Biology subjects

Ruttink, T.

Publications and source records attributed to Ruttink, T..

13 recordsLinked to original sources

bioRxiv2023

Cultivated Robusta coffee meets wild Coffea canephora: Evidence of cultivated-wild hybridisation in the Democratic Republic of the Congo

Background and aimsPlant breeders are increasingly turning to crop wild relatives (CWRs) to ensure global food security amidst a rapidly changing environment. However, CWR populations are confronted with various human-induced threats, including hybridisation with their nearby cultivated crops, posing a significant threat to the wild gene pool. Here, we focussed on Robusta coffee in the Yangambi region in the DR Congo, where cultivated Robusta coffee coexists with its wild relative (Coffea canephora), creating ample opportunity for genetic exchange. We examined the geographical distribution of cultivated Robusta coffee plants and the incidence of hybridisation between cultivated and wild individuals within the rainforest. MethodsWe used Genotyping-by-Sequencing data from 471 C. canephora individuals across various locations, including natural rainforests, home gardens, and the INERA Coffee Collection, a Robusta coffee field genebank. Our analyses involved establishing robust diagnostic fingerprints that differentiate cultivated from wild coffee, identifying cultivated-wild hybrids and mapping their geographical position. Key resultsWe identified cultivated genotypes and cultivated-wild hybrids in zones with clear anthropogenic activity, and where Robusta coffee cultivated in the home gardens may serve as a source for crop-to-wild gene flow. We found relatively few hybrids and backcrosses in the rainforests, which is so far likely not sufficient for the stable incorporation of alleles from the cultivated gene pool into the CWR gene pool. ConclusionsThe cultivation of Robusta coffee in close proximity to its wild gene pool has led to cultivated genotypes and cultivated-wild hybrids appearing within the natural habitats of C. canephora. Yet, given the high overlap between the cultivated and wild gene pool, together with the relatively low frequency of hybridisation, our results indicate that the overall impact in terms of risk of introgression remains limited so far. Nevertheless, it is important to continue monitoring crop-to-wild gene flow to safeguard the wild gene pool of C. canephora.

ecology↗

bioRxiv2023

Characterization of the genetic composition and establishment of a core collection for the INERA Robusta coffee (Coffea canephora) field genebank from the Democratic Republic of the Congo

Cultivation of Robusta coffee is likely to gain importance because of its high disease resistance and climate envelope, but substantial effort must be made to improve its cupping quality and other agronomic traits. Robusta coffee genetic resources conserved in field genebanks can play an important role in this context, but many of them are limited and poorly characterized. In this study, we aimed to characterize the genetic composition of the historically important but until recently neglected INERA Coffee Collection in Yangambi (Democratic Republic of the Congo) and to propose a Robusta core collection. We combined genome-wide GBS marker sets with a novel versatile multiplex amplicon sequencing (HiPlex) screening assay, for the genetic screening of the INERA Coffee Collection and created tools for future genetic screening. A set of 730 coffee shrubs in the collection were genotyped at 86 HiPlex loci and 263 unique genetic fingerprints were identified. Representative fingerprints were compared to Lula cultivars, Luki cultivars, Congolese subgroup A, and wild genotypes growing in the rainforest of the Yangambi region. The majority of the unique genetic fingerprints were identified as Lula cultivars but also four Congolese subgroup A and nine wild genotypes were found in the collection. Twenty-five percent of the unique genetic fingerprints had a hybrid identity which could result from seed-based propagation of the collection or natural cross-pollination. Moreover, eight unique genetic fingerprints were frequently identified as a parent and these may correspond to the ancient selected mother plants, or direct descendants thereof, of which seed material was widely distributed from 1930 to 1960 for the creation of coffee plantations. Finally, to delineate core collections we tested the maximization strategy and genetic distance method for the INERA Coffee Collection, and proposed i) a core collection conserving the loci diversity of the collection, i.e. 100 accessions and ii) a core collection holding the highest genetic distances between the accessions, i.e. 10 accessions. Our study provides a method for the genetic characterization of Robusta coffee collections in general and contributes to the revaluation and exploitation of the Robusta coffee genetic resources in the Democratic Republic of the Congo in particular.

plant biology↗

bioRxiv2023

Industrial chicory genome gives insight into the molecular time table of anther development and male sterility

Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but crucially relies on stable self-incompatibility. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines.

plant biology↗

bioRxiv2022

Multi-location trials and population-based genotyping reveal high diversity and adaptation to breeding environments in a large collection of red clover

Red clover (Trifolium pratense L.) is an outcrossing forage legume that has adapted to a wide range of climatic and growing conditions across Europe. Red clover is valued for its high yield potential and forage quality. The high amount of genetic diversity present in red clover provides an invaluable, but often poorly characterized resource to improve key traits such as yield, quality, and resistance to biotic and abiotic stresses. In this study, we examined the genetic and phenotypic diversity within a diverse set of 395 diploid red clover accessions via genome wide allele frequency fingerprinting and multi-location field trials across Europe. We found that the genetic structure of accessions mostly reflected their geographic origin and only few cases were detected, where breeders integrated foreign genetic resources into their local breeding pools. Phenotypic performance of accessions in the multi-location field trials revealed a very strong accession x location interaction. Notably, breeding material and cultivars generally performed well at the location where they were developed. Local adaptation was especially prominent in Nordic red clover accessions that showed a distinct adaptation to the growing conditions and cutting regime of the North. Our results confirmed that red clover cultivars were bred from regional ecotypes and show a narrow adaptation to regional conditions. Our study can serve as a valuable basis for identifying interesting material that express the desired characteristics and contribute to the adaptation of red clover to future climatic conditions.

genetics↗

bioRxiv2022

Genetic diversity and structure in wild Robusta coffee (Coffea canephora A. Froehner) populations in Yangambi (DR Congo) and their relation with forest disturbance

Degradation and regeneration of tropical forests can strongly affect gene flow in understorey species, resulting in genetic erosion and changes in genetic structure. Yet, these processes remain poorly studied in tropical Africa. Coffea canephora is an economically important species, found in the understorey of tropical rainforests of Central and West Africa, and the genetic diversity harboured in its wild populations is vital for sustainable coffee production worldwide. Here, we aimed to quantify genetic diversity, genetic structure, and pedigree relations in wild C. canephora populations, and we investigated associations between these descriptors and forest disturbance and regeneration. Therefore, we sampled 256 C. canephora individuals within 24 plots across three forest categories in Yangambi (DR Congo), and used genotyping-by-sequencing to identify 18 894 SNPs. Overall, we found high genetic diversity, and no evidence of genetic erosion in C. canephora in disturbed old-growth forest, as compared to undisturbed old-growth forest. Additionally, an overall heterozygosity excess was found in all populations, which was expected for a self-incompatible species. Genetic structure was mainly a result of isolation-by-distance, reflecting geographical location, with low to moderate relatedness at finer scales. Populations in regrowth forest had lower allelic richness than populations in old-growth forest and were characterised by a lower inter-individual relatedness and a lack of isolation-by-distance, suggesting that they originated from different neighbouring populations and were subject to founder effects. Wild Robusta coffee populations in the study area still harbour high levels of genetic diversity, yet careful monitoring of their response to ongoing forest degradation remains required.

ecology↗

bioRxiv2022

SMAP design: A multiplex PCR amplicon and gRNA design tool to screen for natural and CRISPR-induced genetic variation

Multiplex amplicon sequencing is a versatile method to identify genetic variation in natural or mutagenized populations through eco-tilling or multiplex CRISPR screens. Such genotyping screens require reliable and specific primer designs, combined with simultaneous gRNA design for CRISPR screens. Unfortunately, current tools are unable to combine multiplex gRNA and primer design into a high-throughput and easy-to-use manner with high design flexibility. Here, we report the development of a bioinformatics tool called SMAP design to overcome these limitations. We tested SMAP design on several plant and non-plant genomes and obtained designs for more than 80-90% of the target genes, depending on the genome and gene family. We validated the primer designs with Illumina multiplex amplicon sequencing and Sanger sequencing in Arabidopsis and soybean. We also used SMAP design to perform eco-tilling by tilling PCR amplicons across nine candidate genes putatively associated with haploid induction in Cichorium intybus. We screened 60 accessions of chicory and witloof and identified thirteen knockout haplotypes and their carriers. SMAP design is an easy-to-use command-line tool that generates highly specific gRNA and/or primer designs for any number of loci for CRISPR or natural variation screens and is compatible with other SMAP modules for seamless downstream analysis.

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↗

bioRxiv2022

BREEDIT: A novel multiplex genome editing strategy to improve complex quantitative traits in maize (Zea mays L.)

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Though new technologies are being applied to tackle the problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in gene engineering via the CRISPR/Cas technology pave the way to accelerate plant breeding and meet this increasing demand. Here, we present a gene discovery pipeline named BREEDIT that combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought resistance. We induced gene knockouts in 48 growth-related genes using CRISPR/Cas9 and generated a collection of over 1000 gene-edited maize plants. Edited populations displayed, on average, significant increases of 5 to 10% for leaf length and up to 20% for leaf width compared with controls. For each gene family, edits in subsets of genes could be associated with increased traits, allowing us to reduce the gene space needed to focus on for trait improvement. We propose BREEDIT as a gene discovery pipeline which can be rapidly applied to generate a diverse collection of mutants to identify subsets of promising candidates that could be later incorporated in breeding programs.

systems biology↗

bioRxiv2022

Stack Mapping Anchor Points (SMAP): a versatile suite of tools for read-backed haplotyping

Here we present SMAP, a software package that implements a suite of computational tools to extract multi-allelic haplotypes using read-backed haplotyping. SMAP tools first perform accurate read processing and analyze read mapping distributions across sample sets. Then, two complementary modules can be invoked for haplotype calling: SMAP haplotype-sites combines known Single Nucleotide Polymorphisms (SNPs) and/or read mapping position polymorphisms (SMAPs) to reconstruct compressed, read-reference-encoded haplotype strings. In contrast, SMAP haplotype-window works independent of prior knowledge of polymorphisms, groups reads by locus, defines a window enclosed between two custom border sequences, and retains the entire corresponding DNA sequence as haplotype. Haplotype-window is, among many applications, especially useful for high-throughput CRISPR/Cas mutation screens. Either way, SMAP creates a single integrated haplotype call table across all loci and samples. SMAP haplotyping is extremely versatile and can be applied to highly multiplex amplicon sequencing (HiPlex), Shotgun (e.g. whole genome shotgun (WGS) sequencing, probe capture and RNA-Seq), or Genotyping-by-Sequencing (GBS) data; and to Illumina short reads, PacBio and MinION long reads. SMAP creates discrete genotype calls for individuals of any ploidy or quantitative haplotype frequency spectra for Pool-Seq data, and can scale from tens to thousands of loci and/or samples. SMAP, including the source code written in Python is available at https://gitlab.com/truttink/smap, and a detailed user manual and guidelines for accurate read processing is available at https://ngs-smap.readthedocs.io/, under the GNU Affero General Public License v3.0.

genomics↗

bioRxiv2020

Phylogenomic analysis clarifies the evolutionary origin of Coffea arabica L.

Interspecific hybridization events have played a major role in plant speciation, yet, the evolutionary origin of hybrid species often remains enigmatic. Here, we inferred the evolutionary origin of the allotetraploid species Coffea arabica, which is widely cultivated for Arabica coffee production. We estimated genetic distances between C. arabica and all species that are known to be closely related to C. arabica using genotyping-by-sequencing (GBS) data. In addition, we reconstructed a time-calibrated multilabeled phylogenetic tree of 24 species to infer the age of the C. arabica hybridization event. Ancestral states of self-compatibility were also reconstructed to infer the evolution of self-compatibility in Coffea. C. canephora and C. eugenioides were confirmed as the putative progenitor species of C. arabica. These species most likely hybridized between 1.08 million and 543 thousand years ago. We inferred the phylogenetic relationships between C. arabica and its closest relatives and shed new light on the evolution of self-compatibility in Coffea. Furthermore, the age of the hybridization event coincides with periods of environmental upheaval, which may have induced range shifts of the progenitor species that facilitated the emergence of C. arabica.

evolutionary biology↗

bioRxiv2018

Utilization of tissue ploidy level variation in de novo transcriptome assembly of Pinus sylvestris

Compared to angiosperms, gymnosperms lag behind in the availability of assembled and annotated genomes. Most genomic analyses in gymnosperms, especially conifer tree species, rely on the use of de novo assembled transcriptomes. However, the level of allelic redundancy and transcript fragmentation in these assembled transcriptomes, and their effect on downstream applications have not been fully investigated. Here, we assessed three assembly strategies, including the utility of haploid (megagametophyte) tissue during de novo assembly as single-allele guides, for six individuals and five different tissues in Pinus sylvestris. We then contrasted haploid and diploid tissue genotype calls obtained from the assembled transcriptomes to evaluate the extent of paralog mapping. The use of the haploid tissue during assembly increased its completeness without reducing the number of assembled transcripts. Our results suggest that current strategies that rely on available genomic resources as guidance to minimize allelic redundancy are less effective than the application of strategies that cluster redundant assembled transcripts. The strategy yielding the lowest levels of allelic redundancy among the assembled transcriptomes assessed here was the generation of SuperTranscripts with Lace followed by CD-HIT clustering. However, we still observed some levels of heterozygosity (multiple gene fragments per transcript reflecting allelic redundancy) in this assembled transcriptome on the haploid tissue, indicating that further filtering is required before using these assemblies for downstream applications. We discuss the influence of allelic redundancy when these reference transcriptomes are used to select regions for probe design of exome capture baits and for estimation of population genetic diversity.

evolutionary biology↗

bioRxiv2018

Pleistocene climate changes explain large-scale genetic variation in a dominant grassland species, Lolium perenne L.

AimGrasslands have been pivotal in the development of herbivore breeding since the Neolithic and are still nowadays the most widespread agricultural land-use across Europe. However, it remains unclear whether the current large-scale genetic variation of plant species found in natural grasslands of Europe is the result of human activities or natural processes.\n\nLocationEurope.\n\nTaxonLolium perenne L (perennial ryegrass).\n\nMethodsWe reconstructed the phylogeographic history of L. perenne, a dominant grassland species, using 481 natural populations including 11 populations from closely related taxa. We combined the Genotyping-by-Sequencing (GBS) and Pool-sequencing (Pool-seq) methods to obtain high-quality allele frequency calls of ~ 500 k SNP loci. We performed genetic structure analyses and demographic reconstructions based on the site frequency spectrum (SFS). We additionally used the same genotyping protocol to assess the genomic diversity of a set of 32 cultivars representative of the L. perenne cultivars widely used for forage purposes.\n\nResultsExpansion across Europe took place during the Wurm glaciation (12-110 kya), a cooling period that decreased the dominance of trees in favour of grasses. Splits and admixtures in L. perenne fit historical sea level changes in the Mediterranean basin. The development of agriculture in Europe (7-3.5 kya), that caused an increase in the abundance of grasslands, did not have an effect on the demographic patterns of L. perenne. We found little differentiation between modern cultivars and certain natural variants. However, modern cultivars do not represent the wide genetic variation found in natural populations.\n\nMain conclusionsDemographic events in L. perenne can be explained by the changing climatic conditions during the Pleistocene. Natural populations maintain a wide genomic variability at continental scale that has been underused by recent breeding activities. This variability constitutes valuable standing genetic variation for future adaptation of grasslands to climate change, safeguarding the agricultural services they provide.

evolutionary biology↗

bioRxiv2018

A high-quality sequence of Rosa chinensis to elucidate genome structure and ornamental traits

Rose is the worlds most important ornamental plant with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Rose has a complex genome with high heterozygosity and various ploidy levels. Our objectives were (i) to develop the first high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short read sequencing, and anchoring to a high-density genetic map and (ii) to study the genome structure and the genetic basis of major ornamental traits.\n\nWe produced a haploid rose line from R. chinensis Old Blush and generated the first rose genome sequence at the pseudo-molecule scale (512 Mbp with N50 of 3.4 Mb and L75 of 97). The sequence was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features including the pericentromeric regions through annotation of TE families and positioned centromeric repeats using FISH. Genetic diversity was analysed by resequencing eight Rosa species. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and number of flower petals. A rose APETALA2 homologue is proposed to be the major regulator of petals number in rose. This reference sequence is an important resource for studying polyploidisation, meiosis and developmental processes as we demonstrated for flower and prickle development. This reference sequence will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.

genomics↗