RESUMO
The main bottleneck in the application of biotechnological breeding methods to woody species is due to the in vitro regeneration recalcitrance shown by several genotypes. On the other side, woody species, especially grapevine (Vitis vinifera L.), use most of the pesticides and other expensive inputs in agriculture, making the development of efficient approaches of genetic improvement absolutely urgent. Genome editing is an extremely promising technique particularly for wine grape genotypes, as it allows to modify the desired gene in a single step, preserving all the quality traits selected and appreciated in elite varieties. A genome editing and regeneration protocol for the production of transgene-free grapevine plants, exploiting the lipofectamine-mediated direct delivery of CRISPR-Cas9 ribonucleoproteins (RNPs) to target the phytoene desaturase gene, is reported. We focused on Nebbiolo (V. vinifera), an extremely in vitro recalcitrant wine genotype used to produce outstanding wines, such as Barolo and Barbaresco. The use of the PEG-mediated editing method available in literature and employed for highly embryogenic grapevine genotypes did not allow the proper embryo development in the recalcitrant Nebbiolo. Lipofectamines, on the contrary, did not have a negative impact on protoplast viability and plant regeneration, leading to the obtainment of fully developed edited plants after about 5 months from the transfection. Our work represents one of the first examples of lipofectamine use for delivering editing reagents in plant protoplasts. The important result achieved for the wine grape genotype breeding could be extended to other important wine grape varieties and recalcitrant woody species.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Genótipo , Lipídeos , Protoplastos , Vitis , Vitis/genética , Edição de Genes/métodos , Protoplastos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Vinho , Genoma de Planta/genética , Oxirredutases/genética , Oxirredutases/metabolismoRESUMO
Climate change is deeply impacting the food chain production, lowering quality and yield. In this context, the international scientific community has dedicated many efforts to enhancing resilience and sustainability in agriculture. Italy is among the main European producers of several fruit trees; therefore, national research centers and universities undertook several initiatives to maintain the specificity of the 'Made in Italy' label. Despite their importance, fruit crops are suffering from difficulties associated with the conventional breeding approaches, especially in terms of financial commitment, land resources availability, and long generation times. The 'new genomic techniques' (NGTs), renamed in Italy as 'technologies for assisted evolution' (TEAs), reduce the time required to obtain genetically improved cultivars while precisely targeting specific DNA sequences. This review aims to illustrate the role of the Italian scientific community in the use of NGTs, with a specific focus on Citrus, grapevine, apple, pear, chestnut, strawberry, peach, and kiwifruit. For each crop, the key genes and traits on which the scientific community is working, as well as the technological improvements and advancements on the regeneration of local varieties, are presented. Lastly, a focus is placed on the legal aspects in the European and in Italian contexts.
Assuntos
Frutas , Árvores , Árvores/genética , Frutas/genética , Melhoramento Vegetal/métodos , Genoma de Planta , GenômicaRESUMO
The importance of plants as complex entities influenced by genomes of the associated microorganisms is now seen as a new source of variability for a more sustainable agriculture, also in the light of ongoing climate change. For this reason, we investigated through metatranscriptomics whether the taxa profile and behaviour of microbial communities associated with the wood of 20-year-old grapevine plants are influenced by the health status of the host. We report for the first time a metatranscriptome from a complex tissue in a real environment, highlighting that this approach is able to define the microbial community better than referenced transcriptomic approaches. In parallel, the use of total RNA enabled the identification of bacterial taxa in healthy samples that, once isolated from the original wood tissue, displayed potential biocontrol activities against a wood-degrading fungal taxon. Furthermore, we revealed an unprecedented high number of new viral entities (~120 new viral species among 180 identified) associated with a single and limited environment and with potential impact on the whole holobiont. Taken together, our results suggest a complex multitrophic interaction in which the viral community also plays a crucial role in raising new ecological questions for the exploitation of microbial-assisted sustainable agriculture.
Assuntos
Endófitos , Microbiota , Bactérias/genética , Plantas , MadeiraRESUMO
BACKGROUND: Vitis vinifera L. is the most cultivated grapevine species worldwide. Erysiphe necator Sch., the causal agent of grape powdery mildew, is one of the main pathogens affecting viticulture. V. vinifera has little or no genetic resistances against E. necator and the grape industry is highly dependent on agrochemicals. Some Caucasian V. vinifera accessions have been reported to be resistant to E. necator and to have no genetic relationships to known sources of resistance to powdery mildew. The main purpose of this work was the study and mapping of the resistance to E. necator in the Caucasian grapes 'Shavtsitska' and 'Tskhvedianis tetra'. RESULTS: The Caucasian varieties 'Shavtsitska' and 'Tskhvedianis tetra' showed a strong partial resistance to E. necator which segregated in two cross populations: the resistant genotypes delayed and limited the pathogen mycelium growth, sporulation intensity and number of conidia generated. A total of 184 seedlings of 'Shavtsitska' x 'Glera' population were genotyped through the Genotyping by Sequencing (GBS) technology and two high-density linkage maps were developed for the cross parents. The QTL analysis revealed a major resistance locus, explaining up to 80.15% of the phenotypic variance, on 'Shavtsitska' linkage group 13, which was associated with a reduced pathogen infection as well as an enhanced plant necrotic response. The genotyping of 105 Caucasian accessions with SSR markers flanking the QTL revealed that the resistant haplotype of 'Shavtsitska' was shared by 'Tskhvedianis tetra' and a total of 25 Caucasian grape varieties, suggesting a widespread presence of this resistance in the surveyed germplasm. The uncovered QTL was mapped in the region where the Ren1 locus of resistance to E. necator, identified in the V. vinifera 'Kishmish vatkana' and related grapes of Central Asia, is located. The genetic analysis conducted revealed that the Caucasian grapes in this study exhibit a resistant haplotype different from that of Central Asian grape accessions. CONCLUSIONS: The QTL isolated in 'Shavtsitska' and present in the Caucasian V. vinifera varieties could be a new candidate gene of resistance to E. necator to use in breeding programmes. It co-localizes with the Ren1 locus but shows a different haplotype from that of grapevines of Central Asia. We therefore consider that the Caucasian resistance locus, named Ren1.2, contains a member of a cluster of R-genes, of which the region is rich, and to be linked with, or possibly allelic, to Ren1.
Assuntos
Resistência à Doença/genética , Erysiphe/fisiologia , Genes de Plantas , Doenças das Plantas/genética , Vitis/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Cruzamentos Genéticos , Ligação Genética , Técnicas de Genotipagem , Doenças das Plantas/microbiologia , Locos de Características Quantitativas , Vitis/microbiologiaRESUMO
Traditional breeding or genetically modified organisms (GMOs) have for a long time been the sole approaches to effectively cope with biotic and abiotic stresses and implement the quality traits of crops. However, emerging diseases as well as unpredictable climate changes affecting agriculture over the entire globe force scientists to find alternative solutions required to quickly overcome seasonal crises. In this review, we first focus on cisgenesis and genome editing as challenging biotechnological approaches for breeding crops more tolerant to biotic and abiotic stresses. In addition, we take into consideration a toolbox of new techniques based on applications of RNA interference and epigenome modifications, which can be adopted for improving plant resilience. Recent advances in these biotechnological applications are mainly reported for non-model plants and woody crops in particular. Indeed, the characterization of RNAi machinery in plants is fundamental to transform available information into biologically or biotechnologically applicable knowledge. Finally, here we discuss how these innovative and environmentally friendly techniques combined with traditional breeding can sustain a modern agriculture and be of potential contribution to climate change mitigation.
Assuntos
Proteção de Cultivos , Melhoramento Vegetal , Produtos Agrícolas/genética , Edição de Genes , Plantas Geneticamente Modificadas/genéticaRESUMO
Fruits stored at low temperature can exhibit different types of chilling injury. In apple, one of the most serious physiological disorders is superficial scald, which is characterized by discoloration and brown necrotic patches on the fruit exocarp. Although this phenomenon is widely ascribed to the oxidation of α-farnesene, its physiology is not yet fully understood. To elucidate the mechanism of superficial scald development and possible means of prevention, we performed an integrated metabolite screen, including an analysis of volatiles, phenols and lipids, together with a large-scale transcriptome study. We also determined that prevention of superficial scald, through the use of an ethylene action inhibitor, is associated with the triggering of cold acclimation-related processes. Specifically, the inhibition of ethylene perception stimulated the production of antioxidant compounds to scavenge reactive oxygen species, the synthesis of fatty acids to stabilize plastid and vacuole membranes against cold temperature, and the accumulation of the sorbitol, which can act as a cryoprotectant. The pattern of sorbitol accumulation was consistent with the expression profile of a sorbitol 6-phosphate dehydrogenase, MdS6PDH, the overexpression of which in transgenic Arabidopsis thaliana plants confirmed its involvement in the cold acclimation and freezing tolerance.
Assuntos
Ciclopropanos/metabolismo , Etilenos/antagonistas & inibidores , Malus/fisiologia , Doenças das Plantas/imunologia , Reguladores de Crescimento de Plantas/antagonistas & inibidores , Transcriptoma , Aclimatação , Temperatura Baixa , Resistência à Doença , Etilenos/metabolismo , Frutas/genética , Frutas/imunologia , Frutas/metabolismo , L-Iditol 2-Desidrogenase/genética , Malus/genética , Malus/imunologia , Modelos Biológicos , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas , Metabolismo Secundário , Análise de Sequência de RNA , Sorbitol/metabolismoRESUMO
For the viticulture of the future, it will be an essential prerequisite to manage grapevine diseases with fewer chemical inputs. The development and the deployment of novel mildew resistant varieties are considered one of the most promising strategies towards a sustainable viticulture. In this regard, a collection of 102 accessions derived from crossing Vitis hybrids with V. vinifera varieties was studied. In addition to the true-to-type analysis, an exhaustive genetic characterization was carried out at the 11 reliable mildew resistance (R) loci available in the literature to date. Our findings highlight the pyramiding of R-loci against downy mildew in 15.7% and against powdery mildew in 39.2% of the total accessions. The genetic analysis was coupled with a three-year evaluation of disease symptoms in an untreated field in order to assess the impact of the R-loci arrangement on the disease resistance degree at leaf and bunch level. Overall, our results strongly suggest that R-loci pyramiding does not necessarily mean to increase the overall disease resistance, but it guarantees the presence of further barriers in case of pathogens overcoming the first. Moreover, our survey allows the discovery of new mildew resistance sources useful for novel QTL identifications towards marker-assisted breeding.
Assuntos
Ascomicetos/patogenicidade , Doenças das Plantas/microbiologia , Vitis/metabolismo , Vitis/microbiologia , Ascomicetos/genética , Mapeamento Cromossômico , Resistência à Doença/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Locos de Características Quantitativas/genéticaRESUMO
KEY MESSAGE: Data obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads. The reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25 S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S 5 , S 6a , S 6b , S 8 , S 11 , S 23 , S 39 , S 46 , S 50 and S 58 ). Moreover, sequence divergence clearly suggests that alleles S 6a and S 6b , proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles.
Assuntos
Malus/genética , Ribonucleases/genética , Autoincompatibilidade em Angiospermas/genética , Região 3'-Flanqueadora/genética , Região 5'-Flanqueadora/genética , Alelos , Genoma de Planta/genética , Filogenia , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome-wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom(®) genotyping array for apple and discuss its potential applications. The array has been built from the high-depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non-genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom(®) Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom(®) Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple.
Assuntos
Genoma de Planta/genética , Técnicas de Genotipagem/métodos , Malus/genética , Polimorfismo de Nucleotídeo Único/genética , Mapeamento Cromossômico , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genótipo , Desequilíbrio de Ligação , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Apple (Malus x domestica Borkh.) is a model species for studying the metabolic changes that occur at the onset of ripening in fruit crops, and the physiological mechanisms that are governed by the hormone ethylene. In this study, to dissect the climacteric interplay in apple, a multidisciplinary approach was employed. To this end, a comprehensive analysis of gene expression together with the investigation of several physiological entities (texture, volatilome and content of polyphenolic compounds) was performed throughout fruit development and ripening. The transcriptomic profiling was conducted with two microarray platforms: a dedicated custom array (iRIPE) and a whole genome array specifically enriched with ripening-related genes for apple (WGAA). The transcriptomic and phenotypic changes following the application of 1-methylcyclopropene (1-MCP), an ethylene inhibitor leading to important modifications in overall fruit physiology, were also highlighted. The integrative comparative network analysis showed both negative and positive correlations between ripening-related transcripts and the accumulation of specific metabolites or texture components. The ripening distortion caused by the inhibition of ethylene perception, in addition to affecting the ethylene pathway, stimulated the de-repression of auxin-related genes, transcription factors and photosynthetic genes. Overall, the comprehensive repertoire of results obtained here advances the elucidation of the multi-layered climacteric mechanism of fruit ripening, thus suggesting a possible transcriptional circuit governed by hormones and transcription factors.
Assuntos
Etilenos/metabolismo , Ácidos Indolacéticos/metabolismo , Malus/metabolismo , Ciclopropanos , Etilenos/antagonistas & inibidores , Frutas/efeitos dos fármacos , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Malus/efeitos dos fármacos , Malus/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Fruit quality represents a fundamental factor guiding consumers' preferences. Among apple quality traits, volatile organic compounds and texture features play a major role. Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-ToF-MS), coupled with an artificial chewing device, was used to profile the entire apple volatilome of 162 apple accessions, while the fruit texture was dissected with a TAXT-AED texture analyzer. The array of volatile compounds was classed into seven major groups and used in a genome-wide association analysis carried out with 9142 single nucleotide polymorphisms (SNPs). Marker-trait associations were identified on seven chromosomes co-locating with important candidate genes for aroma, such as MdAAT1 and MdIGS. The integration of volatilome and fruit texture data conducted with a multiple factor analysis unraveled contrasting behavior, underlying opposite regulation of the two fruit quality aspects. The association analysis using the first two principal components identified two QTLs located on chromosomes 10 and 2, respectively. The distinction of the apple accessions on the basis of the allelic configuration of two functional markers, MdPG1 and MdACO1, shed light on the type of interplay existing between fruit texture and the production of volatile organic compounds.
Assuntos
Frutas/genética , Estudo de Associação Genômica Ampla , Malus/genética , Odorantes/análise , Locos de Características Quantitativas , Compostos Orgânicos Voláteis/metabolismo , Frutas/fisiologia , Malus/fisiologia , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Varieties resistant to powdery mildew (PM; caused by Podosphaera leucotricha) are a major component of sustainable apple production. Resistance can be achieved by knocking-out susceptibility S-genes to be singled out among members of the MLO (Mildew Locus O) gene family. Candidates are MLO S-genes of phylogenetic clade V up-regulated upon PM inoculation, such as MdMLO11 and 19 (clade V) and MdMLO18 (clade VII). We report the knock-down through RNA interference of MdMLO11 and 19, as well as the complementation of resistance with MdMLO18 in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 reduced PM disease severity by 75%, whereas the knock-down of MdMLO11, alone or in combination with MdMLO19, did not result in any reduction or additional reduction of susceptibility compared with MdMLO19 alone. The test in A. thaliana excluded a role for MdMLO18 in PM susceptibility. Cell wall appositions (papillae) were present in both PM-resistant and PM-susceptible plants, but were larger in resistant lines. No obvious negative phenotype was observed in plants with mlo genes knocked down. Apparently, MdMLO19 plays the pivotal role in apple PM susceptibility and its knock-down induces a very significant level of resistance.
Assuntos
Ascomicetos/fisiologia , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Malus , Proteínas de Plantas , Plantas Geneticamente Modificadas/microbiologia , Técnicas de Silenciamento de Genes , Malus/genética , Malus/metabolismo , Malus/microbiologia , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Interferência de RNARESUMO
UNLABELLED: ASSIsT (Automatic SNP ScorIng Tool) is a user-friendly customized pipeline for efficient calling and filtering of SNPs from Illumina Infinium arrays, specifically devised for custom genotyping arrays. Illumina has developed an integrated software for SNP data visualization and inspection called GenomeStudio (GS). ASSIsT builds on GS-derived data and identifies those markers that follow a bi-allelic genetic model and show reliable genotype calls. Moreover, ASSIsT re-edits SNP calls with null alleles or additional SNPs in the probe annealing site. ASSIsT can be employed in the analysis of different population types such as full-sib families and mating schemes used in the plant kingdom (backcross, F1, F2), and unrelated individuals. The final result can be directly exported in the format required by the most common software for genetic mapping and marker-trait association analysis. ASSIsT is developed in Python and runs in Windows and Linux. AVAILABILITY AND IMPLEMENTATION: The software, example data sets and tutorials are freely available at http://compbiotoolbox.fmach.it/assist/. CONTACT: eric.vandeweg@wur.nl.
Assuntos
Técnicas de Genotipagem/métodos , Polimorfismo de Nucleotídeo Único , Software , Alelos , Animais , HumanosRESUMO
BACKGROUND: The complex dynamics of gene regulation in plants are still far from being fully understood. Among many factors involved, alternative splicing (AS) in particular is one of the least well documented. For many years, AS has been considered of less relevant in plants, especially when compared to animals, however, since the introduction of next generation sequencing techniques the number of plant genes believed to be alternatively spliced has increased exponentially. RESULTS: Here, we performed a comprehensive high-throughput transcript sequencing of ten different grapevine cultivars, which resulted in the first high coverage atlas of the grape berry transcriptome. We also developed findAS, a software tool for the analysis of alternatively spliced junctions. We demonstrate that at least 44% of multi-exonic genes undergo AS and a large number of low abundance splice variants is present within the 131.622 splice junctions we have annotated from Pinot noir. CONCLUSIONS: Our analysis shows that ~70% of AS events have relatively low expression levels, furthermore alternative splice sites seem to be enriched near the constitutive ones in some extent showing the noise of the splicing mechanisms. However, AS seems to be extensively conserved among the 10 cultivars.
Assuntos
Processamento Alternativo/genética , Vitis/genética , Sequenciamento de Nucleotídeos em Larga Escala , Splicing de RNA/genéticaRESUMO
BACKGROUND: Russeting is a disorder developed by apple fruits that consists of cuticle cracking followed by the replacement of the epidermis by a corky layer that protects the fruit surface from water loss and pathogens. Although influenced by many environmental conditions and orchard management practices, russeting is under genetic control. The difficulty in classifying offspring and consequent variable segregation ratios have led several authors to conclude that more than one genetic determinant could be involved, although some evidence favours a major gene (Ru). RESULTS: In this study we report the mapping of a major genetic russeting determinant on linkage group 12 of apple as inferred from the phenotypic observation in a segregating progeny derived from 'Renetta Grigia di Torriana', the construction of a 20 K Illumina SNP chip based genetic map, and QTL analysis. Recombination analysis in two mapping populations restricted the region of interest to approximately 400 Kb. Of the 58 genes predicted from the Golden Delicious sequence, a putative ABCG family transporter has been identified. Within a small set of russeted cultivars tested with markers of the region, only six showed the same haplotype of 'Renetta Grigia di Torriana'. CONCLUSIONS: A major determinant (Ru_RGT) for russeting development putatively involved in cuticle organization is proposed as a candidate for controlling the trait. SNP and SSR markers tightly co-segregating with the Ru_RGT locus may assist the breeder selection. The observed segregations and the analysis of the 'Renetta Grigia di Torriana' haplotypic region in a panel of russeted and non-russeted cultivars may suggest the presence of other determinants for russeting in apple.
Assuntos
Mapeamento Cromossômico , Ligação Genética , Malus/genética , Doenças das Plantas/genética , Locos de Características Quantitativas/genética , Segregação de Cromossomos/genética , Cromossomos de Plantas/genética , Cruzamentos Genéticos , Bases de Dados como Assunto , Estudos de Associação Genética , Loci Gênicos , Marcadores Genéticos , Haplótipos/genética , FenótipoRESUMO
Flavonols are a ubiquitous class of flavonoids that accumulate preferentially in flowers and mature berries. Besides their photo-protective function, they play a fundamental role during winemaking, stabilizing the colour by co-pigmentation with anthocyanins and contributing to organoleptic characteristics. Although the general flavonol pathway has been genetically and biochemically elucidated, the genetic control of flavonol content and composition at harvest is still not clear. To this purpose, the grapes of 170 segregating F1 individuals from a 'Syrah'×'Pinot Noir' population were evaluated at the mature stage for the content of six flavonol aglycons in four seasons. Metabolic data in combination with genetic data enabled the identification of 16 mQTLs (metabolic quantitative trait loci). For the first time, major genetic control by the linkage group 2 (LG 2)/MYBA region on flavonol variation, in particular of tri-hydroxylated flavonols, is demonstrated. Moreover, seven regions specifically associated with the fine control of flavonol biosynthesis are identified. Gene expression profiling of two groups of individuals significantly divergent for their skin flavonol content identified a large set of differentially modulated transcripts. Among these, the transcripts coding for MYB and bZIP transcription factors, methyltranferases, and glucosyltranferases specific for flavonols, proteins, and factors belonging to the UV-B signalling pathway and co-localizing with the QTL regions are proposed as candidate genes for the fine regulation of flavonol content and composition in mature grapes.
Assuntos
Flavonóis/metabolismo , Regulação da Expressão Gênica de Plantas , Pigmentação/genética , Proteínas de Plantas/genética , Vitis/genética , Flavonóis/genética , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Proteínas de Plantas/metabolismo , Locos de Características Quantitativas , Análise de Sequência de DNA , Vitis/metabolismoRESUMO
Both the origin of domesticated apple and the overall phylogeny of the genus Malus are still not completely resolved. Having this as a target, we built a 134,553-position-long alignment including two previously published chloroplast DNAs (cpDNAs) and 45 de novo sequenced, fully colinear chloroplast genomes from cultivated apple varieties and wild apple species. The data produced are free from compositional heterogeneity and from substitutional saturation, which can adversely affect phylogeny reconstruction. Phylogenetic analyses based on this alignment recovered a branch, having the maximum bootstrap support, subtending a large group of the cultivated apple sorts together with all analyzed European wild apple (Malus sylvestris) accessions. One apple cultivar was embedded in a monophylum comprising wild M. sieversii accessions and other Asian apple species. The data demonstrate that M. sylvestris has contributed chloroplast genome to a substantial fraction of domesticated apple varieties, supporting the conclusion that different wild species should have contributed the organelle and nuclear genomes to the domesticated apple.
Assuntos
Genoma de Cloroplastos , Malus/classificação , Malus/genética , Filogenia , Teorema de Bayes , Evolução MolecularRESUMO
BACKGROUND: Fruit quality features resulting from ripening processes need to be preserved throughout storage for economical reasons. However, during this period several physiological disorders can occur, of which superficial scald is one of the most important, due to the development of large brown areas on the fruit skin surface. RESULTS: This study examined the variation in polyphenolic content with the progress of superficial scald in apple, also with respect to 1-MCP, an ethylene competitor interacting with the hormone receptors and known to interfere with this etiology. The change in the accumulation of these metabolites was further correlated with the gene set involved in this pathway, together with two specific VOCs (Volatile Organic Compounds), α-farnesene and its oxidative form, 6-methyl-5-hepten-2-one. Metabolite profiling and qRT-PCR assay showed these volatiles are more heavily involved in the signalling system, while the browning coloration would seem to be due more to a specific accumulation of chlorogenic acid (as a consequence of the activation of MdPAL and MdC3H), and its further oxidation carried out by a polyphenol oxidase gene (MdPPO). In this physiological scenario, new evidence regarding the involvement of an anti-apoptotic regulatory mechanism for the compartmentation of this phenomenon in the skin alone was also hypothesized, as suggested by the expression profile of the MdDAD1, MdDND1 and MdLSD1 genes. CONCLUSIONS: The results presented in this work represent a step forward in understanding the physiological mechanisms of superficial scald in apple, shedding light on the regulation of the specific physiological cascade.
Assuntos
Regulação da Expressão Gênica de Plantas , Malus/genética , Malus/metabolismo , Catecol Oxidase/metabolismo , Ácido Clorogênico/metabolismo , Perfilação da Expressão Gênica , Sesquiterpenos/metabolismoRESUMO
Anthocyanin accumulation is coordinated in plants by a number of conserved transcription factors. In apple (Malus × domestica), an R2R3 MYB transcription factor has been shown to control fruit flesh and foliage anthocyanin pigmentation (MYB10) and fruit skin color (MYB1). However, the pattern of expression and allelic variation at these loci does not explain all anthocyanin-related apple phenotypes. One such example is an open-pollinated seedling of cv Sangrado that has green foliage and develops red flesh in the fruit cortex late in maturity. We used methods that combine plant breeding, molecular biology, and genomics to identify duplicated MYB transcription factors that could control this phenotype. We then demonstrated that the red-flesh cortex phenotype is associated with enhanced expression of MYB110a, a paralog of MYB10. Functional characterization of MYB110a showed that it was able to up-regulate anthocyanin biosynthesis in tobacco (Nicotiana tabacum). The chromosomal location of MYB110a is consistent with a whole-genome duplication event that occurred during the evolution of apple within the Maloideae family. Both MYB10 and MYB110a have conserved function in some cultivars, but they differ in their expression pattern and response to fruit maturity.
Assuntos
Frutas/metabolismo , Duplicação Gênica , Malus/metabolismo , Fenótipo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Antocianinas/biossíntese , Sequência de Bases , Cruzamento , Cromatografia Líquida de Alta Pressão , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Evolução Molecular , Frutas/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Malus/genética , Malus/crescimento & desenvolvimento , Dados de Sequência Molecular , Filogenia , Pigmentação , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Locos de Características Quantitativas , Alinhamento de Sequência , Especificidade da Espécie , Nicotiana/genética , Nicotiana/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Plant breeders utilize marker-assisted selection (MAS) to identify favorable or unfavorable alleles in seedlings early. In this task, they need methods that provide maximum information with minimal input of time and economic resources. Grape breeding aimed at producing cultivars resistant to pathogens employs several resistance loci (Rpv, Ren, and Run) that are ideal for implementing MAS. In this work, a sustainable MAS protocol was developed based on non-purified DNA (crude), multiplex PCR of SSR markers, and capillary electrophoresis, and its application on grapevine seedlings to follow some main resistance loci was described. The optimized protocol was utilized on 8440 samples and showed high efficiency, reasonable throughput (2-3.2 min sample), easy handling, flexibility, and tolerable costs (reduced by at least 3.5 times compared to a standard protocol). The Rpv, Ren, and Run allelic data analysis did not show limitations to loci combination and pyramiding, but segregation distortions were frequent and displayed both low (undesired) and high rates of inheritance. The protocol and results presented are useful tools for grape breeders and beyond, and they can address sustainable changes in MAS. Several progenies generated have valuable pyramided resistance and will be the subject of new studies and implementation in the breeding program.