Genome-wide variants and optimal allelic combinations for citric acid in tomato.

Citric acid (CA) plays a crucial role as a fruit flavor enhancer and serves as a mediator in multiple metabolic pathways in tomato fruit development. Understanding factors influencing CA metabolism is essential for enhancing fruit flavor and CA-mediated biological processes. The accumulation of CA, however, is influenced by a complex interplay of genetic and environmental factors, leading to challenges in accurately predicting and regulating its levels. In this study, we conducted a genome-wide association study (GWAS) on CA, employing six landmark models based on genome-wide variations including structural variants, insertions and deletions, and single nucleotide polymorphisms. The identification of 11 high-confidence candidate genes was further facilitated by leveraging linkage disequilibrium and causal variants associated with CA. The transcriptome data from candidate genes were examined, revealing higher correlations between the expression of certain candidate genes and changes in CA metabolism. Three CA-associated genes exerted a positive regulatory effect on CA accumulation, while the remaining genes exhibited negative impacts based on gene cluster and correlation analyses. The CA content of tomatoes is primarily influenced by improvement sweeps with minimal influence from domestication sweeps in the long-term breeding history, as evidenced by population differentiation and variants distribution. The presence of various causal variants within candidate genes is implicated in the heterogeneity of CA content observed among the tomato accessions. This observation suggests a potential correlation between the number of alternative alleles and CA content. This study offers significant function-based markers that can be utilized in marker-assisted breeding, thereby enhancing their value and applicability.

CRISPR/Cas9-mediated mutations of FANTASTIC FOUR gene family for creating early flowering mutants in tomato.

Flowering time is of great agricultural importance and the timing and extent of flowering usually determines yield and availability of flowers, fruits and seeds. Identification of genes determining flowering has important practical applications for tomato breeding. Here we demonstrate the roles of the FANTASTIC FOUR (FAF) gene family in regulating tomato flowering time. In this plant-specific gene family, SlFAF1/2a shows a constitutive expression pattern during the transition of the shoot apical meristem (SAM) from vegetative to reproductive growth and significantly influences flowering time. Overexpressing SlFAF1/2a causes earlier flowering compared with the transformations of other genes in the FAF family. SlFAF1/2c also positively regulates tomato flowering, although to a lesser extent. The other members of the SlFAF gene family, SlFAF1/2b, SlFAF3/4a and SlFAF3/4b, are negative regulators of tomato flowering and faf1/2b, faf3/4a and faf3/4b single mutants all display early flowering. We generated a series of early flowering mutants using the CRISPR/Cas9 editing system, and the faf1/2b faf3/4a faf3/4b triple mutant flowering earliest compared with other mutants. More importantly, these mutants show no adverse effect on yield. Our results have uncovered the role of the FAF gene family in regulating tomato flowering time and generated early flowering germplasms for molecular breeding.

Multiple-model GWAS identifies optimal allelic combinations of quantitative trait loci for malic acid in tomato.

Malic acid (MA) is an important flavor acid in fruits and acts as a mediator in a series of metabolic pathways. It is important to understand the factors affecting MA metabolism for fruit flavor improvement and to understand MA-mediated biological processes. However, the metabolic accumulation of MA is controlled by complex heredity and environmental factors, making it difficult to predict and regulate the metabolism of MA. In this study, we carried out a genome-wide association study (GWAS) on MA using eight milestone models with two-environment repeats. A series of associated SNP variations were identified from the GWAS, and 15 high-confidence annotated genes were further predicted based on linkage disequilibrium and lead SNPs. The transcriptome data of candidate genes were explored within different tomato organs as well as various fruit tissues, and suggested specific expression patterns in fruit pericarp. Based on the genetic parameters of population differentiation and SNP distribution, tomato MA content has been more influenced by domestication sweeps and less affected by improvement sweeps in the long-term history of tomato breeding. In addition, genotype × environment interaction might contribute to the difference in domestication phenotypic data under different environments. This study provides new genetic insights into how tomato changed its MA content during breeding and makes available function-based markers for breeding by marker-assisted selection.

A 21-bp InDel in the promoter of STP1 selected during tomato improvement accounts for soluble solid content in fruits.

Domestication and improvement are important processes that generate the variation in genome and phonotypes underlying crop improvement. Unfortunately, during selection for certain attributes, other valuable traits may be inadvertently discarded. One example is the decline in fruit soluble solids content (SSC) during tomato breeding. Several genetic loci for SSC have been identified, but few reports on the underlying mechanisms are available. In this study we performed a genome-wide association study (GWAS) for SSC of the red-ripe fruits in a population consisting of 481 tomato accessions with large natural variations and found a new quantitative trait locus, STP1, encoding a sugar transporter protein. The causal variation of STP1, a 21-bp InDel located in the promoter region 1124 bp upstream of the start codon, alters its expression. STP1 Insertion accessions with an 21-bp insertion have higher SSC than STP1 Deletion accessions with the 21-bp deletion. Knockout of STP1 in TS-23 with high SSC using CRISPR/Cas9 greatly decreased SSC in fruits. In vivo and in vitro assays demonstrated that ZAT10-LIKE, a zinc finger protein transcription factor (ZFP TF), can specifically bind to the promoter of STP1 Insertion to enhance STP1 expression, but not to the promoter of STP1 Deletion , leading to lower fruit SSC in modern tomatoes. Diversity analysis revealed that STP1 was selected during tomato improvement. Taking these results together, we identified a naturally occurring causal variation underlying SSC in tomato, and a new role for ZFP TFs in regulating sugar transporters. The findings enrich our understanding of tomato evolution and domestication, and provide a genetic basis for genome design for improving fruit taste.

A mutation in a C2H2-type zinc finger transcription factor contributed to the transition toward self-pollination in cultivated tomato.

The degree of stigma exsertion has a major influence on self-pollination efficiency in tomato, and its improvement is essential for raising productivity and for fixing advantageous traits in cultivated tomato. To study the evolution of stigma exsertion degree in tomato, we searched for genes associated with this trait and other aspects of flower morphology, including the lengths of anthers, styles, and ovaries. We performed a genome-wide association on 277 tomato accessions and discovered a novel stigma exsertion gene (SE3.1). We reannotated the structure of the gene, which encodes a C2H2-type zinc finger transcription factor. A mutation of the lead single nucleotide polymorphism creates a premature termination codon in SE3.1 and an inserted stigma in cultivated tomatoes. SE3.1 is essential for the conversion of flush stigmas to inserted stigmas. This conversion has a major impact on the rate of self-fertilization. Intriguingly, we found that both SE3.1 and Style2.1 contribute to the transition from stigma exsertion to insertion during the domestication and improvement of tomato. Style2.1 controls the first step of exserted stigmas to flush stigmas, and SE3.1 controls the second step of flush stigmas to inserted stigmas. We provide molecular details for the two-step process that controls the transition from stigma exsertion to insertion, which is of great agronomic importance in tomato.

A CCAAT-binding factor, SlNFYA10, negatively regulates ascorbate accumulation by modulating the D-mannose/L-galactose pathway in tomato.

Ascorbic acid (AsA), an important antioxidant and growth regulator, and it is essential for plant development and human health. Specifically, humans have to acquire AsA from dietary sources due to their inability to synthesize it. The AsA biosynthesis pathway in plants has been elucidated, but its regulatory mechanism remains largely unknown. In this report, we biochemically identified a CCAAT-box transcription factor (SlNFYA10) that can bind to the promoter of SlGME1, which encodes GDP-Man-3',5'-epimerase, a pivotal enzyme in the D-mannose/L-galactose pathway. Importantly, SlNFYA10 simultaneously binds to the promoter of SlGGP1, a downstream gene of SlGME1 in the D-mannose/L-galactose pathway. Binding assays in yeast and functional analyses in plants have confirmed that SlNFYA10 exerts a negative effect on the expression of both SlGME1 and SlGGP1. Transgenic tomato lines overexpressing SlNFYA10 show decreased levels of SlGME1 and SlGGP1 abundance and AsA concentration in their leaves and fruits, accompanied by enhanced sensitivity to oxidative stress. Overall, SlNFYA10 is the first CCAAT-binding factor identified to date to negatively regulate the AsA biosynthetic pathway at multiple sites and modulate plant responses to oxidative stress.