Genomic characterization of a nematode tolerance locus in sugar beet.

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

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

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

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

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

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

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

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

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

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

Flavonols are colourless secondary metabolites, primarily regarded as UV-protection pigments that are deposited in plants in their glycosylated forms. The glycosylation of flavonols is mainly catalysed by UDP-sugar-dependent glycosyltransferases (UGTs). Although the structures of flavonol glycosides accumulating in Arabidopsis thaliana are known, many genes involved in the flavonol glycosylation pathway are yet to be discovered. The flavonol glycoside profiles of seedlings from 81 naturally occurring A. thaliana accessions were screened using high performance thin layer chromatography. A qualitative variation in flavonol 3-O-gentiobioside 7-O-rhamnoside (F3GG7R) content was identified. Ler × Col-0 recombinant inbred line mapping and whole genome association mapping led to the identification of a glycoside hydrolase family 1-type gene, At1g60270/BGLU6, that encodes a homolog of acyl-glucose-dependent glucosyltransferases involved in the glycosylation of anthocyanins, possibly localized in the cytoplasm, and that is co-expressed with genes linked to phenylpropanoid biosynthesis. A causal single nucleotide polymorphism introducing a premature stop codon in non-producer accessions was found to be absent in the producers. Several other naturally occurring loss-of-function alleles were also identified. Two independent bglu6 T-DNA insertion mutants from the producer accessions showed loss of F3GG7R. Furthermore, bglu6 mutant lines complemented with the genomic Ler BGLU6 gene confirmed that BGLU6 is essential for production of F3GGR7. We have thus identified an accession-specific gene that causes a qualitative difference in flavonol glycoside accumulation in A. thaliana strains. This gene encodes a flavonol 3-O-glucoside: 6″-O-glucosyltransferase that does not belong to the large canonical family of flavonol glycosyltransferases that use UDP-conjugates as the activated sugar donor substrate.

Elongation-related functions of LEAFY COTYLEDON1 during the development of Arabidopsis thaliana.

The transcription factor LEAFY COTYLEDON1 (LEC1) controls aspects of early embryogenesis and seed maturation in Arabidopsis thaliana. To identify components of the LEC1 regulon, transgenic plants were derived in which LEC1 expression was inducible by dexamethasone treatment. The cotyledon-like leaves and swollen root tips developed by these plants contained seed-storage compounds and resemble the phenotypes produced by increased auxin levels. In agreement with this, LEC1 was found to mediate up-regulation of the auxin synthesis gene YUCCA10. Auxin accumulated primarily in the elongation zone at the root-hypocotyl junction (collet). This accumulation correlates with hypocotyl growth, which is either inhibited in LEC1-induced embryonic seedlings or stimulated in the LEC1-induced long-hypocotyl phenotype, therefore resembling etiolated seedlings. Chromatin immunoprecipitation analysis revealed a number of phytohormone- and elongation-related genes among the putative LEC1 target genes. LEC1 appears to be an integrator of various regulatory events, involving the transcription factor itself as well as light and hormone signalling, especially during somatic and early zygotic embryogenesis. Furthermore, the data suggest non-embryonic functions for LEC1 during post-germinative etiolation.

A unique transcriptome: 1782 positions of RNA editing alter 1406 codon identities in mitochondrial mRNAs of the lycophyte Isoetes engelmannii.

The analysis of the mitochondrial DNA of Isoetes engelmannii as a first representative of the lycophytes recently revealed very small introns and indications for extremely frequent RNA editing. To analyze functionality of intron splicing and the extent of RNA editing in I. engelmannii, we performed a comprehensive analysis of its mitochondrial transcriptome. All 30 groups I and II introns were found to be correctly removed, showing that intron size reduction does not impede splicing. We find that mRNA editing affects 1782 sites, which lead to a total of 1406 changes in codon meanings. This includes the removal of stop codons from 23 of the 25 mitochondrial protein encoding genes. Comprehensive sequence analysis of multiple cDNAs per locus allowed classification of partially edited sites as either inefficiently edited but relevant or as non-specifically edited at mostly low frequencies. Abundant RNA editing was also found to affect tRNAs in hitherto unseen frequency, taking place at 41 positions in tRNA-precursors, including the first identification of U-to-C exchanges in two tRNA species. We finally investigated the four group II introns of the nad7 gene and could identify 27 sites of editing, most of which improve base pairing for proper secondary structure formation.

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

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

IncP-1ß plasmids of Comamonas sp. and Delftia sp. strains isolated from a wastewater treatment plant mediate resistance to and decolorization of the triphenylmethane dye crystal violet.

The application of toxic triphenylmethane dyes such as crystal violet (CV) in various industrial processes leads to large amounts of dye-contaminated sludges that need to be detoxified. Specific bacteria residing in wastewater treatment plants (WWTPs) are able to degrade triphenylmethane dyes. The objective of this work was to gain insights into the genetic background of bacterial strains capable of CV degradation. Three bacterial strains isolated from a municipal WWTP harboured IncP-1ß plasmids mediating resistance to and decolorization of CV. These isolates were assigned to the genera Comamonas and Delftia. The CV-resistance plasmid pKV29 from Delftia sp. KV29 was completely sequenced. In addition, nucleotide sequences of the accessory regions involved in conferring CV resistance were determined for plasmids pKV11 and pKV36 from the other two isolates. Plasmid pKV29 contains typical IncP-1ß backbone modules that are highly similar to those of previously sequenced IncP-1ß plasmids that confer antibiotic resistance, degradative capabilities or mercury resistance. The accessory regions located between the conjugative transfer (tra) and mating pair formation modules (trb) of all three plasmids analysed share common modules and include a triphenylmethane reductase gene, tmr, that is responsible for decolorization of CV. Moreover, these accessory regions encode other enzymes that are dispensable for CV degradation and hence are involved in so-far-unknown metabolic pathways. Analysis of plasmid-mediated degradation of CV in Escherichia coli by ultra-high-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight MS revealed that leuco crystal violet was the first degradation product. Michler's ketone and 4-dimethylaminobenzaldehyde appeared as secondary degradation metabolites. Enzymes encoded in the E. coli chromosome seem to be responsible for cleavage of leuco crystal violet. Plasmid-mediated degradation of triphenylmethane dyes such as CV is an option for the biotechnological treatment of sludges contaminated with these dyes.

Candidate genes within a 143 kb region of the flower sex locus in Vitis.

Wild Vitis species are dioecious plants, while the cultivated counterpart, Vitis vinifera subspec. vinifera, generally shows hermaphroditic flowers. In Vitis the genetic determinants of flower sex have previously been mapped to a region on chromosome 2. In a combined strategy of map-based cloning and the use of the publicly available grapevine reference genome sequence, the structure of the grapevine flower sex locus has been elucidated with the subsequent identification of candidate genes which might be involved in the development of the different flower sex types. In a fine mapping approach, the sex locus in grapevine was narrowed down using a population derived from a cross of a genotype with a Vitis vinifera background ('Schiava Grossa' × 'Riesling') with the male rootstock cv. 'Börner' (V. riparia × V. cinerea). A physical map of 143 kb was established from BAC clones spanning the 0.5 cM region defined by the closest flanking recombination break points. Sequencing and gene annotation of the entire region revealed several candidate genes with a potential impact on flower sex formation. One of the presumed candidate genes, an adenine phosphoribosyltransferase, was analysed in more detail. The results led to the development of a marker for the presence or absence of the female alleles, while the male and hermaphroditic alleles are still to be differentiated. The impact of other candidate genes is discussed, especially with regard to plant hormone actions. The markers developed will permit the selection of female breeding lines which do not require laborious emasculation thus considerably simplifying grapevine breeding. The genetic finger prints displayed that our cultivated grapevines frequently carry a female allele while homozygous hermaphrodites are rare.

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

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

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

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

Genome Sequencing of Musa acuminata Dwarf Cavendish Reveals a Duplication of a Large Segment of Chromosome 2.

Different Musa species, subspecies, and cultivars are currently investigated to reveal their genomic diversity. Here, we compare the genome sequence of one of the commercially most important cultivars, Musa acuminata Dwarf Cavendish, against the Pahang reference genome assembly. Numerous small sequence variants were detected and the ploidy of the cultivar presented here was determined as triploid based on sequence variant frequencies. Illumina sequence data also revealed a duplication of a large segment on the long arm of chromosome 2 in the Dwarf Cavendish genome. Comparison against previously sequenced cultivars provided evidence that this duplication is unique to Dwarf Cavendish. Although no functional relevance of this duplication was identified, this example shows the potential of plants to tolerate such aneuploidies.

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

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

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

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

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

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

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

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

Selective egg cell polyspermy bypasses the triploid block.

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.

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.

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

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.