Repeat turnover meets stable chromosomes: repetitive DNA sequences mark speciation and gene pool boundaries in sugar beet and wild beets.

Sugar beet and its wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNAs are among the fastest evolving parts of the genome, we investigated, if repeatome innovations and losses are linked to chromosomal differentiation and speciation. We traced genome and chromosome-wide evolution across 13 beet species comprising all sections of the genera Beta and Patellifolia. For this, we combined short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive framework that spans the complete scale from DNA to chromosome to genome. Genome sizes and repeat profiles reflect the separation into three gene pools with contrasting evolutionary patterns. Among all repeats, satellite DNAs harbor most genomic variability, leading to fundamentally different centromere architectures, ranging from chromosomal uniformity in Beta and Patellifolia to the formation of patchwork chromosomes in Corollinae/Nanae. We show that repetitive DNAs are causal for the genome expansions and contractions across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably between beet genomes, leading to the evolution of distinct chromosomal setups in the three gene pools, likely contributing to the barriers in beet breeding. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genomic variability, and chromosomal differentiation and provide a theoretical fundament for understanding barriers in any crop breeding effort.

Genomic characterization of a nematode tolerance locus in sugar beet.

BACKGROUND: Infection by beet cyst nematodes (BCN, Heterodera schachtii) causes a serious disease of sugar beet, and climatic change is expected to improve the conditions for BCN infection. Yield and yield stability under adverse conditions are among the main breeding objectives. Breeding of BCN tolerant sugar beet cultivars offering high yield in the presence of the pathogen is therefore of high relevance. RESULTS: To identify causal genes providing tolerance against BCN infection, we combined several experimental and bioinformatic approaches. Relevant genomic regions were detected through mapping-by-sequencing using a segregating F2 population. DNA sequencing of contrasting F2 pools and analyses of allele frequencies for variant positions identified a single genomic region which confers nematode tolerance. The genomic interval was confirmed and narrowed down by genotyping with newly developed molecular markers. To pinpoint the causal genes within the potential nematode tolerance locus, we generated long read-based genome sequence assemblies of the tolerant parental breeding line Strube U2Bv and the susceptible reference line 2320Bv. We analyzed continuous sequences of the potential locus with regard to functional gene annotation and differential gene expression upon BCN infection. A cluster of genes with similarity to the Arabidopsis thaliana gene encoding nodule inception protein-like protein 7 (NLP7) was identified. Gene expression analyses confirmed transcriptional activity and revealed clear differences between susceptible and tolerant genotypes. CONCLUSIONS: Our findings provide new insights into the genomic basis of plant-nematode interactions that can be used to design and accelerate novel management strategies against BCN.

Homoeologous non-reciprocal translocation explains a major QTL for seed lignin content in oilseed rape (Brassica napus L.).

KEY MESSAGE: A homoeologous non-reciprocal translocation was identified in the major QTL for seed lignin content in the low lignin line SGDH14. The lignin biosynthetic gene PAL4 was deleted. Oilseed rape is a major oil crop and a valuable protein source for animal and human nutrition. Lignin is a non-digestible, major component of the seed coat with negative effect on sensory quality, bioavailability and usage of oilseed rape's protein. Hence, seed lignin reduction is of economic and nutritional importance. In this study, the major QTL for reduced lignin content found on chromosome C05 in the DH population SGDH14 x Express 617 was further examined. SGDH14 had lower seed lignin content than Express 617. Harvested seeds from a F2 population of the same cross were additionally field tested and used for seed quality analysis. The F2 population showed a bimodal distribution for seed lignin content. F2 plants with low lignin content had thinner seed coats compared to high lignin lines. Both groups showed a dark seed colour with a slightly lighter colour in the low lignin group indicating that a low lignin content is not necessarily associated with yellow seed colour. Mapping of genomic long-reads from SGDH14 against the Express 617 genome assembly revealed a homoeologous non-reciprocal translocation (HNRT) in the confidence interval of the major QTL for lignin content. A homologous A05 region is duplicated and replaced the C05 region in SGDH14. As consequence several genes located in the C05 region were lost in SGDH14. Thus, a HNRT was identified in the major QTL region for reduced lignin content in the low lignin line SGDH14. The most promising candidate gene related to lignin biosynthesis on C05, PAL4, was deleted.

Complete pan-plastome sequences enable high resolution phylogenetic classification of sugar beet and closely related crop wild relatives.

BACKGROUND: As the major source of sugar in moderate climates, sugar-producing beets (Beta vulgaris subsp. vulgaris) have a high economic value. However, the low genetic diversity within cultivated beets requires introduction of new traits, for example to increase their tolerance and resistance attributes - traits that often reside in the crop wild relatives. For this, genetic information of wild beet relatives and their phylogenetic placements to each other are crucial. To answer this need, we sequenced and assembled the complete plastome sequences from a broad species spectrum across the beet genera Beta and Patellifolia, both embedded in the Betoideae (order Caryophyllales). This pan-plastome dataset was then used to determine the wild beet phylogeny in high-resolution. RESULTS: We sequenced the plastomes of 18 closely related accessions representing 11 species of the Betoideae subfamily and provided high-quality plastome assemblies which represent an important resource for further studies of beet wild relatives and the diverse plant order Caryophyllales. Their assembly sizes range from 149,723 bp (Beta vulgaris subsp. vulgaris) to 152,816 bp (Beta nana), with most variability in the intergenic sequences. Combining plastome-derived phylogenies with read-based treatments based on mitochondrial information, we were able to suggest a unified and highly confident phylogenetic placement of the investigated Betoideae species. Our results show that the genus Beta can be divided into the two clearly separated sections Beta and Corollinae. Our analysis confirms the affiliation of B. nana with the other Corollinae species, and we argue against a separate placement in the Nanae section. Within the Patellifolia genus, the two diploid species Patellifolia procumbens and Patellifolia webbiana are, regarding the plastome sequences, genetically more similar to each other than to the tetraploid Patellifolia patellaris. Nevertheless, all three Patellifolia species are clearly separated. CONCLUSION: In conclusion, our wild beet plastome assemblies represent a new resource to understand the molecular base of the beet germplasm. Despite large differences on the phenotypic level, our pan-plastome dataset is highly conserved. For the first time in beets, our whole plastome sequences overcome the low sequence variation in individual genes and provide the molecular backbone for highly resolved beet phylogenomics. Hence, our plastome sequencing strategy can also guide genomic approaches to unravel other closely related taxa.

Transcriptomic analysis of temporal shifts in berry development between two grapevine cultivars of the Pinot family reveals potential genes controlling ripening time.

BACKGROUND: Grapevine cultivars of the Pinot family represent clonally propagated mutants with major phenotypic and physiological differences, such as different colour or shifted ripening time, as well as changes in important viticultural traits. Specifically, the cultivars 'Pinot Noir' (PN) and 'Pinot Noir Precoce' (PNP, early ripening) flower at the same time, but vary in the beginning of berry ripening (veraison) and, consequently, harvest time. In addition to genotype, seasonal climatic conditions (i.e. high temperatures) also affect ripening times. To reveal possible regulatory genes that affect the timing of veraison onset, we investigated differences in gene expression profiles between PN and PNP throughout berry development with a closely meshed time series and over two separate years. RESULTS: The difference in the duration of berry formation between PN and PNP was quantified to be approximately two weeks under the growth conditions applied, using plant material with a proven PN and PNP clonal relationship. Clusters of co-expressed genes and differentially expressed genes (DEGs) were detected which reflect the shift in the timing of veraison onset. Functional annotation of these DEGs fit to observed phenotypic and physiological changes during berry development. In total, we observed 3,342 DEGs in 2014 and 2,745 DEGs in 2017 between PN and PNP, with 1,923 DEGs across both years. Among these, 388 DEGs were identified as veraison-specific and 12 were considered as berry ripening time regulatory candidates. The expression profiles revealed two candidate genes for ripening time control which we designated VviRTIC1 and VviRTIC2 (VIT_210s0071g01145 and VIT_200s0366g00020, respectively). These genes likely contribute the phenotypic differences observed between PN and PNP. CONCLUSIONS: Many of the 1,923 DEGs show highly similar expression profiles in both cultivars if the patterns are aligned according to developmental stage. In our work, putative genes differentially expressed between PNP and PN which could control ripening time as well as veraison-specific genes were identified. We point out connections of these genes to molecular events during berry development and discuss potential candidate genes which may control ripening time. Two of these candidates were observed to be differentially expressed in the early berry development phase. Several down-regulated genes during berry ripening are annotated as auxin response factors / ARFs. Conceivably, general changes in auxin signaling may cause the earlier ripening phenotype of PNP.

The transition to flowering in winter rapeseed during vernalization.

Flowering time is a major determinant of adaptation, fitness and yield in the allopolyploid species rapeseed (Brassica napus). Despite being a close relative to Arabidopsis thaliana, little is known about the timing of floral transition and the genes that govern this process. Winter, semi-winter and spring type plants have important life history characteristics that differ in vernalization requirements for flowering and are important for growing rapeseed in different regions of the world. In this study, we investigated the timing of vernalization-driven floral transition in winter rapeseed and the effect of photoperiod and developmental age on flowering time and vernalization responsiveness. Microscopy and whole transcriptome analyses at the shoot apical meristems of plants grown under controlled conditions showed that floral transition is initiated within few weeks of vernalization. Certain Bna.SOC1 and Bna.SPL5 homeologs were among the induced genes, suggesting that they are regulating the timing of cold-induced floral transition. Moreover, the flowering response of plants with shorter pre-vernalization period correlated with a delayed expression of Bna.SOC1 and Bna.SPL5 genes. In essence, this study presents a detailed analysis of vernalization-driven floral transition and the aspects of juvenility and dormancy and their effect on flowering time in rapeseed.

Vacuolar sucrose homeostasis is critical for plant development, seed properties, and night-time survival in Arabidopsis.

Most cellular sucrose is present in the cytosol and vacuoles of plant cells; however, little is known about the effect of this sucrose compartmentation on plant properties. Here, we examined the effects of altered intracellular sucrose compartmentation in Arabidopsis thaliana leaves by heterologously expressing the sugar beet (Beta vulgaris) vacuolar sucrose loader BvTST2.1 and by generating lines with reduced vacuolar invertase activity (amiR vi1-2). Heterologous expression of BvTST2.1 led to increased monosaccharide levels in leaves, whereas sucrose levels remained constant, indicating that vacuolar invertase activity in mesophyll vacuoles exceeds sucrose uptake. This notion was supported by analysis of tobacco (Nicotiana benthamiana) leaves transiently expressing BvTST2.1 and the invertase inhibitor NbVIF. However, sucrose levels were strongly elevated in leaf extracts from amiR vi1-2 lines, and experiments confirmed that sucrose accumulated in the corresponding vacuoles. The amiR vi1-2 lines exhibited impaired early development and reduced seed weight. When germinated in the dark, amiR vi1-2 seedlings were less able to convert sucrose into monosaccharides than the wild type. Cold temperatures strongly down-regulated both VI genes, but the amiR vi1-2 lines showed normal frost tolerance. These observations indicate that increased vacuolar sucrose levels fully compensate for the effects of low monosaccharide concentrations on frost tolerance.

The genome of the recently domesticated crop plant sugar beet (Beta vulgaris).

Sugar beet (Beta vulgaris ssp. vulgaris) is an important crop of temperate climates which provides nearly 30% of the world's annual sugar production and is a source for bioethanol and animal feed. The species belongs to the order of Caryophylalles, is diploid with 2n = 18 chromosomes, has an estimated genome size of 714-758 megabases and shares an ancient genome triplication with other eudicot plants. Leafy beets have been cultivated since Roman times, but sugar beet is one of the most recently domesticated crops. It arose in the late eighteenth century when lines accumulating sugar in the storage root were selected from crosses made with chard and fodder beet. Here we present a reference genome sequence for sugar beet as the first non-rosid, non-asterid eudicot genome, advancing comparative genomics and phylogenetic reconstructions. The genome sequence comprises 567 megabases, of which 85% could be assigned to chromosomes. The assembly covers a large proportion of the repetitive sequence content that was estimated to be 63%. We predicted 27,421 protein-coding genes supported by transcript data and annotated them on the basis of sequence homology. Phylogenetic analyses provided evidence for the separation of Caryophyllales before the split of asterids and rosids, and revealed lineage-specific gene family expansions and losses. We sequenced spinach (Spinacia oleracea), another Caryophyllales species, and validated features that separate this clade from rosids and asterids. Intraspecific genomic variation was analysed based on the genome sequences of sea beet (Beta vulgaris ssp. maritima; progenitor of all beet crops) and four additional sugar beet accessions. We identified seven million variant positions in the reference genome, and also large regions of low variability, indicating artificial selection. The sugar beet genome sequence enables the identification of genes affecting agronomically relevant traits, supports molecular breeding and maximizes the plant's potential in energy biotechnology.

The B2 flowering time locus of beet encodes a zinc finger transcription factor.

Sugar beet (Beta vulgaris) is a biennial root crop that grows vegetatively in the first year and starts shoot elongation (bolting) and flowering after exposure to cold temperatures over winter. Early bolting before winter is controlled by the dominant allele of the B locus. Recently, the BOLTING time control 1 (BTC1) gene has been cloned from this locus. BTC1 promotes early bolting through repression of the downstream bolting repressor B. vulgaris flowering locus T1 (BvFT1) and activation of the downstream floral activator BvFT2. We have identified a new bolting locus B2 acting epistatically to B. B2 houses a transcription factor which is diurnally regulated and acts like BTC1 upstream of BvFT1 and BvFT2. It was termed BvBBX19 according to its closest homolog from Arabidopsis thaliana. The encoded protein has two conserved domains with homology to zinc finger B-boxes. Ethyl methanesulfonate-induced mutations within the second B-box caused up-regulation of BvFT1 and complete down-regulation of BvFT2. In Arabidopsis, the expression of FT is promoted by the B-box containing protein CONSTANS (CO). We performed a phylogenetic analysis with B-box genes from beet and A. thaliana but only BvCOL1 clustered with CO. However, BvCOL1 had been excluded as a CO ortholog by previous studies. Therefore, a new model for flowering induction in beet is proposed in which BTC1 and BvBBX19 complement each other and thus acquire a CO function to regulate their downstream targets BvFT1 and BvFT2.

Rapid gene identification in sugar beet using deep sequencing of DNA from phenotypic pools selected from breeding panels.

BACKGROUND: The combination of bulk segregant analysis (BSA) and next generation sequencing (NGS), also known as mapping by sequencing (MBS), has been shown to significantly accelerate the identification of causal mutations for species with a reference genome sequence. The usual approach is to cross homozygous parents that differ for the monogenic trait to address, to perform deep sequencing of DNA from F2 plants pooled according to their phenotype, and subsequently to analyze the allele frequency distribution based on a marker table for the parents studied. The method has been successfully applied for EMS induced mutations as well as natural variation. Here, we show that pooling genetically diverse breeding lines according to a contrasting phenotype also allows high resolution mapping of the causal gene in a crop species. The test case was the monogenic locus causing red vs. green hypocotyl color in Beta vulgaris (R locus). RESULTS: We determined the allele frequencies of polymorphic sequences using sequence data from two diverging phenotypic pools of 180 B. vulgaris accessions each. A single interval of about 31 kbp among the nine chromosomes was identified which indeed contained the causative mutation. CONCLUSIONS: By applying a variation of the mapping by sequencing approach, we demonstrated that phenotype-based pooling of diverse accessions from breeding panels and subsequent direct determination of the allele frequency distribution can be successfully applied for gene identification in a crop species. Our approach made it possible to identify a small interval around the causative gene. Sequencing of parents or individual lines was not necessary. Whenever the appropriate plant material is available, the approach described saves time compared to the generation of an F2 population. In addition, we provide clues for planning similar experiments with regard to pool size and the sequencing depth required.

SMRT sequencing only de novo assembly of the sugar beet (Beta vulgaris) chloroplast genome.

BACKGROUND: Third generation sequencing methods, like SMRT (Single Molecule, Real-Time) sequencing developed by Pacific Biosciences, offer much longer read length in comparison to Next Generation Sequencing (NGS) methods. Hence, they are well suited for de novo- or re-sequencing projects. Sequences generated for these purposes will not only contain reads originating from the nuclear genome, but also a significant amount of reads originating from the organelles of the target organism. These reads are usually discarded but they can also be used for an assembly of organellar replicons. The long read length supports resolution of repetitive regions and repeats within the organelles genome which might be problematic when just using short read data. Additionally, SMRT sequencing is less influenced by GC rich areas and by long stretches of the same base. RESULTS: We describe a workflow for a de novo assembly of the sugar beet (Beta vulgaris ssp. vulgaris) chloroplast genome sequence only based on data originating from a SMRT sequencing dataset targeted on its nuclear genome. We show that the data obtained from such an experiment are sufficient to create a high quality assembly with a higher reliability than assemblies derived from e.g. Illumina reads only. The chloroplast genome is especially challenging for de novo assembling as it contains two large inverted repeat (IR) regions. We also describe some limitations that still apply even though long reads are used for the assembly. CONCLUSIONS: SMRT sequencing reads extracted from a dataset created for nuclear genome (re)sequencing can be used to obtain a high quality de novo assembly of the chloroplast of the sequenced organism. Even with a relatively small overall coverage for the nuclear genome it is possible to collect more than enough reads to generate a high quality assembly that outperforms short read based assemblies. However, even with long reads it is not always possible to clarify the order of elements of a chloroplast genome sequence reliantly which we could demonstrate with Fosmid End Sequences (FES) generated with Sanger technology. Nevertheless, this limitation also applies to short read sequencing data but is reached in this case at a much earlier stage during finishing.

Profiling of extensively diversified plant LINEs reveals distinct plant-specific subclades.

A large fraction of eukaryotic genomes is made up of long interspersed nuclear elements (LINEs). Due to their capability to create novel copies via error-prone reverse transcription, they generate multiple families and reach high copy numbers. Although mammalian LINEs have been well described, plant LINEs have been only poorly investigated. Here, we present a systematic cross-species survey of LINEs in higher plant genomes shedding light on plant LINE evolution as well as diversity, and facilitating their annotation in genome projects. Applying a Hidden Markov Model (HMM)-based analysis, 59 390 intact LINE reverse transcriptases (RTs) were extracted from 23 plant genomes. These fall in only two out of 28 LINE clades (L1 and RTE) known in eukaryotes. While plant RTE LINEs are highly homogenous and mostly constitute only a single family per genome, plant L1 LINEs are extremely diverse and form numerous families. Despite their heterogeneity, all members across the 23 species fall into only seven L1 subclades, some of them defined here. Exemplarily focusing on the L1 LINEs of a basal reference plant genome (Beta vulgaris), we show that the subclade classification level does not only reflect RT sequence similarity, but also mirrors structural aspects of complete LINE retrotransposons, like element size, position and type of encoded enzymatic domains. Our comprehensive catalogue of plant LINE RTs serves the classification of highly diverse plant LINEs, while the provided subclade-specific HMMs facilitate their annotation.

Genome-wide identification and characterisation of R2R3-MYB genes in sugar beet (Beta vulgaris).

BACKGROUND: The R2R3-MYB genes comprise one of the largest transcription factor gene families in plants, playing regulatory roles in plant-specific developmental processes, metabolite accumulation and defense responses. Although genome-wide analysis of this gene family has been carried out in some species, the R2R3-MYB genes in Beta vulgaris ssp. vulgaris (sugar beet) as the first sequenced member of the order Caryophyllales, have not been analysed heretofore. RESULTS: We present a comprehensive, genome-wide analysis of the MYB genes from Beta vulgaris ssp. vulgaris (sugar beet) which is the first species of the order Caryophyllales with a sequenced genome. A total of 70 R2R3-MYB genes as well as genes encoding three other classes of MYB proteins containing multiple MYB repeats were identified and characterised with respect to structure and chromosomal organisation. Also, organ specific expression patterns were determined from RNA-seq data. The R2R3-MYB genes were functionally categorised which led to the identification of a sugar beet-specific clade with an atypical amino acid composition in the R3 domain, putatively encoding betalain regulators. The functional classification was verified by experimental confirmation of the prediction that the R2R3-MYB gene Bv_iogq encodes a flavonol regulator. CONCLUSIONS: This study provides the first step towards cloning and functional dissection of the role of MYB transcription factor genes in the nutritionally and evolutionarily interesting species B. vulgaris. In addition, it describes the flavonol regulator BvMYB12, being the first sugar beet R2R3-MYB with an experimentally proven function.

QTL analysis of flowering time and ripening traits suggests an impact of a genomic region on linkage group 1 in Vitis.

In the recent past, genetic analyses of grapevine focused mainly on the identification of resistance loci for major diseases such as powdery and downy mildew. Currently, breeding programs make intensive use of these results by applying molecular markers linked to the resistance traits. However, modern genetics also allows to address additional agronomic traits that have considerable impact on the selection of grapevine cultivars. In this study, we have used linkage mapping for the identification and characterization of flowering time and ripening traits in a mapping population from a cross of V3125 ('Schiava Grossa' × 'Riesling') and the interspecific rootstock cultivar 'Börner' (Vitis riparia × Vitis cinerea). Comparison of the flowering time QTL mapping with data derived from a second independent segregating population identified several common QTLs. Especially a large region on linkage group 1 proved to be of special interest given the genetic divergence of the parents of the two populations. The proximity of the QTL region contains two CONSTANS-like genes. In accordance with data from other plants such as Arabidopsis thaliana and Oryza sativa, we hypothesize that these genes are major contributors to control the time of flowering in Vitis.

Palaeohexaploid ancestry for Caryophyllales inferred from extensive gene-based physical and genetic mapping of the sugar beet genome (Beta vulgaris).

Sugar beet (Beta vulgaris) is an important crop plant that accounts for 30% of the world's sugar production annually. The genus Beta is a distant relative of currently sequenced taxa within the core eudicotyledons; the genomic characterization of sugar beet is essential to make its genome accessible to molecular dissection. Here, we present comprehensive genomic information in genetic and physical maps that cover all nine chromosomes. Based on this information we identified the proposed ancestral linkage groups of rosids and asterids within the sugar beet genome. We generated an extended genetic map that comprises 1127 single nucleotide polymorphism markers prepared from expressed sequence tags and bacterial artificial chromosome (BAC) end sequences. To construct a genome-wide physical map, we hybridized gene-derived oligomer probes against two BAC libraries with 9.5-fold cumulative coverage of the 758 Mbp genome. More than 2500 probes and clones were integrated both in genetic maps and the physical data. The final physical map encompasses 535 chromosomally anchored contigs that contains 8361 probes and 22 815 BAC clones. By using the gene order established with the physical map, we detected regions of synteny between sugar beet (order Caryophyllales) and rosid species that involves 1400-2700 genes in the sequenced genomes of Arabidopsis, poplar, grapevine, and cacao. The data suggest that Caryophyllales share the palaeohexaploid ancestor proposed for rosids and asterids. Taken together, we here provide extensive molecular resources for sugar beet and enable future high-resolution trait mapping, gene identification, and cross-referencing to regions sequenced in other plant species.

Evolutionary reshuffling in the Errantivirus lineage Elbe within the Beta vulgaris genome.

LTR retrotransposons and retroviruses are closely related. Although a viral envelope gene is found in some LTR retrotransposons and all retroviruses, only the latter show infectivity. The identification of Ty3-gypsy-like retrotransposons possessing putative envelope-like open reading frames blurred the taxonomical borders and led to the establishment of the Errantivirus, Metavirus and Chromovirus genera within the Metaviridae. Only a few plant Errantiviruses have been described, and their evolutionary history is not well understood. In this study, we investigated 27 retroelements of four abundant Elbe retrotransposon families belonging to the Errantiviruses in Beta vulgaris (sugar beet). Retroelements of the Elbe lineage integrated between 0.02 and 5.59 million years ago, and show family-specific variations in autonomy and degree of rearrangements: while Elbe3 members are highly fragmented, often truncated and present in a high number of solo LTRs, Elbe2 members are mainly autonomous. We observed extensive reshuffling of structural motifs across families, leading to the formation of new retrotransposon families. Elbe retrotransposons harbor a typical envelope-like gene, often encoding transmembrane domains. During the course of Elbe evolution, the additional open reading frames have been strongly modified or independently acquired. Taken together, the Elbe lineage serves as retrotransposon model reflecting the various stages in Errantivirus evolution, and allows a detailed analysis of retrotransposon family formation.

Phased grapevine genome sequence of an Rpv12 carrier for biotechnological exploration of resistance to Plasmopara viticola.

The downy mildew disease caused by the oomycete Plasmopara viticola is a serious threat for grapevine and can cause enormous yield losses in viticulture. The quantitative trait locus Rpv12, mediating resistance against P. viticola, was originally found in Asian Vitis amurensis. This locus and its genes were analyzed here in detail. A haplotype-separated genome sequence of the diploid Rpv12-carrier Gf.99-03 was created and annotated. The defense response against P. viticola was investigated in an infection time-course RNA-seq experiment, revealing approximately 600 upregulated Vitis genes during host-pathogen interaction. The Rpv12 regions of the resistance and the sensitivity encoding Gf.99-03 haplotype were structurally and functionally compared with each other. Two different clusters of resistance-related genes were identified within the Rpv12 locus. One cluster carries a set of four differentially expressed genes with three ACCELERATED CELL DEATH 6-like genes. The other cluster carries a set of six resistance gene analogs related to qualitative pathogen resistance. The Rpv12 locus and its candidate genes for P. viticola resistance provide a precious genetic resource for P. viticola resistance breeding. Newly developed co-segregating simple sequence repeat markers in close proximity to the R-genes enable its improved applicability in marker-assisted grapevine breeding.

An improved reference of the grapevine genome reasserts the origin of the PN40024 highly homozygous genotype.

The genome sequence of the diploid and highly homozygous Vitis vinifera genotype PN40024 serves as the reference for many grapevine studies. Despite several improvements to the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, being nearly homozygous, this genome contains several heterozygous regions that are yet to be resolved. Taking the opportunity of improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences, an improved version of the reference, called PN40024.v4, was generated. Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased with a total number decreasing from 2,059 to 640 and a reduction in N bases of 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the number of unplaced scaffolds was reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow for Vitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation of 35,230 genes to date. Finally, we demonstrated that PN40024 resulted from 9 selfings of cv. "Helfensteiner" (cross of cv. "Pinot noir" and "Schiava grossa") instead of a single "Pinot noir". These advances will help maintain the PN40024 genome as a gold-standard reference, also contributing toward the eventual elaboration of the grapevine pangenome.

The complete reference genome for grapevine (Vitis vinifera L.) genetics and breeding.

Grapevine is one of the most economically important crops worldwide. However, the previous versions of the grapevine reference genome tipically consist of thousands of fragments with missing centromeres and telomeres, limiting the accessibility of the repetitive sequences, the centromeric and telomeric regions, and the study of inheritance of important agronomic traits in these regions. Here, we assembled a telomere-to-telomere (T2T) gap-free reference genome for the cultivar PN40024 using PacBio HiFi long reads. The T2T reference genome (PN_T2T) is 69 Mb longer with 9018 more genes identified than the 12X.v0 version. We annotated 67% repetitive sequences, 19 centromeres and 36 telomeres, and incorporated gene annotations of previous versions into the PN_T2T assembly. We detected a total of 377 gene clusters, which showed associations with complex traits, such as aroma and disease resistance. Even though PN40024 derives from nine generations of selfing, we still found nine genomic hotspots of heterozygous sites associated with biological processes, such as the oxidation-reduction process and protein phosphorylation. The fully annotated complete reference genome therefore constitutes an important resource for grapevine genetic studies and breeding programs.

Survey of sugar beet (Beta vulgaris L.) hAT transposons and MITE-like hATpin derivatives.

Genome-wide analyses of repetitive DNA suggest a significant impact particularly of transposable elements on genome size and evolution of virtually all eukaryotic organisms. In this study, we analyzed the abundance and diversity of the hAT transposon superfamily of the sugar beet (B. vulgaris) genome, using molecular, bioinformatic and cytogenetic approaches. We identified 81 transposase-coding sequences, three of which are part of structurally intact but nonfunctional hAT transposons (BvhAT), in a B. vulgaris BAC library as well as in whole genome sequencing-derived data sets. Additionally, 116 complete and 497 truncated non-autonomous BvhAT derivatives lacking the transposase gene were in silico-detected. The 116 complete derivatives were subdivided into four BvhATpin groups each characterized by a distinct terminal inverted repeat motif. Both BvhAT and BvhATpin transposons are specific for species of the genus Beta and closely related species, showing a localization on B. vulgaris chromosomes predominantely in euchromatic regions. The lack of any BvhAT transposase function together with the high degree of degeneration observed for the BvhAT and the BvhATpin genomic fraction contrasts with the abundance and activity of autonomous and non-autonomous hAT transposons revealed in other plant species. This indicates a possible genus-specific structural and functional repression of the hAT transposon superfamily during Beta diversification and evolution.