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.

SlRCM1, which encodes tomato Lutescent1, is required for chlorophyll synthesis and chloroplast development in fruits.

In plants, chloroplasts are the sites at which photosynthesis occurs, and an increased abundance of chloroplasts increases the nutritional quality of plants and the resultant color of fruits. However, the molecular mechanisms underlying chlorophyll synthesis and chloroplast development in tomato fruits remain unknown. In this study, we isolated a chlorophyll-deficient mutant, reduced chlorophyll mutant 1 (rcm1), by ethylmethanesulfonate mutagenesis; this mutant produced yellowish fruits with altered chloroplast development. MutMap revealed that Solyc08g005010 is the causal gene underlying the rcm1 mutant phenotype. A single-nucleotide base substitution in the second exon of SlRCM1 results in premature termination of its translated protein. SlRCM1 encodes a chloroplast-targeted metalloendopeptidase that is orthologous to the BCM1 protein of Arabidopsis and the stay-green G protein of soybean (Glycine max L. Merr.). Notably, the yellowish phenotype of the lutescent1 mutant can be restored with the allele of SlRCM1 from wild-type tomato. In contrast, knockout of SlRCM1 by the CRISPR/Cas9 system in Alisa Craig yielded yellowish fruits at the mature green stage, as was the case for lutescent1. Amino acid sequence alignment and functional complementation assays showed that SlRCM1 is indeed Lutescent1. These findings provide new insights into the regulation of chloroplast development in tomato fruits.

GREEN STRIPE, encoding methylated TOMATO AGAMOUS-LIKE 1, regulates chloroplast development and Chl synthesis in fruit.

Fruit development involves chloroplast development, carotenoid accumulation and fruit coloration. Although genetic regulation of fruit development has been extensively investigated, epigenetic regulation of fruit coloration remains largely unexplored. Here, we report a naturally occurring epigenetic regulation of TAGL1, and its impact on chloroplast development and fruit coloration. We used a genome-wide association study in combination with map-based cloning to identify the GREEN STRIPE (GS) locus, a methylated isoform of TAGL1 regulating diversified chloroplast development and carotenoid accumulation. Nonuniform pigmentation of fruit produced by GS was highly associated with methylation of the TAGL1 promoter, which is linked to a SNP at SL2.50ch07_63842838. High degrees of methylation of the TAGL1 promoter downregulated its expression, leading to green stripes. By contrast, low degrees of methylation led to light green stripes in gs. RNA-seq and ChIP collectively showed that the expression of genes involved with Chl synthesis and chloroplast development were significantly upregulated in green stripes relative to light green stripes. Quantitative PCR and dual luciferase assay confirmed that TAGL1 downregulates expression of SlMPEC, SlPsbQ, and SlCAB, and upregulates expression of PSY1 - genes which are associated with chloroplast development and carotenoid accumulation. Altogether, our findings regarding the GS locus demonstrate that naturally occurring methylation of TAGL1 has diverse effects on plastid development in fruit.

Early Response of Radish to Heat Stress by Strand-Specific Transcriptome and miRNA Analysis.

Radish is a crucial vegetable crop of the Brassicaceae family with many varieties and large cultivated area in China. Radish is a cool season crop, and there are only a few heat tolerant radish varieties in practical production with little information concerning the related genes in response to heat stress. In this work, some physiological parameter changes of young leaves under short-term heat stress were detected. Furthermore, we acquired 1802 differentially expressed mRNAs (including encoding some heat shock proteins, heat shock factor and heat shock-related transcription factors), 169 differentially expressed lncRNAs and three differentially expressed circRNAs (novel_circ_0000265, novel_circ_0000325 and novel_circ_0000315) through strand-specific RNA sequencing technology. We also found 10 differentially expressed miRNAs (ath-miR159b-3p, athmiR159c, ath-miR398a-3p, athmiR398b-3p, ath-miR165a-5p, ath-miR169g-3p, novel_86, novel_107, novel_21 and ath-miR171b-3p) by small RNA sequencing technology. Through function prediction and enrichment analysis, our results suggested that the significantly possible pathways/complexes related to heat stress in radish leaves were circadian rhythm-plant, photosynthesis-antenna proteins, photosynthesis, carbon fixation in photosynthetic organisms, arginine and proline metabolism, oxidative phosphorylation, peroxisome and plant hormone signal transduction. Besides, we identified one lncRNA-miRNA-mRNAs combination responsive to heat stress. These results will be helpful for further illustration of molecular regulation networks of how radish responds to heat stress.