Plant-parasitic nematodes are economically important threats to global crop production. The beet cyst nematode (Heterodera schachtii) is a crucial pest in sugar beet (Beta vulgaris ssp. vulgaris). While all species of the genus Beta are highly susceptible, the three species of the beet wild relative genus Patellifolia are entirely resistant. Recently, we cloned the Hs4 gene from P. procumbens, which confers complete resistance. In this study, we aimed to determine whether putative Hs4 orthologs exist in Beta and Patellifolia species. The Hs4 gene consisted of 4999 bp, with six exons and five introns. Patellifolia species contain highly similar Hs4 homologs. Single nucleotide polymorphisms and insertions/deletions between accessions and species could be detected. We found an exonic integration of three bases, resulting in the addition of one amino acid. Interestingly, this variant was present in single accessions of all three Patellifolia species. Beta vulgaris ssp. vulgaris contains an Hs4 homolog (BvHs4) with 60 % protein identity to Hs4. BvHs4 homologs were present in all Beta species analyzed. Further, we examined the expression patterns of Hs4 and BvHs4 homologs. While Hs4 homologs from Patellifolia species are strongly expressed in roots, BvHs4 homologs are expressed mainly in leaves. When the spatio-temporal expression of Hs4 was examined, no response to nematode inoculation was observed. These results are highly relevant for searching for functional Hs4 alleles and breeding nematode-resistant varieties.

One of the most recent crop species to be domesticated is sugar beet (Beta vulgaris L. ssp. vulgaris Doell.), which was bred for high sucrose content within the last few centuries in Europe. Crop domestication can also lead to the evolution of novel pathogens, which may spread across large geographical distances with their crop host. In this study, we addressed the recent evolution of the fungal pathogen causing the disease Cercospora leaf spot, Cercospora beticola. This pathogen has become increasingly important in sugar beet and table beet production worldwide. We used genome sequences of 326 C. beticola isolates collected from 4 continents from 4 closely related Beta subspecies (3 domesticated and 1 wild). We applied population genomic analyses to identify signatures of population differentiation and host specialization in C. beticola populations derived from the cultivated and wild hosts. We found evidence that C. beticola populations in agro-ecosystems likely originate from sea beet-infecting isolates. Intriguingly, host jumps from wild to cultivated beet occurred in at least 2 independent events as evidenced by our population data of C. beticola from wild beet collected in the Mediterranean and the UK. We explore the occurrence of genetic variants associated with fungicide resistance and virulence and show that standing genetic variation in C. beticola populations from both wild and domesticated plants may serve as a reservoir of functionally important alleles. Overall, our results highlight the ability of C. beticola to invade the agro-ecosystem and establish new populations, demonstrating the rapid adaptation potential of the species.

Seed number per silique (SNPS) is an important determinant for seed yield in rapeseed (Brassica napus). Here, the molecular mechanism of BnaC9.APT5 (Adenine phosphoribosyltransferase 5) regulating SNPS was identified by combining gene function with biochemical analyses. Overexpression (OE) of Arabidopsis Adenine Phosphoribosyltransferase 5 (AtAPT5) and ectopic expression of BnaC9.APT5 in Arabidopsis thaliana increased SNPS by c. 9%. By contrast, the Arabidopsis knockout mutant had 18% fewer seeds per silique. OE of BnaC9.APT5 in rapeseed increased SNPS by c. 11% through upregulating cytokinin (CK) biosynthesis genes. The enzyme activity analysis showed that BnaC9.APT5 converts adenine to adenosine monophosphate. Spraying the Arabidopsis knockout mutant with 6-benzylaminopurine increased SNPS by 34%. The treatment of the Arabidopsis AtAPT5 and rapeseed BnaC9.APT5 OE lines with the CK biosynthesis inhibitor lovastatin reduced SNPS by c. 7% and 9%, respectively. Thus, BnaC9.APT5 controls SNPS via the CK metabolism in rapeseed. A 48-bp InDel in the BnaC9.APT5 promoter was significantly associated with gene expression and SNPS and has been selected during rapeseed 'double-low' quality breeding. Our data provide clear evidence of the role of the BnaC9.APT5 gene for the genetic improvement of modern rapeseed.

The genus Beta encompasses important crops such as sugar, table, fodder, and leaf beets. All cultivated beets are believed to have originated from the wild sea beet, B. vulgaris subsp. maritima. Sugar beet, a recent crop dating back nearly 200 years, was selectively bred for enhanced root yield in combination with high sucrose content. We assembled a Beta diversity panel comprising wild and cultivated beet accessions. Whole-genome sequencing identified 10.3 million SNP markers. Four distinct genetic clusters were identified: table beet, sugar beet, Mediterranean sea beet, and Atlantic sea beet. A phylogenetic analysis revealed that cultivated beet accessions were genetically closer to Mediterranean than to Atlantic sea beet and that cultivated beets producing storage roots share a common ancestor. Cultivated beets exhibited genome regions with reduced nucleotide diversity compared to Mediterranean sea beets, indicating selection signatures. These regions contained putative candidate genes with potential roles in root development, suppression of lateral root formation, flowering time, and sucrose metabolism. A yet unknown sucrose transporter on chromosome 6 showed reduced nucleotide diversity exclusively in sugar beet accessions compared to other Beta types with low sucrose content, suggesting its role in sucrose storage. Within a region of high nucleotide diversity between accessions with contrasting root phenotypes, we found two genes encoding auxin response factors, which play a crucial role in root development. We reason these genes to be significant root thickening regulators in root crops.

The genus Beta encompasses economically important root crops such as sugar and table beet. A Beta diversity set including the wild relative B. vulgaris ssp. maritima was grown in the field, and a large phenotypic diversity was observed. The genomes of 290 accessions were sequenced, and more than 10 million high-quality SNPs were employed to study genetic diversity. A genome-wide association study was performed, and marker-trait associations were found for nine phenotypic traits. The candidate gene within the M locus controlling monogermity on chromosome 4 was previously unknown. The most significant association for monogermity was identified at the end of chromosome 4. Within this region, a non-synonymous mutation within the zinc-finger domain of the WIP2 gene co-segregated with monogermity. This gene plays a regulatory role in AGL8/FUL in Arabidopsis. Intriguingly, commercial hybrids are in a heterozygous state at this position. Thus, the long-sought gene for monogermity was identified in this study. Red and yellow pigmentation due to betalain accumulation in shoots and roots is an important characteristic of table and leaf beets. The strongest associations were found upstream or downstream of two genes encoding Cytochrome P450 and anthocyanin MYB-like transcription factor proteins involved in betalain biosynthesis. Significant associations for Cercospora leaf spot resistance were identified on chromosomes 1, 2, 7, and 9. The associated regions harbor genes encoding proteins with leucine-rich repeats and nucleotide binding sites whose homologs are major constituents of plant-pathogen defense.

Root lesion nematodes of the genus Pratylenchus pose a threat to global cereal production. The traditional identification by counting nematodes under the microscope is laborious and time-consuming. We have established a protocol for quantifying nematodes within cereal roots by RT-qPCR. This method holds promise in selecting resistant plant genotypes among large populations during plant breeding programs.

Quinoa (Chenopodium quinoa Willd.) can offer an alternative for staple food considering its tolerance to abiotic stresses and high seed quality. However, its cultivation in temperate regions has not been successful due to its photoperiod sensitivity and low seed yield. This study investigated the agronomical performance and quality traits of 48 accessions for cultivation in northern Europe. We conducted two-year field trials and phenotyped traits related to phenological development, plant architecture, yield components, seed quality, and disease resistance. The major determinants of seed yield in this study were days to flowering, days to maturity, thousand-kernel weight, and panicle density, while downy mildew susceptibility and stem lodging showed a negative correlation with seed yield. We developed a selection index to enable simultaneous selection based on different important agronomical traits. We evaluated the stability of different accessions over the two years of the experiment. Finally, we provided a list of 10 selected accessions that can be directly integrated and serve as new crossing parents in quinoa breeding programs for temperate regions.

Understanding the regulation of flowering time is crucial for adaptation of crops to new environment. In this study, we examined the timing of floral transition and analysed transcriptomes in leaf and shoot apical meristems of photoperiod-sensitive and -insensitive quinoa accessions. Histological analysis showed that floral transition in quinoa initiates 2-3 weeks after sowing. We found four groups of differentially expressed genes in quinoa genome that responded to plant development and floral transition: (i) 222 genes responsive to photoperiod in leaves, (ii) 1812 genes differentially expressed between accessions under long-day conditions in leaves, (iii) 57 genes responding to developmental changes under short-day conditions in leaves and (iv) 911 genes responding to floral transition within the shoot apical meristem. Interestingly, among numerous candidate genes, two putative FT orthologs together with other genes (e.g. SOC1, COL, AP1) were previously reported as key regulators of flowering time in other species. Additionally, we used coexpression networks to associate novel transcripts to a putative biological process based on the annotated genes within the same coexpression cluster. The candidate genes in this study would benefit quinoa breeding by identifying and integrating their beneficial haplotypes in crossing programs to develop adapted cultivars to diverse environmental conditions.

The ability of a plant parasitic nematode to infect and reproduce within a host plant depends on its genotype and the environmental conditions before and during infection. We studied the culturing conditions of the root lesion nematode Pratylenchus neglectus to produce inoculum for plant infection tests. Nematodes were either cultivated on carrot calli for different periods or directly isolated from the roots of the host plants. After infection of wheat and barley plants in the greenhouse, nematodes were quantified by RT-qPCR and by visual counting of the nematodes. We observed drastically reduced infection rates after long-term (> 96 weeks) cultivation on carrot callus. In contrast, fresh isolates from cereal roots displayed much higher pathogenicity. We recommend using root lesion nematodes cultivated on carrot calli no longer than 48 weeks to guarantee uniform infection rates.

The presence of anti-nutritive compounds like glucosinolates (GSLs) in the rapeseed meal severely restricts its utilization as animal feed. Therefore, reducing the GSL content to <18 µmol/g dry weight in the seeds is a major breeding target. While candidate genes involved in the biosynthesis of GSLs have been described in rapeseed, comprehensive functional analyses are missing. By knocking out the aliphatic GSL biosynthesis genes BnMYB28 and BnCYP79F1 encoding an R2R3 MYB transcription factor and a cytochrome P450 enzyme, respectively, we aimed to reduce the seed GSL content in rapeseed. After expression analyses on single paralogs, we used an ethyl methanesulfonate (EMS) treated population of the inbred winter rapeseed ‘Express617’ to detect functional mutations in the two gene families. Our results provide the first functional analysis by knock-out for the two GSL biosynthesis genes in winter rapeseed. We demonstrate that independent knock-out mutants of the two genes possessed significantly reduced seed aliphatic GSLs, primarily progoitrin. Compared to the wildtype Express617 control plants (36.3 µmol/g DW), progoitrin levels were decreased by 55.3% and 32.4% in functional mutants of BnMYB28 (16.20 µmol/g DW) and BnCYP79F1 (24.5 µmol/g DW), respectively. Our study provides a strong basis for breeding rapeseed with improved meal quality in the future.

Induced mutations are an essential source of genetic variation in plant breeding. EMS mutagenesis has been frequently applied, and mutants have been detected by phenotypic or genotypic screening of large populations. In this study, a rapeseed M 2 population was derived from M 1 parent cultivar “Express” treated with EMS. Whole genomes were sequenced from fourfold (4x) pools of 1,988 M 2 plants representing 497 M 2 families. Detected mutations were not evenly distributed and displayed distinct patterns across the 19 chromosomes with lower mutation rates towards the ends. Mutation frequencies ranged from 32/Mb to 48/Mb. On average, 284,442 single nucleotide polymorphisms per M 2 DNA pool were found resulting from EMS mutagenesis. 55% were C→T and G→A transitions, characteristic for EMS induced (‘canonical’) mutations, whereas the remaining SNPs were ‘non-canonical’ transitions (15%) or transversions (30%). Additionally, we detected 88,725 high confidence insertions and deletions (InDels) per pool. On average, each M 2 plant carried 39,120 canonical mutations, corresponding to a frequency of one mutation per 23.6 kb. Roughly 82% of such mutations were located either 5 kb upstream or downstream (~56%) of gene coding regions or within intergenic regions (26%). The remaining 18% were located within regions coding for genes. All mutations detected by whole-genome sequencing could be verified by comparison with known mutations. Furthermore, all sequences are accessible via the online tool “EMS Brassica” ( http://www.emsbrassica.plantbreeding.uni-kiel.de/ ), which enables direct identification of mutations in any target sequence. The sequence resource described here will further add value for functional gene studies in rapeseed breeding.

Seed weight is a component of seed yield in rapeseed (Brassica napus L.). Although quantitative trait loci (QTL) for seed weight have been reported in rapeseed, only a few causal quantitative trait genes (QTGs) have been identified, resulting in a limitation in understanding of seed weight regulation. We constructed a gene coexpression network at the early seed developmental stage using transcripts of 20,408 genes in QTL intervals and 1017 rapeseed homologs of known genes from other species. Among the 10 modules in this gene coexpression network, modules 1 and 2 were core modules and contained genes involved in source–flow–sink processes such as synthesis and transportation of fatty acid and protein, and photosynthesis. A hub gene SERINE CARBOXYPEPTIDASE-LIKE 19 (SCPL19) was identified by candidate gene association analysis in rapeseed and functionally investigated using Arabidopsis T-DNA mutant and overexpression lines. Our study demonstrates the power of gene coexpression analysis to prioritize candidate genes from large candidate QTG sets and enhances the understanding of molecular mechanism for seed weight at the early developmental stage in rapeseed.

Root lesion nematodes (RLN) of the genus Pratylenchus are causing significant damage in cereal production worldwide. Due to climate change and without efficient and environment-friendly treatments, the damages through RLNs are predicted to increase. Microscopic assessments of RLNs in the field and the greenhouses are time-consuming and laborious. As a result, cereal breeders have mostly ignored this pest. We present a method measuring RLN in infected cereal roots using a standardized PCR approach. Publicly available Pratylenchus neglectus primer combinations were evaluated. An optimal primer combination for RT-qPCR assay was identified to detect and quantify P. neglectus within infected cereal roots. Using the RT-qPCR detection assay, P. neglectus could be clearly distinguished from other plant parasitic nematodes. We could identify P. neglectus DNA in barley and wheat roots as low as 0.863 and 0.916 ng/µl of total DNA, respectively. A single P. neglectus individual was detected in water suspension and within barley and wheat roots. The RT-qPCR detection assay provides a robust and accurate alternative to microscopic nematode identification and quantification. It could be of interest for resistance breeding, where large populations must be screened to detect and quantify P. neglectus in farmer’s fields.

Our study aimed to identify candidate genes for flowering time regulation and photoperiod response in quinoa. We investigated the timing of photoperiod-driven floral transition and analyzed the transcriptomes of leaf and shoot apical meristems in photoperiod-sensitive and -insensitive quinoa accessions. Histology analysis of the apical meristem showed that floral transition in quinoa initiates two to three weeks after sowing. We found four groups of differentially expressed genes responding to plant development and floral transition, which were annotated in the QQ74-V2 reference genome, including (i) 222 genes differentially responding to photoperiod in leaves, (ii) 1,812 genes differentially expressed between accessions under long-day conditions in leaves, (iii) 57 genes responding to developmental changes between weeks under short-day conditions in leaves, and (iv) 911 genes responding to floral transition within the shoot apical meristem. Interestingly, out of the thousands of candidates, two putative FT orthologues and several others have been reported as key regulators of flowering time in other species (e.g., SOC1 , COL , AP1 ). Additionally, we used co-expression networks to associate novel transcripts to a putative biological process based on the annotated genes within the same co-expression cluster. The candidate genes in this study would benefit quinoa breeding by identifying and integrating their beneficial haplotypes in crossing programs to develop adapted cultivars to diverse environmental conditions.

Flower development and plant architecture determine the efficiency of mechanized harvest and seed yield in Brassica napus. Although TERMINAL FLOWER 1 (AtTFL1) is a regulator of flower development in Arabidopsis thaliana, the function and regulatory mechanism of TFL1 orthologs in B. napus remains unclear. Six BnTFL1 paralogs in the genome of the B. napus inbred line ‘K407’ showed steadily increasing expression during vernalization. CRISPR/Cas-induced mutagenesis of up to four BnTFL1 paralogs resulted in early flowering and alteration of plant architecture, whereas seed yield was not altered in BnTFL1 single, double, or triple mutants. Six BnTFL1 paralogs, but not BnaA02.TFL1, showed an additive and conserved effect on regulating flowering time, total and terminal flower number, and plant architecture. BnaA10.TFL1 regulates flower development by interacting with BnaA08.FD through the protein BnaA05.GF14nu, resulting in the transcriptional repression of floral integrator and floral meristem identity genes. These findings about the regulatory network controlling flower development and plant architecture present a promising route to modifying these traits in B. napus.

Quinoa is a pseudocereal originating from the Andean regions. Despite quinoa’s long cultivation history, genetic analysis of this crop is still in its infancy. We aimed to localize quantitative trait loci (QTL) contributing to the phenotypic variation of agronomically important traits. We crossed the Chilean accession PI-614889 and the Peruvian accession CHEN-109, which depicted significant differences in days to flowering, days to maturity, plant height, panicle length, and thousand kernel weight (TKW), saponin content, and mildew susceptibility. We observed sizeable phenotypic variation across F2 plants and F3 families grown in the greenhouse and the field, respectively. We used Skim-seq to genotype the F2 population and constructed a high-density genetic map with 133,923 single nucleotide polymorphism (SNPs). Fifteen QTL were found for ten traits. Two significant QTL, common in F2 and F3 generations, depicted pleiotropy for days to flowering, plant height, and TKW. The pleiotropic QTL harbored several putative candidate genes involved in photoperiod response and flowering time regulation. This study presents the first high-density genetic map of quinoa that incorporates QTL for several important agronomical traits. The pleiotropic loci can facilitate marker-assisted selection in quinoa breeding programs.

All genetic variation results from mutations. Orders of magnitude can increase mutation rates by applying irradiation or chemical treatment. Genome editing offers new perspectives for mutation induction because mutation sites can be precisely targeted even within large plant genomes for the first time. Generally, transgenes after genetic engineering also result in new genetic variation. All single-gene mutations after genome editing, transgenesis, or chemical mutagenesis are inherited according to the Mendelian rules. Thus, offspring can be phenotypically classified into discrete classes, whereas polygenic inheritance results in continuous variation. However, genome-wide studies have blurred the boundaries between the two in recent years. Single-gene knockout mutations can be inherited non-Mendelian, mainly if transcription factor genes are targeted. Even classical Mendelian traits are now believed to be controlled by numerous genes. Therefore, ic.

Quinoa germplasm preserves useful and substantial genetic variation, yet it remains untapped due to a lack of implementation of modern breeding tools. We have integrated field and sequence data to characterize a large diversity panel of quinoa. Whole-genome sequencing of 310 accessions revealed 2.9 million polymorphic high confidence single nucleotide polymorphism (SNP) loci. Highland and Lowland quinoa were clustered into two main groups, with FST divergence of 0.36 and linkage disequilibrium (LD) decay of 6.5 and 49.8 kb, respectively. A genome-wide association study using multi-year phenotyping trials uncovered 600 SNPs stably associated with 17 traits. Two candidate genes are associated with thousand seed weight, and a resistance gene analog is associated with downy mildew resistance. We also identified pleiotropically acting loci for four agronomic traits important for adaptation. This work demonstrates the use of re-sequencing data of an orphan crop, which is partially domesticated to rapidly identify marker-trait association and provides the underpinning elements for genomics-enabled quinoa breeding.

Random mutagenesis is a standard procedure to increase allelic variation in a crop species, especially in countries where the use of genetically modified crops is limited due to legal constraints. The chemical mutagen EMS is used in many species to induce random mutations throughout the genome with high mutation density. The major drawback for functional analysis is a high background mutation load in a single plant that must be eliminated by subsequent backcrossing, a time and resource-intensive activity. Here, we demonstrate that genomic background selection combined with marker-assisted selection is an efficient way to select individuals with reduced background mutations within a short period. We identified BC1 plants with a significantly higher share of the recurrent parent genome, thus saving one backcross generation. Furthermore, spring rapeseed as the recurrent parent in a backcrossing program could accelerate breeding by reducing the generation cycle. Our study depicts the potential for reducing the background mutation load while accelerating the generation cycle in EMS-induced winter oilseed rape populations by integrating genomic background selection.