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|Autori: ||Miculan, Mara|
|Supervisore afferente all'Università: ||MORGANTE, MICHELE|
|Centro di ricerca: ||DIPARTIMENTO SCIENZE AGRARIE E AMBIENTALI - DISA|
|Titolo: ||Detection of somatic variants from next-generation sequencing data in grapevine bud sports|
|Abstract (in inglese): ||The grapevine (Vitis vinifera) is one of the oldest and the most valuable horticultural crops. Sexual crossing has been a major driver of grapevine evolution and, more recently, it has generated thousands of varieties. Somatic variation plays a crucial role in intravarietal grapevine diversity, generating novel interesting phenotypes. Somatic mutations that accidentally happened in buds of vegetatively propagated varieties were frequently noticed and the resulting bud sports were selected for their distinguished phenotype. In this work, we aimed to explore clonal variability to identify DNA mutations and transcriptional changes among genomes within a grapevine variety. Vitis vinifera is an ideal model because there are many clones with visible phenotypic differences and a high quality reference sequence is available (Jaillon et al 2007).
Previous studies of clonal diversity used SSR and AFLP markers that only enabled the identification of a limited number of clones. Thus, we adopted a whole genome scan approach. Illumina next generation sequencing technology was used to resequence four ‘Pinot’ clones (‘Pinot blanc’, ‘Pinot gris’, ’Pinot Meunier’ and ‘Pinot noir’) and two ‘Sangiovese’ clones (commercially called ‘R24’ and ‘VCR23’). Post-processed paired-end reads (2x100bp) were mapped against the PN40024 reference genome obtaining a depth of coverage >35x. Four libraries were of high quality, while the distribution of 16-kmers occurrences in the ‘Pinot gris’ and ‘Pinot noir’ Illumina reads revealed low complexity of the library and suggested to discard those clones for subsequent analyses.
SNPs were first detected in the pairwise comparison ‘Pinot blanc’ and ‘Pinot Meunier’ using the GATK – UnifiedGenotyper tool with default parameters, followed by a quality filtering and by a calibration step. In the filtering step, we removed SNPs in repetitive regions, transposable elements, and regions surrounding microsatellite motifs and INDELs, we removed bad quality SNPs based on GATK internal parameters, SNPs with <0.2 minor allele frequency and positions with low or high coverage (<0.5-fold and >3-fold the average coverage). The calibration step was based on quality scores of a known somatic variation in ‘Pinot Meunier’ in the position chr1:4,897,066 (Boss and Thomas 2002). In the comparison between ‘Pinot blanc’ and ‘Pinot Meunier’ we ended up with a total of 144 putative SNPs, 79 of which were validated as true positive by Sanger resequencing (29 in ‘Pinot blanc’ and 50 in ‘Pinot Meunier’) with a FDR of 0.33 and 0.24, respectively. We performed the pairwise comparison between ‘Sangiovese R24’ and ‘Sangiovese VCR23’ with the same parameters used for ‘Pinot’ clones, ending up with only three putative variant positions. Of these, two SNPs were validated as true positive by Sanger resequencing. In all cases, Sanger resequencing confirmed the chimerical nature of the putative somatic mutation.
Genome scanning for copy number variations larger than 25 kbp was performed by a depth of coverage (DOC) analysis and revealed only the known somatic deletion in ‘Pinot blanc’ in the interval chr2:14,149,000..14,250,000 as compared to ‘Pinot Meunier’. The complementary approach of paired-end mapping (PEM) revealed 11 putative deletions smaller than 25kbp in ‘Pinot blanc’, 19 in ‘Pinot gris’, 15 in ‘Pinot Meunier’, and 5 in ‘Pinot noir’ as unique to each clone and not shared with a set of 20 varieties of Vitis vinifera analysed with the same pipeline. In the comparison of ‘Sangiovese’ clones, the PEM algorithm identified seven putative deletions in ‘Sangiovese VCR23’, not shared with either ‘Sangiovese R24’ or other varieties of Vitis vinifera. No copy number variation larger than 25 kbp was detected by a depth of coverage (DOC) analysis between ‘Sangiovese’ clones.
We also compared the transcriptome of different clones in order to monitor gene expression changes that could be directly or indirectly related to somatic mutations at the DNA level. We obtained RNA-seq of leaf tissues of the same ‘Pinot’ and ‘Sangiovese’ clones analysed by DNA sequencing. Furthermore for ‘Sangiovese’ clones, we sequenced berry transcriptomes at two developmental stages – before ripening (2 weeks after berry set) and at the inception of ripening. More than 30,000 genes were expressed in all clones of both varieties. The vast majority of the predicted genes in the grapevine genome was transcribed at detectable levels in all organs and stages of development investigated. Under the same experimental conditions, leaf transcriptomes were much more variable in pairwise comparisons between ‘Pinot’ clones than between the pair of ‘Sangiovese’ clones. Between the clones of ‘Sangiovese’, the widest differentiation in terms of global transcriptome was detected in berries collected two weeks after fruit set. Genes that showed significant differences in transcriptional levels between clones were in general not correlated with the position of the DNA mutations identified by DNA sequencing.
Through the power of the Next Generation Sequencing technology we have produced a sufficient depth and breadth of sequence coverage to comprehensively discover somatic mutations that allowed us to distinguish four ‘Pinot’ clones and two ‘Sangiovese’ clones analysed in this study. At the DNA level, somatic mutations in two ‘Sangiovese’ genomes appeared to be more rare than those observed among ‘Pinot’ clones, which corresponds to a lower level of phenotypic differentiation between the two ‘Sangiovese’ clones and is in accord with a presumed more recent origin compared to the ‘Pinot’ clones. This analysis provides the first whole-genome estimation of the rate of somatic mutation in grapevine varieties.|
|Parole chiave: ||Vitis vinifera; Clones; Viticulture; Structural variation; Snps|
|MIUR : ||Settore AGR/07 - Genetica Agraria|
|Corso di dottorato: ||Dottorato di ricerca in Scienze e biotecnologie agrarie|
|Ciclo di dottorato: ||25|
|Università di conseguimento titolo: ||Università degli Studi di Udine|
|Luogo di discussione: ||Udine|
|Altre informazioni: ||Struttura di aggregazione: Istituto di Genomica Applicata - Co-supervisor: Gabriele Di Gaspero|
|Citazione: ||Miculan, M. Detection of somatic variants from next-generation sequencing data in grapevine bud sports. (Doctoral Thesis, Università degli Studi di Udine, 2013).|
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