Genotyping-by-sequencing-based high-resolution mapping reveals a single candidate gene for the grapevine veraison locus Ver1.

Veraison marks the transition from berry growth to berry ripening and is a crucial phenological stage in grapevine (Vitis vinifera): the berries become soft and begin to accumulate sugars, aromatic substances, and, in red cultivars, anthocyanins for pigmentation, while the organic acid levels begin to decrease. These changes determine the potential quality of wine. However, rising global temperatures lead to earlier flowering and ripening, which strongly influence wine quality. Here, we combined genotyping-by-sequencing with a bioinformatics pipeline on ∼150 F1 genotypes derived from a cross between the early ripening variety 'Calardis Musqué' and the late-ripening variety 'Villard Blanc'. Starting from 20,410 haplotype-based markers, we generated a high-density genetic map and performed a quantitative trait locus analysis based on phenotypic datasets evaluated over 20 years. Through locus-specific-marker-enrichment and recombinant screening of ∼1000 additional genotypes, we refined the originally postulated 5 Mb veraison locus, Ver1, on chromosome 16 to only 112 kb, allowing us to pinpoint the ethylene response factor (ERF) VviERF027 (VCost.v3 gene ID: Vitvi16g00942, CRIBIv1 gene ID: VIT_16s0100g00400) as veraison candidate gene. Furthermore, the early veraison allele could be traced back to a clonal 'Pinot' variant first mentioned in the 17th century. 'Pinot Precoce Noir' passed this allele over 'Madeleine Royale' to the maternal grandparent 'Bacchus Weiss' and, ultimately, to the maternal parent 'Calardis Musqué'. Our findings are crucial for ripening time control, thereby improving wine quality, and for breeding grapevines adjusted to climate change scenarios that have a major impact on agro-ecosystems in altering crop plant phenology.

Rapid identification of inflorescence type markers by genotyping-by-sequencing of diploid and triploid F1 plants of Hydrangea macrophylla.

BACKGROUND: The ornamental crop Hydrangea macrophylla develops highly attractive lacecap (wild type) or mophead inflorescences. The mophead trait, which is mostly favored by consumers, is recessively inherited by the INFLORESCENCE TYPE locus (INF). If lacecap cultivars are crossed with mophead cultivars, then either 50% or all progenies develop lacecap inflorescences, depending on the zygosity at the INF locus. For most cultivars, the zygosity at the INF locus is unknown. Furthermore, the determination of the inflorescence type in offspring populations is time-consuming, because seedlings flower the first time in the 2nd year after sowing. Within this study, we aimed to develop DNA-based markers that allow to determine the zygosity at the INF locus of prospective parental plants and to predict the inflorescence phenotype of seedlings already in the non-flowering stage. RESULTS: By crossing a mophead and a lacecap cultivar of H. macrophylla, we produced a pseudo-backcross F1 population consisting of 422 plants. These plants segregated into 279 lacecap, 73 mophead, 3 intermediate and 67 non-flowering plants, differing significantly from the expected 1:1 segregation ratio. Surprisingly, 75% of these plants were triploid, although both parents were diploid. We found that the lacecap parent produced unreduced pollen, which induced the formation of triploids. 380 randomly selected F1 plants were genotyped by genotyping-by-sequencing (GbS). Using a genome assembly of cultivar 'Sir Joseph Banks', we performed subsequently a bulk sequence analysis with pooled GbS data of diploid versus mophead plants. We identified directly 2 markers tightly linked with the INF locus, each of them explaining 99.7% of the inflorescence phenotype. Using a collection consisting of 56 diploid, triploid or tetraploid H. macrophylla varieties, we detected 6 sequence variants for one of these markers. Two variants were associated with the mophead phenotype. Furthermore, we found by marker analysis a co-segregation between the mophead and the non-flowering trait, which indicates a major flowering time locus next to the INF locus. CONCLUSION: Through bulk sequence analysis of pooled GbS data from diploid and polyploid F1 plants, we identify rapidly tightly linked markers for the inflorescence type, a dominant-recessively inherited trait in the non-model plant species H. macrophylla.

Evolutionary modes of emergence of short interspersed nuclear element (SINE) families in grasses.

Short interspersed nuclear elements (SINEs) are non-autonomous transposable elements which are propagated by retrotransposition and constitute an inherent part of the genome of most eukaryotic species. Knowledge of heterogeneous and highly abundant SINEs is crucial for de novo (or improvement of) annotation of whole genome sequences. We scanned Poaceae genome sequences of six important cereals (Oryza sativa, Triticum aestivum, Hordeum vulgare, Panicum virgatum, Sorghum bicolor, Zea mays) and Brachypodium distachyon to examine the diversity and evolution of SINE populations. We comparatively analyzed the structural features, distribution, evolutionary relation and abundance of 32 SINE families and subfamilies within grasses, comprising 11 052 individual copies. The investigation of activity profiles within the Poaceae provides insights into their species-specific diversification and amplification. We found that Poaceae SINEs (PoaS) fall into two length categories: simple SINEs of up to 180 bp and dimeric SINEs larger than 240 bp. Detailed analysis at the nucleotide level revealed that multimerization of related and unrelated SINE copies is an important evolutionary mechanism of SINE formation. We conclude that PoaS families diversify by massive reshuffling between SINE families, likely caused by insertion of truncated copies, and provide a model for this evolutionary scenario. Twenty-eight of 32 PoaS families and subfamilies show significant conservation, in particular either in the 5' or 3' regions, across Poaceae species and share large sequence stretches with one or more other PoaS families.

Short interspersed nuclear elements (SINEs) are abundant in Solanaceae and have a family-specific impact on gene structure and genome organization.

Short interspersed nuclear elements (SINEs) are highly abundant non-autonomous retrotransposons that are widespread in plants. They are short in size, non-coding, show high sequence diversity, and are therefore mostly not or not correctly annotated in plant genome sequences. Hence, comparative studies on genomic SINE populations are rare. To explore the structural organization and impact of SINEs, we comparatively investigated the genome sequences of the Solanaceae species potato (Solanum tuberosum), tomato (Solanum lycopersicum), wild tomato (Solanum pennellii), and two pepper cultivars (Capsicum annuum). Based on 8.5 Gbp sequence data, we annotated 82 983 SINE copies belonging to 10 families and subfamilies on a base pair level. Solanaceae SINEs are dispersed over all chromosomes with enrichments in distal regions. Depending on the genome assemblies and gene predictions, 30% of all SINE copies are associated with genes, particularly frequent in introns and untranslated regions (UTRs). The close association with genes is family specific. More than 10% of all genes annotated in the Solanaceae species investigated contain at least one SINE insertion, and we found genes harbouring up to 16 SINE copies. We demonstrate the involvement of SINEs in gene and genome evolution including the donation of splice sites, start and stop codons and exons to genes, enlargement of introns and UTRs, generation of tandem-like duplications and transduction of adjacent sequence regions.

Diversification, evolution and methylation of short interspersed nuclear element families in sugar beet and related Amaranthaceae species.

Short interspersed nuclear elements (SINEs) are non-autonomous non-long terminal repeat retrotransposons which are widely distributed in eukaryotic organisms. While SINEs have been intensively studied in animals, only limited information is available about plant SINEs. We analysed 22 SINE families from seven genomes of the Amaranthaceae family and identified 34 806 SINEs, including 19 549 full-length copies. With the focus on sugar beet (Beta vulgaris), we performed a comparative analysis of the diversity, genomic and chromosomal organization and the methylation of SINEs to provide a detailed insight into the evolution and age of Amaranthaceae SINEs. The lengths of consensus sequences of SINEs range from 113 nucleotides (nt) up to 224 nt. The SINEs show dispersed distribution on all chromosomes but were found with higher incidence in subterminal euchromatic chromosome regions. The methylation of SINEs is increased compared with their flanking regions, and the strongest effect is visible for cytosines in the CHH context, indicating an involvement of asymmetric methylation in the silencing of SINEs.

Next-generation sequencing reveals differentially amplified tandem repeats as a major genome component of Northern Europe's oldest Camellia japonica.

Northern Europe's oldest and largest Camellia japonica growing at the Pillnitz Castle (Germany) for over 200 years is of botanical and cultural importance and is a reference for C. japonica molecular scale analysis. In order to provide a fundament for genome analysis of the genus Camellia, we characterize the C. japonica tandem repeat fraction, constituting 12.5 % of the Pillnitz camellia's genome. A genomic library of the Pillnitz C. japonica was produced and Illumina sequenced to generate 36 Gb of paired-end reads. We performed graph-based read clustering implemented in the RepeatExplorer pipeline to estimate the C. japonica repeat fraction of 73 %. This enabled us to identify and characterize the most prominent satellite DNAs, Camellia japonica satellite 1-4 (CajaSat1-CajaSat4), and the 5S ribosomal DNA (rDNA) by bioinformatics, fluorescent in situ and Southern hybridization. Within the Camellia genus, satellite spreading, array expansion and formation of higher-order structures highlight different modes of repeat evolution. The CajaSat satellites localize at prominent chromosomal sites, including (peri)centromeres and subtelomeres of all chromosomes, thus serving as chromosomal landmarks for their identification. This work provides an insight into the C. japonica chromosome organization and significantly expands the Camellia genomic knowledge, also with respect to the tea plant Camellia sinensis.

Targeted identification of short interspersed nuclear element families shows their widespread existence and extreme heterogeneity in plant genomes.

Short interspersed nuclear elements (SINEs) are non-long terminal repeat retrotransposons that are highly abundant, heterogeneous, and mostly not annotated in eukaryotic genomes. We developed a tool designated SINE-Finder for the targeted discovery of tRNA-derived SINEs. We analyzed sequence data of 16 plant genomes, including 13 angiosperms and three gymnosperms and identified 17,829 full-length and truncated SINEs falling into 31 families showing the widespread occurrence of SINEs in higher plants. The investigation focused on potato (Solanum tuberosum), resulting in the detection of seven different SolS SINE families consisting of 1489 full-length and 870 5' truncated copies. Consensus sequences of full-length members range in size from 106 to 244 bp depending on the SINE family. SolS SINEs populated related species and evolved separately, which led to some distinct subfamilies. Solanaceae SINEs are dispersed along chromosomes and distributed without clustering but with preferred integration into short A-rich motifs. They emerged more than 23 million years ago and were species specifically amplified during the radiation of potato, tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum). We show that tobacco TS retrotransposons are composite SINEs consisting of the 3' end of a long interspersed nuclear element integrated downstream of a nonhomologous SINE family followed by successfully colonization of the genome. We propose an evolutionary scenario for the formation of TS as a spontaneous event, which could be typical for the emergence of SINE families.

Chemical imitation of yeast fermentation by the drosophilid-pollinated deceptive trap-flower Aristolochia baetica (Aristolochiaceae).

Deceptive flowers, unlike in mutualistic pollination systems, mislead their pollinators by advertising rewards which ultimately are not provided. Although our understanding of deceptive pollination systems increased in recent years, the attractive signals and deceptive strategies in the majority of species remain unknown. This is also true for the genus Aristolochia, famous for its deceptive and fly-pollinated trap flowers. Representatives of this genus were generally assumed to be oviposition-site mimics, imitating vertebrate carrion or mushrooms. However, recent studies found a broader spectrum of strategies, including kleptomyiophily and imitation of invertebrate carrion. A different deceptive strategy is presented here for the western Mediterranean Aristolochia baetica L. We found that this species is mostly pollinated by drosophilid flies (Drosophilidae, mostly Drosophila spp.), which typically feed on fermenting fruit infested by yeasts. The flowers of A. baetica emitted mostly typical yeast volatiles, predominantly the aliphatic compounds acetoin and 2,3-butandiol, and derived acetates, as well as the aromatic compound 2-phenylethanol. Analyses of the absolute configurations of the chiral volatiles revealed weakly (acetoin, 2,3-butanediol) to strongly (mono- and diacetates) biased stereoisomer-ratios. Electrophysiological (GC-EAD) experiments and lab bioassays demonstrated that most of the floral volatiles, although not all stereoisomers of chiral compounds, were physiologically active and attractive in drosophilid pollinators; a synthetic mixture thereof successfully attracted them in field and lab bioassays. We conclude that A. baetica chemically mimics yeast fermentation to deceive its pollinators. This deceptive strategy (scent chemistry, pollinators, trapping function) is also known from more distantly related plants, such as Arum palaestinum Boiss. (Araceae) and Ceropegia spp. (Apocynaceae), suggesting convergent evolution. In contrast to other studies working on floral scents in plants imitating breeding sites, the present study considered the absolute configuration of chiral compounds.

Development and application of SINE-based markers for genotyping of potato varieties.

Potato variety discrimination based on morphological traits is laborious and influenced by the environment, while currently applied molecular markers are either expensive or time-consuming in development or application. SINEs, short interspersed nuclear elements, are retrotransposons with a high copy number in plant genomes representing a potential source for new markers. We developed a marker system for potato genotyping, designated inter-SINE amplified polymorphism (ISAP). Based on nine potato SINE families recently characterized (Wenke et al. in Plant Cell 23:3117-3128, 2011), we designed species-specific SINE primers. From the resulting 153 primer combinations, highly informative primer sets were selected for potato variety analysis regarding number of bands, quality of the banding pattern, and the degree of polymorphism. Fragments representing ISAPs can be separated by conventional agarose gel electrophoresis; however, automation with a capillary sequencer is feasible. Two selected SINE families, SolS-IIIa and SolS-IV, were shown to be highly but differently amplified in Solanaceae, Solaneae tribe, including wild and cultivated potatoes, tomato, and eggplant. Fluorescent in situ hybridization demonstrated the genome-wide distribution of SolS-IIIa and SolS-IV along potato chromosomes, which is the basis for genotype discrimination and differentiation of somaclonal variants by ISAP markers.

The Ty1-copia families SALIRE and Cotzilla populating the Beta vulgaris genome show remarkable differences in abundance, chromosomal distribution, and age.

Long terminal repeat (LTR) retrotransposons are major components of plant genomes influencing genome size and evolution. Using two separate approaches, we identified the Ty1-copia retrotransposon families Cotzilla and SALIRE in the Beta vulgaris (sugar beet) genome. While SALIRE elements are similar to typical Ty1-copia retrotransposons, Cotzilla elements belong to a lineage called Sireviruses. Hallmarks of Cotzilla retrotransposons are the existence of an additional putative env-like open reading frame upstream of the 3'LTR, an extended gag region, and a frameshift separating the gag and pol genes. Detected in a c ( 0 ) t-1 DNA library, Cotzilla elements belong to the most abundant retrotransposon families in B. vulgaris and are relatively homogenous and evolutionarily young. In contrast, the SALIRE family has relatively few copies, is diverged, and most likely ancient. As revealed by fluorescent in situ hybridization, SALIRE elements target predominantly gene-rich euchromatic regions, while Cotzilla retrotransposons are abundant in the intercalary and pericentromeric heterochromatin. The analysis of two retrotransposons from the same subclass contrasting in abundance, age, sequence diversity, and localization gives insight in the heterogeneity of LTR retrotransposons populating a plant genome.

Analysis of a c0t-1 library enables the targeted identification of minisatellite and satellite families in Beta vulgaris.

BACKGROUND: Repetitive DNA is a major fraction of eukaryotic genomes and occurs particularly often in plants. Currently, the sequencing of the sugar beet (Beta vulgaris) genome is under way and knowledge of repetitive DNA sequences is critical for the genome annotation. We generated a c0t-1 library, representing highly to moderately repetitive sequences, for the characterization of the major B. vulgaris repeat families. While highly abundant satellites are well-described, minisatellites are only poorly investigated in plants. Therefore, we focused on the identification and characterization of these tandemly repeated sequences. RESULTS: Analysis of 1763 c0t-1 DNA fragments, providing 442 kb sequence data, shows that the satellites pBV and pEV are the most abundant repeat families in the B. vulgaris genome while other previously described repeats show lower copy numbers. We isolated 517 novel repetitive sequences and used this fraction for the identification of minisatellite and novel satellite families. Bioinformatic analysis and Southern hybridization revealed that minisatellites are moderately to highly amplified in B. vulgaris. FISH showed a dispersed localization along most chromosomes clustering in arrays of variable size and number with exclusion and depletion in distinct regions. CONCLUSION: The c0t-1 library represents major repeat families of the B. vulgaris genome, and analysis of the c0t-1 DNA was proven to be an efficient method for identification of minisatellites. We established, so far, the broadest analysis of minisatellites in plants and observed their chromosomal localization providing a background for the annotation of the sugar beet genome and for the understanding of the evolution of minisatellites in plant genomes.

An abundant and heavily truncated non-LTR retrotransposon (LINE) family in Beta vulgaris.

We describe a non-LTR retrotransposon family,BvL, of the long interspersed nuclear elements L1 clade isolated from sugar beet (Beta vulgaris). Characteristic molecular domains of three full-length BvL elements were determined in detail, showing that coding sequences are interrupted and most likely non-functionally. In addition,eight highly conserved endonuclease regions were defined by comparison with other plant LINEs. The abundant BvL family is widespread within the genus Beta, however, the vast majority of BvL copies are extremely 50 truncated indicating an error-prone reverse transcriptase activity. The dispersed distribution of BvL copies on all sugar beet chromosomes with exclusion of most heterochromatic regions was shown by fluorescent in situ hybridization. The analysis of BvL 30 end sequences and corresponding flanking regions, respectively, revealed the preferred integration of BvL into A/T-rich regions of the sugar beet genome, but no specific target sequences.

A BAC library of Beta vulgaris L. for the targeted isolation of centromeric DNA and molecular cytogenetics of Beta species.

We constructed a sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) library of the monosomic addition line PAT2. This chromosomal mutant carries a single additional chromosome fragment (minichromosome) derived from the wild beet Beta patellaris. Restriction analysis of the mutant line by pulsed-field gel electrophoresis was used to determine HindIII as a suitable enzyme for partial digestion of genomic DNA to generate large-insert fragments which were cloned into the vector pCC1. The library consists of 36,096 clones with an average insert size of 120 kb, and 2.2% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents 5.7 genome equivalents providing the probability of 99.67% that any sequence of the PAT2 genome can be found in the library. Hybridization to high-density filters was used to isolate 89 BACs containing arrays of the centromere-associated satellite repeats pTS5 and pTS4.1. Using the identified BAC clones in fluorescent in situ hybridization experiments with PAT2 and Beta patellaris chromosome spreads their wild beet origin and centromeric localization was demonstrated. Multi-colour FISH with differently labelled satellite repeats pTS5 and pTS4.1 was used to investigate the large-scale organization of the centromere of the PAT2 minichromosome in detail. FISH studies showed that the centromeric satellite pTS5 is flanked on both sides by pTS4.1 arrays and the arms of the minichromosome are terminated by the Arabidopsis-type telomeric sequences. FISH with a BAC, selected from high-density filters after hybridization with an RFLP marker of the genetic linkage group I, demonstrated that it is feasible to correlate genetic linkage groups with chromosomes. Therefore, the PAT2 BAC library provides a useful tool for the characterization of Beta centromeres and a valuable resource for sugar beet genome analysis.

Automated detection of the HER2 gene amplification status in Fluorescence in situ hybridization images for the diagnostics of cancer tissues.

The human epidermal growth factor receptor 2 (HER2) gene amplification status is a crucial marker for evaluating clinical therapies of breast or gastric cancer. We propose a deep learning-based pipeline for the detection, localization and classification of interphase nuclei depending on their HER2 gene amplification state in Fluorescence in situ hybridization (FISH) images. Our pipeline combines two RetinaNet-based object localization networks which are trained (1) to detect and classify interphase nuclei into distinct classes normal, low-grade and high-grade and (2) to detect and classify FISH signals into distinct classes HER2 or centromere of chromosome 17 (CEN17). By independently classifying each nucleus twice, the two-step pipeline provides both robustness and interpretability for the automated detection of the HER2 amplification status. The accuracy of our deep learning-based pipeline is on par with that of three pathologists and a set of 57 validation images containing several hundreds of nuclei are accurately classified. The automatic pipeline is a first step towards assisting pathologists in evaluating the HER2 status of tumors using FISH images, for analyzing FISH images in retrospective studies, and for optimizing the documentation of each tumor sample by automatically annotating and reporting of the HER2 gene amplification specificities.

Diversity of a complex centromeric satellite and molecular characterization of dispersed sequence families in sugar beet (Beta vulgaris).

BACKGROUND AND AIMS: The aim of this work was the identification and molecular characterization of novel sugar beet (Beta vulgaris) repetitive sequences to unravel the impact of repetitive DNA on size and evolution of Beta genomes via amplification and diversification. METHODS: Genomic DNA and a pool of B. vulgaris repetitive sequences were separately used as probes for a screening of high-density filters from a B. vulgaris plasmid library. Novel repetitive motifs were identified by sequencing and further used as probes for Southern analyses in the genus Beta. Chromosomal localization of the repeats was analysed by fluorescent in situ hybridization on chromosomes of B. vulgaris and two other species of the section Beta. KEY RESULTS: Two dispersed repetitive families pDvul1 and pDvul2 and the tandemly arranged repeat family pRv1 were isolated from a sugar beet plasmid library. The dispersed repetitive families pDvul1 and pDvul2 were identified in all four sections of the genus Beta. The members of the pDvul1 and pDvul2 family are scattered over all B. vulgaris chromosomes, although amplified to a different extent. The pRv1 satellite repeat is exclusively present in species of the section Beta. The centromeric satellite pBV1 by structural variations of the monomer and interspersion of pRv1 units forms complex satellite structures, which are amplified in different degrees on the centromeres of 12 chromosomes of the three species of the Beta section. CONCLUSIONS: The complexity of the pBV1 satellite family observed in the section Beta of the genus Beta and, in particular, the strong amplification of the pBV1/pRv1 satellite in the domesticated B. vulgaris indicates the dynamics of centromeric satellite evolution during species radiation within the genus. The dispersed repeat families pDvul1 and pDvul2 might represent derivatives of transposable elements.

Inter-SINE Amplified Polymorphism (ISAP) for rapid and robust plant genotyping.

The unambiguous differentiation of crop genotypes is often laborious or expensive. A rapid, robust, and cost-efficient marker system is required for routine genotyping in plant breeding and marker-assisted selection. We describe the Inter-SINE Amplified Polymorphism (ISAP) system that is based on standard molecular methods resulting in genotype-specific fingerprints at high resolution. These markers are derived from Short Interspersed Nuclear Elements (SINEs) which are dispersed repetitive sequences present in most if not all plant genomes and can be efficiently extracted from plant genome sequences. The ISAP method was developed on potato as model plant but is also transferable to other plant species.