Conserved Regulatory Pathways for Stock-Scion Healing Revealed by Comparative Analysis of Arabidopsis and Tomato Grafting Transcriptomes.

Many plants can successfully join root and shoot sections at cut surfaces when severed at the stem. Graft healing is complex and conserved in diverse taxonomic groups with different vascular structures. Herein, we compared transcriptome data from autografted and separated stem sections of Arabidopsis thaliana and tomato (Solanum lycopersicum) to explore changes related to graft healing. Using orthologous gene pairs identified between the two species, temperal expression patterns of evolutionary associated genes in grafted top and bottom, separated top and bottom, and intact stems were exhibited. Genes with expression preference indicate functional diversification of genes related to anatomical structure and cellular development in the two species. Expression profiles of the variable genes revealed common pathways operating during graft healing, including phenylpropanoid metabolism, response to oxygen-containing compounds, xylan, and cell wall biogenesis, mitosis and the cell cycle, carboxylic acid catabolism, and meristem structural organization. In addition, vascular differentiation related NAC domain transcription factors and genome-wide members in Arabidopsis and tomato were used for phylogenetic and expression analysis. Expression differences were largely consistent with sequence differences, reflecting high similarity for protein-coding and regulatory regions of individual clades. NAC proteins mainly clustered in accordance with their reported functions in xylem differentiation or cambium formation. The putative conserved mechanisms suggested by conserved genes and functions could help to expand graft healing theory to a wider range of species, and temporal fluctuations in common pathways imply conserved biological processes during graft healing.

B-class MADS-box TM6 is a candidate gene for tomato male sterile-1526.

KEY MESSAGE: Tomato male sterile-1526 locus was fine-mapped to an interval of 44.6 kb, and a B-class MADS-box gene TM6 was identified as the candidate gene. Male sterile lines have been widely used for hybrid seed production in many crop plants. The tomato male sterile-1526 (ms-1526) mutant displays abnormal stamens and exerted stigmas and is suitable for practical use. In this study, the ms-1526 locus was fine-mapped to a 44.6 kb interval that contained four putative genes. Thereinto, Solyc02g084630 encodes tomato B-class MADS-box gene TM6 (syn. TDR6), which plays an important role in stamen development. Sequencing revealed that there was a 12.7 kb deletion in the ms-1526 region, where the promoter and first four exons of the TM6 gene were absent. ms-1547, an allele of ms-1526, also contained the same deletion in the TM6 gene. And the other allele ms-15 mutant contained a single-nucleotide polymorphism (SNP, C to A) in the coding region of the TM6 gene, which led to a missense mutation (G to W). The codominant insertion/deletion (InDel) marker MS26D and codominant derived cleaved amplified polymorphic sequence (dCAPS) marker MS15C were developed based on the deletion and SNP, respectively. A real-time quantitative reverse-transcription PCR showed that expression of the TM6 gene was barely detectable in the flowers of the ms-1526 and ms-1547 mutants. In addition, other floral organ identity genes, pollen development marker genes, and pistil marker genes were differentially expressed between wild type and mutant flowers. These findings may facilitate functional analysis of the TM6 gene and help in the marker-assisted selection of ms-15 and its alleles in tomato breeding.

Fine mapping and molecular marker development of the Sm gene conferring resistance to gray leaf spot (Stemphylium spp.) in tomato.

KEY MESSAGE: The tomato gray leaf spot resistance gene Sm was fine-mapped in a 185-kb region through a map-based cloning strategy and genome-wide association study; a candidate gene was proved to be involved in Sm-mediated resistance through transient gene silencing. Gray leaf spot, caused by Stemphylium spp., is a warm weather foliar disease in tomato (Solanum lycopersicum L). Resistance against gray leaf spot is conferred by a single incompletely dominant gene (Sm) located on chromosome 11. This study aimed to map and identify molecular marker tightly linked to the Sm gene for the use of marker-assisted selection in breeding. Using an F2 population derived from a cross between the resistant line '9706' and the susceptible line 'Heinz 1706', the Sm gene was mapped to a 185-kb interval between two markers, InDel343 and InDel-FT-32 on chromosome 11, which was consistent with the result of a genome-wide association study using 289 diverse accessions. An ORF predicted in this region was proved to be involved in Sm-mediated resistance through transient gene silencing and seems to be a good candidate of the Sm locus. To clone the Sm gene, a bacterial artificial chromosome (BAC) library was screened and one BAC clone B80B15 containing the predicted ORF was identified. The analysis of sequence and structure characteristics demonstrated that the candidate gene was not a typical type resistance gene. Additionally, a co-dominant marker Sm-InDel, which produced a 122-bp or 140-bp fragment for resistant or susceptible alleles, respectively, was developed. This marker was validated in 289 germplasm and could be used in marker-assisted selection for gray leaf spot resistance.

A putative R3 MYB repressor is the candidate gene underlying atroviolacium, a locus for anthocyanin pigmentation in tomato fruit.

Anthocyanins are potential health-promoting compounds in the human diet. The atv (atroviolacium) locus, derived from the wild tomato species Solanum cheesmaniae, has been shown to enhance anthocyanin pigmentation in tomato fruit when it co-exists with either the Aft (Anthocyanin fruit) or the Abg (Aubergine) locus. In the present study, the atv locus was fine-mapped to an approximately 5.0-kb interval on chromosome 7. A putative R3 MYB repressor was identified in this interval and is hereby designated as SlMYBATV. The allele of SlMYBATV underlying the atv locus harbored a 4-bp insertion in its coding region, which is predicted to result in a frame-shift and premature protein truncation. The other candidate R3 MYB and R2R3 MYB repressors of anthocyanin biosynthesis were also identified in tomato via a genome-wide search. Transcriptional analysis showed that most of the structural genes and several regulatory genes of anthocyanin biosynthesis were up-regulated in the tomato SlMYBATV mutant lines. These findings may facilitate the elucidation of the molecular mechanisms underlying anthocyanin pigmentation in tomato fruit and help in the marker-assisted selection of anthocyanin-enriched tomato cultivars.

Fine Mapping of a Gene (ER4.1) that Causes Epidermal Reticulation of Tomato Fruit and Characterization of the Associated Transcriptome.

The hydrophobic cuticle that covers the surface of tomato (Solanum lycopersicum) fruit plays key roles in development and protection against biotic and abiotic stresses, including water loss, mechanical damage, UV radiation, pathogens, and pests. However, many details of the genes and regulatory mechanisms involved in cuticle biosynthesis in fleshy fruits are not well understood. In this study, we describe a novel tomato fruit phenotype, characterized by epidermal reticulation (ER) of green fruit and a higher water loss rate than wild type (WT) fruit. The ER phenotype is controlled by a single gene, ER4.1, derived from an introgressed chromosomal segment from the wild tomato species S. pennellii (LA0716). We performed fine mapping of the single dominant gene to an ~300 kb region and identified Solyc04g082540, Solyc04g082950, Solyc04g082630, and Solyc04g082910as potential candidate genes for the ER4.1 locus, based on comparative RNA-seq analysis of ER and WT fruit peels. In addition, the transcriptome analysis revealed that the expression levels of genes involved in cutin, wax and flavonoid biosynthesis were altered in the ER fruit compared with WT. This study provides new insights into the regulatory mechanisms and metabolism of the fruit cuticle.

The Tomato Hoffman's Anthocyaninless Gene Encodes a bHLH Transcription Factor Involved in Anthocyanin Biosynthesis That Is Developmentally Regulated and Induced by Low Temperatures.

Anthocyanin pigments play many roles in plants, including providing protection against biotic and abiotic stresses. Many of the genes that mediate anthocyanin accumulation have been identified through studies of flowers and fruits; however, the mechanisms of genes involved in anthocyanin regulation in seedlings under low-temperature stimulus are less well understood. Genetic characterization of a tomato inbred line, FMTT271, which showed no anthocyanin pigmentation, revealed a mutation in a bHLH transcription factor (TF) gene, which corresponds to the ah (Hoffman's anthocyaninless) locus, and so the gene in FMTT271 at that locus was named ah. Overexpression of the wild type allele of AH in FMTT271 resulted in greater anthocyanin accumulation and increased expression of several genes in the anthocyanin biosynthetic pathway. The expression of AH and anthocyanin accumulation in seedlings was shown to be developmentally regulated and induced by low-temperature stress. Additionally, transcriptome analyses of hypocotyls and leaves from the near-isogenic lines seedlings revealed that AH not only influences the expression of anthocyanin biosynthetic genes, but also genes associated with responses to abiotic stress. Furthermore, the ah mutation was shown to cause accumulation of reactive oxidative species and the constitutive activation of defense responses under cold conditions. These results suggest that AH regulates anthocyanin biosynthesis, thereby playing a protective role, and that this function is particularly important in young seedlings that are particularly vulnerable to abiotic stresses.

Detection of an inversion in the Ty-2 region between S. lycopersicum and S. habrochaites by a combination of de novo genome assembly and BAC cloning.

KEY MESSAGE: A chromosomal inversion associated with the tomato Ty - 2 gene for TYLCV resistance is the cause of severe suppression of recombination in a tomato Ty - 2 introgression line. Among tomato and its wild relatives inversions are often observed, which result in suppression of recombination. Such inversions hamper the transfer of important traits from a related species to the crop by introgression breeding. Suppression of recombination was reported for the TYLCV resistance gene, Ty-2, which has been introgressed in cultivated tomato (Solanum lycopersicum) from the wild relative S. habrochaites accession B6013. Ty-2 was mapped to a 300-kb region on the long arm of chromosome 11. The suppression of recombination in the Ty-2 region could be caused by chromosomal rearrangements in S. habrochaites compared with S. lycopersicum. With the aim of visualizing the genome structure of the Ty-2 region, we compared the draft de novo assembly of S. habrochaites accession LYC4 with the sequence of cultivated tomato ('Heinz'). Furthermore, using populations derived from intraspecific crosses of S. habrochaites accessions, the order of markers in the Ty-2 region was studied. Results showed the presence of an inversion of approximately 200 kb in the Ty-2 region when comparing S. lycopersicum and S. habrochaites. By sequencing a BAC clone from the Ty-2 introgression line, one inversion breakpoint was identified. Finally, the obtained results are discussed with respect to introgression breeding and the importance of a priori de novo sequencing of the species involved.

The Ph-3 gene from Solanum pimpinellifolium encodes CC-NBS-LRR protein conferring resistance to Phytophthora infestans.

KEY MESSAGE: Ph-3 is the first cloned tomato gene for resistance to late blight and encodes a CC-NBS-LRR protein. Late blight, caused by Phytophthora infestans, is one of the most destructive diseases in tomato. The resistance (R) gene Ph-3, derived from Solanum pimpinellifolium L3708, provides resistance to multiple P. infestans isolates and has been widely used in tomato breeding programmes. In our previous study, Ph-3 was mapped into a region harbouring R gene analogues (RGA) at the distal part of long arm of chromosome 9. To further narrow down the Ph-3 interval, more recombinants were identified using the flanking markers G2-4 and M8-2, which defined the Ph-3 gene to a 26 kb region according to the Heinz1706 reference genome. To clone the Ph-3 gene, a bacterial artificial chromosome (BAC) library was constructed using L3708 and one BAC clone B25E21 containing the Ph-3 region was identified. The sequence of the BAC clone B25E21 showed that only one RGA was present in the target region. A subsequent complementation analysis demonstrated that this RGA, encoding a CC-NBS-LRR protein, was able to complement the susceptible phenotype in cultivar Moneymaker. Thus this RGA was considered the Ph-3 gene. The predicted Ph-3 protein shares high amino acid identity with the chromosome-9-derived potato resistance proteins against P. infestans (Rpi proteins).

Establishment of an in vitro method for evaluating whitefly resistance in tomato.

An accurate and simple evaluation method is crucial for identifying whitefly resistance in tomato breeding. We developed an in vitro method for evaluating resistance of tomato leaves and tested this on wild and cultivated tomato varieties. We found that young leaves observed for whitefly oviposition after 8 hours provided appropriate comparative conditions. This method effectively distinguished resistance among tomato cultivars and wild species and also demonstrated significant difference in oviposition rates among leaf positions on susceptible cultivars. The in vitro test was as precise as in vivo test using intact plants and had advantages over in vivo test, and can be used for evaluating resistance in large populations.

Alfalfa benefits from Medicago truncatula: the RCT1 gene from M. truncatula confers broad-spectrum resistance to anthracnose in alfalfa.

Alfalfa is economically the most important forage legume worldwide. A recurrent challenge to alfalfa production is the significant yield loss caused by disease. Although knowledge of molecular mechanisms underlying host resistance should facilitate the genetic improvement of alfalfa, the acquisition of such knowledge is hampered by alfalfa's tetrasomic inheritance and outcrossing nature. However, alfalfa is congeneric with the reference legume Medicago truncatula, providing an opportunity to use M. truncatula as a surrogate to clone the counterparts of many agronomically important genes in alfalfa. In particular, the high degree of sequence identity and remarkably conserved genome structure and function between the two species enables M. truncatula genes to be used directly in alfalfa improvement. Here we report the map-based cloning of RCT1, a host resistance (R) gene in M. truncatula that confers resistance to multiple races of Colletotrichum trifolii, a hemibiotrophic fungal pathogen that causes anthracnose disease of alfalfa. RCT1 is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant R genes and confers broad-spectrum anthracnose resistance when transferred into susceptible alfalfa plants. Thus, RCT1 provides a novel resource to develop anthracnose-resistant alfalfa cultivars and contributes to our understanding of host resistance against the fungal genus Colletotrichum. This work demonstrates the potential of using M. truncatula genes for genetic improvement of alfalfa.