Abstract
BackgroundQTL-seq, in combination with bulked segregant analysis and next-generation sequencing (NGS), is used to identify loci in small plant genomes, but is technically challenging to perform in species with large genomes, such as barley. A combination of exome sequencing and QTL-seq (exome QTL-seq) was used to map the mono-factorial Mendelian locus black lemma and pericarp (Blp) and QTLs for resistance to net blotch disease, a common disease of barley caused by the fungus Pyrenophora teres, which segregated in a population of 100 doubled haploid barley lines.MethodsThe provisional exome sequences were prepared by ordering the loci of expressed genes based on the genome information and concatenating genes with intervals of 200-bp spacer "N" for each chromosome. The QTL-seq pipeline was used to analyze short reads from the exome-captured library.ResultsIn this study, short NGS reads of bulked total DNA samples from segregants with extreme trait values were subjected to exome capture, and the resulting exome sequences were aligned to the reference genome. SNP allele frequencies were compared to identify the locations of genes/QTLs responsible for the trait value differences between lines. For both objective traits examined, exome QTL-seq identified the monogenic Mendelian locus and associated QTLs. These findings were validated using conventional mapping approaches.ConclusionsExome QTL-seq broadens the utility of NGS-based gene/QTL mapping in organisms with large genomes.
Highlights
Quantitative trait loci (QTL)-seq, in combination with bulked segregant analysis and next-generation sequencing (NGS), is used to identify loci in small plant genomes, but is technically challenging to perform in species with large genomes, such as barley
Construction of the pseudo reference sequence As a result of adding a reference sequence step to the QTL-seq pipeline, ca. 3.2 million Haruna Nijo RNAseq reads were mapped onto the provisional exome sequences (PESs), and a total of 25,451 Single nucleotide polymorphism (SNP) was detected between the RNA-seq reads and the PESs (Additional file 2: Table S3), in which the nucleotides from Morex were replaced with those from Haruna Nijo
Exome QTL-seq in barley provides opportunities for the direct mapping of a trait onto the genome, and for generating markers that can be used to narrow down the position of a particular locus in the genome
Summary
QTL-seq, in combination with bulked segregant analysis and next-generation sequencing (NGS), is used to identify loci in small plant genomes, but is technically challenging to perform in species with large genomes, such as barley. Bulked segregant analysis (BSA) has been combined with whole-genome sequencing (WGS) to rapidly identify causative nucleotide changes in a given mutant (MutMap [1]) and quantitative trait loci (QTLs) (QTL-seq [2]) in rice. These methods are suitable for studies of organisms with relatively small genomes (e.g., genome sizes of
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