Progress 09/01/00 to 08/31/04
Outputs
This project has been completed with the development of four BAC libraries in common bean in phylogenetically related Phaseolus accessions, three in Phaseolus vulgaris and one in P. lunatus. The three P. vulgaris libraries were established in a Peruvian wild accession, representing the ancestral gene pool of the species, a Mexican wild accession (G02771, representing the wild Mesoamerican gene pool), and a Mesoamerican breeding line, BAT93. The P. lunatus library is considered to be the outlier for control purposes. All the libraries have a coverage of 8-10 genome equivalents, with the exception of the BAT93 library, with a coverage of 20 genome equivalents. Furthermore, the frequency of empty and chloroplast sequence-containing clones is below 1 percent. These libraries have been screened for sequences belonging to two multigene families coding for seed proteins, the phaseolin and APA families. One positive clone of some 155 kb, from G02771, was sequenced in its
entirety. It reveals the three subfamilies of the APA family, namely the arcelin, phytohaemagglutinin, and alpha-amylase inhibitor subfamilies, as predicted from phenotypic analyses of the G02771 accession. This set of phylogenetically ordered BAC libraries is one of the first sets to our knowledge. The BAT93 library has been distributed to colleagues for further investigations into the evolution of disease resistance gene clusters and in situ hybridizations, respectively.
Impacts
The APA locus is an insect resistance locus that has increased in size and resistance functionalities over a brief evolutionary time scale. Materials from this project will help us understand how new resistances appear in plants. This may help us design new resistance genes and understand how resistance genes breakdown after some time. The array of BAC libraries will also help us understand how other important traits evolve over short evolutionary times (including since domestication), including growth habit, disease resistances, and seed protein levels
Publications
- McClean P, Gepts P, Kami J. 2004. Genomics and genetic diversity in common bean. In: Wilson RF, Stalker HT, Brummer EC (eds.), Legume crop genomics. AOCS Press, Champaign, IL: pp. 60 82.
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Progress 01/01/03 to 12/31/03
Outputs
In Phaseolus sp., resistance of seeds to predation by bruchid insects is largely due to members of the APA gene family. Alpha-amylase inhibitors, Phytohaemagglutinins, and Arcelins represent sub-families within this grouping. The family consists of a small, as yet undetermined number of genes that are inherited as a single locus. New members of this family have presumably arisen through gene duplication and diversification during the evolution of the Phaseolus vulgaris complex. The most recent evolutionary event, the appearance of the arcelins, appears to have occurred some 500,000 years ago. Common bean (Phaseolus vulgaris) consists of two major geographic gene pools, Andean and Mesoamerican. These two pools were originally derived from a common ancestor located in the Andes mountains from Ecuador and northern Peru. The DGD-1962 accession is a presumed ancestral type from which the two gene pools originally diverged. A more distantly related species is the lima bean
(P. lunatus), which belongs to a different clade of the genus Phaseolus altogether. To examine the evolutionary changes in the genome structure of the APA gene family, BAC libraries were developed in four representative Phaseolus genotypes (3 in P. vulgaris and 1 in P. lunatus) that typify the evolution of the gene family and Phaseolus sp. as a whole. The BAC libraries are essential in order to examine the evolutionary changes in the genes and the effects evolution has had on the surrounding genomic regions. The results of this investigation will be compared to a similar examination of the Phaseolin gene family. Phaseolin is the major seed protein in Phaseolus spp. This protein is also coded as a small, multi-gene family that has co-evolved with the divergence of the Phaseolus complex and the APA family, but, presumably, as the result of different selective pressures. The BAC libraries were developed using the method of Peterson et al. (2000), but with a novel nuclei isolation
procedure based on the method of Kuehl (1964). This novel procedure yielded large quantities of very high molecular weight DNA with a minimal amount of plastid DNA contamination. The average genome coverage of the libraries represents about 98 percent probability of locating a specific sequence in the library. Although the number of APA clones in the BAT93 and DGD1962 accessions and the Phaseolin clones in the three P. vulgaris accessions approximates the number of genome equivalents, the number of positives in the G02771 accession is about twice that of the other two varieties. G02771 is thought to contain the most recent duplication of the APA locus. BAC end sequences of many of the G02771 clones do not have homologous sequences in the GenBank database whereas the end sequences of BAT93 and DGD1962 clones do have previously identified homologues. This set of BAC libraries is currently the only phylogenetically ordered set of libraries available. The well-established phylogeny of the
Phaseolus complex in combination with these libraries will greatly enhance our ability to study the evolution and domestication of the Phaseolus genus.
Impacts
The APA locus is a insect resistance locus that has increased in size and resistance functionalities over an evolutionary time scale. The goal of this project is to determine how plants generate new sequences with novel resistances at the molecular level. This may help us design new resistance genes and understand how resistance genes breakdown after a while.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
In this year, substantial progress has been made towards the overall goal of the project. Two new BAC libraries have been developed, in genotypes DGD1962 and G02771. DGD1962 is a representative of the presumed wild common ancestor of the two major geographic gene pools. G02771 is a wild bean from Mexico, the progenitor of the Mesoamerican domesticates. Each of these has some 60,000 clones, which represents some 10x coverage of the bean genome. These two libraries complement the BAT93 library developed earlier in this project. The latter library has been shared with a collaborator in France, who is also interested in studying the evolution of multigene families. Furthermore, a small subsample of 1000 clones has been fingerprinted using an automated capillary DNA sequencing procedure. The BAT93 library has been screened with a probe for the APA (arcelin-phytohaemagglutinin-alpha-amylase inhibitor locus) as well as with several RFLP probes linked to genes for
domestication. For each probe, several clones have been identified consistent with the 5x genome coverage of this library. These clones have also been fingerprinted and will form the nucleus of contigs to in development around these loci. One BAC clone of some 130 kb and related to the APA locus has been sent to Belgium for sequencing with a collaborator.
Impacts
The APA locus is a insect resistance locus that has increased in size and resistance functionalities over an evolutionary time scale. The goal of this project is to determine how plants generate new sequences with novel resistances at the molecular level. This may help us design new resistance genes and understand how resistance genes breakdown after a while.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
During the past year a bacterial artificial chromosome (BAC) library was developed for genotype BAT93. This particular genotype is one of the parents of the core mapping population of common bean, the recombinant inbred population BJ (BAT93 x Jalo EEP558). It is also a multiple disease resistant genotype with resistances to anthracnose, rust, bean common mosaic virus, and common bacterial blight. The library currently contains 40,000 clones. The analysis of the insert size in a sample of 222 clones showed that the average size is 95 kb and the median size is 112 kb. The sizes ranged between 10 and 152 kb. Attempts to increase the average size were unsuccessful. However, procedural modifications will be made to decrease the frequency of the smallest clones. This library has been replicated by hand-picking. A preliminary screening has identified clones containing APA genes. A second library is now being developed in the genotype DGD1962. Development of the first clones
has been successful. Additional clones are being produced with the goal of generating a library of some 30,000 clones. Concurrently, the size of the APA locus was determined to be around 180 kb, which suggests that only a small number of clones will be needed to develop a contig of this locus. To increase the density of PCR-based markers around the APA locus and elsewhere in the genome, RFLP probes are being converted into sequence-tagged sites (STS) so that they can be uses as PCR-based markers. The sequences of the first clones have been deposited in GenBank. Our final goal is to convert all the clones mapped in the BJ RI into STS markers.
Impacts
The APA locus is a insect resistance locus that has increased in size and resistance functionalities over an evolutionary time scale. The goal of this project is to determine how plants generate new sequences with novel resistances at the molecular level. This may help us design new resistance genes and understand how resistance genes breakdown after a while.
Publications
- No publications reported this period
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