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[Purpose] Observe the difference between potato (Solanum tuberosum ssp. andigena) WT and BRC1b RNAi axillary buds in response to the transition from long-day to short-day conditions. The time course includes four time points: Long days, after 2 days in short days, 1 week in short days and 2 weeks in short days., [Methods] stem were flash-frozen in N2(l) one hour after dawn and the axillary buds were dissected in the cold room. The four axillary buds bellow the third visible node counting from the apex (those most likely to produce tubers in RNAi line) were collected. RNA was extracted with FavorPrep™ Plant Total RNA Mini Kit from FAVORGEN. DNA was degraded in the column with RNase-free DNase I (Roche). Three biological replicates were used and each replicate is a pool of axillary buds from 4 plants.

The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy.

Both inter- and intra-specific diversity has been described for trichome patterning in fruits, which is presumably involved in plant adaptation. However, the mechanisms underlying this developmental trait have been hardly addressed. Here we examined natural populations of Arabidopsis (Arabidopsis thaliana) that develop trichomes in fruits and pedicels, phenotypes previously not reported in the Arabidopsis genus. Genetic analyses identified five loci, MALAMBRUNO 1-5 (MAU1-5), with MAU2, MAU3, and MAU5 showing strong epistatic interactions that are necessary and sufficient to display these traits. Functional characterization of these three loci revealed cis-regulatory mutations in TRICHOMELESS1 and TRIPTYCHON, as well as a structural mutation in GLABRA1. Therefore, the multiple mechanisms controlled by three MYB transcription factors of the core regulatory network for trichome patterning have jointly been modulated to trigger trichome development in fruits. Furthermore, analyses of worldwide accessions showed that these traits and mutations only occur in a highly differentiated relict lineage from the Iberian Peninsula. In addition, these traits and alleles were associated with low spring precipitation, which suggests that trichome development in fruits and pedicels might be involved in climatic adaptation. Thus, we show that the combination of synergistic mutations in a gene regulatory circuit has driven evolutionary innovations in fruit trichome patterning in Arabidopsis.

The control of carbon allocation, storage and usage is critical for plant growth and development and is exploited for both crop food production and CO2 capture. Potato tubers are natural carbon reserves in the form of starch that have evolved to allow propagation and survival over winter. They form from stolons, below ground, where they are protected from adverse environmental conditions and animal foraging. We show that BRANCHED1b (BRC1b) acts as a tuberization repressor in aerial axillary buds, which prevents buds from competing in sink strength with stolons. BRC1b loss of function leads to ectopic production of aerial tubers and reduced underground tuberization. In aerial axillary buds, BRC1b promotes dormancy, abscisic acid responses and a reduced number of plasmodesmata. This limits sucrose accumulation and access of the tuberigen protein SP6A. BRC1b also directly interacts with SP6A and blocks its tuber-inducing activity in aerial nodes. Altogether, these actions help promote tuberization underground.

BACKGROUND: Monoterpenes and C13-norisoprenoids are key terpenoid compounds for wine aroma. The enzyme encoded by VviDXS1 participates in terpenoid biosynthesis in grapevine fruits and gain-of-function mutations in this gene lead to characteristic muscat aroma. OBJECTIVE: To assess for VviDXS1 contribution to aroma variation in Northwestern Iberian wine cultivars, we resequenced this gene in 111 cultivars and compared grape juice terpenic composition in 12 of them. METHODS: VviDXS1 was capture-targeted for resequencing with Illumina paired-end reads, SAMtools was used for variant calling and gene haplotypes were reconstructed with PHASE. Monoterpenes and C13-norisoprenoids were quantified in free and glycosidically-bound forms from grape juice by gas chromatography-mass spectrometry. RESULTS: Terpenic composition discriminated between muscat, terpenic and neutral profiles across cultivars. While the terpenic profile of Loureira and Albariño white cultivars was not associated with muscat-like mutations, Albariño carries a V34L substitution in VviDXS1 that is also present in other aromatic cultivars and was not reported before. Tempranillo and Cabernet Sauvignon red cultivars accumulated higher levels of C13-norisoprenoids, which was not associated with specific variation in VviDXS1. CONCLUSIONS: Apart from the uncharacterized substitution present in Albariño, findings suggest that terpenoid pathway-related genes other than VviDXS1 could contribute to the aromatic attributes of these cultivars.

Grapevine is one of the most valuable fruit crops in the world. Adverse environmental conditions reduce fruit quality and crop yield, so understanding the genetic and molecular mechanisms determining crop yield components is essential to optimize grape production. The analysis of a diverse collection of grapevine cultivars allowed us to evaluate the relationship between fruit set-related components of yield, including the incidence of reproductive disorders such as coulure and millerandage. The collection displayed a great phenotypic variation that we surveyed in a genetics association study using 15,309 single nucleotide polymorphisms (SNPs) detected in the sequence of 289 candidate genes scattered across the 19 grapevine linkage groups. After correcting statistical models for population structure and linkage disequilibrium effects, 164 SNPs from 34 of these genes were found to associate with fruit set-related traits, supporting a complex polygenic determinism. Many of them were found in the sequence of different putative MADS-box transcription factors, a gene family related with plant reproductive development control. In addition, we observed an additive effect of some of the associated SNPs on the phenotype, suggesting that advantageous alleles from different loci could be pyramided to generate superior cultivars with optimized fruit production.

Seedlessness is greatly prized by consumers of fresh grapes. While stenospermocarpic seed abortion determined by the SEED DEVELOPMENT INHIBITOR (SDI) locus is the usual source of seedlessness in commercial grapevine (Vitis vinifera) cultivars, the underlying sdi mutation remains unknown. Here, we undertook an integrative approach to identify the causal mutation. Quantitative genetics and fine-mapping in two ‘Crimson Seedless’-derived F1 mapping populations confirmed the major effect of the SDI locus and delimited the sdi mutation to a 323-kb region on chromosome 18. RNA-sequencing comparing seed traces of seedless and seeds of seeded F1 individuals identified processes triggered during sdi-determined seed abortion, including the activation of salicylic acid-dependent autoimmunity. The RNA-sequencing data set was investigated for candidate genes, and while no evidence for causal cis-acting regulatory mutations was detected, deleterious nucleotide changes in coding sequences of the seedless haplotype were predicted in two genes within the sdi fine-mapping interval. Targeted resequencing of the two genes in a collection of 124 grapevine cultivars showed that only the point variation causing the arginine-197-to-leucine substitution in the seed morphogenesis regulator gene AGAMOUS-LIKE11 (VviAGL11) was fully linked with stenospermocarpy. The concurrent postzygotic variation identified for this missense polymorphism and seedlessness phenotype in seeded somatic variants of the original stenospermocarpic cultivar supports a causal effect. We postulate that seed abortion caused by this amino acid substitution in VviAGL11 is the major cause of seedlessness in cultivated grapevine. This information can be exploited to boost seedless grape breeding.

Cluster compactness is a trait with high agronomic relevance, affecting crop yield and grape composition. Rachis architecture is a major component of cluster compactness determinism, and is a target trait toward the breeding of grapevine varieties less susceptible to pests and diseases. Although its genetic basis is scarcely understood, a preliminary result indicated a possible involvement of the VviUCC1 gene. The aim of this study was to characterize the VviUCC1 gene in grapevine and to test the association between the natural variation observed for a series of rachis architecture traits and the polymorphisms detected in the VviUCC1 sequence. This gene encodes an uclacyanin plant-specific cell-wall protein involved in fiber formation and/or lignification processes. A high nucleotide diversity in the VviUCC1 gene promoter and coding regions was observed, but no critical effects were predicted in the protein domains, indicating a high level of conservation of its function in the cultivated grapevine. After correcting statistical models for genetic stratification and linkage disequilibrium effects, marker-trait association results revealed a series of single nucleotide polymorphisms (SNPs) significantly associated with cluster compactness and rachis traits variation. Two of them (Y-984 and K-88) affected two common cis-transcriptional regulatory elements, suggesting an effect on phenotype via gene expression regulation. This work reinforces the interest of further studies aiming to reveal the functional effect of the detected VviUCC1 variants on grapevine rachis architecture.

Grape (Vitis vinifera) color somatic variants that can be used to develop new grapevine cultivars occasionally appear associated with deletion events of uncertain origin. To understand the mutational mechanisms generating somatic structural variation in grapevine, we compared the Tempranillo Blanco (TB) white berry somatic variant with its black berry ancestor, Tempranillo Tinto. Whole-genome sequencing uncovered a catastrophic genome rearrangement in TB that caused the hemizygous deletion of 313 genes, including the loss of the functional copy for the MYB transcription factors required for anthocyanin pigmentation in the berry skin. Loss of heterozygosity and decreased copy number delimited interspersed monosomic and disomic regions in the right arm of linkage groups 2 and 5. At least 11 validated clustered breakpoints involving intrachromosomal and interchromosomal translocations between three linkage groups flanked the deleted fragments, which, according to segregation analyses, are phased in a single copy of each of the affected chromosomes. These hallmarks, along with the lack of homology between breakpoint joins and the randomness of the order and orientation of the rearranged fragments, are all consistent with a chromothripsis-like pattern generated after chromosome breakage and illegitimate rejoining. This unbalanced genome reshuffling has additional consequences in reproductive development. In TB, lack of sexual transmission of rearranged chromosomes associates with low gamete viability, which compromises fruit set and decreases fruit production. Our findings show that catastrophic genome rearrangements arise spontaneously and stabilize during plant somatic growth. These dramatic rearrangements generate new interesting phenotypes that can be selected for the improvement of vegetatively propagated plant species.

Computational tools are essential in the process of designing a CRISPR/Cas experiment for the targeted modification of an organism’s genome. Among other functionalities, these tools facilitate the design of a guide-RNA (gRNA) for a given nuclease that maximizes its binding to the intended genomic site, while avoiding binding to undesired sites with similar sequences in the genome of the organism of interest (off-targets). Due to the popularity of this methodology and the rapid pace at which it evolves and changes, new computational tools show up constantly. This rapid turnover, together with the intrinsic high death-rate of bioinformatics tools, mean that many of the published tools become unavailable at some point. Consequently, the traditional ways to inform the community about the landscape of available tools, i.e., reviews in the scientific literature, are not adequate for this fast-moving field. To overcome these limitations, we have developed “WeReview: CRISPR Tools,” a live, on-line, user-updatable repository of computational tools to assist researchers in designing CRISPR/Cas experiments. In its web site users can find an updated comprehensive list of tools and search for those fulfilling their specific needs, as well as proposing modifications to the data associated with the tools or the incorporation of new ones.

Seedlessness is a relevant trait in grapevine cultivars intended for fresh consumption or raisin production. Previous DNA marker analysis indicated that Corinto bianco (CB) is a parthenocarpic somatic variant of the seeded cultivar Pedro Ximenes (PX). This study compared both variant lines to determine the basis of this parthenocarpic phenotype. At maturity, CB seedless berries were 6-fold smaller than PX berries. The macrogametophyte was absent from CB ovules, and CB was also pollen sterile. Occasionally, one seed developed in 1.6% of CB berries. Microsatellite genotyping and flow cytometry analyses of seedlings generated from these seeds showed that most CB viable seeds were formed by fertilization of unreduced gametes generated by meiotic diplospory, a process that has not been described previously in grapevine. Microarray and RNA-sequencing analyses identified 1958 genes that were differentially expressed between CB and PX developing flowers. Genes downregulated in CB were enriched in gametophyte-preferentially expressed transcripts, indicating the absence of regular post-meiotic germline development in CB. RNA-sequencing was also used for genetic variant calling and 14 single-nucleotide polymorphisms distinguishing the CB and PX variant lines were detected. Among these, CB-specific polymorphisms were considered as candidate parthenocarpy-responsible mutations, including a putative deleterious substitution in a HAL2-like protein. Collectively, these results revealed that the absence of a mature macrogametophyte, probably due to meiosis arrest, coupled with a process of fertilization-independent fruit growth, caused parthenocarpy in CB. This study provides a number of grapevine parthenocarpy-responsible candidate genes and shows how genomic approaches can shed light on the genetic origin of woody crop somatic variants.