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BACKGROUND AND AIMS:The advent of molecular breeding is advocated to improve the productivity and sustainability of second-generation bioenergy crops. Advanced molecular breeding in bioenergy crops relies on the ability to massively sample the genetic diversity. Genotyping-by-sequencing has become a widely adopted method for cost-effective genotyping. It basically requires no initial investment for design as compared with array-based platforms which have been shown to offer very robust assays. The latter, however, has the drawback of being limited to analyse only the genetic diversity accounted during selection of a set of polymorphisms and design of the assay. In contrast, genotyping-by-sequencing with random sampling of genomic loci via restriction enzymes or random priming has been shown to be fast and convenient but lacks the ability to target specific regions of the genome and to maintain high reproducibility across laboratories. METHODS:Here we present a first adoption of single-primer enrichment technology (SPET) which provides a highly efficient and scalable system to obtain targeted sequence-based large genotyping data sets, bridging the gaps between array-based systems and traditional sequencing-based protocols. To fully explore SPET performance, we conducted a benchmark study in ten Zea mays lines and a large-scale study of a natural black poplar population of 540 individuals with the aim of discovering polymorphisms associated with biomass-related traits. KEY RESULTS:Our results showed the ability of this technology to provide dense genotype information on a customized panel of selected polymorphisms, while yielding hundreds of thousands of untargeted variable sites. This provided an ideal resource for association analysis of natural populations harbouring unexplored allelic diversities and structure such as in black poplar. CONCLUSION:The improvement of sequencing throughput and the development of efficient library preparation methods has made it feasible to carry out targeted genotyping-by-sequencing experiments cost-competitively with either random complexity reduction systems or traditional array-based platforms, while maintaining the key advantages of both technologies.

Wood is a renewable resource that can be employed for the production of second generation biofuels by enzymatic saccharification and subsequent fermentation. Knowledge on how the saccharification potential is affected by genotype-related variation of wood traits and drought is scarce. Here, we used three Populus nigra L. genotypes from habitats differing in water availability to (i) investigate the relationships between wood anatomy, lignin content and saccharification and (ii) identify genes and co-expressed gene clusters related to genotype and drought-induced variation in wood traits and saccharification potential. The three poplar genotypes differed in wood anatomy, lignin content and saccharification potential. Drought resulted in reduced cambial activity, decreased vessel and fiber lumina, and increased the saccharification potential. The saccharification potential was unrelated to lignin content as well as to most wood anatomical traits. RNA sequencing of the developing xylem revealed that 1.5% of the analyzed genes were differentially expressed in response to drought, while 67% differed among the genotypes. Weighted gene correlation network analysis identified modules of co-expressed genes correlated with saccharification potential. These modules were enriched in gene ontology terms related to cell wall polysaccharide biosynthesis and modification and vesicle transport, but not to lignin biosynthesis. Among the most strongly saccharification-correlated genes, those with regulatory functions, especially kinases, were prominent. We further identified transcription factors whose transcript abundances differed among genotypes, and which were co-regulated with genes for biosynthesis and modifications of hemicelluloses and pectin. Overall, our study suggests that the regulation of pectin and hemicellulose metabolism is a promising target for improving wood quality of second generation bioenergy crops. The causal relationship of the identified genes and pathways with saccharification potential needs to be validated in further experiments.

BackgroundSecond generation (2G) bioenergy from lignocellulosic feedstocks has the potential to develop as a sustainable source of renewable energy; however, significant hurdles still remain for large-scale commercialisation. Populus is considered as a promising 2G feedstock and understanding the genetic basis of biomass yield and feedstock quality are a research priority in this model tree species.ResultsWe report the first coppiced biomass study for 714 members of a wide population of European black poplar (Populus nigra L.), a native European tree, selected from 20 river populations ranging in latitude and longitude between 40.5 and 52.1°N and 1.0 and 16.4°E, respectively. When grown at a single site in southern UK, significant Site of Origin (SO) effects were seen for 14 of the 15 directly measured or derived traits including biomass yield, leaf area and stomatal index. There was significant correlation (p

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 33.2 Mb and 62.9 Mb 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.

Consensus linkage maps are important tools in crop genomics. We have assembled a high-density tetraploid wheat consensus map by integrating 13 data sets from independent biparental populations involving durum wheat cultivars (Triticum turgidum ssp. durum), cultivated emmer (T. turgidum ssp. dicoccum) and their ancestor (wild emmer, T. turgidum ssp. dicoccoides). The consensus map harboured 30 144 markers (including 26 626 SNPs and 791 SSRs) half of which were present in at least two component maps. The final map spanned 2631 cM of all 14 durum wheat chromosomes and, differently from the individual component maps, all markers fell within the 14 linkage groups. Marker density per genetic distance unit peaked at centromeric regions, likely due to a combination of low recombination rate in the centromeric regions and even gene distribution along the chromosomes. Comparisons with bread wheat indicated fewer regions with recombination suppression, making this consensus map valuable for mapping in the A and B genomes of both durum and bread wheat. Sequence similarity analysis allowed us to relate mapped gene-derived SNPs to chromosome-specific transcripts. Dense patterns of homeologous relationships have been established between the A- and B-genome maps and between nonsyntenic homeologous chromosome regions as well, the latter tracing to ancient translocation events. The gene-based homeologous relationships are valuable to infer the map location of homeologs of target loci/QTLs. Because most SNP and SSR markers were previously mapped in bread wheat, this consensus map will facilitate a more effective integration and exploitation of genes and QTL for wheat breeding purposes.

High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker-trait associations in mapping experiments. We developed a genotyping array including about 90,000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence-absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.

High-density single nucleotide polymorphism (SNP) genotyping arrays are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships between individuals in populations and studying marker-trait associations in mapping experiments. We developed a genotyping array including about 90,000 gene-associated SNPs and used it to characterize genetic variation in allohexaploid and allotetraploid wheat populations. The array includes a significant fraction of common genome-wide distributed SNPs that are represented in populations of diverse geographical origin. We used density-based spatial clustering algorithms to enable high-throughput genotype calling in complex data sets obtained for polyploid wheat. We show that these model-free clustering algorithms provide accurate genotype calling in the presence of multiple clusters including clusters with low signal intensity resulting from significant sequence divergence at the target SNP site or gene deletions. Assays that detect low-intensity clusters can provide insight into the distribution of presence-absence variation (PAV) in wheat populations. A total of 46 977 SNPs from the wheat 90K array were genetically mapped using a combination of eight mapping populations. The developed array and cluster identification algorithms provide an opportunity to infer detailed haplotype structure in polyploid wheat and will serve as an invaluable resource for diversity studies and investigating the genetic basis of trait variation in wheat.

Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes--a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes--and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement.

Although Citrus is the most globally significant tree fruit, its domestication history is poorlyunderstood. Cultivated citrus types are believed to comprise selections from and/or hybrids of several wild progenitor species, but the identities of these progenitors, and their contribution to modern cultivars, remain controversial. Here we report the genomes of a collection of mandarins, pummelos, and oranges, including a high quality reference sequence from a haploid Clementine mandarin. By comparative genome analysis we show that these cultivated types can be derived from two progenitor species. Cultivated pummelos represent selections from a single progenitor species C. maxima. Unexpectedly, however, we find that cultivated mandarins are introgressions of C. maxima into a distinct second population that we identify with the ancestral wild mandarin species C. reticulata. Sweet and sour oranges are found to be interspecific hybrids. Sweet orange, the most widely cultivated citrus, arose as the offspring of previously admixed individuals. In contrast, sour (or Seville) orange is an F1 hybrid of pure C. maxima and C. reticulata parents, implying that wild mandarins were part of the early breeding germplasm.Surprisingly, we also find that a wild Chinese mandarin from Mangshan, China showssubstantial sequence divergence from C. reticulata and appears to represent a distinct taxon.Understanding the relationships and phylogeny of cultivated citrus through genome analysis will clarify taxonomic relationships and enable previously inconceivable opportunities for sequence-directed genetic improvement.Citrus are widely consumed worldwide as juice or fresh fruit, providing important sources ofvitamin C and other health-promoting compounds. Global production in 2012 exceeded 86million metric tons, with an estimated value of US$9 billion (http://www.fas.usda.gov/psdonline/circulars/citrus.pdf). The very narrow genetic diversity of cultivated citrus makes it highly vulnerable to disease outbreaks, including citrus greening disease (also known as Huanglongbing) that is rapidly spreading throughout the world's major citrus producing regions1. Understanding the population genomics and domestication of citrus will enable strategies for improvements to citrus including resistance to greening and otherdiseases. The domestication and distribution of edible citrus types began several thousand years ago in Southeast Asia and spread globally following ancient land and sea routes. The lineages that gave rise to most modern cultivated varieties, however, are lost in undocumented antiquity, and their identities remain controversial2, 3. Several features of Citrus biology and cultivation make deciphering these origins difficult. Cultivated varieties are typically propagated clonally by grafting and through asexual seed production (apomixis via nucellar polyembryony) to maintain desirable combinations of traits (Fig. 1). Thus many important cultivar groups have characteristic basic genotypes that presumably arose through interspecific hybridization and/or successive introgressive hybridizations of wild ancestral species. These domestication events predated the global expansion of citrus cultivation by hundreds or perhaps thousands of years, with no record of the domestication process. Diversity within such groups arises through accumulated somatic mutations, generally without sexual recombination, either as limb sports on trees or variants among apomictic seedling progeny.Two wild species are believed to have contributed to domesticated pummelos, mandarins and oranges. Based on morphology and genetic markers, pummelos have generally been identified with the wild species C. maxima (Burm.) Merrill that is indigenous to Southeast Asia. Although mandarins are similarly widely identified with the species C. reticulata Blanco 4-6, wild populations of C. reticulata have not been definitively described. Various authors have taken dif

Abstract Background Flavonoid 3',5'-hydroxylases (F3'5'Hs) and flavonoid 3'-hydroxylases (F3'Hs) competitively control the synthesis of delphinidin and cyanidin, the precursors of blue and red anthocyanins. In most plants, F3'5'H genes are present in low-copy number, but in grapevine they are highly redundant. Results The first increase in F3'5'H copy number occurred in the progenitor of the eudicot clade at the time of the γ triplication. Further proliferation of F3'5'Hs has occurred in one of the paleologous loci after the separation of Vitaceae from other eurosids, giving rise to 15 paralogues within 650 kb. Twelve reside in 9 tandem blocks of ~35-55 kb that share 91-99% identity. The second paleologous F3'5'H has been maintained as an orphan gene in grapevines, and lacks orthologues in other plants. Duplicate F3'5'Hs have spatially and temporally partitioned expression profiles in grapevine. The orphan F3'5'H copy is highly expressed in vegetative organs. More recent duplicate F3'5'Hs are predominately expressed in berry skins. They differ only slightly in the coding region, but are distinguished in the structure of the promoter. Differences in cis-regulatory sequences of promoter regions are paralleled by temporal specialisation of gene transcription during fruit ripening. Variation in anthocyanin profiles consistently reflects changes in the F3'5'H mRNA pool across different cultivars. More F3'5'H copies are expressed at high levels in grapevine varieties with 93-94% of 3'5'-OH anthocyanins. In grapevines depleted in 3'5'-OH anthocyanins (15-45%), fewer F3'5'H copies are transcribed, and at lower levels. Conversely, only two copies of the gene encoding the competing F3'H enzyme are present in the grape genome; one copy is expressed in both vegetative and reproductive organs at comparable levels among cultivars, while the other is transcriptionally silent. Conclusions These results suggest that expansion and subfunctionalisation of F3'5'Hs have increased the complexity and diversification of the fruit colour phenotype among red grape varieties.

BackgroundNucleotide binding site-leucine rich repeat (NBS-LRR)-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes.ResultsWe analyzed the expression patterns of approximately 170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST) representation, massively parallel signature sequencing (MPSS), microarray analysis, rapid amplification of cDNA ends (RACE) PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA) treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler) after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH), a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for alternative splicing was found for at least 12 genes, 11 of which encode TIR-NBS-LRR proteins. There was no obvious correlation between expression pattern, phylogenetic relationship or genomic location of the NBS-LRR-encoding and related genes studied.ConclusionTranscripts of many NBS-LRR-encoding and related genes were defined. Most were present at low levels and exhibited tissue-specific expression patterns. Expression data are consistent with most Arabidopsis NBS-LRR-encoding and related genes functioning in plant defense responses but do not preclude other biological roles.

Minor varieties represent a significant part of the grapevine germplasm (Vitis vinifera L. subsp. vinifera) in Croatia. During the long history of grape cultivation in insular, coastal and continental Croatia, numerous local varieties were cultivated, many of which are still used today. There are also several populations of wild grapevine (Vitis vinifera subsp. sylvestris Hegi Gmel) that have survived until the present in natural sites. Here, we developed a single primer enrichment technology (SPET) panel consisting of 61,308 probes for targeted sequencing of the V. vinifera gene space. In doing so, we examined a total of 28,092 gene models, 88.2% of all predicted genes, interrogating an average of 19 Mb nucleotides per individual genome, which corresponds to 4% of the haploid genome length. This is the first time that SPET-based sequencing has been applied to Croatian grapevine germplasm to generate multilocus genotype data of 126 cultivated accessions and 50 wild specimens. We identified 531,900 variants sites, 208,802 of which reside in coding sequences, 140,836 in introns, 118,416 in UTRs, and 63,424 in the nearby intergenic space that revealed the presence of 33 clonally propagated specimens representing both synonymies and clones within local varieties (22) as well as synonymies between local and international varieties (11), mostly from neighbouring countries. To investigate possible contribution of wild grapevine (Vitis vinifera subsp. sylvestris Hegi Gmel) to the development of present cultivars, four wild grapevine populations were included. None of the cultivated varieties showed close kinship with local wild specimens. This study opens new possibilities for studying the genetic diversity of Croatian grapevine germplasm and provides additional information with respect to SSR genotyping and phenotyping. [ABSTRACT FROM AUTHOR]