Graci, Salvatore (2024) High-throughput genotyping platforms to identify candidate genes for heat stress response in a thermotolerant tomato genotype and its exploitation for breeding purposes. [Tesi di dottorato]

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Abstract

Climate change is a major threat to global food security, directly impacting food systems by reducing the production. As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperatures, inducing the production of reactive oxygen species (ROS), which imply oxidative stress and cell death. Tomato (S. lycopersicum) is an important crop distributed worldwide, but it is highly sensitive to heat stress (HS) depending on the genotype. Genetic improvement of heat tolerance will be critical for the sustainable production of tomato. During the last years, we selected the thermotolerant E42 genotype, which showed stable yield, high fruit quality and high number of flowers and fruits produced under HS, even when grown with one-month late transplant to increase exposure to high temperatures during the reproduction stage. In this thesis, focusing on the reproductive stage, we conducted a genomic investigation of this genotype by using four different high-throughput technologies (GBS, SPET, whole genome resequencing and RNA seq) to identify candidate genes for the HS response. In addition, we developed two breeding programs aimed at converting the E42 plant habitus from determinate to indeterminate to allow its eligibility as variety for the tomato “da mensa” fresh market and constituting superior hybrids tolerant to high temperatures and showing high fruit quality traits, by using E42 as parental line. As a first step, a list of 393 heat-related genes for key traits involved in the final yield, including Hsfs, Hsps, flower-, pollen- and fruit set-related genes was identified (Chapter 2). On one hand, the investigation of the E42 genome through two untargeted (GBS) and targeted (SPET) sequencing technologies allowed us to find a high number of polymorphisms mostly mapping on chromosomes 1, 4, 7 and 12. Moreover, eight missense mutations having MODERATE impact on the translated protein and mapping on eight heat-related genes from the list of 393 were identified (Chapter 3). On the other hand, the investigation performed with whole genome resequencing data confirmed the distribution of the SNP and InDel variants across the E42 genome and, more specifically, allowed to identify 18 highly polymorphic regions. In addition, the phylogenetic analysis evidenced the strong relationship between the referred genotype and the S. pimpinellifolium wild species, suggesting that the high variability detected in the E42 genome could be related to its origin. From this work, a list of 35 candidate genes presenting variants with HIGH and/or MODERATE impact on the translated protein, mapping on E42 polymorphic regions and some of these colocalizing in QTLs controlling flowering in tomato was identified (Chapter 4). Some of these could contribute to explain the constitutive high number of flower production of E42, such as the FT-like gene (Solyc11g008650) that shared 100% identity with the one of the LA2093 S. pimpinellifolium accession and is already reported to be involved in day-neutral flowering time in tomato. However, heat tolerance is a quantitative trait, and many genes are involved in this response, whose expression could vary among different genotypes. In this context, we decided to study the regulatory network of the HS response in tomato by investigating RNA-seq data publicly available and the distribution and variability of heat stress elements (HSE) in the E42 promotor regions (Chapter 5). HSE were found in the promoters of 106 heat-related genes out of the 393 identified and reported in Chapter 2; by using publicly tomato RNA seq data and a gene co-expression network (GCN) analysis we could identify the interactions among these genes. The integration of these results permitted to prioritize a final subset of 13 candidate genes involving two Hsfs, nine Hsps and two GELPs. These genes, showed HSE sequences in the promoter, interact with Hsfs and presented variants with HIGH and/or MODERATE impact on the coding sequence. Among the selected genes, the LeHsp100 (Solyc02g088610) was always highlighted by also using the approaches in Chapters 3 and 4, it belongs to the network specifically involved in the HS response and presents two HSE motifs in the promoter that could be bound from four Hsfs, thus regulating its expression. However, the presence of four missense variants in the coding region of the gene allows the translation of a different protein that could alter the HS response. In addition, it also interacts with several target genes such as Hsps, flower-related genes like Single Flower Truss (SFT), Falsiflora (FA), pollen-related genes like GELPs, fruit set-related genes like SlDELLA. Some of these genes also showed polymorphisms in the coding regions, and this could result in an improved or decreased response to the abiotic stress through an altered gene regulation or protein functions. This evidence suggest that the LeHsp100 may represents a strong candidate. Altogether, these features may contribute to regulate the E42 HS response. In addition, to provide thermotolerant plant materials for the fresh market, two breeding programs were developed. The backcross program speeded up through the use of a designed SP_CAPS marker allowed to convert E42 habitus from determinate to indeterminate (Chapter 6). Ten backcross combinations were obtained by crossing the referred genotype with five indeterminate plants, and the evaluation of the BC3 progenies permitted to identify two offspring combinations (E20xE42 BC3 and E36xE42 BC3) best performing in terms of both yield-related and fruit quality traits under HS. In addition, GBS and SPET data of the parental lines were analyzed to investigate in the future the linkage drag in the backcross progenies: genes close to the SP gene and showing variants with HIGH and MODERATE impact in the donor parents were identified. Within the second breeding program, five hybrids were obtained by crossing E42 with three thermotolerant indeterminate genotypes (Chapter 7). These F1 combinations were evaluated in two environments under high temperatures, thus selecting one hybrid (E36xE42 F1) with superior traits in terms of both heat tolerance and fruit quality and also evidencing heterosis respect to the parental lines, while a second hybrid (E42xPDVIT F1) was selected only for its high fruit quality. Moreover, SPET data of the parental lines allowed to identify genes of interest in response to biotic and abiotic stresses that will be heterozygous in the F1 hybrids or showing homozygous alternative variants that could explain their superior performances. Comprehensively, results obtained in this thesis provided both novel insights to understand the E42 heat tolerance mechanisms and new plant materials able to face current and future climate changes.

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