Detection of somatic variants from next-generation sequencing data in grapevine bud sports

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 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.Struttura di aggregazione: Istituto di Genomica Applicata - Co-supervisor: Gabriele Di GasperoopenDottorato di ricerca in Scienze e biotecnologie agrarieembargoed_20141009Miculan, Mar

Genome wide association study of agronomic and seed traits in a world collection of proso millet (Panicum miliaceum L.)

The climate crisis threatens sustainability of crop production worldwide. Crop diversification may enhance food security while reducing the negative impacts of climate change. Proso millet (Panicum milaceum L.) is a minor cereal crop which holds potential for diversification and adaptation to different environmental conditions. In this study, we assembled a world collection of proso millet consisting of 88 varieties and landraces to investigate its genomic and phenotypic diversity for seed traits, and to identify marker-trait associations (MTA)

Transcriptome and metabolite profiling reveals that prolonged drought modulates the phenylpropanoid and terpenoid pathway in white grapes (Vitis vinifera L.)

Background: Secondary metabolism contributes to the adaptation of a plant to its environment. In wine grapes, fruit secondary metabolism largely determines wine quality. Climate change is predicted to exacerbate drought events in several viticultural areas, potentially affecting the wine quality. In red grapes, water deficit modulates flavonoid accumulation, leading to major quantitative and compositional changes in the profile of the anthocyanin pigments; in white grapes, the effect of water deficit on secondary metabolism is still largely unknown Results: In this study we investigated the impact of water deficit on the secondary metabolism of white grapes using a large scale metabolite and transcript profiling approach in a season characterized by prolonged drought. Irrigated grapevines were compared to non-irrigated grapevines that suffered from water deficit from early stages of berry development to harvest. A large effect of water deficit on fruit secondary metabolism was observed. Increased concentrations of phenylpropanoids, monoterpenes, and tocopherols were detected, while carotenoid and flavonoid accumulations were differentially modulated by water deficit according to the berry developmental stage. The RNA- sequencing analysis carried out on berries collected at three developmental stages—before, at the onset, and at late ripening—indicated that water deficit affected the expression of 4,889 genes. The Gene Ontology category secondary metabolic process was overrepresented within up-regulated genes at all the stages of fruit development considered, and within down-regulated genes before ripening. Eighteen phenylpropanoid, 16 flavonoid, 9 carotenoid, and 16 terpenoid structural genes were modulated by water deficit, indicating the transcriptional regulation of these metabolic pathways in fruit exposed to water deficit. An integrated network and promoter analyses identified a transcriptional regulatory module that encompasses terpenoid genes, transcription factors, and enriched drought- responsive elements in the promoter regions of those genes as part of the grapes response to drought Conclusion: Our study reveals that grapevine berries respond to drought by modulating several secondary metabolic pathways, and particularly, by stimulating the production of phenylpropanoids, the carotenoid zeaxanthin, and of volatile organic compounds such as monoterpenes, with potential effects on grape and wine antioxidant potential, composition, and sensory feature

Data-driven, participatory characterization of farmer varieties discloses teff breeding potential under current and future climates

In smallholder farming systems, traditional farmer varieties of neglected and underutilized species (NUS) support the livelihoods of millions of growers and consumers. NUS combine cultural and agronomic value with local adaptation, and transdisciplinary methods are needed to fully evaluate their breeding potential. Here, we assembled and characterized the genetic diversity of a representative collection of 366 Ethiopian teff (Eragrostis tef) farmer varieties and breeding materials, describing their phylogenetic relations and local adaptation on the Ethiopian landscape. We phenotyped the collection for its agronomic performance, involving local teff farmers in a participatory variety evaluation. Our analyses revealed environmental patterns of teff genetic diversity and allowed us to identify 10 genetic clusters associated with climate variation and with uneven spatial distribution. A genome-wide association study was used to identify loci and candidate genes related to phenology, yield, local adaptation, and farmers' appreciation. The estimated teff genomic offset under climate change scenarios highlighted an area around lake Tana where teff cropping may be most vulnerable to climate change. Our results show that transdisciplinary approaches may efficiently propel untapped NUS farmer varieties into modern breeding to foster more resilient and sustainable cropping systems

Fine mapping and identification of a candidate gene for a major locus controlling maturity date in peach

BACKGROUND: Maturity date (MD) is a crucial factor for marketing of fresh fruit, especially those with limited shelf-life such as peach (Prunus persica L. Batsch): selection of several cultivars with differing MD would be advantageous to cover and extend the marketing season. Aims of this work were the fine mapping and identification of candidate genes for the major maturity date locus previously identified on peach linkage group 4. To improve genetic resolution of the target locus two F(2) populations derived from the crosses Contender x Ambra (CxA, 306 individuals) and PI91459 (NJ Weeping) x Bounty (WxBy, 103 individuals) were genotyped with the Sequenom and 9K Illumina Peach Chip SNP platforms, respectively. RESULTS: Recombinant individuals from the WxBy F(2) population allowed the localisation of maturity date locus to a 220 kb region of the peach genome. Among the 25 annotated genes within this interval, functional classification identified ppa007577m and ppa008301m as the most likely candidates, both encoding transcription factors of the NAC (NAM/ATAF1, 2/CUC2) family. Re-sequencing of the four parents and comparison with the reference genome sequence uncovered a deletion of 232 bp in the upstream region of ppa007577m that is homozygous in NJ Weeping and heterozygous in Ambra, Bounty and the WxBy F(1) parent. However, this variation did not segregate in the CxA F(2) population being the CxA F(1) parent homozygous for the reference allele. The second gene was thus examined as a candidate for maturity date. Re-sequencing of ppa008301m, showed an in-frame insertion of 9 bp in the last exon that co-segregated with the maturity date locus in both CxA and WxBy F(2) populations. CONCLUSIONS: Using two different segregating populations, the map position of the maturity date locus was refined from 3.56 Mb to 220 kb. A sequence variant in the NAC gene ppa008301m was shown to co-segregate with the maturity date locus, suggesting this gene as a candidate controlling ripening time in peach. If confirmed on other genetic materials, this variant may be used for marker-assisted breeding of new cultivars with differing maturity date

Data-driven, participatory characterization of farmer varieties discloses teff breeding potential under current and future climates

In smallholder farming systems, traditional farmer varieties of neglected and underutilized species (NUS) support the livelihoods of millions of growers and consumers. NUS combine cultural and agronomic value with local adaptation, and transdisciplinary methods are needed to fully evaluate their breeding potential. Here, we assembled and characterized the genetic diversity of a representative collection of 366 Ethiopian teff (Eragrostis tef) farmer varieties and breeding materials, describing their phylogenetic relations and local adaptation on the Ethiopian landscape. We phenotyped the collection for its agronomic performance, involving local teff farmers in a participatory variety evaluation. Our analyses revealed environmental patterns of teff genetic diversity and allowed us to identify 10 genetic clusters associated with climate variation and with uneven spatial distribution. A genome-wide association study was used to identify loci and candidate genes related to phenology, yield, local adaptation, and farmers’ appreciation. The estimated teff genomic offset under climate change scenarios highlighted an area around lake Tana where teff cropping may be most vulnerable to climate change. Our results show that transdisciplinary approaches may efficiently propel untapped NUS farmer varieties into modern breeding to foster more resilient and sustainable cropping systems

Molecular Diversity and Landscape Genomics of the Crop Wild Relative Triticum urartu Across the Fertile Crescent

Modern plant breeding can benefit from the allelic variation existing in natural populations of crop wild relatives that evolved under natural selection in varying pedoclimatic conditions. In this study, next-generation sequencing was used to generate 1.3 million genome-wide SNPs on ex situ collections of Triticum urartu L., the wild donor of the Ausub-genome of modern wheat. A set of 75,511 high quality SNPs were retained to describe 298 T. urartu accessions collected throughout the Fertile Crescent. Triticum urartu showed a complex pattern of genetic diversity, with two main genetic groups distributed sequentially from West to East. The incorporation of geographic information of sampling points showed that genetic diversity did correlate to geographic distance (R2= 0.19), separating samples from Jordan and Lebanon, to samples from Syria and Southern Turkey, to samples from Eastern Turkey, Iran, and Iraq. The wild emmer genome was used to derive SNPs physical positions on the 7 chromosomes of the Ausub-genome, allowing to describe a relatively slow linkage disequilibrium decay in the collection. Outlier loci were described on the basis of geographical distribution of the T. urartu accessions, identifying a hotspot of directional selection on chromosome 4A. Bioclimatic variation was derived from grid data and put in relation to allelic variation with a genome-wide association approach, identifying several marker-environment associations (MEAs). Fifty-seven MEAs were associated with altitude and temperature measures, while 358 were associated with rainfall measures. The most significant MEAs and outlier loci were used to identify genomic loci with adaptive potential, some already reported in wheat, including dormancy and frost resistance loci. We advocate the application of genomics and landscape genomics on ex situ collections of crop wild relatives to efficiently identify promising alleles and genetic materials to be incorporated in modern crop breeding. This article is protected by copyright. All rights reserved