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.

MdWOX4-2 modulated MdLBD41 functioning in adventitious shoot of apple (Malus domestica).

Apple (Malus domestica Borkh.) is not only an important fruit crop distributed worldwide, but also a common model plant. However, the lack of efficient genetic transformation procedures for apples limits the in-depth studies of their gene functions. Although leaf-regenerated adventitious shoots (LRAS) are a prerequisite for successful genetic transformation of apple, little is known about the underlying molecular mechanism of LRAS. Here, we identified the WUSCHEL-related homeobox (WOX) transcription factor in apple, MdWOX4-2, which was a transcriptional activator. Gene expression as well as morphological and histological observations revealed that MdWOX4-2 is involved in the development of LRAS. Overexpression of MdWOX4-2 conferred higher regenerative capacity in transgenic tobacco (Nicotiana tabacum) as compared to the wild type (WT). The combined results of the yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), dual luciferase assays, and transient transactivation assay, revealed that MdWOX4-2 directly bound to and activated the MdLBD41 promoter. Moreover, transgenic experiments further demonstrated that MdLBD41 could significantly enhance the formation of adventitious shoot in transgenic tobacco. Collectively, our findings demonstrate that MdWOX4-2 is important for regulating the LRAS development by activating MdLBD41.

The Artificial Promoter rMdAG2I Confers Flower-specific Activity in Malus.

Genetic modifications of floral organs are important in the breeding of Malus species. Flower-specific promoters can be used to improve floral organs specifically, without affecting vegetative organs, and therefore developing such promoters is highly desirable. Here, we characterized two paralogs of the Arabidopsis thaliana gene AGAMOUS (AG) from Malus domestica (apple): MdAG1 and MdAG2. We then isolated the second-intron sequences for both genes, and created four artificial promoters by fusing each intron sequence to a minimal 35S promoter sequence in both the forward and reverse directions. When transferred into tobacco (Nicotiana benthamiana) by Agrobacterium tumefaciens-mediated stable transformation, one promoter, rMdAG2I, exhibited activity specifically in flowers, whereas the other three also showed detectable activity in vegetative organs. A test of the four promoters' activities in the ornamental species Malus micromalus by Agrobacterium-mediated transient transformation showed that, as in tobacco, only rMdAG2I exhibited a flower-specific expression pattern. Through particle bombardment transformation, we demonstrated that rMdAG2I also had flower-specific activity in the apple cultivar 'Golden Delicious'. The flower-specific promoter rMdAG2I, derived from M. domestica, thus has great potential for use in improving the floral characteristics of ornamental plants, especially the Malus species.