RESUMO
To preserve their varietal attributes, established grapevine cultivars (Vitis vinifera L. ssp. vinifera) must be clonally propagated, due to their highly heterozygous genomes. Malbec is a France-originated cultivar appreciated for producing high-quality wines and is the offspring of cultivars Prunelard and Magdeleine Noire des Charentes. Here, we have built a diploid genome assembly of Malbec, after trio binning of PacBio long reads into the two haploid complements inherited from either parent. After haplotype-aware deduplication and corrections, complete assemblies for the two haplophases were obtained with a very low haplotype switch-error rate (<0.025). The haplophase alignment identified > 25% of polymorphic regions. Gene annotation including RNA-seq transcriptome assembly and ab initio prediction evidence resulted in similar gene model numbers for both haplophases. The annotated diploid assembly was exploited in the transcriptomic comparison of four clonal accessions of Malbec that exhibited variation in berry composition traits. Analysis of the ripening pericarp transcriptome using either haplophases as a reference yielded similar results, although some differences were observed. Particularly, among the differentially expressed genes identified only with the Magdeleine-inherited haplotype as reference, we observed an over-representation of hypothetically hemizygous genes. The higher berry anthocyanin content of clonal accession 595 was associated with increased abscisic acid responses, possibly leading to the observed overexpression of phenylpropanoid metabolism genes and deregulation of genes associated with abiotic stress response. Overall, the results highlight the importance of producing diploid assemblies to fully represent the genomic diversity of highly heterozygous woody crop cultivars and unveil the molecular bases of clonal phenotypic variation.
RESUMO
Grapevine cultivars are clonally propagated to preserve their varietal attributes. However, genetic variations accumulate due to the occurrence of somatic mutations. This process is anthropically influenced through plant transportation, clonal propagation and selection. Malbec is a cultivar that is well-appreciated for the elaboration of red wine. It originated in Southwestern France and was introduced in Argentina during the 1850s. In order to study the clonal genetic diversity of Malbec grapevines, we generated whole-genome resequencing data for four accessions with different clonal propagation records. A stringent variant calling procedure was established to identify reliable polymorphisms among the analyzed accessions. The latter procedure retrieved 941 single nucleotide variants (SNVs). A reduced set of the detected SNVs was corroborated through Sanger sequencing, and employed to custom-design a genotyping experiment. We successfully genotyped 214 Malbec accessions using 41 SNVs, and identified 14 genotypes that clustered in two genetically divergent clonal lineages. These lineages were associated with the time span of clonal propagation of the analyzed accessions in Argentina and Europe. Our results show the usefulness of this approach for the study of the scarce intra-cultivar genetic diversity in grapevines. We also provide evidence on how human actions might have driven the accumulation of different somatic mutations, ultimately shaping the Malbec genetic diversity pattern.
Assuntos
Variação Genética , Genoma de Planta , Genótipo , Vitis/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Anthocyanin profiles are commonly used for grapevine cultivar identification because it is currently accepted that this trait is closely related to their genetic characteristics. Nevertheless, the extent of the variation for the anthocyanin profiles among clones of the same cultivar has not yet been studied in depth. The relative concentration of anthocyanins of 131 Malbec clones grown in the same vineyard was investigated by HPLC-DAD and the use of comprehensive statistic procedures. Complementarily, the expression level of structural and regulatory genes was studied via real time polymerase chain reaction. Significant variation was identified among the profiles of the clones, mainly due to variations in the amounts of malvidin derivatives. Finally, the differential expression in F3'5'H, OMT1 and AM2 genes seems to be related to the malvidin content variation. This work shows the existence of variation for the anthocyanin profiles among clones from the same grapevine cultivar and the putative involvement of genes related to hydroxylation, methylation, and transport of anthocyanins on the basis of such variation.
Assuntos
Antocianinas/análise , Expressão Gênica , Vitis/química , Vitis/genética , Células Clonais , Frutas/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Extratos Vegetais/química , Especificidade da Espécie , Vitis/classificaçãoRESUMO
El presente estudio se realizo durante el mes de noviembre del año 2000 en acción coordinada entre los servicios de Gineco-obstetricia y de laboratorio Clínico del Hospital 11 Essalud de la Ciudad de Jaén. En un paciente de 30 años con problemas de infertilidad conyugal se aplicaron técnicas de laboratorio especiales en este tipo de problemas se evalúo el semen del paciente, a quien se le explico la metodología de trabajo, aceptando participar en el mismo. El Examen mostró ausencia completa de espermatozoides, lo que señalaba que se estaba frente a un caso de 220 azooospermia. El Ph del semen fue de 9, que es mayor a lo descrito por otros autores. Se observo además en escasovolumen de semen (2 mi) en este paciente. Se puede concluir que la utilización de técnicas sencillas y de bajo costo pueden contribuir al diagnostico precoz de infertilidad masculina y que la participación de los pacientes de los equipos multidisciplinarios por el abordaje de infertilidad conyugal permitirán establecer los patrones epidemiológicos en la ciudad de Jaén del Departamento de Cajamarca - Perú.
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