Comparative metabolic profiling of new genetic multiple flavonoid r2r3-myb mutants show that different types of R2R3–MYBs activate the early flavonoid biosynthesis genes. A revised model for flavonoid biosynthesis in Arabidopsis thaliana is proposed that integrates the regulatory roles of these R2R3–MYBs across early and late biosynthetic steps. Flavonoids are a large group of specialized plant metabolites. Their biosynthesis is mainly transcriptionally regulated by a sophisticated network of different transcription factors from various families, with R2R3–MYB factors being the main determinant of specific flavonoid class formation. The early biosynthetic steps, leading to the formation of non-visible flavonoids, have been proposed to be regulated by three R2R3–MYBs, PRODUCTION OF FLAVONOL GYLCOSIDE1-3 (PFG1-3), while the later biosynthetic steps leading to the formation of visible anthocyanin and proanthocyanidin pigments are reported to be regulated by four R2R3–MYBs, PRODUCTION OF ANTHOCYANIN PIGMENT1-4 (PAP1-4) and TRANSPARENT TESTA2 (TT2), respectively. Several studies have indicated that this model for the transcriptional regulation of flavonoid biosynthesis may be incomplete. To address this issue, especially regarding the regulation of the early biosynthesis genes by PAP1-4 and TT2, we generated several multiple r2r3-myb mutant lines. We characterized the pfg1-3, pfg1-3 tt2 and pfg1-3 pap1-4 mutants and did comparative metabolite profiling. This revealed that only the pfg1-3 tt2 mutant was deficient in proanthocyanidins and only the pfg1-3 pap1-4 mutant was deficient in anthocyanins. We demonstrate that PAP and TT2 R2R3–MYBs are also capable of activating the early biosynthesis genes required for dihydroflavonol formation. Our results provide evidence that the traditional view of distinct branch-specific R2R3–MYB regulators in flavonoid biosynthesis is overly simplistic. We, therefore, propose a revised model for the transcriptional regulation of flavonoid biosynthesis.

Flavonoids are specialized metabolites that play important roles in plants, including interactions with the environment. The high structural diversity of this metabolite group is largely due to enzyme-mediated modifications of flavonoid core skeletons. In particular, glycosylation with different sugars is very common. In this study, the functions of the Arabidopsis thaliana glycoside hydrolase family 1-type glycosyltransferase proteins BGLU1, BGLU3, and BGLU4 were investigated, using a reverse genetics approach and untargeted metabolic fingerprinting. We screened for metabolic differences between A. thaliana wild type, loss-of-function mutants, and overexpression lines and partially identified differentially accumulating metabolites, which are putative products and/or substrates of the BGLU enzymes. Our study revealed that the investigated BGLU proteins are glycosyltransferases involved in the glycosylation of already glycosylated flavonoids using different substrates. While BGLU1 appears to be involved in the rhamnosylation of a kaempferol diglycoside in leaves, BGLU3 and BGLU4 are likely involved in the glycosylation of quercetin diglycosides in A. thaliana seeds. In addition, we present evidence that BGLU3 is a multifunctional enzyme that catalyzes other metabolic reactions with more complex substrates. This study deepens our understanding of the metabolic pathways and enzymes that contribute to the high structural diversity of flavonoids.

Transcription factors control gene expression during development and in response to a broad range of internal and external stimuli. They regulate promoter activity by directly binding cis-regulatory elements in DNA. The angiosperm Arabidopsis (Arabidopsis thaliana) contains more than 1,500 annotated transcription factors, each containing a DNA-binding domain that is used to define transcription factor families. Analyzing the binding motifs of 686 and the binding sites of 335 Arabidopsis transcription factors, as well as motifs of 92 transcription factors from other plants, we identified a constrained vocabulary of 74 conserved motifs spanning 50 families in plants. Among 21 transcription factor families, we found 1 core motif for all analyzed members and between 2% and 72% overlapping binding sites. Five families show conservation of the motif along phylogenetic clades. Five families, including the C2H2 zinc finger family, show high diversity among motifs in plants, suggesting potential for the neofunctionalization of duplicated transcription factors based on the motif recognized. We tested whether conserved motifs remained conserved since at least 450 million years ago by determining the binding motifs of 17 transcription factors from 11 families in Marchantia (Marchantia polymorpha) using amplified DNA affinity purification sequencing. We detected nearly identical binding motifs as predicted from the angiosperm data. Our findings show a large repertoire of overlapping binding sites within a transcription factor family and species and a high degree of binding motif conservation for at least 450 million years, indicating more potential for evolution in cis- rather than trans-regulatory elements.

Drought is a major factor of yield loss in annual crops. It triggers a wide range of physiological and molecular changes which are mediated by a suite of transcription factors from at least four different protein families. In the literature, the observed phenotypic changes and the number of transcriptome changes upon drought in Arabidopsis thaliana varies widely. To resolve the apparent variation, we conducted a phenotyping and transcriptomics experiment with progressive drought which is initially mild and escalates to strong but still sublethal drought followed by rewatering. Phenotypic data is analyzed with machine learning methods and connected to transcriptome data. The phenotypic data show that drought stress manifests in distinct stages. The transcriptional analysis shows one threshold program and gradual expression programs among typical drought responsive regulons over time and during recovery. Plant aging prior to senescence and the drought response overlap to a large degree and drought stressed plants rejuvenate transcriptionally before returning to the control aging program. The phenotypic traits are associated with different transcript abundances again reflecting multiple overlaying programs. Transcripts with high explanatory power of phenotypes are biomarkers and not causal for the phenotype. Significance statement Large scale phenotyping and transcriptomics during progressive drought demonstrate that the drought response in the annual Arabidopsis thaliana is controlled by multiple transcriptional programs, mostly with gradual onset, that overlap with the plant aging program. Transcripts with high explanatory power for phenotypes are biomarkers and not causal. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Objectives Despite the advances in genomics, repetitive DNAs (repeats) are still difficult to sequence, assemble, and identify. This is due to their high abundance and diversity, with many repeat families being unique to the organisms in which they were described. In sugar beet, repeats make up a significant portion of the genome (at least 53%), with many repeats being restricted to the beet genera, Beta and Patellifolia. Over the course of over 30 years and many repeat-based studies, over a thousand reference repeat sequences for beet genomes have been identified and many experimentally characterized (e.g. physically located on the chromosomes). Here, we present the collection of these reference repeat sequences for beets. Data description The BeetRepeats_v1.0 resource is a comprehensive compilation of all characterized repeat families, including satellite DNAs, ribosomal DNAs, transposable elements and endogenous viruses. The genomes covered are those of sugar beet and closely related wild beets (genera Beta and Patellifolia) as well as Chenopodium quinoa and Spinacia oleracea (all belonging to the Amaranthaceae). The reference sequences are in fasta format and comprise well-characterized repeats from both repeat categories (dispersed/mobile as well as tandemly arranged). The database is suitable for the RepeatMasker and RepeatExplorer2 pipelines and can be used directly for any repeat annotation and repeat polymorphism detection purposes.

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.

Hydrangea serrata, also knowen as the Japanese tea hortensia, is known for its sweet taste and health properties of bevarages produced from this plant. The H. serrata 3,4-dihydroisocoumarins, hydrangenol and phyllodulcin harbour a variety of biological activities and pharmacological properties. Therefore, a detailed understanding of dihydroisocoumarin biosynthesis in H. serrata is of major interest. Their biosynthesis is assumed to be enhanced by elicitors and mediated by polyketide synthases like in cases of phenylpropanoid derived phytoalexins. A de-novo transcriptome assembly of leaves and roots from the aluminium chloride treatment group versus the control group alongside with annotation was generated. Secondary plant metabolites were analysed by LC-MS. It revealed that a terpene synthase and a triterpenoid synthase gene as well as lignin biosynthesis encoding genes were upregulated in roots. Many genes for transporters, glycosyl, and other transferases as well as glycosylases were found to be differentially expressed in both organs. As no differentially expressed polyketide synthase gene homolog was found, the relative leaf and root 3,4-dihydroisocoumarin content was analysed by LC-MS measurement. Although Hydrangea species are known for their aluminium detoxification using phenylpropanoid-derived compounds, the levels of 3,4- dihydroisocoumarins were not enhanced. In this metabolite analysis, an organ- specific accumulation profile of hydrangenol, phyllodulcin, hydrangeic acid and their mono- and di-glycosides was figured out.

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.

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.

Predicting gene expression from promoter sequence requires understanding of the different signal integration points within a promoter. Sequence-specific transcription factors (TFs) binding to their cognate TF binding motifs control gene expression in eukaryotes by activating and repressing transcription. Their interplay generates complex expression patterns in reaction to environmental conditions and developmental cues. We hypothesized that signals are not only integrated by different TFs binding various positions in a promoter, but also by single TF binding motifs onto which multiple TFs can bind. Analyzing 2,190 binding motifs, we identified only 76 core TF binding motifs in plants. Twenty-one TF protein families act highly specific and bind a single conserved motif. Four TF families are classified as semi-conserved as they bind up to four motifs within a family, with divisions along phylogenetic groups. Five TF families bind diverse motifs. Expression analyses revealed high competition within TF families for the same binding motif. The results show that singular binding motifs act as signal integrators in plants where a combination of binding affinity and TF abundance likely determine the output.

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.

Plasmopara viticola, the grapevine downy mildew pathogen, causes severe losses in viticulture if not counteracted by fungicide sprays that need to be repeatedly applied during each growing season. To reduce the amount of plant protection, modern grapevine breeding generates fungus-resistant grapevine cultivars by introgression of resistance loci from wild Vitis spec. sources. However, the presence of only a single resistance locus may provoke the emergence of pathogen races able to overcome the resistance trait of the host. Therefore, a combination of several, independently acting resistance loci is required for sustainable genetic resistance. Quite little is known about the resistance-conferring genes within the various grapevine resistance loci. To ameliorate this situation and make stacking of resistance loci more efficient, the Rpv12 locus originating from the Asian Vitis amurensis was sequenced and characterized. The complete genome of breeding line Gf.99-03, carrying Rpv12 in heterozygous state, was analyzed. Haplotypes were resolved by assigning the reads to one of the parents of Gf.99-03 using trio binning. Annotation of the resulting genomic sequences was based on RNA-Seq data and predicted gene models.The haplotype carrying the Rpv12 locus, delimited by markers UDV-014 and UDV-370 on chromosome 14 (Venuti et al., 2013), diverges strongly from the susceptible haplotype as well as from the reference genome PN40024 12X.v2. It was found to contain two important gene clusters. One cluster includes pathogen-inducible genes similar to the gene ACCELERATED CELL DEATH 6 (A. thaliana) likely involved in hypersensitive response upon pathogen attack. The second cluster comprises positional resistance candidate genes corresponding to typical NLRs (nucleotide binding site, leucine rich repeats), hypothesized to be involved in pathogen perception and cellular defense signalling.

Most crop plants, including sugar beet ( Beta vulgaris subsp. vulgaris ), suffer from domestication bottlenecks and low genetic diversity caused by extensive selection for few traits. However, crop wild relatives (CWRs) harbour useful traits relevant for crop improvement, including enhanced adaptation to biotic and abiotic stresses. Especially polyploids are interesting from an evolutionary perspective as genes undergo reorganisation after the polyploidisation event. Through neo-and subfunctionalisation, novel functions emerge, which enable plants to cope with changing environments and extreme/harsh conditions. Particularly in the face of climate change, specific stress and pathogen resistances or tolerances gain importance. To introduce such traits into breeding material, CWRs have already been identified as an important source for sustainable breeding. The identification of genes underlying traits of interest is crucial for crop improvement. For beets, the section Corollinae contains the tetraploid species Beta corolliflora (2n=4x=36) that harbours salt and frost tolerances as well as a wealth of pathogen resistances. The number of beneficial traits of B. corolliflora is increased compared to those of the known diploids in this section (all 2n=2x=18). Nevertheless, neither the parental relationships of B. corolliflora have been resolved, nor are genomic resources available to steer sustainable, genomics-informed breeding. To benefit from the resources offered by polyploid beet wild relatives, we generated a comprehensive pangenome dataset including B. corolliflora , Beta lomatogona , and Beta macrorhiza , as well as a more distant wild beet Patellifolia procumbens (2n=2x=18). Joined analyses with publicly available genome sequences of two additional wild beets allowed the identification of genomic regions absent from cultivated beet, providing a sequence database harbouring traits relevant for future breeding endeavours. In addition, we present strong evidence for the parental relationship of the B. corolliflora wild beet as an autotetraploid emerging from B. macrorhiza .

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.

AbstractThe downy mildew disease caused by the oomycetePlasmopara viticolais a serious threat for grapevine and can cause enormous yield losses in viticulture. The quantitative trait locusRpv12,mediating resistance againstP. viticola, was originally found in AsianVitis amurensis. This locus and its genes were analyzed here in detail. A haplotype-separated genome sequence of the diploidRpv12-carrier Gf.99-03 was created and annotated. The defense response againstP. viticolawas investigated in an infection time-course RNA-Seq experiment, revealing approximately 600 up-regulatedVitisgenes during host-pathogen interaction. TheRpv12regions of the resistance conferring and the sensitivity encoding Gf.99-03 haplotypes were structurally and functionally compared to each other. Two different clusters of resistance-related genes were identified within theRpv12locus. One cluster carries a set of four differentially expressed genes with threeACCELERATED CELL DEATH 6-like genes. The other cluster carries a set of six resistance gene analogues related to qualitative pathogen resistance. TheRpv12locus and its candidate genes forP. viticolaresistance provide a precious genetic resource forP. viticolaresistance breeding. Newly developed co-segregating simple sequence repeat markers in close proximity to theR-genes enable its improved applicability in marker-assisted grapevine breeding.

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.

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.

AbstractThe phylloxera resistant rootstock cultivar ‘Börner’ is an interspecific hybrid derived fromVitis ripariaandV. cinereaand a valuable resource forVitisdisease resistances. We created a fully phased, high-quality ‘Börner’ genome sequence named BoeRC using long PacBio reads. Comprehensive gene annotation of both ‘Börner’ haplotypes, designated BoeRip and BoeCin, was applied to describe the phylloxera resistance locusRdv1. Using a mapping population derived from a susceptibleV. viniferabreeding line and ‘Börner’, theRdv1locus was further delimited.Rdv1, which is derived fromV. cinereaand included in the haplotype BoeCin, was compared with sequences of phylloxera-susceptible and phylloxera-tolerant cultivars. Between flanking regions that display high synteny, we detected and precisely characterized a diverse sequence region that covers between 202 to 403 kbp in different haplotypes. In BoeCin, five putative disease resistance genes were identified that represent likely candidates for conferring resistance to phylloxera.

The phylloxera resistant rootstock cultivar ‘Börner’ is an interspecific hybrid derived from Vitis riparia and V. cinerea and a valuable resource for Vitis disease resistances. We created a fully phased, high-quality ‘Börner’ genome sequence named BoeRC using long PacBio reads. Comprehensive gene annotation of both ‘Börner’ haplotypes, designated BoeRip and BoeCin, was applied to describe the phylloxera resistance locus Rdv1 . Using a mapping population derived from a susceptible V. vinifera breeding line and ‘Börner’, the Rdv1 locus was further delimited. Rdv1 , which is derived from V. cinerea and included in the haplotype BoeCin, was compared with sequences of phylloxera-susceptible and phylloxera-tolerant cultivars. Between flanking regions that display high synteny, we detected and precisely characterized a diverse sequence region that covers between 202 to 403 kbp in different haplotypes. In BoeCin, five putative disease resistance genes were identified that represent likely candidates for conferring resistance to phylloxera.### Competing Interest StatementThe authors have declared no competing interest.

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.