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Interspecific hybridization was initiated between wild P. acutifolius accession G40199 and P. vulgaris to introgress resistance to Acanthoscelides obtectus (bean seed weevil). F1 interspecific hybrids were recovered by embryo rescue and maintained until flowering. Mostly sterile hybrids were backcrossed twice to common bean cultivar ICA Pijao to recover sufficient fertility for the lines to reproduce without assistance. The inheritance of a 33 kDa seed storage protein from accession G40199 was studied in an F2 intraspecific population from a cross of G40199 and a cultivated Brown Tepary accession. G40199 possessed the protein but Brown Tepary did not. The protein was inherited as a single dominant gene in a F2 population of 116 individuals. Following these observations, interspecific hybrids were progeny tested for the introgression of a 33 kDa protein from accession G40199. Backcross interspecific introgression lines were used as bridge parents to transfer the 33 kDa protein into the large red seeded cultivar Rojo adapted to Tanzania, and into a phaseolin null backcross breeding line of Rojo with the objective of introgressing resistance to A. obtectus into a Sub-Saharan Africa variety. Genomic, proteomic and phylogenetic characterization of genes associated with the 33 kDa proteins were conducted in order to identify the mechanism of resistance found in accession G40199. Genomic DNA encoding a family of lectin-like seed storage proteins (the complex APA locus) was amplified by PCR using primers for arcelin, phytohaemagglutinin and alpha amylase inhibitors. The PCR products were used as molecular markers and co-segregated with the 33 kDa protein. Analyses of mRNA expression identified two arcelin variants, ARL-3pa and ARL-4pa, as expressed in G40199 and its derived interspecific hybrids. MS-MS proteomic analysis of seed protein profiles in G40199 demonstrated the presence of protein peptides with amino acid sequences corresponding to the two arcelin variants and phytohaemagglutinin (PHA) protein subunits. Essentially similar protein peptides were observed in the introgression lines. Phylogenetic analysis demonstrated the difference of the two arcelin variants in G40199 that clustered with P. acutifolius arcelin-like proteins and were separated from those of P. vulgaris. Seeds from G40199 and BC2F3 interspecific hybrid lines were subjected to an A. obtectus feeding trial at OSU. Introgression lines expressing the homozygous 33 kDa protein demonstrated significant bruchid resistance. This resistance was exhibited as delayed insect emergence with a mean of 63 days for 50% F1 adults after inoculation compared to 44 days to 50% adult for ICA Pijao. In addition, reduced size and weight of adults, and reduced number of adults was observed in a period of 72 days after bruchid infestation. G40199 was completely resistant with no emerging adults. The 33 kDa protein which is linked to co-expression of ARL-3pa, ARL-4pa and PHA protein subunits is a contributing factor to the observed resistance to A. obtectus among interspecific hybrids. |
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