Dataset Information

The Potential of HTS Approaches for Accurate Genotyping in Grapevine (Vitis vinifera L.).

ABSTRACT: The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price.

SUBMITTER: Kunej U

PROVIDER: S-EPMC7464945 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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The Potential of HTS Approaches for Accurate Genotyping in Grapevine (<i>Vitis vinifera</i> L.).

Kunej Urban U Dervishi Aida A Laucou Valérie V Jakše Jernej J Štajner Nataša N

Genes 20200810 8

The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences an ...[more]

PMID: 32785184

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Project description:UnlabelledCrown gall disease of grapevine is caused by virulent Agrobacterium strains and establishes a suitable habitat for agrobacteria and, potentially, other bacteria. The microbial community associated with grapevine plants has not been investigated with respect to this disease, which frequently results in monetary losses. This study compares the endophytic microbiota of organs from grapevine plants with or without crown gall disease and the surrounding vineyard soil over the growing seasons of 1 year. Amplicon-based community profiling revealed that the dominating factor causing differences between the grapevine microbiota is the sample site, not the crown gall disease. The soil showed the highest microbial diversity, which decreased with the distance from the soil over the root and the graft union of the trunk to the cane. Only the graft union microbiota was significantly affected by crown gall disease. The bacterial community of graft unions without a crown gall hosted transient microbiota, with the three most abundant bacterial species changing from season to season. In contrast, graft unions with a crown gall had a higher species richness, which in every season was dominated by the same three bacteria (Pseudomonas sp., Enterobacteriaceae sp., and Agrobacterium vitis). For in vitro-cultivated grapevine plantlets, A. vitis infection alone was sufficient to cause crown gall disease. Our data show that microbiota in crown galls is more stable over time than microbiota in healthy graft unions and that the microbial community is not essential for crown gall disease outbreak.ImportanceThe characterization of bacterial populations in animal and human diseases using high-throughput deep-sequencing technologies, such as 16S amplicon sequencing, will ideally result in the identification of disease-specific microbiota. We analyzed the microbiota of the crown gall disease of grapevine, which is caused by infection with the bacterial pathogen Agrobacterium vitis. All other Agrobacterium species were found to be avirulent, even though they lived together with A. vitis in the same crown gall tumor. As has been reported for human cancer, the crown gall tumor also hosted opportunistic bacteria that are adapted to the tumor microenvironment. Characterization of the microbiota in various diseases using amplicon sequencing may help in early diagnosis, to serve as a preventative measure of disease in the future.

Project description:BackgroundVegetative buds provide plants in temperate environments the possibility for growth and reproduction when environmental conditions are favorable. In grapevine, crucial developmental events take place within buds during two growing seasons in consecutive years. The first season, the shoot apical meristem within the bud differentiates all the basic elements of the shoot including flowering transition in lateral primordia and development of inflorescence primordia. These events practically end with bud dormancy. The second season, buds resume shoot growth associated to flower formation and development. Gene expression has been previously monitored at specific stages of bud development but has never been followed along the two growing seasons.ResultsGene expression changes were analyzed along the bud annual cycle at eight different time points. Principal Components Analysis (PCA) revealed that the main factors explaining the global gene expression differences were the processes of bud dormancy and active growth as well as stress responses. Accordingly, non dormant buds showed an enrichment in functional categories typical of actively proliferating and growing cells together with the over abundance of transcripts belonging to stress response pathways. Differential expression analyses performed between consecutive time points indicated that major transcriptional changes were associated to para/endodormancy, endo/ecodormancy and ecodormancy/bud break transitions. Transcripts encoding key regulators of reproductive development were grouped in three major expression clusters corresponding to: (i) transcripts associated to flowering induction, (ii) transcripts associated to flower meristem specification and initiation and (iii) transcripts putatively involved in dormancy. Within this cluster, a MADS-box gene (VvFLC2) and other transcripts with similar expression patterns could participate in dormancy regulation.ConclusionsThis work provides a global view of major transcriptional changes taking place along bud development in grapevine, highlighting those molecular and biological functions involved in the main events of bud development. As reported in other woody species, the results suggest that genes regulating flowering could also be involved in dormancy regulatory pathways in grapevine.

Project description:BackgroundSingle-nucleotide polymorphisms (SNPs) are the most abundant type of DNA sequence polymorphisms. Their higher availability and stability when compared to simple sequence repeats (SSRs) provide enhanced possibilities for genetic and breeding applications such as cultivar identification, construction of genetic maps, the assessment of genetic diversity, the detection of genotype/phenotype associations, or marker-assisted breeding. In addition, the efficiency of these activities can be improved thanks to the ease with which SNP genotyping can be automated. Expressed sequence tags (EST) sequencing projects in grapevine are allowing for the in silico detection of multiple putative sequence polymorphisms within and among a reduced number of cultivars. In parallel, the sequence of the grapevine cultivar Pinot Noir is also providing thousands of polymorphisms present in this highly heterozygous genome. Still the general application of those SNPs requires further validation since their use could be restricted to those specific genotypes.ResultsIn order to develop a large SNP set of wide application in grapevine we followed a systematic re-sequencing approach in a group of 11 grape genotypes corresponding to ancient unrelated cultivars as well as wild plants. Using this approach, we have sequenced 230 gene fragments, what represents the analysis of over 1 Mb of grape DNA sequence. This analysis has allowed the discovery of 1573 SNPs with an average of one SNP every 64 bp (one SNP every 47 bp in non-coding regions and every 69 bp in coding regions). Nucleotide diversity in grape (pi = 0.0051) was found to be similar to values observed in highly polymorphic plant species such as maize. The average number of haplotypes per gene sequence was estimated as six, with three haplotypes representing over 83% of the analyzed sequences. Short-range linkage disequilibrium (LD) studies within the analyzed sequences indicate the existence of a rapid decay of LD within the selected grapevine genotypes. To validate the use of the detected polymorphisms in genetic mapping, cultivar identification and genetic diversity studies we have used the SNPlextrade mark genotyping technology in a sample of grapevine genotypes and segregating progenies.ConclusionThese results provide accurate values for nucleotide diversity in coding sequences and a first estimate of short-range LD in grapevine. Using SNPlextrade mark genotyping we have shown the application of a set of discovered SNPs as molecular markers for cultivar identification, linkage mapping and genetic diversity studies. Thus, the combination a highly efficient re-sequencing approach and the SNPlextrade mark high throughput genotyping technology provide a powerful tool for grapevine genetic analysis.

Project description:BACKGROUND: In traditional vine areas, the production should present a typicity that partly depends on the grapevine variety. Therefore, vine improvement is considered difficult because of the limited choice in the natural variability of the cultivars within the limits of their characteristics. A possibility to circumvent this problem is the use of somatic variability. In vitro somatic embryogenesis and organogenesis can lead to genotypic and phenotypic variations, described as somaclonal variation, that could be useful for the selection of improved grapevine genotypes. RESULTS: In order to study tissue culture-induced variation of grapevine, we have analysed 78 somaclones obtained from somatic embryos of two distinct cultivars using molecular marker techniques. SSRs were only useful to verify the conservation of the microsatellite genotype between the somaclones and the respective mother clones. AFLP polymorphism between mother clones and somaclones was 1.3-2.8 times higher to that found between clones. However, a majority of the somaclones (45/78) exhibited only few changes. Seven and five somaclones of 'Chardonnay 96' and 'Syrah 174', respectively, which covered at least all polymorphic loci found in AFLP analysis were used for MSAP study. All of the 120 polymorphic fragments were found only in the somaclones. The percentage of full methylation at CCGG recognition sites was slightly higher in somaclones due to more polymorphic bands generated after cleavage by EcoRI/HpaII. Different digestion patterns revealed different methylation status, especially different levels of de-methylation, that are the consequence of the in vitro culture. CONCLUSION: MSAP highlights DNA methylation variation in somaclones compared to mother clones and, therefore, is a powerful tool for genotypic characterisation of somatic embryo-derived grapevines. The detection of the same polymorphic bands in numerous somaclones of different cultivars suggests the possibility of hot spots of DNA methylation variation. SSR profiles of the 'Chardonnay' and 'Syrah' somaclones were the same as of the respective mother clones. The somaclones exhibited a higher AFLP variation than clones obtained via traditional clonal selection in the field. Therefore, somatic embryogenesis through in vitro culture technique could be useful for the selection of improved cultivars with subtle changes but conserving their main characteristics.

Dataset Information

The Potential of HTS Approaches for Accurate Genotyping in Grapevine (Vitis vinifera L.).

Publications

The Potential of HTS Approaches for Accurate Genotyping in Grapevine (<i>Vitis vinifera</i> L.).

Similar Datasets

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