Fabio Marroni

The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution

Rosaceae is the most important fruit-producing clade, and its key commercially relevant genera (Fragaria, Rosa, Rubus and Prunus) show broadly diverse growth habits, fruit types and compact diploid genomes. Peach, a diploid Prunus species, is one of the best genetically characterized deciduous trees. Here we describe the high-quality genome sequence of peach obtained from a completely homozygous genotype. We obtained a complete chromosome-scale assembly using Sanger whole-genome shotgun methods. We predicted 27,852 protein-coding genes, as well as noncoding RNAs. We investigated the path of peach domestication through whole-genome resequencing of 14 Prunus accessions. The analyses suggest major genetic bottlenecks that have substantially shaped peach genome diversity. Furthermore, comparative analyses showed that peach has not undergone recent whole-genome duplication, and even though the ancestral triplicated blocks in peach are fragmentary compared to those in grape, all seven paleosets of paralogs from the putative paleoancestor are detectable.

Breeding with rare defective alleles (BRDA): a natural Populus nigra HCT mutant with modified lignin as a case study

Next-generation (NG) sequencing in a natural population of Populus nigra revealed a mutant with a premature stop codon in the gene encoding hydroxycinnamoyl-CoA : shikimate hydroxycinnamoyl transferase1 (HCT1), an essential enzyme in lignin biosynthesis. The lignin composition of P. nigra trees homozygous for the defective allele was compared with that of heterozygous trees and trees without the defective allele. The lignin was characterized by phenolic profiling, lignin oligomer sequencing, thioacidolysis and NMR. In addition, HCT1 was heterologously expressed for activity assays and crosses were made to introduce the mutation in different genetic backgrounds. HCT1 converts p-coumaroyl-CoA into p-coumaroyl shikimate. The mutant allele, PnHCT1-Δ73, encodes a truncated protein, and trees homozygous for this recessive allele have a modified lignin composition characterized by a 17-fold increase in p-hydroxyphenyl units. Using the lignin pathway as proof of concept, we illustrated that the capture of rare defective alleles is a straightforward approach to initiate reverse genetics and accelerate tree breeding. The proposed breeding strategy, called breeding with rare defective alleles (BRDA), should be widely applicable, independent of the target gene or the species.

Large-scale detection of rare variants via pooled multiplexed next-generation sequencing: towards next-generation Ecotilling

Common variants, such as those identified by genome-wide association scans, explain only a small proportion of trait variation. Growing evidence suggests that rare functional variants, which are usually missed by genome-wide association scans, play an important role in determining the phenotype. We used pooled multiplexed next-generation sequencing and a customized analysis workflow to detect mutations in five candidate genes for lignin biosynthesis in 768 pooled Populus nigra accessions. We identified a total of 36 non-synonymous single nucleotide polymorphisms, one of which causes a premature stop codon. The most common variant was estimated to be present in 672 of the 1536 tested chromosomes, while the rarest was estimated to occur only once in 1536 chromosomes. Comparison with individual Sanger sequencing in a selected sub-sample confirmed that variants are identified with high sensitivity and specificity, and that the variant frequency was estimated accurately. This proposed method for identification of rare polymorphisms allows accurate detection of variation in many individuals, and is cost-effective compared to individual sequencing.

Structural variation and genome complexity: is dispensable really dispensable?

Structural variants (SVs) such as copy number variants (CNVs) and presence/absence variants (PAVs) substantially contribute to genetic variation and have an important effect on phenotypic diversity. Since unbalanced SVs are by definition sequences present only in some individuals, they have therefore been referred to as dispensable genome and are not necessary for survival, even though they may provide an important contribution to phenotypic diversity within the species. However, some multi-copy sequences of the dispensable genomes (e.g., multigene families) may be needed in a given proportion by each individual, thus belonging to a conditionally dispensable portion of the pan-genome. Another interesting aspect reported by recent studies is that the rate at which SVs are formed might be influenced by the mating system and by common environmental stresses. In conclusion the dispensable genome plays an important role in genome evolution and in the complex interplay between the genome and the environment.

Nucleotide diversity and linkage disequilibrium in Populus nigra cinnamyl alcohol dehydrogenase (CAD4) gene

Cinnamyl alcohol dehydrogenase (CAD) is involved in the biosynthesis of lignin, a component of plant cell wall which negatively impacts paper pulp processing and biomass fermentation to ethanol. Transgenic poplars with depressed CAD activity show structural alterations of lignin. Natural CAD mutants have been identified in several plants; however, no natural CAD mutants have been identified in poplar. We surveyed the natural genetic variation in CAD4, a gene coding for CAD, in 360 poplar trees from Western Europe. We measured linkage disequilibrium (LD) between single-nucleotide polymorphisms (SNPs), performed neutrality tests and estimated diversity indexes, and investigated their dependence from sample size. We identified 45 SNPs, six of which caused an amino acid substitution. Our results suggest a short span of LD in Populus nigra CAD4 gene. We identified carriers of different nonsynonymous SNPs in CAD4; those subjects are candidate to be used in classical breeding programs to obtain carriers of different combinations of functional polymorphisms. We showed that use of small sample size might lead to biased estimates of LD, neutrality tests, and diversity indexes.

Characterization of the Poplar Pan-Genome by Genome-Wide Identification of Structural Variation

Many recent studies have emphasized the important role of structural variation (SV) in determining human genetic and phenotypic variation. In plants, studies aimed at elucidating the extent of SV are still in their infancy. Evidence has indicated a high presence and an active role of SV in driving plant genome evolution in different plant species. With the aim of characterizing the size and the composition of the poplar pan-genome, we performed a genome-wide analysis of structural variation in three intercrossable poplar species: Populus nigra, Populus deltoides, and Populus trichocarpa. We detected a total of 7,889 deletions and 10,586 insertions relative to the P. trichocarpa reference genome, covering respectively 33.2u2009Mb and 62.9u2009Mb of genomic sequence, and 3,230 genes affected by copy number variation (CNV). The majority of the detected variants are inter-specific in agreement with a recent origin following separation of species. Insertions and deletions (INDELs) were preferentially located in low-gene density regions of the poplar genome and were, for the majority, associated with the activity of transposable elements. Genes affected by SV showed lower-than-average expression levels and higher levels of dN/dS, suggesting that they are subject to relaxed selective pressure or correspond to pseudogenes. Functional annotation of genes affected by INDELs showed over-representation of categories associated with transposable elements activity, while genes affected by genic CNVs showed enrichment in categories related to resistance to stress and pathogens. This study provides a genome-wide catalogue of SV and the first insight on functional and structural properties of the poplar pan-genome.

The Quest for Rare Variants: Pooled Multiplexed Next Generation Sequencing in Plants

Next generation sequencing (NGS) instruments produce an unprecedented amount of sequence data at contained costs. This gives researchers the possibility of designing studies with adequate power to identify rare variants at a fraction of the economic and labor resources required by individual Sanger sequencing. As of today, few research groups working in plant sciences have exploited this potentiality, showing that pooled NGS provides results in excellent agreement with those obtained by individual Sanger sequencing. The aim of this review is to convey to the reader the general ideas underlying the use of pooled NGS for the identification of rare variants. To facilitate a thorough understanding of the possibilities of the method, we will explain in detail the possible experimental and analytical approaches and discuss their advantages and disadvantages. We will show that information on allele frequency obtained by pooled NGS can be used to accurately compute basic population genetics indexes such as allele frequency, nucleotide diversity, and Tajima’s D. Finally, we will discuss applications and future perspectives of the multiplexed NGS approach.

Genome-wide analysis of LTR-retrotransposon expression in leaves of Populus × canadensis water-deprived plants

Retrotransposons represent a major component of plant genomes; however, large-scale studies on their expression are rare. Massively parallel sequencing offers new analytical possibilities enabling a comprehensive study of retrotransposon RNA transcription. We evaluated the expression of long terminal repeat-retrotransposons in leaves of two sister hybrids Populus × canadensis (P. deltoides × P. nigra), subjected to moderate or severe water deprivation by mapping Illumina RNA-Seq reads onto a set of 958 unique full-length retrotransposons of P. trichocarpa. Detectable levels of transcription were ascertained for 140 retrotransposons in 1 hybrid and 182 in the other. The two hybrids showed different retrotransposon expression levels, and these differences reduced at increasing drought levels. The number of expressed Gypsy elements in control and water-deprived plants was higher than those of Copia, as were their expression levels. The two hybrids showed different retrotransposon expression patterns following water deprivation. Such variations between hybrids were related to differential expression of a few genes involved in chromatin methylation and remodeling. Overall, our data indicate that even in genetically close individuals, large differences can occur in retrotransposon expression, with possible consequences for genome differentiation.

Merino and Merino-derived sheep breeds: a genome-wide intercontinental study

BackgroundMerino and Merino-derived sheep breeds have been widely distributed across the world, both as purebred and admixed populations. They represent an economically and historically important genetic resource which over time has been used as the basis for the development of new breeds. In order to examine the genetic influence of Merino in the context of a global collection of domestic sheep breeds, we analyzed genotype data that were obtained with the OvineSNP50 BeadChip (Illumina) for 671 individuals from 37 populations, including a subset of breeds from the Sheep HapMap dataset.ResultsBased on a multi-dimensional scaling analysis, we highlighted four main clusters in this dataset, which corresponded to wild sheep, mouflon, primitive North European breeds and modern sheep (including Merino), respectively. The neighbor-network analysis further differentiated North-European and Mediterranean domestic breeds, with subclusters of Merino and Merino-derived breeds, other Spanish breeds and other Italian breeds. Model-based clustering, migration analysis and haplotype sharing indicated that genetic exchange occurred between archaic populations and also that a more recent Merino-mediated gene flow to several Merino-derived populations around the world took place. The close relationship between Spanish Merino and other Spanish breeds was consistent with an Iberian origin for the Merino breed, with possible earlier contributions from other Mediterranean stocks. The Merino populations from Australia, New Zealand and China were clearly separated from their European ancestors. We observed a genetic substructuring in the Spanish Merino population, which reflects recent herd management practices.ConclusionsOur data suggest that intensive gene flow, founder effects and geographic isolation are the main factors that determined the genetic makeup of current Merino and Merino-derived breeds. To explain how the current Merino and Merino-derived breeds were obtained, we propose a scenario that includes several consecutive migrations of sheep populations that may serve as working hypotheses for subsequent studies.

Reduction of heterozygosity (ROH) as a method to detect mosaic structural variation

Dipartimento di Scienze agroalimentari, ambientali e animali, Universit a di Udine, Udine, Italy Istituto di Genomica Applicata (IGA), Udine, Italy IGA Technology Services, Udine, Italy Parco Tecnologico Padano, Lodi, Italy Institute of Biosciences and Bioresources, National Research Council, Sesto Fiorentino (Firenze), Italy Department of Mathematics and Computer Science, University of Udine, Udine, Italy