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Prioritization of vigor QTL-associated genes for future genome-directed Vitis breeding Priorización de genes asociados a QTLs de vigor para futuros planes de mejoramiento dirigido en Vitis

Vigor control in grapevine may become especially important under climate change. A better understanding of gene-phenotype relationships is required in order to exploit plant genomics for breeding purposes. This research aims to use quantitative trait loci (QTLs) for vigor identified in the progeny from a cross of Ramsey (Vitis champinii) × Riparia Gloire (V. riparia). Genes located 700 kb up and downstream from each QTL position were interrogated for functional enrichment through ShinyGO online tool, based on the gene ontology annotation of Vitis vinifera PN40024. Key biological processes like phloem and xylem development, cell cycle, response to hormones, amino acid transport, tissue development, sugar metabolism, nitrogen transport, and stress/immune responses, showed functional enrichment. Integral response to light and auxin might be required for fine molecular tuning of vegetative growth in Vitis. Fifty out of 1318 candidate genes were prioritized, reducing their amount to a manageable number of candidates genes for further directed breeding strategies.

* This authors contributed equally to this study.

Introduction
The understanding of genotype-phenotype associations constitute a major challenge for plant scientists. The causal association between variation of a certain trait and genotypic differences is the foundation for developing targeted strategies to be used in molecular breeding (1).
Vigor is considered as the propensity to assimilate, store, and/or use non-structural carbohydrates to produce large canopies. It is associated with high metabolism and rapid shoot growth (17). The main processes that account for vigor are: 1-carbon assimilation or photosynthesis, 2-cell growth, by division and expansion, and 3-leaf area development, for the reception of CO 2 and light. All these complex processes contribute to what is known as a highly quantitative trait.
There is increasing interest in understanding the genetic basis of grapevine vigor and biomass production, given its substantial impact on yield, water management, plant health, and fruit quality. Advances in understanding the genetic bases of complex traits through genetic mapping and quantitative trait locus (QTL) analysis have linked certain complex phenotypes to specific regions of chromosomes, and helped to identify the action, number, and precise location of these regions. The Ramsey (Vitis champinii) × Riparia Gloire (V. riparia) linkage map was developed by Lowe and Walker (2006) with the goal of mapping traits like biotic resistance, drought tolerance and vigor. We recently reported QTLs for vigor in this progeny that are associated with leaf area, specific leaf area, field canopy biomass and several partitioning indices (12). However, the genes behind these quantitative traits have not been identified. Here, we analyze the function of genes surrounding the QTLs based on gene location and the Gene Ontology (GO) annotation of the V. vinifera genome. GO is a computational representation of the functions of protein and non-coding RNA molecules produced by genes from many different organisms. It can be used for interpreting large-scale molecular biology experiments to gain insight into the structure, function, and dynamics of an organism. This approach was proposed by Correa et al. (2014) when identifying QTLs and candidate genes associated with cluster architecture in grapevines, and later by Ritcher et al. (2019), when approaching cluster architecture and revealing genes enriched by physical projection of the QTLs onto the PN40024 reference genome sequence.
In this paper, we communicate the results of a GO functional enrichment to prioritize candidate genes responsible for vigor that interrogated 1318 genes obtained from the QTL mapping of the Ramsey x Riparia Gloire progeny. We also attempt to elucidate the putative role of genes in terms of physiological and biochemical processes and identify priority genes for further targeted breeding approaches. Revista de la Facultad de Ciencias Agrarias -UNCuyo | Tomo 53-2 -Año 2021

Materials and Methods
Six highly significant QTLs for vigor components from the Ramsey x Riparia Gloire map were selected based on their LOD values above the genome wide significance level (12). The BLAST server against the V. vinifera PN40024 genome (https://urgi.versailles.inra. fr/blast/) was then used to locate the hybridization regions of PCR primers targeting the simple sequence repeat (SSR) markers linked to the selected QTLs (table 1, page 30-31). The coordinates 700 kb upstream and downstream of primer hybridization on the reference sequence were used to identify the genes within the region flanked by those limits. The 700 kb limit was established considering that candidate genes should be no farther than 3-4 cM from the marker, since the V. vinifera genome has been estimated at about 475 Mb and 1 cM equals 200-300 kb (3). For that purpose, a script was developed to search for annotated genes flanking the QTL regions in the annotation file of the V. vinifera PN40024 genome.
The annotated genes found with the script were then used to interrogate for functional enrichment through ShinyGO online tool version 0.61 (8). For the set of genes contained in the QTL regions associated with vigor, the occurrence of associated gene ontology terms (GO terms) was statistically evaluated for overrepresentation through an hypergeometric test retrieving those GOterms with statistical significance (False Discovery Rate < 0.05) when comparing their percentage of occurrence with the percentage of each GOterm in the whole annotated genome. Integral plots that associate gene locations in chromosomes with enriched GO categories were performed with shinyCircos (22).

Results
In this work, GO functional enrichment allowed the identification of genes involved in key processes related to vigor and prioritization reduced the number of candidate genes from 1318 to 50.
Our results show that associations between overrepresented GO terms and vigor helped to rank candidate genes, based on their putative function. Phloem and xylem development, cell cycle, response to hormones, tissue development, aminoacid and nitrogen transport, sugar metabolism and immune responses, all showed functional enrichment (figure 1 page 32; table 1, page 30-31). On chromosome 1, most identified genes encode for aminoacid transmembrane transporters (figure 1 page 32; table 1, page 30-31). In addition, the single gene found on chromosome 3 influences the enrichment for functions related to transport of organic acids and nitrogen compounds.
Two important transcription factors (TFs) related to photomorphogenesis (TF 104879018) and auxins (TF 104879021) were identified on chromosome 4 (figure 1 page 32; table 1, page 30-31). TFs are especially interesting because they control the expression of several genes. Genes in chromosome 10 are involved in biotic and abiotic stress responses (figure 1 page 32; table 1, page 30-31). Another transcription factor related to this processes, identified in table 1 (page 30-31) as 100243518, appears significant in this chromosome. On chromosomes 14 and 19, we found the most varied group of genes in terms of function (figure 1 page 32; table 1, page 30-31), including, once again, genes encoding for nitrogen transport, that are key for growth, cell cycle regulation and development (13,19). Many genes encode for proteins involved in phloem and xylem development. This was the function with major enrichment (figure 1 page 32; table 1, page 30-31). It represents a key aspect for growth, as transport of water, nutrients, proteins and sugars is vital for the plant to develop.

Discussion
Although the analysis used the V. vinifera PN40024 genome, due to its superior characterization and annotation, sequence alignment with V. riparia Gloire (9)    As mentioned, on chromosome 1, most identified genes encode for aminoacid transmembrane transporters ( figure 1; table 1, page 30-31). Transmembrane aminoacid transport is associated with enhanced growth and high rates of protein synthesis (20). Nitrogen can be taken up from soil in various forms, being one of them amino acids, and is of considerable importance in vigor control, yield and berry quality (7). The fact that these genes are closely located, suggests some kind of structural regulation. It has been observed that root elongation and enlargement in the rootstock 110R, partly depend on transcriptomic regulations of sugar and protein transporter genes SWEET and NRT1/PTR in roots. This was found to facilitate carbohydrate and nitrogen accumulation, providing essential energy to 110R roots under drought (21).
In relation to the two important TFs related to photomorphogenesis and auxins identified on chromosome 4, they are particularly intriguing given that they control the expression of several genes. The particularity of having both TFs in the same chromosomal region increases the probability of a synergistic response through coregulation. Indeed, the coordination between TFs in response to light and auxins was well established in Arabidopsis thaliana by Halliday, et al. (2009), who showed that light regulates growth of distant tissues from the site of light exposure through auxin production. Something similar was observed in A. thaliana by Hornitschek, et al. (2012), where phytochrome interacting factors 4 and 5 (PIF4 and PIF5) regulated elongation growth by controlling the expression of genes that encode for auxin biosynthesis and signaling. Interestingly, TF 104879018 in our study is homologous to HY5 of A. thaliana, which is found downstream in the signaling cascade of PIF1/PIF3 (10). HY5 promotes growth, especially through photosynthesis induction and higher nutrient uptake by roots. The other prioritized TF 104879021, is a homolog to the auxin-responsive protein IAA28 of A. thaliana, which plays a role in regulation of lateral root growth. In grapevines, kaolin, a mineral that reflects radiation from the leaf surface, produced an increase in IAA content. This treatment also caused higher values of stomatal conductance, net CO 2 assimilation rate, intrinsic water use efficiency, and a slight decrease in ABA (5). These results might be supported by the same mechanism connecting growth hormones and light interception. Therefore, vigor in grapevines may partly depend on promotion of photosynthesis, lateral root development and nitrate uptake, and these processes may be associated through the expression of genes downstream-regulated by TF 104879018 and TF 104879021.
Regarding the genes and TF found in chromosome 10, involved in biotic and abiotic stress responses, some authors have shown significant correlation between vigor and tolerance/ susceptibility to diseases that could induce different defense responses in the host plant (2). Vigorous plants may have developed stronger immune responses to defend themselves.
Considering our findings in chromosomes 14 and 19, many genes encode for proteins involved in phloem and xylem development. This function showed the major enrichment (figure 1 page 32; table 1, page 30-31), representing a key aspect for growth, as transport of water, nutrients, proteins and sugars is vital for the plant to develop. These functions are tightly correlated to auxins and soluble carbohydrates seasonal dynamics, since cambium activity and xylem/phloem development respond to this signaling in woody species. It has been observed that IAA and soluble carbohydrate dynamics directly affect xylem and phloem formation in trees (6). In addition, gibberellins increase carbon allocation to different organs by inducing accumulation of non-structural carbohydrates in leaves, enhancement of phloem area and expression of sugar transporters (16).
Our results may lead to deeper gene selection strategies, aiming at choosing a smaller number of genes. Candidates with the smallest enrichment FDR values that are associated with QTLs that explain the highest percentages of variation, constitute interesting targets. For quantitative characters, where positive feedbacks cause large effects, significant explanatory effects from 10% to 20%, may result in impressive effects. This strategy considers that both approaches, QTL mapping and GO enrichment, work at different levels. While QTLs indentify regions on chromosomes containing genes encoding for a certain trait, the enrichment process takes into account all the genes involved in a pathway related to that trait. In processes where numerous genes are involved, a meaningful change should include many of them. Consequently, transcription factors could produce phenotype differences even at higher FDR values. In our work, this last effort selected 16 genes and 4 TRFs as plausible candidates for further breeding studies (table 1, page 30-31). Further functional genomic studies should weigh the importance of these selected genes on the final phenotype.

Conclusions
In conclusion, this analysis allowed the detection of plausible candidate genes encoding for the components of key processes governing vegetative growth in Vitis. The analysis allowed the reduction of candidate gene number based on marker proximity and functional enrichment, clearly demonstrating a suitable shortcut for target-directed genome-guided breeding strategies. This approach is particularly useful when a map is not densely saturated. Revista de la Facultad de Ciencias Agrarias -UNCuyo | Tomo 53-2 -Año 2021 Two TFs, which potentially enhance growth by relating light response to hormone activation, and then to photosynthesis and morphogenesis, are strong candidates for targeted breeding. The nitrogen transport encoding genes would allow this light/hormone promoted growth by facilitating amino acid/protein synthesis and transport. Phloem and xylem related genes would also be part of this process by enabling water and nutrient transport. All these functions need to be tightly correlated, since auxins and soluble carbohydrates seasonal dynamics play key roles in tissue growth, cambium activity and xylem/phloem development. Gene characterization in individuals of the Ramsey × Riparia Gloire progeny will be the topic for future research.