Innovative Solutions

InnoVine: Combining innovation in vineyard management and genetics for a sustainable European Viticulture

InnoVine is investigating innovative systems of production for the European wine industry. Innovation in viticulture addresses consumers’ demands for quality wines and beverage safety, citizens’ requests for eco-friendly production and winegrowers’ quest for vineyard management practices that mitigate the impact of climate change. The activities include:

• Modelling of berry composition in response to imposed stresses
• Designing optimized vineyard practices to reduce the use of pesticides
• Screening of grape germplasm for accessions with resistance to biotic and abiotic stresses, in particular pathogenic fungi, drought and heat.
• Development of portable devices for proximal sensing of vine status and berry traits
• Implementation of decision support systems (DSS) for sustainable pest management, irrigation, and management of the vine canopy

The InnoVine consortium includes 27 partners from 7 European countries. Half of the partnership is public, mainly research institutions, and half is composed of private organizations and companies.

Thanks to InnoVine, IGA has investigated:

- the durability of fungal resistances historically used by grape breeders

- new sources of mildew resistance in wild and cultivated grapevines from the Caucasus

- the genome of resistant accessions by DNA genotyping and sequencing

Funded by EU FP7 Knowledge Based Bio-Economy (KBBE) program

Status: InnoVine, completed

GenTree: Optimising the management and sustainable use of forest genetic resources in Europe

IGA's commitment to innovative solutions in agriculture extends to forestry.

The goal of GenTree is to provide the European forestry sector with better knowledge, methods and tools for optimising the management and sustainable use of forest genetic resources in the context of climate change and in response to evolving demands for forest products and services. To this end, GenTree will

• design innovative strategies for dynamic conservation of genetic resources in European forests
• broaden the range of genetic resources available to European breeding programmes
• define new forest management scenarios and policy frameworks to adapt forests and forestry to changing environmental conditions and societal demands

GenTree focuses on economically and ecologically important tree species in Europe, growing in a wide range of habitats and covering different societal uses and values. The expected output will include: new knowledge on phenotypic and genotypic diversity across environmental gradients in Europe, data collection and curation for information systems of in-situ and ex-situ genetic collections; innovative strategies for conservation, breeding and exchange of forest reproductive material.

With its subsidiary IGA Technology Services, IGA has genotyped 576 accessions of Betula pendula, 720 of Fagus sylvatica, 624 of Picea abies, 624 of Pinus pinaster, 624 of Pinus sylvestris, 574 of Populus nigra and 572 of Quercus petraea using the in-house developed SPET targeted sequencing.

Funded by EU Horizon 2020 - SOCIETAL CHALLENGES - Food security, sustainable agriculture and forestry, marine, maritime and inland water research, and the bioeconomy

Status: GenTree current project

Identification of rare genetic variants

Next-generation sequencing is used for the identification of rare allelic variants in pooled DNAs.

IGA has borrowed this approach from cancer research and optimized the procedure for the quest for rare variants in plant populations. DNA targets are amplified from several individuals, and DNA barcoding retains the individual information in pooled sequencing. The following article guides you for designing your own experiment through all steps: pooling strategy, target enrichment, multiplexing, sequencing depth and appropriate bioinformatics.

Marroni et al (2012) The quest for rare variants: pooled multiplexed next generation sequencing in plants. Frontiers in Plant Science doi: 10.3389/fpls.2012.00133. eCollection 2012.

Variants that control complex traits are used in plant breeding for crop improvement. An illustrative example comes from black poplar, an important feedstock for the production of biofuels. Allelic variants of lignin biosynthetic genes in natural populations of poplar revealed trees with altered lignin composition, thus removing a major limiting factor in the conversion of plant biomass to biofuels.

Marroni et al (2011) Large-scale detection of rare variants via pooled multiplexed next-generation sequencing: towards next-generation Ecotilling. Plant Journal 67:736-745

Pooled NGS is also applied to study demography and selection in natural populations of Norway spruce. IGA has compared the European populations of Norway spruce that evolved northerly in the Fennoscandian region and southerly in the Alpine mountain range. We investigated DNA sequence variation in 88 genes associated with phenology and photoperiodism.

Chen et al (2016) Identifying genetic signatures of natural selection using pooled population sequencing in Picea abies. G3: Genes, Genomes, Genetics (Bethesda) doi:10.1534/g3.116.028753

Breeding with rare defective alleles (BRDA)

IGA has used NGS to detect mutations in five genes for lignin biosynthesis in 768 trees of black poplar, sampled in forests from across Western Europe in France, Italy, Germany, Spain, and The Netherlands. A total of 36 non-synonymous single nucleotide polymorphisms were identified. The rarest validated variant was estimated to occur only once in 1536 chromosomes.

One mutation causes a premature stop codon in the gene encoding hydroxycinnamoyl-CoA : shikimate hydroxycinnamoyl transferase1 (HCT1), an essential enzyme in lignin biosynthesis. The mutant allele encodes a truncated protein. One naturally occurring poplar tree is homozygous for this recessive allele and its lignin composition is 17-fold enriched in p-hydroxyphenyl units, compared to trees that encode the full-length HCT1 enzyme.

This proof-of-concept in poplar led us to propose a breeding strategy, called 'breeding with rare defective alleles' (BRDA), as a tool for conventional breeding based on the capture of rare defective alleles from wild germplasm. Naturally occurring variants are identified in wild populations and their rare useful alleles are introduced into the breeding germplasm.

Vanholme et al (2013) Breeding with rare defective alleles (BRDA): a natural Populus nigra HCT mutant with modified lignin as a case study. New Phytologist 198:765-776

MAGIC maize populations for corn improvement

Dissecting the architecture of complex traits into Mendelian loci and identifying the causal genes are still limiting steps on the path of crop improvement. Linkage mapping in biparental populations or association mapping on diversity panels restrict our ability to discover specific genetic determinants in a single mapping study. The availability of populations with high genetic diversity and recombination density overcomes the limitation of either approach. Multi-parent cross designs (MpCD) bridge the advantages of either approaches and dramatically increase mapping resolution and power by incorporating greater genetic diversity and by increasing the number of crossing generations.

We have produced the first balanced multi-parental population in maize, a tool that provides high diversity and dense recombination events to improve our understanding of the genetic basis of quantitative traits and guide predictive crop breeding.

A total of 1,636 MAGIC maize recombinant inbred lines were derived from eight genetically diverse founder lines. Their genomes are evenly differentiated mosaics of the eight founders, with mapping power and resolution strengthened by high minor allele frequencies and a fast decay of linkage disequilibrium. We demonstrate MAGIC maize's value in identifying the genetic bases of complex traits of agronomic relevance by illustrating the case-studies for grain yield and flowering time.

Dell'Acqua et al (2015) Genetic properties of the MAGIC maize population: a new platform for high definition QTL mapping in Zea mays. Genome Biology 16:167

Holland et al (2015) MAGIC maize: a new resource for plant genetics. Genome Biology 16:163

Genotyping-by-sequencing

IGA is using different methods of genotyping-by-sequencing (GBS) for the construction of high density genetic maps and genetic diversity analysis. This unprecedented marker saturation opens the door to new applications in plant breeding, genome analysis and trait dissection.

These procedures can be applied to any plant species, even in the absence of a reference genome. IGA-subsidiary IGATech uses this know-how to offer a service of GBS and genetic mapping to breeding companies and plant scientists. See the application of this technology to two case studies: kiwifruit and the huge 16 gigabase genome of Cedrus atlantica.

Scaglione et al (2015) A RAD-based linkage map of kiwifruit (Actinidia chinensis Pl.) as a tool to improve the genome assembly and to scan the genomic region of the gender determinant for the marker-assisted breeding. Tree Genetics & Genomes 11: 115

Karam et al (2015) Genomic exploration and molecular marker development in a large and complex conifer genome using RADseq and mRNAseq. Molecular Ecology Resources 15:601-612

Genomics information and bioinformatics tools developed in the frame of research projects are made publicly available by IGA.

You can explore sequenced genomes using blast search pages and genome browsers for grapevine, peach and poplar. IGA genome browsers contain basic tracks available from collaborative information systems and annotation tracks that have been computed at IGA.

Bioinformatic tools produced by IGA are "open source". Have a look at available software tools.

ERNE

ERNE (Extended Randomized Numerical alignEr) is a short string alignment package whose goal is to provide an all-inclusive set of tools to handle short (NGS-like) reads. ERNE comprises ERNE-MAP (align short DNA or RNA reads against a genome), ERNE-DMAP (distributed version of the aligner), ERNE- BS5 (align short reads treated with bisulfite against a genome), ERNE-FILTER (a quality trimming and contamination filter for de novo assembly that can also be used for alignments), and ERNE-VISUAL (graphical user interface).

ERNE is an open source project. The source code and executables for GNU/Linux, Windows, and OSX are available on SourceForge.

COREFINDER

COREFINDER is a software for sorting out a core collection from a large database of individual molecular profiles. Read the tutorial or download the software

SmartFinder

SMaRTFinder is a command line tool to search biosequences for structured motifs (or structured models); that is lists of patterns separated by distance intervals. It reads a sequence in FASTA format and a structured model specification, either from a file or from the standard input, then searches for exact/approximate occurrences of the structured model (in one or both strands, if the sequence is DNA) and outputs a file in GFF format.

LadderFinder

LadderFinder is a program designed to solve a problem in DNA genotyping. It helps in the construction of allelic ladders that drive the sizing and binning of SSR fragments produced by PCR. The algorithm scans a spreadsheet containing a database of individual profiles and finds a minimum set of individuals that covers the maximum number of alleles represented in the database, with all alleles represented only once. The last constraint is imposed to make homogeneous the ladder peaks in the pherograms. Given such a constraint, the best solution may not include all alleles.

matchfinder

Matchfinder computes a maximal matching of a graph. A matching of a graph G is a set of pairwise non-adjacent edges of G. A maximal matching for G is a matching of maximum cardinality with respect to G. Notice that there can be many different maximal matchings for a single graph. For instance, the graph G=( V={1, 2, 3}, E={(1,2), (2,3)} has two maximal matchings: M={(1,2)} and M={(2,3)}.

Genome Assemblies Merger

GAM is a free parallel software tool to integrate two different assemblies and improve the overall quality of the genome sequences by merging them. It discovers and aligns similar sequences by using the positions of placed reads. An ordered sequence of reads contiguous on the same contig is called a frame. When the same frame is shared by both original assemblies, GAM dubs it block.

GAM deduces block orders from assembly read orders and, by using them, builds a graph of assemblies whose nodes represent blocks and whose edges represents block orders. Since cycles in the graph of assemblies denote conflicting block order in assemblies, GAM avoids merging sequences containing blocks belonging to them.

χ-SCAN

χ-SCAN is a software package to identify mosaic (and germline) structural variants (SVs) using whole-genome resequencing data. χ-scan computes deviations from the expected allele frequency in a population of cells, using counts of aligned reads covering heterozygous SNP sites, to detect signals of reduction of heterozygosity (ROH), which can be caused (among others) by events of deletion, CNV, or chromosomal replacement. χ- SCAN offers a number of statistical tests to assess the significance of the detected ROHs.

Marroni et al (2017) Reduction of heterozygosity (ROH) as a method to detect mosaic structural variation. Plant Biotechnology Journal doi: 10.1111/pbi.12691

In the frame of the Rural Development Programme 2014-2020 of the Region Friuli Venezia Giulia (FVG), IGA has been funded for gathering research institutions and grape nurseries into a consortium.

This consortium is aimed at planning, drafting and submitting a joint research project to a forthcoming call of the FVG Rural Development Programme 2014-2020, Sub-measure 16.1 Support for European Innovation Partnerships (EIP), in order to address the following needs of local nurseries:

• improve disease control measures to prevent the spread of flavescence doree phytoplasma, grapevine fanleaf virus, and grapevine pinot gris virus
• implement laboratory protocols for the detection and identification these causal agents in grape plant material

More information in Italian language is provided here in compliance with local laws.

Funded by the European Agricultural Fund for Rural Development (EAFRD) and the Regional Administration of Friuli Venezia Giulia

Status: current project