia esculenta lectin and larval development of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae)

Bruchid larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil and the Mexican bean weevil, are pests that damage stored seeds. Plant lectins have been implicated as antibiosis factors against insects, particularly the cowpea weevil, Callosobruchus maculatus. Talisia esculenta lectin (TEL) was tested for anti-insect activity against C. maculatus and Zabrotes subfasciatus larvae. TEL produced ca. 90% mortality to these bruchids when incorporated in an artificial diet at a level of 2% (w/w). The LD(50) and ED(50) for TEL was ca. 1% (w/w) for both insects. TEL was not digested by midgut preparations of C. maculatus and Z. subfasciatus. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

Insecticidal activity of an alpha-amylase inhibitor-like protein resembling a putative precursor of alpha-amylase inhibitor in the common bean, Phaseolus vulgaris L.

alpha-Amylase inhibitor (alphaAI) in the common bean, Phaseolus vulgaris L., protects seeds from insect pests such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (C. chinensis). Cultivars which lack alphaAI still show resistance to both bruchids. These cultivars have a glycoprotein that reacts with anti-alphaAI-1 antibodies. The glycoprotein with a molecular mass of 29 kDa (Gp29) was purified and the encoding gene was isolated. The primary structure of Gp29 is the same as alpha-amylase inhibitor-like protein (AIL) from which the encoding gene has already been isolated. AIL resembles a putative precursor of alphaAI, even though it does not form the active inhibitor. However, AIL has some inhibitory effect on the growth of C. maculatus but not C. chinensis. The presence of AIL alone is insufficient to explain the bruchid resistance of common bean cultivars lacking alpha-AI. Common bean seeds appear to contain several factors responsible for the bruchid resistance.     

Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests

Plant cystatins show great potential as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevils Acanthoscelides obtectus and Zabrotes subfasciatus, which display major activities of digestive cysteine proteinases in midguts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna unguiculata) seeds, was expressed in Escherichia coli and purified with a Ni-NTA agarose column. It strongly inhibited papain and proteinases from midguts of both A. obtectus and Z. subfasciatus bruchids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N-terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabilization of the enzyme-inhibitor complex are absent in the partial N-terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The application of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed.     

Bean [alpha]-Amylase Inhibitor Confers Resistance to the Pea Weevil (Bruchus pisorum) in Transgenic Peas (Pisum sativum L.)

Bruchid larvae cause major losses of grain legume crops through-out the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing in the field. We transferred the cDNA encoding the [alpha]-amylase inhibitor ([alpha]-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacterium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The [alpha]-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T5 seed generation, and [alpha]-AI accumulated in the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2% [alpha]-AI. In the T5 seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although [alpha]-AI also inhibits human [alpha]-amylase, cooked peas should not have a negative impact on human energy metabolism.     

Altering protein composition by genetically removing phaseolin from common bean seeds containing arcelin or phytohemagglutinin

 Arcelin seed proteins of common bean (Phaseolus vulgaris L.) confer resistance to bruchid pests, and in vitro results suggested that greater resistance could be achieved by increasing the concentration of arcelin. We created backcross lines having arcelin alleles (SMARC lines), or alleles for the related protein phytohemagglutinin (PHA) (SMPHA lines), and a null allele for phaseolin to determine if seeds lacking phaseolin would contain increased quantities of arcelin or PHA proteins. To test the affects of genetically removing phaseolin, SMARC and SMPHA lines were derived as pairs of phaseolin-containing and phaseolin-null lines. Parental, SMARC, and SMPHA lines were grown in a replicated greenhouse trial and measured for days-to-flower, days-to-maturity, seed weight, and for quantities of phaseolin, arcelin dimer, PHA, and total proteins. There were no differences between pairs of phaseolin and phaseolin-null lines for days-to-flower, seed weight or total protein, and inconsistent differences for days-to-maturity. Arcelin concentrations were significantly increased in two of four pairs of SMARC lines, and PHA concentration was significantly greater in four of five pairs of SMPHA lines. These or other changes in the seed protein composition in phaseolin null lines may improve resistance to bruchids. Received: 2 April 1997 / Accepted: 20 May 1997     

Genetic mapping of microsatellite markers around the arcelin bruchid resistance locus in common bean

The deployment in common beans (Phaseolus vulgaris L.) of arcelin-based bruchid resistance could help reduce post-harvest storage losses to the Mexican bean weevil [(Zabrotes subfasciatus (Boheman)]. Arcelin is a member of the arcelin-phytohemagglutinin-α-amylase inhibitor (APA) family of seed proteins, which has been extensively studied but not widely used in bean breeding programs. The purpose of this study was to evaluate microsatellite markers for genetic analysis of arcelin-based bruchid resistance and to determine the orientation of markers and the rate of recombination around the APA locus. A total of 10 previously developed microsatellites and 22 newly developed markers based on a sequenced BAC from the APA locus were screened for polymorphism and of these 15 were mapped with an F₂ population of 157 individuals resulting from a susceptible × resistant cross of SEQ1006 × RAZ106 that segregated for both the arcelin 1 allele and resistance to the bruchid, Z. subfasciatus. Microsatellites derived from APA gene sequences were linked within 0.8 cM of each other and were placed relative to the rest of the b04 linkage group. In a comparison of genetic to physical distance on the BAC sequence, recombination was found to be moderate with a ratio of 125 kb/cM, but repressed within the APA locus itself. Several markers were predicted to be very effective for genetic studies or marker-assisted selection, based on their significant associations with bruchid resistance and on low adult insect emergence and positions flanking the arcelin and phytohemagglutinin genes.