RESUMO
BACKGROUND: An interesting seed protein family with a role in preventing insect herbivory is the multi-gene, APA family encoding the alpha-amylase inhibitor, phytohemagglutinin and arcelin proteins of common bean (Phaseolus vulgaris). Variability for this gene family exists and has been exploited to breed for insect resistance. For example, the arcelin locus has been successfully transferred from wild to cultivated common bean genotypes to provide resistance against the bruchid species Zabrotes subfasciatus although the process has been hampered by a lack of genetic tools for and understanding about the locus. In this study, we analyzed linkage disequilibrium (LD) between microsatellite markers at the APA locus and bruchid resistance in a germplasm survey of 105 resistant and susceptible genotypes and compared this with LD in other parts of the genome. RESULTS: Microsatellite allele diversity was found to vary with each of the eight APA-linked markers analyzed, and two markers within the APA locus were found to be diagnostic for bruchid resistance or susceptibility and for the different arcelin alleles inherited from the wild accessions. Arc1 was found to provide higher levels of resistance than Arc5 and the markers in the APA locus were highly associated with resistance showing that introgression of this gene-family from wild beans provides resistance in cultivated beans. LD around the APA locus was found to be intermediate compared to other regions of the genome and the highest LD was found within the APA locus itself for example between the markers PV-atct001 and PV-ag004. CONCLUSIONS: We found the APA locus to be an important genetic determinant of bruchid resistance and also found that LD existed mostly within the APA locus but not beyond it. Moderate LD was also found for some other regions of the genome perhaps related to domestication genes. The LD pattern may reflect the introgression of arcelin from the wild into the cultivated background through breeding. LD and association studies for the arcelin gene, linked genes and other members of the APA family are essential for breaking linkage drag while maintaining high levels of bruchid resistance in common bean.
Assuntos
Loci Gênicos/genética , Inseticidas/metabolismo , Desequilíbrio de Ligação/genética , Phaseolus/genética , Proteínas de Plantas/genética , Alelos , Animais , Besouros/fisiologia , Marcadores Genéticos , Genoma de Planta/genética , Genótipo , Repetições de Microssatélites/genética , Tamanho do Órgão/genética , Fenótipo , Dinâmica PopulacionalRESUMO
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-alpha-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 F(2) population of 157 individuals resulting from a susceptible x resistant cross of SEQ1006 x 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.
Assuntos
Mapeamento Cromossômico , Glicoproteínas/genética , Imunidade Inata/genética , Repetições de Microssatélites/genética , Phaseolus/genética , Doenças das Plantas/genética , Lectinas de Plantas/genética , Gorgulhos/fisiologia , Animais , DNA de Plantas/genética , Phaseolus/imunologia , Phaseolus/microbiologia , Fenótipo , Doenças das Plantas/imunologia , Sementes/genética , Sementes/imunologia , Sementes/microbiologiaRESUMO
Microsatellite markers are useful genetic tools for a wide array of genomic analyses although their development is time-consuming and requires the identification of simple sequence repeats (SSRs) from genomic sequences. Screening of non-enriched, small-insert libraries is an effective method of SSR isolation that can give an unbiased picture of motif frequency. Here we adapt high-throughput protocols for the screening of plasmid-based libraries using robotic colony picking and filter preparation. Seven non-enriched genomic libraries from common bean genomic DNA were made by digestion with four frequently cutting restriction enzymes, double digestion with a frequently cutting restriction enzyme and a less frequently cutting restriction enzyme, or sonication. Library quality was compared and three of the small-insert libraries were selected for further analysis. Each library was plated and picked into 384-well plates that were used to create high-density filter arrays of over 18 000 clones each, which were screened with oligonucleotide probes for various SSR motifs. Positive clones were found to have low redundancy. One hundred SSR markers were developed and 80 were tested for polymorphism in a standard parental survey. These microsatellite markers derived from non-SSR-enriched libraries should be useful additions to previous markers developed from enriched libraries.