ABSTRACT
BACKGROUND: Genomic methods for identifying causative variants for trait loci applicable to a wide range of germplasm are required for plant biologists and breeders to understand the genetic control of trait variation. RESULTS: We implemented Cas9-targeted sequencing for fine-mapping in apple, a method combining CRISPR-Cas9 targeted cleavage of a region of interest, followed by enrichment and long-read sequencing using the Oxford Nanopore Technology (ONT). We demonstrated the capability of this methodology to specifically cleave and enrich a plant genomic locus spanning 8 kb. The repeated mini-satellite motif located upstream of the Malus × domestica (apple) MYB10 transcription factor gene, causing red fruit colouration when present in a heterozygous state, was our exemplar to demonstrate the efficiency of this method: it contains a genomic region with a long structural variant normally ignored by short-read sequencing technologiesCleavage specificity of the guide RNAs was demonstrated using polymerase chain reaction products, before using them to specify cleavage of high molecular weight apple DNA. An enriched library was subsequently prepared and sequenced using an ONT MinION flow cell (R.9.4.1). Of the 7,056 ONT reads base-called using both Albacore2 (v2.3.4) and Guppy (v3.2.4), with a median length of 9.78 and 9.89 kb, respectively, 85.35 and 91.38%, aligned to the reference apple genome. Of the aligned reads, 2.98 and 3.04% were on-target with read depths of 180 × and 196 × for Albacore2 and Guppy, respectively, and only five genomic loci were off-target with read depth greater than 25 × , which demonstrated the efficiency of the enrichment method and specificity of the CRISPR-Cas9 cleavage. CONCLUSIONS: We demonstrated that this method can isolate and resolve single-nucleotide and structural variants at the haplotype level in plant genomic regions. The combination of CRISPR-Cas9 target enrichment and ONT sequencing provides a more efficient technology for fine-mapping loci than genome-walking approaches.
ABSTRACT
Foram estudadas 164 gestantes primigestas de baixo nivel socioeconomico com idade variavel entre 12 e 25 anos. Dois grupos foram constituidos de acordo com a idade das gestantes: Grupo I: 80 gestantes com idade de 12 ate 17 anos. Grupo II: 84 gestantes com idade de 18 a 25 anos. Os resultados maternos e fetais foram comparados. A analise dos resultados mostrou ser mais frequente no grupo I a hipertensao (Grupo I, 46,3% e grupo II, 16,7%), o tabagismo (grupo I, 50,0% e grupo II 38,1%) o parto prematuro (grupo I 21,2% e grupo II, 14,3%) e a mortalidade perinatal (grupo I 5,0% e grupo II 2,4%). Concluem serem necessarios servicos assistenciais proprios para adolescentes a fim de orienta-las sexualmente oferecendo-lhes amparo psicologico e emocional bem como uma adequada assistencia pre-natal
