Biology subjects

Koelliker, R.

Publications and source records attributed to Koelliker, R..

12 recordsLinked to original sources

bioRxiv2026

Near-complete, haplotype-resolved genome assembly of common buckwheat (Fagopyrum esculentum Moench)

Common buckwheat (Fagopyrum esculentum Moench) is a globally cultivated pseudocereal with a high nutritional quality and economic value. Due to its self-incompatibility, common buckwheat exhibits a high level of heterozygosity, making genome assembly challenging. Consequently, reference-level haplotype-resolved assemblies of common buckwheat are scarce, hindering research and genomics-assisted breeding. Here, we present a near-complete, chromosome-level, haplotype-resolved assembly of a common buckwheat F1 genotype (named Tuka), generated using a trio-binning approach that integrated parental Illumina short-read data with PacBio HiFi and Hi-C data from Tuka. The Tuka assembly comprises two haplomes, Tuka_h1 and Tuka_h2, both showing high contiguity (contig N50 of 76.68 Mb and 84.57 Mb, respectively), high completeness (assembly sizes of 1.28 Gb and 1.23 Gb with BUSCO scores of 96.9% and 96.8%, respectively), high base-level accuracy (QV of 59.08 and 63.03, respectively), and few gaps (35 and 30, respectively). This near-complete assembly of Tuka serves as a valuable genomic resource for common buckwheat, enabling advanced genomic analyses and accelerating research and breeding using state-of-the-art genomic tools.

genomics↗

bioRxiv2025

Candidate genes for stem rust resistance in Italian ryegrass revealed by nested association mapping

Stem rust, caused by Puccinia graminis ssp. graminicola, is a major disease affecting the outcrossing species Italian ryegrass (Lolium multiflorum Lam.), leading to substantial reductions in seed yield. Until now, knowledge on the genetic control of stem rust resistance in Italian ryegrass has been limited to a few quantitative trait loci identified in bi-parental mapping populations. To discover novel resistance sources for use in breeding programs, appropriate plant populations, reliable phenotyping methods and advanced genomic tools are essential. In this study, we utilized a previously established F2 nested association mapping (NAM) population comprising 708 individuals derived from 24 founder plants exhibiting high variation in stem rust resistance. Phenotypic evaluation was conducted under natural inoculation in three location-by-year combinations. By integrating reduced-representation sequencing of the NAM population with whole-genome sequencing of the founder plants, we identified 3,199,253 SNP markers for association mapping. The high SNP marker density, together with the strong detection power of the NAM population, enabled the identification of four novel candidate genes. Two of these genes, located on chromosomes 6 and 7, encode receptor-like serine/threonine kinases that are known to play a role in stem rust resistance in other crops. Within the serine/threonine kinase gene Chr7.32208, two superior haplotypes were identified that can be directly implemented as selection criteria in breeding programs. The novel stem rust resistance candidate genes reported here provide promising targets for functional validation and the improvement of stem rust resistance in Italian ryegrass breeding. Key messageField phenotyping of a previously established NAM population for stem rust resistance, combined with high-density genotyping, enabled the identification of novel sources of stem rust resistance in Italian ryegrass.

plant biology↗

bioRxiv2025

The first nested association mapping (NAM) population for outbreeding Italian ryegrass reveals candidate genes for seed shattering and related traits

Nested association mapping (NAM) populations are a powerful tool for investigating the genetic control of agronomically important traits and have been successfully used in many inbreeding crops. Here, we present the first NAM population established in an outcrossing forage grass species, Italian ryegrass (Lolium multiflorum Lam.), to dissect the genetic control of seed shattering. The NAM population was based on 23 diverse and one common founder plants and consisted of 708 F2 individuals. Reduced-representation sequencing (ddRAD) of the 708 F2 individuals, combined with whole genome sequencing data of the 24 founder plants, yielded a total of 3,199,253 SNPs that were used for population structure analysis, parentship analysis and genome-wide association studies. Phenotypic data for seed shattering and seed yield-related traits, collected in three year x location environments, showed high phenotypic variance within the NAM population. A total of seven QTL were identified for seed shattering, seed yield, spike length, flag leaf length and flowering time. Within these QTL regions, one candidate gene for seed shattering and three candidate genes for flowering time were identified. For seed shattering, the significant SNP association within the gene chr7.26897, known to be in involved in ripening-related pathways, explained 10.03% of the phenotypic variance. These candidate genes identified provide valuable targets for functional validation and demonstrate the effectiveness of NAM populations for elucidating the genetic architecture of complex traits in outcrossing forage grasses.

plant biology↗

bioRxiv2025

Genetic diversity unveiled: cost-effective methods for grassland species

AbstractPermanent grasslands are predominantly composed of allogamous plant species that exhibit high levels of plant genetic diversity (PGD) within their populations. Grasslands with high PGD are more resilient to environmental stress and constitute valuable reservoirs of genetic resources for plant breeding. Therefore, monitoring PGD is the basis for detecting changes in PGD and correspondingly intervening. However, PGD monitoring is often neglected in biodiversity reports due to difficulties in taking representative samples and in using standardised and affordable indicators of PGD. Here we successfully applied two common approaches, multispecies amplicon sequencing (MSAS) and genotyping-by-sequencing (GBS), to assess PGD of agronomically relevant grassland species. Using MSAS, we were able to taxonomically distinguish five species (Dactylis glomerata L., Festuca pratensis Huds., Lolium perenne L., Trifolium pratense L., Trifolium repens L.) from multispecies samples and differentiate accessions within species, with fixation index (F ST) values ranging from 0.014 for T. repens to 0.089 for L. perenne. Based on an extended L. perenne sample set containing mixtures of two cultivars at different ratios, mixtures containing both cultivars at 50% separated from the corresponding cultivars according to this ratio using MSAS and GBS. Furthermore, GBS enabled separation of samples containing two cultivars at a 75:25-ratio from the corresponding cultivars and the 50:50-ratio samples. These results indicate complementing applications of the two approaches in PGD monitoring. While we anticipate that MSAS with its cost-effectiveness could be applied to large-scale PGD monitoring, GBS with its lower detection limit could be applied to studies where cultivar composition shifts are of interest.

genetics↗

bioRxiv2024

Chromosome-level haplotype-resolved genome assembly provides insights into the highly heterozygous genome of Italian ryegrass (Lolium multiflorum Lam.)

Italian ryegrass (Lolium multiflorum Lam.) is an important forage grass, providing a major source of feed for ruminants in temperate regions. Due to its highly heterozygous and repeat-rich genome, high-quality chromosome-level genome assemblies are scarce for Italian ryegrass. Here, we sequenced the genome of a genotype from the Italian ryegrass cultivar Rabiosa (hereafter referred to as Rabiosa), and we obtained Oxford Nanopore Technologies long reads ([~]60-fold coverage), Illumina short reads ([~]85-fold coverage) and high-throughput chromosome conformation capture data ([~]60-fold coverage). With Rabiosa as the parent, we constructed an F1 population consisting of 305 individuals, which were genotyped by reduced representation sequencing for linkage map construction and quantitative trait loci (QTL) analysis. Using whole-genome sequencing data of Rabiosa and the genetic linkage map, we first generated a chromosome-level unphased haploid assembly (scaffold N50 of 338.75 Mb, total BUSCO score of 94.60%). Then, based on the unphased assembly and a reference-based phasing approach, we generated a chromosome-level haplotype-resolved assembly containing both haplotypes (scaffold N50 of [~]250 Mb and total BUSCO score of [~]90% for each haplotype). Between the two haplotypes of Rabiosa, we observed a highly collinear gene order at chromosome level and a high sequence variation at local level. With a graph-based reference built from the unphased and the haplotype-resolved assemblies of Rabiosa, we conducted a QTL analysis, and two QTL significantly associated with stem rust resistance were detected. The genome assemblies of Rabiosa will serve as invaluable genomic resources to facilitate genomic applications in forage grass research and breeding.

genomics↗

bioRxiv2023

High-resolution bulked segregant analysis enables candidate gene identification for bacterial wilt resistance in Italian ryegrass

Bacterial wilt, caused by Xanthomonas translucens pv. graminis (Xtg), is a serious disease of economically important forage grasses, including Italian ryegrass (Lolium multiflorum Lam.). A major QTL for resistance to Xtg was previously identified, but the precise location as well as the genetic factors underlying the resistance are yet to be determined. To this end, we applied a bulked segregant analysis (BSA) approach, using whole-genome deep sequencing of pools of the most resistant and most susceptible individuals of a large (n = 7,484) biparental F2 population segregating for resistance to Xtg. Using chromosome-level genome sequences as references, we were able to define a [~]300 kb region highly associated to resistance on pseudo-chromosome 4. Further investigation of this region revealed multiple genes with a known role in disease resistance, including genes encoding for Pik2-like disease resistance proteins, cysteine-rich kinases, and RGA4- and RGA5-like disease resistance proteins. Investigation of allele frequencies in the pools and comparative genome analysis in the grandparents of the F2 population revealed that some of these genes contain variants with allele frequencies that correspond to the expected heterozygosity in the resistant grandparent. This study emphasizes the efficacy combining BSA studies in very large populations with whole genome deep sequencing and high-quality genome sequences to pinpoint regions associated with a binary trait of interest and accurately define a small set of candidate genes. Furthermore, markers identified in this region hold significant potential for marker-assisted breeding strategies to breed resistance to Xtg in Italian ryegrass cultivars more efficiently.

plant biology↗

bioRxiv2023

An improved chromosome-level genome assembly of perennial ryegrass (Lolium perenne L.)

This work is an update and extension of the previously published article "Ultralong Oxford Nanopore Reads Enable the Development of a Reference-Grade Perennial Ryegrass Genome Assembly", by Frei et al.. The published genome assembly of the doubled haploid perennial ryegrass (Lolium perenne L.) genotype Kyuss marked a milestone for forage grass research and breeding. However, order and orientation errors may exist in the pseudo-chromosomes of Kyuss, since barley (Hordeum vulgare L.), which diverged 30 million years ago from perennial ryegrass, was used as the reference to scaffold Kyuss. To correct for structural errors possibly present in the published Kyuss assembly, we de novo assembled the genome again and generated 50-fold coverage high-throughput chromosome conformation capture (Hi-C) data to assist pseudo-chromosome construction. The resulting new chromosome-level assembly showed improved quality with high contiguity (contig N50 = 120 Mb), high completeness (total BUSCO score = 99%), high base-level accuracy (QV = 50) and correct pseudo-chromosome structure (validated by Hi-C contact map). This new assembly will serve as a better reference genome for Lolium spp. and greatly benefit the forage and turf grass research community.

genomics↗

bioRxiv2023

Higher seed yield through selection for reduced seed shattering in Italian ryegrass (Lolium multiflorum Lam.)

Seed shattering, i.e., the loss of seeds at ripening stage shortly before or during seed harvest, is strongly reducing seed yield in Italian ryegrass (Lolium multiflorum Lam.). The aim of this study was to evaluate the possibility to reduce seed shattering within breeding germplasm via recurrent phenotypic selection on spaced plants. Starting from a founder population of 300 plants serving as F0 population, two cycles of phenotypic selection for high and low seed shattering were applied and compared to randomly selected individuals on spaced plant level and in plot trials. Comparison of the five resulting populations in a spaced plant trial revealed a significant effect of selection, with lowest seed shattering (15.3%) observed in the population selected twice for decreased shattering (15.3%) and highest seed shattering (47.9%) for the population selected twice for increased shattering. The same ranking of the five F2 populations was observed in a subsequent trial with sown plots. Thus, using the method presented here, recurrent selection on single spaced plants allows to efficiently reduce seed shattering and, therefore, to increase seed yield in swards.

plant biology↗

bioRxiv2023

High genomic plasticity and unique features of Xanthomonas translucens pv. graminis revealed through comparative analysis of complete genome sequences

BackgroundXanthomonas translucens pv. graminis (Xtg) is a major bacterial pathogen of economically important forage grasses, causing severe yield losses. So far, genomic resources for this pathovar consisted mostly of draft genome sequences, and only one complete genome sequence was available, preventing comprehensive comparative genomic analyses. Such comparative analyses are essential in understanding the mechanisms involved in the virulence of pathogens and to identify virulence factors involved in pathogenicity. ResultsIn this study, we produced high-quality, complete genome sequences of four strains of Xtg, complementing the recently obtained complete genome sequence of the Xtg pathotype strain. These genomic resources allowed for a comprehensive comparative analysis, which revealed a high genomic plasticity with many chromosomal rearrangements, although the strains were highly related, with 99.9 to 100% average nucleotide identity. A high number of transposases were exclusively found in Xtg and corresponded to 413 to 457 insertion/excision transposable elements per strain. These mobile genetic elements are likely to be involved in the observed genomic plasticity and may play an important role in the adaptation of Xtg. The pathovar was found to lack a type IV secretion system, and it possessed the smallest set of type III effectors in the species. However, three XopE and XopX family effectors were found, while in the other pathovars of the species two or less were present. Additional genes that were specific to the pathovar were identified, including a unique set of minor pilins of the type IV pilus, 17 TonB-dependent receptors (TBDRs), and 11 degradative enzymes. ConclusionThese results suggest a high adaptability of Xtg, conferred by the abundance of mobile genetic elements, which may have led to the loss of many features. Conserved features that were specific to Xtg were identified, and further investigation will help to determine genes that are essential to pathogenicity and host adaptation of Xtg.

plant biology↗

bioRxiv2022

Phenotypic variation and quantitative trait loci for resistance to southern anthracnose and clover rot in red clover

Red clover (Trifolium pratense L.) is an important forage legume of temperate regions, particularly valued for its high yield potential and its high forage quality. Despite substantial breeding progress during the last decades, continuous improvement of cultivars is crucial to ensure yield stability in view of newly emerging diseases or changing climatic conditions. The high amount of genetic diversity present in red clover ecotypes, landraces and cultivars provides an invaluable, but often unexploited resource for the improvement of key traits such as yield, quality, and resistance to biotic and abiotic stresses. A collection of 397 red clover accessions was genotyped using a pooled genotyping-by-sequencing approach with 200 plants per accession. Resistance to the two most pertinent diseases in red clover production, southern anthracnose caused by Colletotrichum trifolii, and clover rot caused by Sclerotinia trifoliorum, was assessed using spray inoculation. The mean survival rate for southern anthracnose was 22.9% and the mean resistance index for clover rot was 34.0%. Genome-wide association analysis revealed several loci significantly associated with resistance to southern anthracnose and clover rot. Most of these loci are in coding regions. One quantitative trait locus (QTL) on chromosome 1 explained 16.8% of the variation in resistance to southern anthracnose. For clover rot resistance we found eight QTL, explaining together 80.2% of the total phenotypic variation. The SNPs associated with these QTL provide, once validated, a promising resource for marker-assisted selection in existing breeding programs, facilitating the development of novel cultivars with increased resistance against two devastating fungal diseases of red clover. Key messageHigh variability for and candidate loci associated with resistance to southern anthracnose and clover rot in a worldwide collection of red clover provide a first basis for genomics-assisted breeding.

plant biology↗

bioRxiv2021

A multispecies amplicon sequencing approach for genetic diversity assessment in grassland plant species

Grasslands are widespread and economically relevant ecosystems at the basis of sustainable roughage production. Plant genetic diversity (PGD; i.e., within-species diversity) is related to many beneficial effects to the ecosystem functioning of grasslands. The monitoring of PGD in temperate grasslands is complicated by the multiplicity of species present and by a shortage of methods for large-scale assessment. However, the continuous advancement of high-throughput DNA sequencing approaches have improved the prospects of broad, multispecies PGD monitoring. Among them, amplicon sequencing stands out as a robust and cost-effective method. Here we report a set of twelve multispecies primer pairs that can be used for high-throughput PGD assessment in multiple grassland plant species. The loci targeted by the amplicons were selected and tested in two phases: a "discovery phase" based on a sequence capture assay (611 target nuclear loci assessed in 16 grassland plant species), which resulted in the selection of eleven loci; and a "validation phase", in which the selected loci were targeted and sequenced using twelve multispecies primers in test populations of Dactylis glomerata L., Lolium perenne L., Festuca pratensis Huds., Trifolium pratense L. and T. repens L. The resulting multispecies amplicons had overall nucleotide diversities per species ranging from 5.19 x 10-3 to 1.29 x 10-2, which is in the range of flowering-related genes but slightly lower than pathogen resistance genes. We conclude that the methodology, the DNA sequence resources, and the amplicon-specific primer pairs reported in this study provide the basis for large-scale, multispecies PGD monitoring in grassland plants.

ecology↗

bioRxiv2021

Evidence for high intergenic sequence variation in heterozygous Italian ryegrass (Lolium multiflorum Lam.) genome revealed by a high-quality draft diploid genome assembly

BackgroundOver the last decade, progress in DNA sequencing technologies and assembly methods allowed plant scientists to move beyond the use of model organisms and work directly on the genomes of the major crops. Forage grass research can also benefit from this revolution, enabling progress in population genetic studies, functional biology, and genomics-assisted breeding. Due to its large genome size and high repeat content, so far only incomplete and fragmented assemblies are available for the grasses of the Lolium and Festuca species complex. FindingsHere, we report a highly contiguous draft assembly of Italian ryegrass (L. multiflorum Lam.), spanning 4.5 Gb and with a N50 of 3 Mb, containing ~70,000 gene models. Thanks to its relatedness to barley, 78% of the assembly was anchored on seven pseudomolecules. The high heterozygosity of the plant allowed obtaining a diploid assembly - i.e. across 95% of the assembly, both alleles were assembled on separate sequences. This feature allowed unraveling a very high amount of intergenic sequence variation between allelic sequences. ConclusionsWe present a nearly complete genome assembly of a genotype used in contemporary Swiss forage grass breeding programs. This work shows how genomic research has improved, allowing to decode the genetic code of large, complex, and heterozygous plants. It also allows the functional characterization of the ryegrass gene repertoire and the large-scale development of molecular markers. Furthermore, it paves the road for a reference-based characterization and exploitation of the genetic variation within the Lolium and Festuca species complex.

genomics↗