Análise da diversidade genética e identificação de citros (Citrus spp) com marcadores moleculares e High Resolution Melting (HRM)
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Data
2024-12-12
Autores
Britto, Karolinni Bianchi
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Universidade Federal do Espírito Santo
Resumo
Citrus farming plays a crucial role in national fruit production, encompassing the cultivation of several species and varieties of the Citrus genus. Citrus growers face challenges related to seedling certification, due to the similarity between the leaves of the cultivars and the possibility of exchange between them, since the morphological characteristics manifest themselves in a subtle and variable way. The selection of molecular markers for citrus is a challenge due to the low molecular variability observed. Thus, the objective of this study was to identify Single Nucleotide Polymorphisms (SNPs) through DArTSeq technology, which combines the Diversity Arrays Technology marker system with next-generation sequencing technology, and from these markers, develop rapid identification kits using the High Resolution Melting (HRM) technique, which is sensitive and low-cost. The methodology, results and discussion were written in three chapters. The first chapter presents a review “Beyond the peel: exploring Citrus diversity through DArTSeq”. The study used DArTSeq technology to analyze genetic variation in 93 accessions of nine citrus species, resulting in the identification of 64,442 SNPs and 69,963 SilicoDArT markers, which after filtering were reduced to 9,073 and 3,496, respectively. The various analyses revealed eight clusters and six genetic groups. The research also established an important basis for future investigations in citrus genetics. The second chapter presents a patent filed with the National Institute of Industrial Property (INPI), entitled “Oligonucleotide primers and method for genetic discrimination and certification of Pera orange (Citrus sinensis) cultivars of agricultural interest using the High Resolution Melting (HRM) technique”. A kit, with seven pairs of primers for HRM analysis, was developed to identify eight Pera orange cultivars. The third chapter presents a patent filed with the INPI, entitled “Oligonucleotide primers and method for genetic discrimination and certification of citrus rootstocks (Family Rutacea) of agricultural interest using the High Resolution Melting (HRM) technique”. A kit with three pairs of primers for HRM analysis was developed to identify seven genotypes of citrus rootstocks, five species and two hybrids. This thesis contributes to analyses of the genetic diversity and population structure of citrus, reflecting the relationships both between and within species. The identification of the cultivars was performed using oligonucleotide primer kits using the HRM technique. In addition, these kits developed will assist in the rapid identification of Pera orange cultivars and citrus rootstocks by HRM, and can be implemented in certification. Thus, the proposed markers proved to be effective in discriminating citrus genotypes, overcoming the limitations of morphological identification, especially during the initial phases of the plant, offering a significant advance for the citrus sector and promoting greater security for producers by ensuring the authenticity of the propagation material
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Palavras-chave
Citricultura , Identificação molecular , Polimorfismo , Certificação , Citriculture , Molecular identification , Polymorphism , Certification