Abstract
BackgroundCultivated 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).ResultsWith 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.ConclusionsThe 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.
Highlights
Cultivated chrysanthemum is an outcrossing hexaploid (2n = 6× = 54) with a disputed mode of inheritance
Lower van Geest et al BMC Genomics (2017) 18:585 single nucleotide polymorphism (SNP) densities are expected in expressed sequences, which is an advantage in highly heterozygous polyploid species, as polymorphisms in flanking regions interact with marker assays
Most of the markers had a dosage of RNA sequencing, assembly, and alignment RNA-seq resulted in an average of 100.4 M reads for the deep-sequenced parents of the large population
Summary
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. Lower van Geest et al BMC Genomics (2017) 18:585 single nucleotide polymorphism (SNP) densities are expected in expressed sequences, which is an advantage in highly heterozygous polyploid species, as polymorphisms in flanking regions interact with marker assays. RNA-seq gives a representation of the transcriptome that helps building resources useful for other analyses
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