RESUMEN
Resolution of the two haplotypes present in an individual that is heterozygous at a locus has been a difficult problem for nucleotide sequence-based population genetic studies. Here, we demonstrate a method in which allele-specific polymerase chain reaction (AS-PCR) and computational phasing are combined for relatively high-throughput, efficient resolution of phase in resequencing studies. Using data from multiple loci that were fully experimentally phased, we demonstrate that the popular computational tool PHASE can accurately phase heterozygous individuals with common SNPs (single nucleotide polymorphisms) and/or common haplotypes. However, we also demonstrate that experimental phasing with AS-PCR can efficiently supplement computational phasing, providing a rapid means to phase individuals with rare SNPs or haplotypes and with heterozygous insertion/deletion polymorphisms. By following simple stepwise procedures, AS-PCR can result in much more efficient and accurate experimental phasing of haplotypes than is possible with traditional methods such as cloning.
RESUMEN
Recombination occurs through both homologous crossing over and homologous gene conversion during meiosis. The contribution of recombination relative to mutation is expected to be dramatically reduced in inbreeding organisms. We report coalescent-based estimates of the recombination parameter (rho) relative to estimates of the mutation parameter (theta) for 18 genes from the highly self-fertilizing grass, wild barley, Hordeum vulgare ssp. spontaneum. Estimates of rho/theta are much greater than expected, with a mean rho/theta approximately 1.5, similar to estimates from outcrossing species. We also estimate rho with and without the contribution of gene conversion. Genotyping errors can mimic the effect of gene conversion, upwardly biasing estimates of the role of conversion. Thus we report a novel method for identifying genotyping errors in nucleotide sequence data sets. We show that there is evidence for gene conversion in many large nucleotide sequence data sets including our data that have been purged of all detectable sequencing errors and in data sets from Drosophila melanogaster, D. simulans, and Zea mays. In total, 13 of 27 loci show evidence of gene conversion. For these loci, gene conversion is estimated to contribute an average of twice as much as crossing over to total recombination.
Asunto(s)
Conversión Génica , Variación Genética , Haplotipos , Mutación , Recombinación Genética , Animales , Drosophila/genética , Drosophila melanogaster/genética , Genes de Plantas , Genética de Población , Hordeum/genética , Zea mays/genéticaRESUMEN
High levels of inbreeding cause populations to become composed of homozygous, inbred lines. High levels of homozygosity limit the effectiveness of recombination, and therefore, retard the rate of decay of linkage (gametic phase) disequilibrium (LD) among mutations. Inbreeding and recombination interact to shape the expected pattern of LD. The actual extent of nucleotide sequence level LD within inbreeding species has only been studied in Arabidopsis, a weedy species whose global range has recently expanded. In the present study, we examine the levels of LD within and between 18 nuclear genes in 25 accessions from across the geographic range of wild barley, a species with a selfing rate of approximately 98%. In addition to examination of intralocus LD, we employ a resampling method to determine whether interlocus LD exceeds expectations. We demonstrate that, for the majority of wild barley loci, intralocus LD decays rapidly, i.e., at a rate similar to that observed in the outcrossing species, Zea mays (maize). Excess interlocus LD is observed at 15% of two-locus combinations; almost all interlocus LD involves loci with significant geographic structuring of mutational variation.
Asunto(s)
Hordeum/genética , Desequilibrio de Ligamiento , ADN de Plantas/genética , Fertilización , Genes de Plantas , Variación Genética , Haplotipos , Endogamia , Datos de Secuencia Molecular , Zea mays/genéticaRESUMEN
A central goal of evolutionary genetics is to trace the causal pathway between mutations at particular genes and adaptation at the phenotypic level. The proximate objective is to identify adaptations through the analysis of molecular sequence data from specific candidate genes or their regulatory elements. In this paper, we consider the molecular evolution of floral color in the morning glory genus (Ipomoea) as a model for relating molecular and phenotypic evolution. To begin, flower color variation usually conforms to simple Mendelian transmission, thus facilitating genetic and molecular analyses. Population genetic studies of flower color polymorphisms in the common morning glory (Ipomoea purpurea) have shown that some morphs are subject to complex patterns of selection. Striking differences in floral color and morphology are also associated with speciation in the genus Ipomoea. The molecular bases for these adaptive shifts can be dissected because the biosynthetic pathways that determine floral pigmentation are well understood and many of the genes of flavonoid biosynthesis have been isolated and extensively studied. We present a comparative analysis of the level of gene expression in Ipomoea for several key genes in flavonoid biosynthesis. Specifically we ask: how frequently are adaptive shifts in flower color phenotypes associated with changes in regulation of gene expression versus mutations in structural genes? The results of this study show that most species differences in this crucial phenotype are associated with changes in the regulation of gene expression.
Asunto(s)
Adaptación Fisiológica/genética , Flores/fisiología , Regulación de la Expresión Génica de las Plantas , Ipomoea/genética , Filogenia , Pigmentación/fisiología , Northern Blotting , ADN Complementario/genética , Flavonoides/biosíntesis , Hibridación in Situ , Ipomoea/fisiología , Funciones de Verosimilitud , Modelos Genéticos , Mutación/genética , Análisis de Secuencia de ADNRESUMEN
Mutations arise in a single individual and at a single point in time and space. The geographic distribution of mutations reflects both historical population size and frequency of migration. We employ coalescence-based methods to coestimate effective population size, frequency of migration, and level of recombination compatible with observed genealogical relationships in sequence data from nine nuclear genes in wild barley (Hordeum vulgare ssp. spontaneum), a highly self-fertilizing grass species. In self-fertilizing plants, gamete dispersal is severely limited; dissemination occurs primarily through seed dispersal. Also, heterozygosity is greatly reduced, which renders recombination less effective at randomizing genetic variation and causes larger portions of the genome to trace a similar history. Despite these predicted effects of this mating system, the majority of loci show evidence of recombination. Levels of nucleotide variation and the patterns of geographic distribution of mutations in wild barley are highly heterogeneous across loci. Two of the nine sampled loci maintain highly diverged, geographic region-specific suites of mutations. Two additional loci include region-specific haplotypes with a much shallower coalescence. Despite inbreeding, sessile growth habit, and the observation of geographic structure at almost half of sampled loci, parametric estimates of migration suggest that seed dispersal is sufficient for migration across the approximately 3,500-km range of the species. Recurrent migration is also evident based on the geographic distribution of mutational variation at some loci. At one locus a single haplotype has spread rapidly enough to occur, unmodified by mutation, across the range of the species.