Altered hippocampal epigenetic regulation underlying reduced cognitive development in response to early life environmental insults
Schachtschneider, Kyle M. ; Welge, Michael E. ; Auvil, Loretta S. ; Chaki, Sulalita ; Rund, Laurie A. ; Madsen, Ole ; Elmore, Monica R.P. ; Johnson, Rodney W. ; Groenen, Martien A.M. ; Schook, Lawrence B. - \ 2020
Genes 11 (2020)2. - ISSN 2073-4425
Cognitive development - DNA methylation - Hippocampus - Machine learning - Porcine biomedical models - RNA-seq
The hippocampus is involved in learning and memory and undergoes significant growth and maturation during the neonatal period. Environmental insults during this developmental timeframe can have lasting effects on brain structure and function. This study assessed hippocampal DNA methylation and gene transcription from two independent studies reporting reduced cognitive development stemming from early life environmental insults (iron deficiency and porcine reproductive and respiratory syndrome virus (PRRSv) infection) using porcine biomedical models. In total, 420 differentially expressed genes (DEGs) were identified between the reduced cognition and control groups, including genes involved in neurodevelopment and function. Gene ontology (GO) terms enriched for DEGs were associated with immune responses, angiogenesis, and cellular development. In addition, 116 differentially methylated regions (DMRs) were identified, which overlapped 125 genes. While no GO terms were enriched for genes overlapping DMRs, many of these genes are known to be involved in neurodevelopment and function, angiogenesis, and immunity. The observed altered methylation and expression of genes involved in neurological function suggest reduced cognition in response to early life environmental insults is due to altered cholinergic signaling and calcium regulation. Finally, two DMRs overlapped with two DEGs, VWF and LRRC32, which are associated with blood brain barrier permeability and regulatory T-cell activation, respectively. These results support the role of altered hippocampal DNA methylation and gene expression in early life environmentally-induced reductions in cognitive development across independent studies.
Usability of reference-free transcriptome assemblies for detection of differential expression: a case study on Aethionema arabicum dimorphic seeds
Wilhelmsson, Per K.I. ; Chandler, Jake O. ; Fernandez-Pozo, Noe ; Graeber, Kai ; Ullrich, Kristian K. ; Arshad, Waheed ; Khan, Safina ; Hofberger, Johannes ; Buchta, Karl ; Edger, Patrick P. ; Pires, J.C. ; Schranz, Eric ; Leubner-Metzger, Gerhard ; Rensing, Stefan A. - \ 2019
Wageningen University and Research
Aethionema arabicum - Dimorphic seeds - Reference and reference-free - RNA-seq - Transciptome
Background RNA-sequencing analysis is increasingly utilized to study gene expression in non-model organisms without sequenced genomes. Aethionema arabicum (Brassicaceae) exhibits seed dimorphism as a bet-hedging strategy â producing both a less dormant mucilaginous (M+) seed morph and a more dormant non-mucilaginous (NM) seed morph. Here, we compared de novo and reference-genome based transcriptome assemblies to investigate Ae. arabicum seed dimorphism and to evaluate the reference-free versus -dependent approach for identifying differentially expressed genes (DEGs). Results A de novo transcriptome assembly was generated using sequences from M+ and NM Ae. arabicum dry seed morphs. The transcripts of the de novo assembly contained 63.1% complete Benchmarking Universal Single-Copy Orthologs (BUSCO) compared to 90.9% for the transcripts of the reference genome. DEG detection used the strict consensus of three methods (DESeq2, edgeR and NOISeq). Only 37% of 1533 differentially expressed de novo assembled transcripts paired with 1876 genome-derived DEGs. Gene Ontology (GO) terms distinguished the seed morphs: the terms translation and nucleosome assembly were overrepresented in DEGs higher in abundance in M+ dry seeds, whereas terms related to mRNA processing and transcription were overrepresented in DEGs higher in abundance in NM dry seeds. DEGs amongst these GO terms included ribosomal proteins and histones (higher in M+), RNA polymerase II subunits and related transcription and elongation factors (higher in NM). Expression of the inferred DEGs and other genes associated with seed maturation (e.g. those encoding late embryogenesis abundant proteins and transcription factors regulating seed development and maturation such as ABI3, FUS3, LEC1 and WRI1 homologs) were put in context with Arabidopsis thaliana seed maturation and indicated that M+ seeds may desiccate and mature faster than NM. The 1901 transcriptomic DEG set GO-terms had almost 90% overlap with the 2191 genome-derived DEG GO-terms. Conclusions Whilst there was only modest overlap of DEGs identified in reference-free versus -dependent approaches, the resulting GO analysis was concordant in both approaches. The identified differences in dry seed transcriptomes suggest mechanisms underpinning previously identified contrasts between morphology and germination behaviour of M+ and NM seeds.
Usability of reference-free transcriptome assemblies for detection of differential expression : A case study on Aethionema arabicum dimorphic seeds
Wilhelmsson, Per K.I. ; Chandler, Jake O. ; Fernandez-Pozo, Noe ; Graeber, Kai ; Ullrich, Kristian K. ; Arshad, Waheed ; Khan, Safina ; Hofberger, Johannes A. ; Buchta, Karl ; Edger, Patrick P. ; Pires, J.C. ; Schranz, M.E. ; Leubner-Metzger, Gerhard ; Rensing, Stefan A. - \ 2019
BMC Genomics 20 (2019)1. - ISSN 1471-2164
Aethionema arabicum - Dimorphic seeds - Reference and reference-free - RNA-seq - Transcriptome
Background: RNA-sequencing analysis is increasingly utilized to study gene expression in non-model organisms without sequenced genomes. Aethionema arabicum (Brassicaceae) exhibits seed dimorphism as a bet-hedging strategy - producing both a less dormant mucilaginous (M+) seed morph and a more dormant non-mucilaginous (NM) seed morph. Here, we compared de novo and reference-genome based transcriptome assemblies to investigate Ae. arabicum seed dimorphism and to evaluate the reference-free versus -dependent approach for identifying differentially expressed genes (DEGs). Results: A de novo transcriptome assembly was generated using sequences from M+ and NM Ae. arabicum dry seed morphs. The transcripts of the de novo assembly contained 63.1% complete Benchmarking Universal Single-Copy Orthologs (BUSCO) compared to 90.9% for the transcripts of the reference genome. DEG detection used the strict consensus of three methods (DESeq2, edgeR and NOISeq). Only 37% of 1533 differentially expressed de novo assembled transcripts paired with 1876 genome-derived DEGs. Gene Ontology (GO) terms distinguished the seed morphs: the terms translation and nucleosome assembly were overrepresented in DEGs higher in abundance in M+ dry seeds, whereas terms related to mRNA processing and transcription were overrepresented in DEGs higher in abundance in NM dry seeds. DEGs amongst these GO terms included ribosomal proteins and histones (higher in M+), RNA polymerase II subunits and related transcription and elongation factors (higher in NM). Expression of the inferred DEGs and other genes associated with seed maturation (e.g. those encoding late embryogenesis abundant proteins and transcription factors regulating seed development and maturation such as ABI3, FUS3, LEC1 and WRI1 homologs) were put in context with Arabidopsis thaliana seed maturation and indicated that M+ seeds may desiccate and mature faster than NM. The 1901 transcriptomic DEG set GO-terms had almost 90% overlap with the 2191 genome-derived DEG GO-terms. Conclusions: Whilst there was only modest overlap of DEGs identified in reference-free versus -dependent approaches, the resulting GO analysis was concordant in both approaches. The identified differences in dry seed transcriptomes suggest mechanisms underpinning previously identified contrasts between morphology and germination behaviour of M+ and NM seeds.
Phenotypic plasticity and the evolution of azole resistance in Aspergillus fumigatus; An expression profile of clinical isolates upon exposure to itraconazole
Hokken, Margriet W.J. ; Zoll, Jan ; Coolen, Jordy P.M. ; Zwaan, Bas J. ; Verweij, Paul E. ; Melchers, Willem J.G. - \ 2019
BMC Genomics 20 (2019)1. - ISSN 1471-2164
Aspergillus fumigatus - Azole resistance - Itraconazole - Phenotypic plasticity - RNA-seq
Background: The prevalence of azole resistance in clinical and environmental Aspergillus fumigatus isolates is rising over the past decades, but the molecular basis of the development of antifungal drug resistance is not well understood. This study focuses on the role of phenotypic plasticity in the evolution of azole resistance in A. fumigatus. When A. fumigatus is challenged with a new stressful environment, phenotypic plasticity may allow A. fumigatus to adjust their physiology to still enable growth and reproduction, therefore allowing the establishment of genetic adaptations through natural selection on the available variation in the mutational and recombinational gene pool. To investigate these short-term physiological adaptations, we conducted time series transcriptome analyses on three clinical A. fumigatus isolates, during incubation with itraconazole. Results: After analysis of expression patterns, we identified 3955, 3430, 1207, and 1101 differentially expressed genes (DEGs), after 30, 60, 120 and 240 min of incubation with itraconazole, respectively. We explored the general functions in these gene groups and we identified 186 genes that were differentially expressed during the whole time series. Additionally, we investigated expression patterns of potential novel drug-efflux transporters, genes involved in ergosterol and phospholipid biosynthesis, and the known MAPK proteins of A. fumigatus. Conclusions: Our data suggests that A. fumigatus adjusts its transcriptome quickly within 60 min of exposure to itraconazole. Further investigation of these short-term adaptive phenotypic plasticity mechanisms might enable us to understand how the direct response of A. fumigatus to itraconazole promotes survival of the fungus in the patient, before any "hard-wired" genetic mutations arise.
Transcriptional effects of cadmium on iron homeostasis differ in calamine accessions of Noccaea caerulescens
Halimaa, Pauliina ; Blande, Daniel ; Baltzi, Erol ; Aarts, Mark G.M. ; Granlund, Lars ; Keinänen, Markku ; Kärenlampi, Sirpa O. ; Kozhevnikova, Anna D. ; Peräniemi, Sirpa ; Schat, Henk ; Seregin, Ilya V. ; Tuomainen, Marjo ; Tervahauta, Arja I. - \ 2019
The Plant Journal 97 (2019)2. - ISSN 0960-7412 - p. 306 - 320.
cadmium - Illumina - iron deficiency - IRT1 - Noccaea caerulescens - RNA-seq - spectral imaging - Thlaspi caerulescens - transcriptome
Calamine accessions of the zinc/cadmium/nickel hyperaccumulator, Noccaea caerulescens, exhibit striking variation in foliar cadmium accumulation in nature. The Ganges accession (GA) from Southern France displays foliar cadmium hyperaccumulation (>1000 μg g−1 DW), whereas the accession La Calamine (LC) from Belgium, with similar local soil metal composition, does not (<100 μg g−1 DW). All calamine accessions are cadmium hypertolerant. To find out the differences between LC and GA in their basic adaptation mechanisms, we bypassed the cadmium excluding phenotype of LC by exposing the plants to 50 μm cadmium in hydroponics, achieving equal cadmium accumulation in the shoots. The iron content increased in the roots of both accessions. GA exhibited significant decreases in manganese and zinc contents in the roots and shoots, approaching those in LC. Altogether 702 genes responded differently to cadmium exposure between the accessions, 157 and 545 in the roots and shoots, respectively. Cadmium-exposed LC showed a stress response and had decreased levels of a wide range of photosynthesis-related transcripts. GA showed less changes, mainly exhibiting an iron deficiency-like response. This included increased expression of genes encoding five iron deficiency-regulated bHLH transcription factors, ferric reduction oxidase FRO2, iron transporters IRT1 and OPT3, and nicotianamine synthase NAS1, and decreased expression of genes encoding ferritins and NEET (a NEET family iron-sulfur protein), which is possibly involved in iron transfer, distribution and/or management. The function of the IRT1 gene in the accessions was compared. We conclude that the major difference between the two accessions is in the way they cope with iron under cadmium exposure.
Comparative transcriptome analysis of Ethiopian indigenous chickens from low and high altitudes under heat stress condition reveals differential immune response
Park, W. ; Srikanth, K. ; Lim, D. ; Park, M. ; Hur, T. ; Kemp, S. ; Dessie, T. ; Kim, M.S. ; Lee, S.R. ; Pas, M.F.W. te; Kim, J.M. ; Park, J.E. - \ 2019
Animal Genetics 50 (2019)1. - ISSN 0268-9146 - p. 42 - 53.
highlands - lowlands - RNA-seq - thermal tolerance
Ethiopia is an ecologically diverse country; the low altitude regions are hot and humid whereas the high altitude regions are cooler. In this study we analyzed the transcriptome response of high altitude (Addis Ababa) and low altitude (Awash) chickens to heat stress conditions that are prevalent in the low altitude regions. The chickens were free ranged for 20 h in an enclosure in Awash, and then the heart, breast muscle and spleen tissues were collected at 6:00 am, 12:00 noon and 6:00 pm to follow a daily circadian cycle. Through RNA-sequencing analysis, we identified differentially expressed genes (DEGs) that were significant (q < 0.05). These DEGs were subjected to protein–protein interaction (PPI) network and gene co-expression network (GCN) analyses to understand their role. KEGG pathway analysis and Gene Ontology analysis of all the identified DEGs and the genes identified from the PPI network and GCN analyses revealed that several immune-related pathways, such as proteasome, focal adhesion, influenza A, the ErbB signaling pathway and glycerophospholipid metabolism, were enriched in response to heat stress. These results suggest that the high altitude chickens were under heat stress and might be immunologically susceptible. Our findings will help in developing a genetic approach to mitigate production loss due to heat stress.
Aspergillus fumigatus - Aspergillus fumigatus differential expression upon itraconazole addition
Hokken, Margriet W.J. ; Zoll, Jan ; Coolen, Jordy P.M. ; Zwaan, Bas ; Verweij, Paul E. ; Melchers, Willem J.G. - \ 2018
Radboud University Medical Centre
PRJNA482512 - 482512 - Aspergillus fumigatus - Itraconazole - Azole resistance - Phenotypic plasticity - RNA-seq
The filamentous fungus Aspergillus fumigatus is an opportunistic pathogen which causes life-threatening diseases in immunocompromised patients. Resistance of A. fumigatus against azole class compounds continues to pose a threat to human health worldwide. How this fungus maintains fitness before any resistance causing mutations arise is not well understood. In this study, we use RNA-seq to demonstrate how A. fumigatus exerts its phenotypic plasticity when exposed to itraconazole, and which important cellular processes contribute to its adaptation to a new, stressful environment containing azole compounds. We conducted an in vitro assay in which we exposed 24h old mycelia of clinical A. fumigatus isolates grown at 37°C, to sublethal concentrations of itraconazole which might permit gradual adaptation of the fungus to the new, stressful environment. Mycelia were then harvested after 30, 60, 120 or 240 minutes respectively, and relative expression was compared to the mycelia harvested directly after 24h without the addition of itraconazole
RNA Sequencing of Stentor Cell Fragments Reveals Transcriptional Changes during Cellular Regeneration
Onsbring, Henning ; Jamy, Mahwash ; Ettema, Thijs J.G. - \ 2018
Current Biology 28 (2018)8. - ISSN 0960-9822 - p. 1281 - 1288.e3.
cell damage repair - cell regeneration - ciliate - microbial eukaryotes - protist - RNA-seq - single-cell transcriptomics - Stentor
While ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 upregulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement, and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many upregulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes, such as dual-specific tyrosine-(Y)-phosphorylation-regulated kinases and MORN domain-containing proteins that seemingly act as key regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies [1, 2], our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities. Onsbring et al. sequence transcriptomes of individual bisections of regenerating cells of the giant heterotrichous ciliate Stentor polymorphus. Their differential expression analysis reveals that protein phosporylation, microtubule-based processes, and genes involved in the cell cycle are important for cellular regeneration.
Molecular Investigation of the Ciliate Spirostomum semivirescens, with First Transcriptome and New Geographical Records
Hines, Hunter N. ; Onsbring, Henning ; Ettema, Thijs J.G. ; Esteban, Genoveva F. - \ 2018
Protist 169 (2018)6. - ISSN 1434-4610 - p. 875 - 886.
anaerobic respiration - Heterotrich. - Protist - RNA-seq - stop codon - symbiotic algae
The ciliate Spirostomum semivirescens is a large freshwater protist densely packed with endosymbiotic algae and capable of building a protective coating from surrounding particles. The species has been rarely recorded and it lacks any molecular investigations. We obtained such data from S. semivirescens isolated in the UK and Sweden. Using single-cell RNA sequencing of isolates from both countries, the transcriptome of S. semivirescens was generated. A phylogenetic analysis identified S. semivirescens as a close relative to S. minus. Additionally, rRNA sequence analysis of the green algal endosymbiont revealed that it is closely related to Chlorella vulgaris. Along with the molecular species identification, an analysis of the ciliates’ stop codons was carried out, which revealed a relationship where TGA stop codon frequency decreased with increasing gene expression levels. The observed codon bias suggests that S. semivirescens could be in an early stage of reassigning the TGA stop codon. Analysis of the transcriptome indicates that S. semivirescens potentially uses rhodoquinol-dependent fumarate reduction to respire in the oxygen-depleted habitats where it lives. The data also shows that despite large geographical distances (over 1,600 km) between the sampling sites investigated, a morphologically-identical species can share an exact molecular signature, suggesting that some ciliate species, even those over 1 mm in size, could have a global biogeographical distribution.
Nucleus-specific expression in the multinuclear mushroom-forming fungus Agaricus bisporus reveals different nuclear regulatory programs
Gehrmann, Thies ; Pelkmans, Jordi F. ; Ohm, Robin A. ; Vos, Aurin M. ; Sonnenberg, Anton S.M. ; Baars, Johan J.P. ; Wösten, Han A.B. ; Reinders, Marcel J.T. ; Abeel, Thomas - \ 2018
Proceedings of the National Academy of Sciences of the United States of America 115 (2018)17. - ISSN 0027-8424 - p. 4429 - 4434.
fungi - heterokaryon - Nuclear-specific expression - quantification - RNA-seq
Many fungi are polykaryotic, containing multiple nuclei per cell. In the case of heterokaryons, there are different nuclear types within a single cell. It is unknown what the different nuclear types contribute in terms of mRNA expression levels in fungal heterokaryons. Each cell of the mushroom Agaricus bisporus contains two to 25 nuclei of two nuclear types originating from two parental strains. Using RNA-sequencing data, we assess the differential mRNA contribution of individual nuclear types and its functional impact. We studied differential expression between genes of the two nuclear types, P1 and P2, throughout mushroom development in various tissue types. P1 and P2 produced specific mRNA profiles that changed through mushroom development. Differential regulation occurred at the gene level, rather than at the locus, chromosomal, or nuclear level. P1 dominated mRNA production throughout development, and P2 showed more differentially up-regulated genes in important functional groups. In the vegetative mycelium, P2 up-regulated almost threefold more metabolism genes and carbohydrate active enzymes (cazymes) than P1, suggesting phenotypic differences in growth. We identified widespread transcriptomic variation between the nuclear types of A. bisporus. Our method enables studying nucleus-specific expression, which likely influences the phenotype of a fungus in a polykaryotic stage. Our findings have a wider impact to better understand gene regulation in fungi in a heterokaryotic state. This work provides insight into the transcriptomic variation introduced by genomic nuclear separation.
Enzyme activities at different stages of plant biomass decomposition in three species of fungusgrowing termites
Costa, Rafael R. da; Hu, Haofu ; Pilgaard, Bo ; Sabine, Sabine M. ; Schückel, Julia ; Pedersen, Kristine S.K. ; Kračun, Stjepan K. ; Busk, Peter K. ; Harholt, Jesper ; Sapountzis, Panagiotis ; Lange, Lene ; Aanen, Duur K. ; Poulsen, Michael - \ 2018
Applied and Environmental Microbiology 84 (2018)5. - ISSN 0099-2240
AZCL - Chromogenic substrates - HPLC - Macrotermes - Odontotermes - Peptide pattern recognition - Plant substrate - RNA-seq - Symbiosis - Termitomyces
Fungus-growing termites rely on mutualistic fungi of the genus Termitomyces and gut microbes for plant biomass degradation. Due to a certain degree of symbiont complementarity, this tripartite symbiosis has evolved as a complex bioreactor, enabling decomposition of nearly any plant polymer, likely contributing to the success of the termites as one of the main plant decomposers in the Old World. In this study, we evaluated which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We found a diversity of active enzymes at different stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant substrate. However, preliminary fungal RNA sequencing (RNA-seq) analyses suggest that this likely transport is supplemented with enzymes produced in situ. Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mixture of plant material, fungal spores, and enzymes, is likely the key to the extraordinarily efficient plant decomposition in fungus-growing termites.
Aethionema arabicum - Aethionema arabicum Raw sequence reads
Wilhelmsson, Per K.I. ; Chandler, Jake O. ; Fernandez-Pozo, Noe ; Graeber, Kai ; Ullrich, Kristian K. ; Arshad, Waheed ; Khan, Safina ; Hofberger, Johannes ; Buchta, Karl ; Edger, Patrick P. ; Pires, Chris ; Schranz, Eric ; Leubner-Metzger, Gerhard ; Rensing, Stefan A. - \ 2017
University of Marburg
PRJNA413671 - 413671 - Aethionema arabicum - Dimorphic seeds - Reference and reference-free - RNA-seq - transcriptome
Investigation of seed dimorphism in Aethionema arabicum.
Construction of a high-density genetic map from RNA-Seq data for an Arabidopsis bay-0 × shahdara ril population
Serin, Elise A.R. ; Snoek, L.B. ; Nijveen, Harm ; Willems, Leo A.J. ; Jiménez-Gómez, Jose M. ; Hilhorst, Henk W.M. ; Ligterink, Wilco - \ 2017
Frontiers in Genetics Livestock Genomics 8 (2017)DEC. - ISSN 1664-8021
Arabidopsis - Genetic map - Genotyping by sequencing - QTL mapping - Resolution - RIL population - RNA-seq
High-density genetic maps are essential for high resolution mapping of quantitative traits. Here, we present a new genetic map for an Arabidopsis Bayreuth × Shahdara recombinant inbred line (RIL) population, built on RNA-seq data. RNA-seq analysis on 160 RILs of this population identified 30,049 single-nucleotide polymorphisms (SNPs) covering the whole genome. Based on a 100-kbp window SNP binning method, 1059 bin-markers were identified, physically anchored on the genome. The total length of the RNA-seq genetic map spans 471.70 centimorgans (cM) with an average marker distance of 0.45 cM and a maximum marker distance of 4.81 cM. This high resolution genotyping revealed new recombination breakpoints in the population. To highlight the advantages of such high-density map, we compared it to two publicly available genetic maps for the same population, comprising 69 PCR-based markers and 497 gene expression markers derived from microarray data, respectively. In this study, we show that SNP markers can effectively be derived from RNA-seq data. The new RNA-seq map closes many existing gaps in marker coverage, saturating the previously available genetic maps. Quantitative trait locus (QTL) analysis for published phenotypes using the available genetic maps showed increased QTL mapping resolution and reduced QTL confidence interval using the RNA-seq map. The new high-density map is a valuable resource that facilitates the identification of candidate genes and map-based cloning approaches.
Conclusive evidence for hexasomic inheritance in chrysanthemum based on analysis of a 183 k SNP array
Geest, Geert van; Voorrips, Roeland E. ; Esselink, Danny ; Post, Aike ; Visser, Richard G.F. ; Arens, Paul - \ 2017
BMC Genomics 18 (2017). - ISSN 1471-2164
Allelic expression bias - Disomic - Hexaploid - Polyploid - Polysomic - RNA-seq - SNP array - SNP retrieval
Background: Cultivated chrysanthemum is an outcrossing hexaploid (2n = 6× = 54) with a disputed mode of inheritance. In this paper, we present a single nucleotide polymorphism (SNP) selection pipeline that was used to design an Affymetrix Axiom array with 183 k SNPs from RNA sequencing data (1). With this array, we genotyped four bi-parental populations (with sizes of 405, 53, 76 and 37 offspring plants respectively), and a cultivar panel of 63 genotypes. Further, we present a method for dosage scoring in hexaploids from signal intensities of the array based on mixture models (2) and validation of selection steps in the SNP selection pipeline (3). The resulting genotypic data is used to draw conclusions on the mode of inheritance in chrysanthemum (4), and to make an inference on allelic expression bias (5). Results: With use of the mixture model approach, we successfully called the dosage of 73,936 out of 183,130 SNPs (40.4%) that segregated in any of the bi-parental populations. To investigate the mode of inheritance, we analysed markers that segregated in the large bi-parental population (n = 405). Analysis of segregation of duplex x nulliplex SNPs resulted in evidence for genome-wide hexasomic inheritance. This evidence was substantiated by the absence of strong linkage between markers in repulsion, which indicated absence of full disomic inheritance. We present the success rate of SNP discovery out of RNA sequencing data as affected by different selection steps, among which SNP coverage over genotypes and use of different types of sequence read mapping software. Genomic dosage highly correlated with relative allele coverage from the RNA sequencing data, indicating that most alleles are expressed according to their genomic dosage. Conclusions: The large population, genotyped with a very large number of markers, is a unique framework for extensive genetic analyses in hexaploid chrysanthemum. As starting point, we show conclusive evidence for genome-wide hexasomic inheritance.