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

Siadjeu C, Pucker B, Viehöver P, Albach DC, Weisshaar B. High Contiguity De Novo Genome Sequence Assembly of Trifoliate Yam (Dioscorea dumetorum) Using Long Read Sequencing. Genes. 2020;11(3): 274.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

Natural variation in flavonol accumulation in Arabidopsis is determined by the flavonol glucosyltransferase BGLU6.

Ishihara H, Tohge T, Viehöver P, Fernie AR, Weisshaar B, Stracke R. Natural variation in flavonol accumulation in Arabidopsis is determined by the flavonol glucosyltransferase BGLU6. Journal of Experimental Botany. 2016;67(5):1505-1517

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

Ries D, Holtgräwe D, Viehöver P, Weisshaar B. Rapid gene identification in sugar beet using deep sequencing of DNA from phenotypic pools selected from breeding panels. BMC Genomics. 2016;17(1): 236.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

Genome Sequences of Both Organelles of the Grapevine Rootstock Cultivar ‘Börner’

Frommer B, Holtgräwe D, Hausmann L, et al. Genome Sequences of Both Organelles of the Grapevine Rootstock Cultivar ‘Börner’. Microbiology Resource Announcements. 2020;9(15): e01471-19.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

Analysis of the Rpv12 locus in a haplotype‑separated grapevine genome sequence

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

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

Holtgräwe D, Rosleff Soerensen T, Hausmann L, et al. 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. Frontiers in Plant Science. 2020;11: 156.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

Characterization of genes and alleles involved in the control of flowering time in grapevine.

Kamal N, Ochßner I, Schwandner A, et al. Characterization of genes and alleles involved in the control of flowering time in grapevine. PLoS One. 2019;14(7): e0214703.Grapevine (Vitis vinifera) is one of the most important perennial crop plants in worldwide. Understanding of developmental processes like flowering, which impact quality and quantity of yield in this species is therefore of high interest. This gets even more important when considering some of the expected consequences of climate change. Earlier bud burst and flowering, for example, may result in yield loss due to spring frost. Berry ripening under higher temperatures will impact wine quality. Knowledge of interactions between a genotype or allele combination and the environment can be used for the breeding of genotypes that are better adapted to new climatic conditions. To this end, we have generated a list of more than 500 candidate genes that may play a role in the timing of flowering. The grapevine genome was exploited for flowering time control gene homologs on the basis of functional data from model organisms like A. thaliana. In a previous study, a mapping population derived from early flowering GF.GA-47-42 and late flowering ‘Villard Blanc’ was analyzed for flowering time QTLs. In a second step we have now established a workflow combining amplicon sequencing and bioinformatics to follow alleles of selected candidate genes in the F1 individuals and the parental genotypes. Allele combinations of these genes in individuals of the mapping population were correlated with early or late flowering phenotypes. Specific allele combinations of flowering time candidate genes within and outside of the QTL regions for flowering time on chromosome 1, 4, 14, 17, and 18 were found to be associated with an early flowering phenotype. In addition, expression of many of the flowering candidate genes was analyzed over consecutive stages of bud and inflorescence development indicating functional roles of these genes in the flowering control network

Twenty-Five Years of Propagation in Suspension Cell Culture Results in Substantial Alterations of the Arabidopsis Thaliana Genome

Pucker B, Rückert C, Stracke R, Viehöver P, Kalinowski J, Weisshaar B. Twenty-Five Years of Propagation in Suspension Cell Culture Results in Substantial Alterations of the Arabidopsis Thaliana Genome. Genes. 2019;10(9): 671.Arabidopsis thaliana is one of the best studied plant model organisms. Besides cultivation in greenhouses, cells of this plant can also be propagated in suspension cell culture. At7 is one such cell line that was established about 25 years ago. Here, we report the sequencing and the analysis of the At7 genome. Large scale duplications and deletions compared to the Columbia-0 (Col-0) reference sequence were detected. The number of deletions exceeds the number of insertions, thus indicating that a haploid genome size reduction is ongoing. Patterns of small sequence variants differ from the ones observed between A. thaliana accessions, e.g., the number of single nucleotide variants matches the number of insertions/deletions. RNA-Seq analysis reveals that disrupted alleles are less frequent in the transcriptome than the native ones

A de novo Genome Sequence Assembly of the Arabidopsis thaliana Accession Niederzenz-1 Displays Presence/Absence Variation and Strong Synteny

Pucker B, Holtgräwe D, Rosleff Sörensen T, Stracke R, Viehöver P, Weisshaar B. A de novo Genome Sequence Assembly of the Arabidopsis thaliana Accession Niederzenz-1 Displays Presence/Absence Variation and Strong Synteny. PLoS One. 2016;11(10): e0164321.Arabidopsis thaliana is the most important model organism for fundamental plant biology. The genome diversity of different accessions of this species has been intensively studied, for example in the 1001 genome project which led to the identification of many small nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). In addition, presence/absence variation (PAV), copy number variation (CNV) and mobile genetic elements contribute to genomic differences between A. thaliana accessions. To address larger genome rearrangements between the A. thaliana reference accession Columbia-0 (Col-0) and another accession of about average distance to Col-0, we created a de novo next generation sequencing (NGS)-based assembly from the accession Niederzenz-1 (Nd-1). The result was evaluated with respect to assembly strategy and synteny to Col-0. We provide a high quality genome sequence of the A. thaliana accession (Nd-1, LXSY01000000). The assembly displays an N50 of 0.590 Mbp and covers 99% of the Col-0 reference sequence. Scaffolds from the de novo assembly were positioned on the basis of sequence similarity to the reference. Errors in this automatic scaffold anchoring were manually corrected based on analyzing reciprocal best BLAST hits (RBHs) of genes. Comparison of the final Nd-1 assembly to the reference revealed duplications and deletions (PAV). We identified 826 insertions and 746 deletions in Nd-1. Randomly selected candidates of PAV were experimentally validated. Our Nd-1 de novo assembly allowed reliable identification of larger genic and intergenic variants, which was difficult or error-prone by short read mapping approaches alone. While overall sequence similarity as well as synteny is very high, we detected short and larger (affecting more than 100 bp) differences between Col-0 and Nd-1 based on bi-directional comparisons. The de novo assembly provided here and additional assemblies that will certainly be published in the future will allow to describe the pan-genome of A. thaliana

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

Funder: Universität Bielefeld (3146)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