Generation and characterisation of an Arabidopsis thaliana f3h/fls1/ans triple mutant that accumulates eriodictyol derivatives.

BACKGROUND: Flavonoids are plant specialised metabolites, which derive from phenylalanine and acetate metabolism. They possess a variety of beneficial characteristics for plants and humans. Several modification steps in the synthesis of tricyclic flavonoids cause for the amazing diversity of flavonoids in plants. The 2-oxoglutarate-dependent dioxygenases (2-ODDs) flavanone 3-hydroxylase (F3H, synonym FHT), flavonol synthase (FLS) and anthocyanidin synthase (ANS, synonym leucoanthocyanidin dioxygenase (LDOX)), catalyse oxidative modifications to the central C ring. They are highly similar and have been shown to catalyse, at least in part, each other's reactions. FLS and ANS have been identified as bifunctional enzymes in many species, including Arabidopsis thaliana, stressing the capability of plants to bypass missing or mutated reaction steps on the way to flavonoid production. However, little is known about such bypass reactions and the flavonoid composition of plants lacking all three central flavonoid 2-ODDs. RESULTS: To address this issue, we generated a f3h/fls1/ans mutant, as well as the corresponding double mutants and investigated the flavonoid composition of this mutant collection. The f3h/fls1/ans mutant was further characterised at the genomic level by analysis of a nanopore DNA sequencing generated genome sequence assembly and at the transcriptomic level by RNA-Seq analysis. The mutant collection established, including the novel double mutants f3h/fls1 and f3h/ans, was used to validate and analyse the multifunctionalities of F3H, FLS1, and ANS in planta. Metabolite analyses revealed the accumulation of eriodictyol and additional glycosylated derivatives in mutants carrying the f3h mutant allele, resulting from the conversion of naringenin to eriodictyol by flavonoid 3'-hydroxylase (F3'H) activity. CONCLUSIONS: We describe the in planta multifunctionality of the three central flavonoid 2-ODDs from A. thaliana and identify a bypass in the f3h/fls1/ans triple mutant that leads to the formation of eriodictyol derivatives. As (homo-)eriodictyols are known as bitter taste maskers, the annotated eriodictyol (derivatives) and in particular the observations made on their in planta production, could provide valuable insights for the creation of novel food supplements.

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.

Three R2R3-MYB transcription factors from banana (Musa acuminata) activate structural anthocyanin biosynthesis genes as part of an MBW complex.

OBJECTIVE: Bananas are one of the most popular fruits in the world, providing food security and employment opportunities in several developing countries. Increasing the anthocyanin content of banana fruit could improve the health-promoting properties. Anthocyanin biosynthesis is largely regulated at the transcriptional level. However, relatively little is known about the transcriptional activation of anthocyanin biosynthesis in banana. RESULTS: We analysed the regulatory activity of three Musa acuminata MYBs that were predicted by bioinformatic analysis to transcriptionally regulate anthocyanin biosynthesis in banana. MaMYBA1, MaMYBA2 and MaMYBPA2 did not complement the anthocyanin-deficient phenotype of the Arabidopsis thaliana pap1/pap2 mutant. However, co-transfection experiments in A. thaliana protoplasts showed that MaMYBA1, MaMYBA2 and MaMYBPA2 function as components of a transcription factor complex with a bHLH and WD40 protein, the so called MBW complex, resulting in the activation of the A. thaliana ANTHOCYANIDIN SYNTHASE and DIHYDROFLAVONOL 4-REDUCTASE promoters. The activation potential of MaMYBA1, MaMYBA2 and MaMYBPA2 was increased when combined with the monocot Zea mays bHLH ZmR instead of the dicot AtEGL3. This work paves the path towards decoding the MBW complex-mediated transcriptional activation of anthocyanin biosynthesis in banana. It will also facilitate research towards increased anthocyanin content in banana and other monocot crops.

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.

Mapping-by-Sequencing Reveals Genomic Regions Associated with Seed Quality Parameters in Brassica napus.

Rapeseed (Brassica napus L.) is an important oil crop and has the potential to serve as a highly productive source of protein. This protein exhibits an excellent amino acid composition and has high nutritional value for humans. Seed protein content (SPC) and seed oil content (SOC) are two complex quantitative and polygenic traits which are negatively correlated and assumed to be controlled by additive and epistatic effects. A reduction in seed glucosinolate (GSL) content is desired as GSLs cause a stringent and bitter taste. The goal here was the identification of genomic intervals relevant for seed GSL content and SPC/SOC. Mapping by sequencing (MBS) revealed 30 and 15 new and known genomic intervals associated with seed GSL content and SPC/SOC, respectively. Within these intervals, we identified known but also so far unknown putatively causal genes and sequence variants. A 4 bp insertion in the MYB28 homolog on C09 shows a significant association with a reduction in seed GSL content. This study provides insights into the genetic architecture and potential mechanisms underlying seed quality traits, which will enhance future breeding approaches in B. napus.

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.

Characterization of the Brassica napus Flavonol Synthase Gene Family Reveals Bifunctional Flavonol Synthases.

Flavonol synthase (FLS) is a key enzyme for the formation of flavonols, which are a subclass of the flavonoids. FLS catalyzes the conversion of dihydroflavonols to flavonols. The enzyme belongs to the 2-oxoglutarate-dependent dioxygenases (2-ODD) superfamily. We characterized the FLS gene family of Brassica napus that covers 13 genes, based on the genome sequence of the B. napus cultivar Express 617. The goal was to unravel which BnaFLS genes are relevant for seed flavonol accumulation in the amphidiploid species B. napus. Two BnaFLS1 homeologs were identified and shown to encode bifunctional enzymes. Both exhibit FLS activity as well as flavanone 3-hydroxylase (F3H) activity, which was demonstrated in vivo and in planta. BnaFLS1-1 and -2 are capable of converting flavanones into dihydroflavonols and further into flavonols. Analysis of spatio-temporal transcription patterns revealed similar expression profiles of BnaFLS1 genes. Both are mainly expressed in reproductive organs and co-expressed with the genes encoding early steps of flavonoid biosynthesis. Our results provide novel insights into flavonol biosynthesis in B. napus and contribute information for breeding targets with the aim to modify the flavonol content in rapeseed.

Functional Characterisation of Banana (Musa spp.) 2-Oxoglutarate-Dependent Dioxygenases Involved in Flavonoid Biosynthesis.

Bananas (Musa) are non-grass, monocotyledonous, perennial plants that are well known for their edible fruits. Their cultivation provides food security and employment opportunities in many countries. Banana fruits contain high levels of minerals and phytochemicals, including flavonoids, which are beneficial for human nutrition. To broaden the knowledge on flavonoid biosynthesis in this major crop plant, we aimed to identify and functionally characterise selected structural genes encoding 2-oxoglutarate-dependent dioxygenases, involved in the formation of the flavonoid aglycon. Musa candidates genes predicted to encode flavanone 3-hydroxylase (F3H), flavonol synthase (FLS) and anthocyanidin synthase (ANS) were assayed. Enzymatic functionalities of the recombinant proteins were confirmed in vivo using bioconversion assays. Moreover, transgenic analyses in corresponding Arabidopsis thaliana mutants showed that MusaF3H, MusaFLS and MusaANS were able to complement the respective loss-of-function phenotypes, thus verifying functionality of the enzymes in planta. Knowledge gained from this work provides a new aspect for further research towards genetic engineering of flavonoid biosynthesis in banana fruits to increase their antioxidant activity and nutritional value.

Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis.

BACKGROUND: Experimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades. RESULTS: We sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days. CONCLUSIONS: Long read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations.

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.

A novel, robust and mating-competent Chlamydomonas reinhardtii strain with an enhanced transgene expression capacity for algal biotechnology.

In the future, algae biotechnology could play an important role in sustainable development, especially with regard to the production of valuable chemicals. Among the established laboratory strains with efficient transgene expression, there are none that have demonstrated the required robustness for industrial applications, which generally require growth at larger scale. Here, we created a robust and mating-competent cell line of the green microalga Chlamydomonas reinhardtii, which also possesses a high transgene expression capacity. This strain shows a comparably high resistance to shear stress by accumulating increased amounts of biomass under these conditions. As a proof-of-concept, a high phototrophic productivity of cadaverine from CO2 and nitrate was demonstrated by efficiently expressing a bacterial l-lysine decarboxylase. In contrast to other established strains, this novel chassis strain for phototrophic production schemes is equipped with the traits required for industrial applications, by combining mating-competence, cell wall-mediated robustness and high level transgene expression.

Functional and evolutionary analysis of the Arabidopsis 4R-MYB protein SNAPc4 as part of the SNAP complex.

Transcription initiation of the genes coding for small nuclear RNA (snRNA) has been extensively analyzed in humans and fruit fly, but only a single ortholog of a snRNA-activating protein complex (SNAPc) subunit has so far been characterized in plants. The genome of the model plant Arabidopsis thaliana encodes orthologs of all three core SNAPc subunits, including A. thaliana SNAP complex 4 (AtSNAPc4)-a 4R-MYB-type protein with four-and-a-half adjacent MYB repeat units. We report the conserved role of AtSNAPc4 as subunit of a protein complex involved in snRNA gene transcription and present genetic evidence that AtSNAPc4 is an essential gene in gametophyte and zygote development. We present experimental evidence that the three A. thaliana SNAPc subunits assemble into a SNAP complex and demonstrate the binding of AtSNAPc4 to snRNA promoters. In addition, co-localization studies show a link between AtSNAPc4 accumulation and Cajal bodies, known to aggregate at snRNA gene loci in humans. Moreover, we show the strong evolutionary conservation of single-copy 4R-MYB/SNAPc4 genes in a broad range of eukaryotes and present additional shared protein features besides the MYB domain, suggesting a conservation of the snRNA transcription initiation machinery along the course of the eukaryotic evolution.

Reference-based QUantification Of gene Dispensability (QUOD).

BACKGROUND: Dispensability of genes in a phylogenetic lineage, e.g. a species, genus, or higher-level clade, is gaining relevance as most genome sequencing projects move to a pangenome level. Most analyses classify genes as core genes, which are present in all investigated individual genomes, and dispensable genes, which only occur in a single or a few investigated genomes. The binary classification as 'core' or 'dispensable' is often based on arbitrary cutoffs of presence/absence in the analysed genomes. Even when extended to 'conditionally dispensable', this concept still requires the assignment of genes to distinct groups. RESULTS: Here, we present a new method which overcomes this distinct classification by quantifying gene dispensability and present a dedicated tool for reference-based QUantification Of gene Dispensability (QUOD). As a proof of concept, sequence data of 966 Arabidopsis thaliana accessions (Ath-966) were processed to calculate a gene-specific dispensability score for each gene based on normalised coverage in read mappings. We validated this score by comparison of highly conserved Benchmarking Universal Single Copy Orthologs (BUSCOs) to all other genes. The average scores of BUSCOs were significantly lower than the scores of non-BUSCOs. Analysis of variation demonstrated lower variation values between replicates of a single accession than between iteratively, randomly selected accessions from the whole dataset Ath-966. Functional investigations revealed defense and antimicrobial response genes among the genes with high-dispensability scores. CONCLUSIONS: Instead of classifying a gene as core or dispensable, QUOD assigns a dispensability score to each gene. Hence, QUOD facilitates the identification of candidate dispensable genes, associated with high dispensability scores, which often underlie lineage-specific adaptation to varying environmental conditions.

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.

RNA-Seq Time Series of Vitis vinifera Bud Development Reveals Correlation of Expression Patterns with the Local Temperature Profile.

Plants display sophisticated mechanisms to tolerate challenging environmental conditions and need to manage their ontogenesis in parallel. Here, we set out to generate an RNA-Seq time series dataset throughout grapevine (Vitis vinifera) early bud development. The expression of the developmental regulator VviAP1 served as an indicator of the progression of development. We investigated the impact of changing temperatures on gene expression levels during the time series and detected a correlation between increased temperatures and a high expression level of genes encoding heat-shock proteins. The dataset also allowed the exemplary investigation of expression patterns of genes from three transcription factor (TF) gene families, namely MADS-box, WRKY, and R2R3-MYB genes. Inspection of the expression profiles from all three TF gene families indicated that a switch in the developmental program takes place in July which coincides with increased expression of the bud dormancy marker gene VviDRM1.

The R2R3-MYB gene family in banana (Musa acuminata): Genome-wide identification, classification and expression patterns.

The R2R3-MYB genes comprise one of the largest transcription factor gene families in plants, playing regulatory roles in plant-specific developmental processes, defense responses and metabolite accumulation. To date MYB family genes have not yet been comprehensively identified in the major staple fruit crop banana. In this study, we present a comprehensive, genome-wide analysis of the MYB genes from Musa acuminata DH-Pahang (A genome). A total of 285 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. Organ- and development-specific expression patterns were determined from RNA-Seq data. For 280 M. acuminata MYB genes for which expression was found in at least one of the analysed samples, a variety of expression patterns were detected. The M. acuminata R2R3-MYB genes were functionally categorised, leading to the identification of seven clades containing only M. acuminata R2R3-MYBs. The encoded proteins may have specialised functions that were acquired or expanded in Musa during genome evolution. This functional classification and expression analysis of the MYB gene family in banana establishes a solid foundation for future comprehensive functional analysis of MaMYBs and can be utilized in banana improvement programmes.

Genome Sequences of Both Organelles of the Grapevine Rootstock Cultivar 'Börner'.

Genomic long reads of the interspecific grapevine rootstock cultivar 'Börner' (Vitis riparia GM183 × Vitis cinerea Arnold) were used to assemble its chloroplast and mitochondrion genome sequences. We annotated 133 chloroplast and 172 mitochondrial genes, including the RNA editing sites. The organelle genomes in 'Börner' were maternally inherited from Vitis riparia.

High Contiguity De Novo Genome Sequence Assembly of Trifoliate Yam (Dioscorea dumetorum) Using Long Read Sequencing.

Trifoliate yam (Dioscorea dumetorum) is one example of an orphan crop, not traded internationally. Post-harvest hardening of the tubers of this species starts within 24 h after harvesting and renders the tubers inedible. Genomic resources are required for D. dumetorum to improve breeding for non-hardening varieties as well as for other traits. We sequenced the D. dumetorum genome and generated the corresponding annotation. The two haplophases of this highly heterozygous genome were separated to a large extent. The assembly represents 485 Mbp of the genome with an N50 of over 3.2 Mbp. A total of 35,269 protein-encoding gene models as well as 9941 non-coding RNA genes were predicted, and functional annotations were assigned.

A Partially Phase-Separated Genome Sequence Assembly of the Vitis Rootstock 'Börner' (Vitis riparia × Vitis cinerea) and Its Exploitation for Marker Development and Targeted Mapping.

Grapevine breeding has become highly relevant due to upcoming challenges like climate change, a decrease in the number of available fungicides, increasing public concern about plant protection, and the demand for a sustainable production. Downy mildew caused by Plasmopara viticola is one of the most devastating diseases worldwide of cultivated Vitis vinifera. In modern breeding programs, therefore, genetic marker technologies and genomic data are used to develop new cultivars with defined and stacked resistance loci. Potential sources of resistance are wild species of American or Asian origin. The interspecific hybrid of Vitis riparia Gm 183 x Vitis cinerea Arnold, available as the rootstock cultivar 'Börner,' carries several relevant resistance loci. We applied next-generation sequencing to enable the reliable identification of simple sequence repeats (SSR), and we also generated a draft genome sequence assembly of 'Börner' to access genome-wide sequence variations in a comprehensive and highly reliable way. These data were used to cover the 'Börner' genome with genetic marker positions. A subset of these marker positions was used for targeted mapping of the P. viticola resistance locus, Rpv14, to validate the marker position list. Based on the reference genome sequence PN40024, the position of this resistance locus can be narrowed down to less than 0.5 Mbp on chromosome 5.