Epigenetic modifications, such as DNA methylation, influence phenotypic plasticity and affect numerous plant traits. Genome-wide DNA methylation patterns differ among cell types, species, and developmental stages. Still, it remains poorly understood in non-model plants. Thus, the potential of epigenetic breeding approaches targeting DNA methylation in crop plants is not fully realised. This study focuses on sugar beet (Beta vulgaris L. ssp. vulgaris) and comprises the first long read-based reference DNA methylome for this species, generated using Oxford Nanopore Technologies (ONT) sequencing.The detection of 5-methyl cytosine (5mC) in the three sequence contexts (CG, CHG, and CHH) was performed with DeepSignal-plant. The 5mCartograph tool was developed to provide a detailed overview of 5mC methylation probabilities: A genome-wide 5mC reference methylome was established for the genotype KWS2320, including gene- and repeat-specific analyses and the identification of 2,088 gene body methylated (gbM) genes, while 10,839 genes remained unmethylated.Genome-wide analysis re-detected more than 99% of the 204.8 Mio reference cytosines, based on ONT read sets of at least 17.5 × mapped genome coverage. Of these cytosines, 14.5% were classified as ‘highly methylated’ in young sugar beet leaves. Methylation levels followed typical plant patterns, being highest in CG (89.8%), followed by CHG (62.8%) and CHH (10.1%) context.This detailed methylome provides a robust foundation for future studies - such as epi-pangenome generation - and supports potential breeding applications in crop improvement.

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.

Crassulacean acid metabolism (CAM) is an adaptation to environments where water availability is seasonal or extremely low. It serves to ensure plant survival and/or maintain productivity in these adverse environments. CAM has repeatedly evolved in many plant lineages, although it requires a large and complex set of enzymes, transporters, and regulatory processes to control metabolite flux and pools. To test the potential levels at which CAM is regulated, we analyzed the CAM plant Kalanchoë laxiflora and compared with the genomes and transcriptomes of other CAM plants across a wide phylogenetic range. We show that CAM-associated transcripts and proteins did not exhibit a binary on/off pattern in abundance between day and night in K. laxiflora. Instead, K. laxiflora and many CAM plants displayed shared amino acid changes among proteins compared to C3 plants, especially in starch metabolism. Phosphoproteomics identified differential phosphorylation in K. laxiflora proteins between day and night. Taken together, our results demonstrate that CAM photosynthesis is regulated at both the transcript and protein levels.

Light as a substrate for photosynthesis may be a boon or a bane. To thrive, photosynthetic organisms must constantly respond to changing light and CO2 conditions by balancing energy harvest and consumption in a highly dynamic way. Two major safeguard measures of photoacclimation, that is photoprotection and carbon concentrating mechanism, underlie tight transcriptional control, leading to expression changes under high light and limited CO2 with different dynamics for both systems. Here, by using a consensus gene regulatory network inferred by employing a compendium of 1,869 RNA-seq datasets, we identified and validated in vivo eight candidate transcription factors (TFs) that contribute to photoacclimation in Chlamydomonas reinhardtii . Target gene analyses indicate that the TFs act individually in associated pathways but also influence each other in expression, and function as network parts with partial redundancy with respect to photoprotection. The analyses unveil that stress responses in Chlamydomonas are mediated by a complex, interconnected network of TFs rather than a hierarchical system where multiple regulators can influence each other and target gene expression and thereby mitigate the effects of loss.

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.

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.

Photosynthesis by which plants convert carbon dioxide to sugars using the energy of light is fundamental to life as it forms the basis of nearly all food chains. Surprisingly, our knowledge about its transcriptional regulation remains incomplete. Effort for its agricultural optimization have mostly focused on post-translational regulatory processes 1–3 but photosynthesis is regulated at the post-transcriptional 4 and the transcriptional level 5 . Stacked transcription factor mutations remain photosynthetically active 5,6 and additional transcription factors have been difficult to identify possibly due to redundancy 6 or lethality. Using a random forest decision tree-based machine learning approach for gene regulatory network calculation 7 we determined ranked candidate transcription factors and validated five out of five tested transcription factors as controlling photosynthesis in vivo . The detailed analyses of previously published and newly identified transcription factors suggest that photosynthesis is transcriptionally regulated in a partitioned, non-hierarchical, interlooped network.

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 .

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.

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 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.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Polyploidization, the increase in genome copies, is considered a major driving force for speciation. We have recently provided the first direct in planta evidence for polyspermy induced polyploidization. Capitalizing on a novel sco1-based polyspermy assay, we here show that polyspermy can selectively polyploidize the egg cell, while rendering the genome size of the ploidy-sensitive central cell unaffected. This unprecedented result indicates that polyspermy can bypass the triploid block, which is an established postzygotic polyploidization barrier. In fact, we here show that most polyspermy-derived seeds are insensitive to the triploid block suppressor admetos. The robustness of polyspermy-derived plants is evidenced by the first transcript profiling of triparental plants and our observation that these idiosyncratic organisms segregate tetraploid offspring within a single generation. Polyspermy-derived triparental plants are thus comparable to triploids recovered from interploidy crosses. Our results expand current polyploidization concepts and have important implications for plant breeding. eLife digest Ever since Darwin published his most famous book on the theory of evolution, scientists have sought to identify the mechanisms that drive the formation of new species. This is especially true for plant biologists who have long been fascinated by the extraordinary diversity of flowering plants. Many species of flowering plant first evolved after a dramatic increase in the DNA content of an individual plant, a process termed polyploidization. Most explanations for polyploidization involve a pollen grain making sperm that mistakenly contain two sets of chromosomes rather than one. Yet, it is difficult to reconcile this explanation with an important aspect of plant reproduction – the so-called “triploid block”. Fertilization in flowering plants is more complicated than in animals. While one sperm fertilizes the egg cell to make the plant embryo, a second sperm from the same pollen grain must fertilize another cell to form the endosperm, the tissue that will nourish the embryo as it develops. This means that sperm with twice the normal number of chromosomes would affect the DNA content of both the embryo and the endosperm. Yet, an endosperm that receives extra paternal DNA typically halts the development of the seed via a process known as the triploid block, meaning it was not clear how often this process would actually result in a polyploid plant. In 2017, researchers reported that plants can, on rare occasions, generate polyploid offspring via a different route: the fertilization of one egg with two sperm rather than one. Now, Mao et al. – who include several researchers involved in the 2017 study – show that this process, termed “polyspermy”, can introduce extra copies of DNA into just the egg cell, meaning it can bypass the triploid block of the endosperm. The experiments involved a model plant called Arabidopsis, and a screen of over 55,000 seeds identified about a dozen with embryos that had three parents, one mother and two fathers. Notably, most of these three-parent embryos developed in seeds that contained endosperm with the regular number of chromosomes and hence escaped the triploid block. These new results show that polyspermy provides plants with a means to essentially sneak extra copies of DNA ‘behind the back’ of the DNA-sensitive endosperm and into the next generation. They also give new insight in how polyploidization may have shaped the evolution of flowering plants and have important implications for agriculture where the breeding of new “hybrid” crops has often been limited by incompatibilities in the endosperm. Introduction The evolutionary history of flowering plants is characterized by recurrent polyploidization events (Comai, 2005; De Bodt et al., 2005; Otto and Whitton, 2000; Van de Peer et al., 2017). Polyploids are generally assumed to arise from unreduced gametes or somatic doubling, i.e. from defects during meiosis or mitosis (Kreiner et al., 2017; Mason and Pires, 2015; Ramsey and Schemske, 1998; Sattler et al., 2016; Spoelhof et al., 2017; Tayalé and Parisod, 2013). In addition, recent in planta and in vitro assays have provided the first direct evidence that viable polyploid plants can arise from polyspermy, the fusion of one egg cell with supernumerary sperm (Nakel et al., 2017; Toda et al., 2016). In fact, this previous work indicates that a single Arabidopsis plant can generate several polyspermy-induced triploid seedlings (Nakel et al., 2017). The currently favored polyploidization scenario involves the formation of unreduced male gametes and the natural occurrence of such sperm has been reported for several species (Kreiner et al., 2017; Mason and Pires, 2015; Ramsey, 2007). Consequently, triploid plants are assumed to function as an important bridge towards polyploidization (Comai, 2005; Felber and Bever, 1997; Ramsey and Schemske, 1998) and field studies have identified both auto and allopolyploid triploids (Kyrkjeeide et al., 2019; Lee et al., 2001; Marques et al., 2018; Meng et al., 2018; Schinkel et al., 2017; Sree Rangasamy, 1972). However, the generation of triploid plants via unreduced male gametes is limited by the triploid block, which is a postzygotic hybridization barrier operating in many plants species (Dilkes et al., 2008; Köhler et al., 2010; Marks, 1966; Ramsey and Schemske, 1998; Scott et al., 1998). The triploid block is explained by the unique reproductive mode of flowering plants, which involves fertilization of two female gametes, the egg and the central cell. The required sperm cell pair is typically delivered by a single pollen tube. While the fertilized egg cell gives rise to the embryo, the fertilized central cell develops into embryo-nourishing endosperm (Russell, 1992). Fertilization involving unreduced sperm consequently not only affects the ploidy status of the egg cell but also introduces additional paternal chromosome copies to the endosperm, and it is this latter tissue, which commonly mounts the triploid block that is manifested by seed abortion (Köhler et al., 2010). In Arabidopsis thaliana, the effect of the triploid block is accession-dependent, being highly penetrant e.g. in Col-0, but less strict in Ler and C24 (Dilkes et al., 2008; Scott et al., 1998). A complete triploid block has been reported in many taxa (Ramsey and Schemske, 1998; Schinkel et al., 2017; Sekine et al., 2013; Stoute et al., 2012). In light of this fatal consequence, it has been suggested that there are ways to overcome this hybridization barrier (Köhler et al., 2010). Making use of a two-component in planta assay, we here show that polyspermy can selectively polyploidize the egg cell, while rendering the genome size of the ploidy-sensitive endosperm unaffected. By introducing the triploid block suppressor admetos, we in addition show, that this unprecedented reproductive mode bypasses the triploid block. Results Establishment of a triparental embryo detection assay Consistent with animal nomenclature, the term polyspermy is used alone when referring to egg cell polyspermy. Central cell polyspermy is specified as such. During flowering plant fertilization, both egg and central cell fuse in a coordinated manner with a single sperm each (Hamamura et al., 2011; Kawashima and Berger, 2011). In order to address whether during polyspermy egg cell fertilization is still coupled to the fertilization of the central cell, we aimed at analyzing endosperm in developing seeds that contain polyspermy-derived embryos. To ease the screening process, we established a novel polyspermy-detection assay termed HIPODSCO1, which can efficiently and unambiguously detect the rare event of egg cell polyspermy already in developing seeds. HIPODSCO1 capitalizes on the pale green appearance of developing seeds defective for the gene SNOWY COTYLEDON 1 (SCO1) (Ruppel and Hangarter, 2007) (Figure 1—figure supplement 1) and a bipartite SCO1 complementation system, provided by two different pollen donors (Figure 1A). Pollen donor one contains the synthetic GAL4 transcription factor under the control of the RPS5a promoter. Pollen donor two contains a functional copy of tdTOMATO tagged SCO1 under the control of the GAL4 responsive UAS enhancer sequence. Seeds that contain a monospermy-derived embryo inherit an incomplete complementation system and will consequently be rendered pale green due to the lack of functional SCO1. By contrast, combinations of both constructs, which can only result from polyspermy, will give rise to green seeds with a positive tdTOMATO fluorescence signal (Figure 1B). It should be noted that this assay only detects polyspermy if the two sperm are derived from different pollen donors. Monopaternal polyspermy, which only delivers a single HIPODSCO1 component, does not rescue seed color. This scenario is expected to account for 50% of all polyspermy events, and escapes detection. Figure 1 with 1 supplement see all Download asset Open asset Establishment of a detection assay for polyspermy-derived embryos. (A) Illustration of HIPODSCO1. The assay is based on the UAS-GAL4 two-component system whereby a synthetic transcription factor mGAL4 expressed under the control of the ubiquitous RPS5a promoter activates the tdTOMATO-tagged SCO1 gene. These two components were combined with the sco1 mutant to generate pollen donor 1 and 2 (PD1 and PD2), respectively. (B) Pollen of PD1 and PD2 (blue, yellow) are applied to the stigma of a sco1 gynoecium (green). Gamete fusion involving two sperm from two different pollen donors leads to transactivation of the SCO1 gene resulting in dark green seeds and fluorescence of tdTOMATO in the embryo, while monospermy-derived seeds remain pale green with no fluorescence. (C) Silique and seed analysis of sco1 mutants containing only pRPS5a::mGAL4-VP16, (upper panel), only pUAS::SCO1-tdTOMATO (middle panel), and both pRPS5a::mGAL4-VP16 and pUAS::SCO1-tdTOMATO (lower panel). Scale bars, 500 μm and 100 μm in left and right panel, respectively. To test the system, we compared seed color of sco1 mutant plants expressing either one of the constructs with seeds containing both, the GAL4 activator and the UAS reporter line. This experiment confirmed that only the presence of both constructs complemented the defect resulting in dark green seeds, which exhibited a tdTOMATO signal (Figure 1C). Polyspermy can selectively polyploidize the egg cell The novel HIPODSCO1 assay enabled us to screen for seeds that contain polyspermy-derived embryos at an advanced seed developmental stage. We processed a total of 56,493 seeds seven days after pollination (DAP) and identified 10 normally developed seeds with a change in color (Figure 2A, Figure 2—figure supplement 1A). To determine whether the candidate embryos were indeed of triparental origin, we microscopically inspected the developing seeds and found that all 10 embryos exhibited a tdTOMATO signal (Figure 2B, Figure 2—figure supplement 1A). This implies that the embryo inherited two rather than one paternal copy. To identify a corresponding shift in embryo ploidy, we carried out a chromosome spread assay. Chromosome counts are technically challenging when performed on subfractions of individual seeds and some chromosomes escape detection. However, comprehensive controls and the fact that parental chromosome contributions are quantal in nature make the assay robust and reliable. Notably all embryos showed a triploid profile (Figure 2C, Figure 2—figure supplement 1B). This finding is comparable to the results obtained from triploid embryos segregated from an interploidy cross between diploid and tetraploid plants and contrasts with the diploid profile detected in embryos recovered from a regular cross involving haploid gametes (Figure 2C). To substantiate this result we introduced a GFP-tagged centromere-localized CENH3 reporter into PD1 (De Storme et al., 2016). In this complementary experiment we screened 10,774 seeds by HIPODSCO1 and recovered three green seeds containing tdTOMATO positive embryos (Figure 2—figure supplement 1A). In all seeds we detected between 11 and 15 GFP foci indicative of triploid embryos (Figure 2E; Figure 2—source data 1). Together, the analysis confirms the triparental origin of embryos in seeds with dark green color, establishing HIPODSCO1 as a powerful novel tool to identify polyspermy-derived embryos already in developing seeds. In order to determine whether egg cell polyspermy is concomitant with central cell polyspermy, we assessed the ploidy of the endosperm in developing seeds containing polyspermy-induced triparental embryos. The central cell of many flowering plants, including Arabidopsis thaliana, is homodiploid and generates a triploid nurturing tissue after sperm fusion. In fact, we detected between 12 and 15 chromosomes in the endosperm of seeds recovered from a cross involving diploid plants. By contrast, more than 15 chromosomes are detected in control interploidy crosses between diploid female and tetraploid male (Figure 2D). Remarkably, in the 10 developing seeds containing triparental embryos we detected between 11 and 15 chromosomes, which is characteristic of a triploid endosperm (Figure 2D, Figure 2—figure supplement 1C). The result was substantiated by a complementary experiment involving the recombinant CENH3-GFP reporter, which detected a triploid profile in the endosperm of three analyzed seeds (Figure 2F; Figure 2—source data 1). Notably, at this advanced seed developmental stage, we recovered one abnormal seed from the HIPODSCO1 assay containing an underdeveloped triploid heart-stage embryo and tetraploid endosperm (Figure 2—figure supplement 1D), characteristic of triploid block-induced seed abortion (Dilkes et al., 2008; Kradolfer et al., 2013; Scott et al., 1998). Figure 2 with 1 supplement see all Download asset Open asset HIPODSCO1 identifies developing seeds harboring polyspermy-derived embryos. (A and B) Bright- and fluorescence light images of seeds from different crosses seven days after pollination (DAP). Upper panel (HIPODSCO1): sco1 × pRPS5a::mGAL4-VP16/+ sco1 (PD1) × pUAS::SCO1-tdTOMATO/+ sco1 (PD2); middle panel: pUAS::SCO1-tdTOMATO/+ sco1 × pRPS5a::mGAL4-VP16/+ sco1; lower panel: sco1 × sco1. Asterisk indicates polyspermy-induced complementation of a sco1 seed. (C and D) DAPI-stained chromosome spreads of embryo (EM) (C) and endosperm (EN) (D) resulting from different crosses. Left panel: HIPODSCO1-rescued embryo segregating from cross between sco1 × pRPS5a::mGAL4-VP16/+ sco1 (PD1) × pUAS::SCO1-tdTOMATO/+ sco1 (PD2); middle panel: pUAS::SCO1-tdTOMATO/+ sco1 × pRPS5a::mGAL4-VP16/+ sco1; right panel: sco1 × wild type (4n). (E and F) Chromosome counting through centromere-targeted CENH3-GFP of embryo (E) and endosperm (F) resulting from different crosses. Left panel: rescued embryo segregating from cross between sco1 × pRPS5a::mGAL4-VP16/+ p35S::CENH3-GFP sco1 (PD1 with CENH3-GFP) × pUAS::SCO1-tdTOMATO/+ sco1 (PD2); middle panel: pUAS::SCO1-tdTOMATO/+ sco1 × pRPS5a::mGAL4-VP16/+ p35S::CENH3-GFP sco1 ; right panel: wild type (4n) × pRPS5a::mGAL4-VP16/+ p35S::CENH3-GFP sco1 . The numbers in parenthesis indicate the average counted chromosomes from all analyzed cells, from left to right, (C) n = 11, 11, 9, (D) n = 20, 6, 9, (E), n = 82, 57, 78, (F), n = 12, 26, 6. Scale bars, 200 μm (A), 100 μm (B), 1 μm (C–E). Figure 2—source data 1 Chromosome counting through centromere-targeted CENH3-GFP in embryo and endosperm resulting from different crosses. https://cdn.elifesciences.org/articles/52976/elife-52976-fig2-data1-v1.xlsx Download elife-52976-fig2-data1-v1.xlsx Together our data indicate that egg cell polyspermy can occur independent of central cell polyspermy. Such selective polyploidization of the egg cell implies that polyspermy has the potential to bypass the triploid block. Most polyspermy-induced polyploidization events are insensitive to the triploid block suppressor admetos To further substantiate our findings, we established a functional assay to determine the potential of polyspermy in bypassing this reproductive barrier. It was previously shown that mutations in the paternally expressed imprinted gene ADMETOS (ADM) suppress the triploid block in Arabidopsis thaliana (Kradolfer et al., 2013). In fact, interploidy crosses between diploid and tetraploid plants lead to a 15.6 fold increase in fertile triploid seeds when the tetraploid pollen donor segregated the adm-1 allele (Figure 3A–C, Figure 3—source data 1). If polyspermy would equally trigger the triploid block, we would expect a similar increase in polyspermy frequencies when using adm-1 segregating pollen donors. In order to identify polyspermy-derived seedlings, we made use of the previously established HIPOD assay that works analogous to the HIPODSCO1 system introduced above, but positively selects triparental seedlings on the basis of herbicide resistance (Nakel et al., 2017). A total of 116,279 and 113,777 seeds were harvested from three independent HIPOD experiments using either adm-1 or wild-type segregating pollen donors, respectively. Out of these, 47 herbicide resistant seedlings segregated from pollen donor with adm-1 background while 27 were recovered from wild type (Figure 3D–F, Figure 3—source data 1 ). This corresponds to an almost two fold increase in the adm-1 segregating approach (Figure 3C), which is more than eight times lower than the effect observed in the interploidy cross. Figure 3 Download asset Open asset Polyspermy-induced polyploidization is partially insensitive of adm-mediated triploid block repression. (A and B) Mature seed (A) and corresponding 9 day old seedling (B) from one silique of wild-type pollinated with diploid wild-type pollen and diploid adm-1 pollen. (C) Effect of the triploid block repressor adm on interploidy cross- recovered triploids (2n × 4n) and polyspermy- derived triploids. Shown is the ratio of triploid plants recovered from two crosses involving either adm or wild-type pollen donors (adm/WT). The data are means ± SEM (n = 3 experiments). (D) Herbicide-treated offspring of triparental triploid (TT) plants recovered from HIPOD with (TTWT) or without (TTadm) ADMETOS segregating pollen donors. Lower panel, herbicide-sensitive offspring of biparental diploid wild type (BD). (E and F) YFP fluorescence (E) and flow-cytometric analysis (F) of TTWT, TTadm and BD plants corresponding to the categories shown in (D). Scale bars, 1 mm (A), 5 mm (B, D), 50 μm (E). Figure 3—source data 1 Comparison of viable triploids recovered from polyspermy and interploidy crosses (2n × 4n). https://cdn.elifesciences.org/articles/52976/elife-52976-fig3-data1-v1.xlsx Download elife-52976-fig3-data1-v1.xlsx Previous results suggested that the egg cell block is stricter than the central cell block (Grossniklaus, 2017; Scott et al., 2008) and fertilization of the two female gametes during monospermy has been shown to occur in a coordinated fashion (Kawashima and Berger, 2011; Hamamura et al., 2011). Our unprecedented finding that most polyspermy-derived embryos develop in the presence of a monospermy-derived endosperm show that polyspermy enables selective polyploidization of the egg cell and concomitant bypassing the triploid block. Polyspermy-derived triparental plants are comparable to triploids generated by interploidy crosses The transcript profile of triploid plants has been characterized previously and remarkably few differences were found with respect to their cognate diploid controls (Hou et al., 2018). Polyspermy-induced plants differ from triploids derived from interploidy crosses as they inherit two sperm cytoplasms and, as shown in this work, mostly develop in a seed characterized by identical ploidies in embryo and endosperm. Given their special mode of origin, we aimed to compare the transcript profile of polyspermy-induced triparental triploids (TT) with that of biparental triploids (BT). In addition, we compared the transcriptome profile of BT plants with that of biparental diploids (BD) to identify ploidy-dependent changes in the transcriptional landscape. We used ein3 mutants as pollen acceptor as they have previously been shown to attract supernumerary pollen tubes (Völz et al., 2013). We performed RNAseq on five plants 18 days after sowing (DAS) each from BD, BT, and TT (Figure 4—figure supplement 1A). We chose this early state as we expected potential differences to become established during seed development, which differs in the three settings. In order to assess the quality of the transcriptome data, and to identify potential transcriptome-wide differences between the mRNA profiles of the three groups of plants, we performed a hierarchical clustering, a two-dimensional principal component analysis (PCA), and a two-dimensional multidimensional scaling (MDS) analysis of the fifteen 21,450-dimensional normalized expression profiles. We found that Spearman’s correlation coefficient c was greater than 0.94 for all of the 105 pairs of profiles (Figure 4—figure supplement 1B). Notably, the sub-trees of the dendrogram obtained by hierarchical average linkage clustering did not correspond to the five biological replicates from the same genotype (Figure 4A), and the 15 samples showed a high overlap in the PCA (Figure 4B) and MDS (Figure 4—figure supplement 1C) plots. These results not only reflect the high quality of the transcriptome data but also suggest a high similarity of the 15 transcriptome profiles compared. Figure 4 with 4 supplements see all Download asset Open asset Characterization of triparental triploid plants. (A) Dendrogram from hierarchical clustering of the 3 × 5 biological replicates of biparental diploid (BD), biparental triploid (BT), and triparental triploid (TT) recovered from polyspermy plant samples. The 15 × 15 matrix of Spearman’s correlation coefficients c was computed from the fifteen regularized log transformed expression profiles. The dendrogram was computed by average linkage clustering (UPGMA) of the 15 × 15 dissimilarity matrix with elements 1 – c. (B) Two-dimensional principle component analysis (PCA) of the fifteen 21,450-dimensional regularized log transformed expression profiles. The points represent the biological replicates, while the shapes resulting from connecting biological replicates from the same genotype highlight the similarity between the transcriptome profiles. (C) Distribution of ploidy level in the aneuploidy swarms produced by a triparental triploid plant. 2.0 in DNA index represents near diploids, 3.0, near triploids, and 4.0, near tetraploids. The gray areas indicate the intermediate ploidies. Each orange triangle represents an F2 plant derived from a triparental triploid. (D) Flow cytometric analysis of tetraploid progeny plants in F3 generation. 2n and 4n represent diploid and tetraploid controls. (E) Analysis of flowering time window of triparental triploid plants recovered from a three accession cross (TT3), Col-0, Ler and C24 during bolting stage. Scale bar, 1 cm. The black bold line represents the flowering period of different accessions. The three red bold lines bordered by the gray dashed lines label the day gaps between flowering termination of the parents and flowering initiation in the TT3. We first addressed, if there were genes with ploidy-specific expression changes by comparing transcript profiles of BD and BT. This comparison did not yield a single gene with a statistically significant differential expression. This result is in support of previous transcriptome profiling approaches that have uncovered remarkably few changes in plants with different ploidy (Pignatta et al., 2010; Riddle et al., 2010; Stupar et al., 2007; Wang et al., 2006; Yu et al., 2010). We next compared the transcript profiles of BT and TT in order to identify specific expression changes potentially associated with polyspermy. Interestingly, also this approach did not yield genes with statistically significant differential expression. The similarity in the overall transcriptional profiles between TT and BT plants is reflected by strong similarities in various life-history traits, including flower organ size, cell size, and even fertility (Figure 4—figure supplement 2). Please note that the normalized expression data presented here does not allow any conclusions on alterations in transcriptome size, i.e. changes affecting the total number of transcripts per cell (Coate and Doyle, 2015). However, the data suggests that Arabidopsis responds in a transcriptionally balanced fashion to the inheritance of supernumerary genomes and seed homoploidy. Along these lines, also the ability to generate tetraploid offspring within a single generation, a parameter that has been described previously for interploidy cross-induced triploids was maintained: To assess whether polyspermy-derived triploids can segregate stable polyploid offspring, we harvested the seeds of polyspermy-derived triparental plants and propagated them in two successive generations. The progeny of polyspermy-derived triploids segregates a complex swarm of karyotypes, similar to what has previously been described for interploidy crosses (Henry et al., 2005). On the basis of flow cytometric analysis, we grouped the plants into five different categories: near-diploids, 2n-3n, near-triploids, 3n-4n and near-tetraploids (Figure 4C, Figure 4—figure supplement 3A). Already in the F2 generation 5 out of 109 plants were found to fall into the near-tetraploids category, while 22 plants segregated a diploid-like profile (Figure 4C, Figure 4—figure supplement 3B). To determine whether any of the high-ploidy plants represented a genuine tetraploid, we collected seeds and determined the ploidy of 20 offspring per individual F2 plant. Flow cytometric analysis revealed that 3 out of 5 near-tetraploid F2 plants segregated exclusively plants that exhibit a ploidy profile characteristic to 4n plants (Figure 4D). This result was confirmed in the F4 generation, which, again, revealed a homogenous tetraploid ploidy profile. Together these results show that polyspermy-derived triploid plants have the potential to generate stable tetraploid and diploid offspring within a single generation. Polyspermy-derived three accession hybrids are reproductively isolated from their parental lines We previously combined three distinct Arabidopsis accessions in a three parent cross (Nakel et al., 2017) and noticed that the resulting triploid hybrids initiate flowering later than their parents, an effect which was previously described also for two-accession hybrids (Groszmann et al., 2014; Moore and Lukens, 2011) (Figure 4—figure supplement 4A). To address whether this phenotype has the potential to reduce gene flow, we compared flowering time between the parental line and triparental three accession hybrids, henceforth referred to as TT3. We found that flowering is induced more than one month later in TT3 than in the parental lines, and the flowering period of TT3 is completely isolated from the parents (Figure 4E). Under our plant growth conditions, Ler and Col-0 start to flower 24.4 ± 0.7 and 26.6 ± 1.0 DAS, and flowering terminates 44.9 ± 1.2 and 47.1 ± 0.9 DAS, respectively. This corresponds to a flowering period of around 20 days. The C24 flowering window is comparable but flower initiation is delayed by five days (31.8 ± 1.2 DAS). By contrast, TT3 plants initiate flowering only after 64.6 ± 5.4 days, which is around 20, 17 and 15 days after the respective parental lines have terminated their flowering phase (Figure 4E; Figure 4—figure supplement 4B). Even though these data are obtained under optimized growth condition, the results suggest that polyspermy-derived triploid three accession hybrids are reproductively isolated from the parental plants in the first generation. Discussion The triploid block is an established and widely distributed postzygotic hybridization barrier. In light of its fatal consequence, it has been suggested that there are ways to overcome this hybridization barrier (Köhler et al., 2010). We here established a novel polyspermy detection assay that allows to identify and characterize developing embryos resulting from supernumerary sperm fusion. With this tool we were able to show that most polyspermy-derived plants develop from seeds resulting from selective egg cell polyploidization. In those seeds, supernumerary paternal copies are only transmitted to the embryo, thereby bypassing the triploid block of the endosperm. Our results expand previous polyploidization concepts, which state that the increase in genome copies is caused by infrequent meiotic or mitotic defects. In fact, the currently favored route towards polyploid plants involves unreduced male gametes; however, this scenario introduces supernumerary paternal copies also to the endosperm, which is not tolerated in many plants resulting in seed abortion (Dilkes et al., 2008; Ramsey and Schemske, 1998; Scott et al., 1998; Stoute et al., 2012). In fact, this endosperm-related triploid block is considered a means of reproductive isolation (Köhler et al., 2010; Ramsey and Schemske, 1998). Plant polyploidization via polyspermy, by contrast, often affects the embryo-derived seed fraction only and hence has the potential to bypass the triploid block. It will be a challenge for the future to determine whether and to what extent polyspermy is relevant in nature and contributed to the evolution of polyploid plants. From an evolutionary and agricultur

This is an updated version of https://doi.org/10.4119/unibi/2936278. Additional filter steps have been applied to generate this high confidence variant set. Please see https://doi.org/10.1101/691923 for a description of the data set and applied methods.

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.

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

Sample data for testing pipeline functionality. (ZIP 18664 kb)

Molecular markers are a highly valuable tool for creating genetic maps. Like in many other crops, sugar beet (Beta vulgaris L.) breeding is increasingly supported by the application of such genetic markers. Single nucleotide polymorphism (SNP) based markers have a high potential for automated analysis and high-throughput genotyping. We developed a bioinformatics workflow that uses Sanger and 2nd-generation sequence data for detection, evaluation and verification of new transcript-associated SNPs from sugar beet. RNAseq data from one parent of an established mapping population were produced by 454-FLX sequencing and compared to Sanger ESTs derived from the other parent. The workflow established for SNP detection considers the quality values of both types of reads, provides polymorphic alignments as well as selection criteria for reliable SNP detection and allows painless generation of new genetic markers within genes. We obtained a total of 14,323 genic SNPs and InDels. According to empirically optimised settings for the quality parameters, we classified these SNPs into four usability categories. Validation of a subset of the in silico detected SNPs by genotyping the mapping population indicated a high success rate of the SNP detection. Finally, a total of 307 new markers were integrated with existing data into a new genetic map of sugar beet which offers improved resolution and the integration of terminal markers.