Abstract
Haplotype phasing maize genetic variants is important for genome interpretation, population genetic analysis and functional analysis of allelic activity. We performed an isoform-level phasing study using two maize inbred lines and their reciprocal crosses, based on single-molecule, full-length cDNA sequencing. To phase and analyze transcripts between hybrids and parents, we developed IsoPhase. Using this tool, we validated the majority of SNPs called against matching short-read data from embryo, endosperm and root tissues, and identified allele-specific, gene-level and isoform-level differential expression between the inbred parental lines and hybrid offspring. After phasing 6907 genes in the reciprocal hybrids, we annotated the SNPs and identified large-effect genes. In addition, we identified parent-of-origin isoforms, distinct novel isoforms in maize parent and hybrid lines, and imprinted genes from different tissues. Finally, we characterized variation in cis- and trans-regulatory effects. Our study provides measures of haplotypic expression that could increase accuracy in studies of allelic expression.
Highlights
Haplotype phasing maize genetic variants is important for genome interpretation, population genetic analysis and functional analysis of allelic activity
Using IsoPhase, we demonstrated that haplotype phasing by full-length transcript sequencing can reveal allele-specific expression (ASE) in maize reciprocal hybrids
We pooled the reads from all four lines and processed them using the IsoSeq[3] workflow (Fig. 1); 76.3% (3,739,812) of reads were classified as fulllength insert transcripts based on the presence of barcoded primers and poly(A) tails (Supplementary Tables 1 and 2)
Summary
Haplotype phasing maize genetic variants is important for genome interpretation, population genetic analysis and functional analysis of allelic activity. We performed an isoform-level phasing study using two maize inbred lines and their reciprocal crosses, based on singlemolecule, full-length cDNA sequencing. To phase and analyze transcripts between hybrids and parents, we developed IsoPhase Using this tool, we validated the majority of SNPs called against matching short-read data from embryo, endosperm and root tissues, and identified allele-specific, gene-level and isoform-level differential expression between the inbred parental lines and hybrid offspring. ASEs were studied using short-read RNAseq, which could quantify alleles at the SNP level but was unable to provide full-length haplotype information[8,9]. We developed a tool called IsoPhase to phase the allelic isoforms in the hybrids based on the single-molecule transcriptome datasets. Using IsoPhase, we demonstrated that haplotype phasing by full-length transcript sequencing can reveal ASE in maize reciprocal hybrids. This technique allows the assessment of biased allelic expression and isoform expression
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