Aliberti, Antonietta (2018) Exploiting genetic and genomic resources to increase the ascorbic acid content in the tomato fruit. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Lingua: English
Title: Exploiting genetic and genomic resources to increase the ascorbic acid content in the tomato fruit
Creators:
CreatorsEmail
Aliberti, Antoniettaantonietta.aliberti@unina.it
Date: 11 December 2018
Number of Pages: 140
Institution: Università degli Studi di Napoli Federico II
Department: Agraria
Dottorato: Scienze agrarie e agroalimentari
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
nomeemail
D'Urso, Guidoguido.durso@unina.it
Tutor:
nomeemail
Barone, AmaliaUNSPECIFIED
Date: 11 December 2018
Number of Pages: 140
Uncontrolled Keywords: S. pennellii introgression lines; fruit quality; RNA-seq; molecular markers; IL assembly; candidate gene identification; functional gene validation
Settori scientifico-disciplinari del MIUR: Area 07 - Scienze agrarie e veterinarie > AGR/07 - Genetica agraria
Date Deposited: 03 Jan 2019 17:16
Last Modified: 16 Jun 2020 10:22
URI: http://www.fedoa.unina.it/id/eprint/12627

Abstract

The wild tomato species such as Solanum pennellii are an important source of genes that were lost during tomato selection and cultivation. The S. pennellii Introgression lines (ILs), which carry defined homozygous segments of the wild genome in the cultivated genetic background of cv.M82, are an important genetic resource to map quantitative traits loci (QTLs), such as those controlling plant yield and fruit quality, and to exploit the genetic diversity present in the wild species. Two introgression lines (IL12-4 and IL7-3) harbouring QTLs for ascorbic acid content were previously identified in the laboratory of Structural and Functional Genomics at the Department of Agricultural Sciences of University of Naples Federico II. These two ILs showed increased content of antioxidant compounds in the fruit compared to the cultivated S. lycopersicum cv. M82. Afterwards, sub-lines with reduced sizes of the introgressed region were obtained from IL7-3 and IL12-4. The first aim of the present thesis was to perform the phenotypic selection and characterization of the S. pennellii sub-lines in different environmental conditions. In the fruit of the different sub-lines, the level of soluble solids content in terms of °Brix, firmness and ascorbic acid was highly variable in three different environmental conditions. The sub-lines R182 and B27, deriving from IL7-3 and IL12-4, respectively, were selected for their better performances in terms of fruit quality since they exhibited a significantly higher firmness, °Brix and ascorbic acid content compared to M82. Moreover, the two sub-lines also showed a production comparable to that of the control line M82 in all the environment conditions tested. The second aim of the present thesis was to identify candidate genes involved in determing the high level of ascorbic acid in the fruit and mapping in the introgressed regions of the sub-lines R182 and B27. Since today the only reference genome used to investigate gene positions and functions in tomato still remains that of the first completely sequenced genome, i.e. that of S. lycopersicum cv. Heinz, the first step to reach this second aim was to reassembly the S. pennellii IL7-3 and IL12-4 genomes. In order to reassemble the genome of M82 and of the ILs, several resources were used. These consisted of Next Generation Sequence resources available today, such as the sequence data of the S. pennellii and different RNA-seq data related to the IL populations. By using an integrated bioinformatic approach a new reference genome and annotation for IL12-4 and the IL7-3 was built. Moreover, to confirm the reliability of the IL7-3 assembly and define the size of S. pennellii introgression region in the sub-line R182, a group of species-specific molecular markers were designed based on polymorphisms found comparing the genomes of the cultivated and the wild species. Finally, to identify candidate genes mapping in the wild regions better defined in the present thesis, a transcriptomic approach with RNA-Seq was carried out. Transcriptome analyses allowed identifying three candidate genes in the two sub-lines: the Solyc12g098480 encoding for the UDP-glucoronic-acid-4-epimerase in the wild introgressed region of B27 and the Solyc07go49310 and Solyc07g049290 corresponding to two Major facilitator superfamily (MFS) proteins in in the wild introgressed region of R182. The third aim of the present thesis was to carry out the functional validation of candidate genes potentially involved in the control of ascorbic acid content in the red ripe fruit. The first gene selected was the UDP-glucoronic-acid-4-epimerase gene mapping in the introgressed region of the sub-line B27. Its role in increasing ascorbic acid in this sub-line fruit was investigated through its over-expression in tomato fruit by stable and transient techniques. In addition, the genotyping and phenotyping evaluation of CRISP/Cas9 knock-out mutants for a non-canonical uORF carried by two GDP-L-galactosephosphorylases (GGP1, Solyc06g073320 and GGP2, Solyc02g091510) allowed demonstrating that these two genes are involved in the regulation of the ascorbate biosynthesis in tomato. In conclusion, the results obtained in the present thesis, allowed selecting two elite sub-lines that in the near future could be used as breeding material to improve tomato fruit for nutritional traits. In addition, the results achieved allowed increasing knowledge about genes involved in the control of ascorbic acid content in tomato fruit. Indeed, three candidate genes were identified exploiting the genomics resources available for tomato, and other two genes have been functionally validated. The transfer of these genes by conventional and innovative strategies will aid in the future the creation of new improved varieties.

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