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Allele-specific RNA-seq of single pollen precursors

ABSTRACT: Single pollen precursors were isolated from maize anthers, covering 3 weeks of development from before meiosis through mature pollen. Starting material was staged by anther length and microscopy, and anther stages were densely sampled throughout pollen development. All samples were from an F1 hybrid between A188 and B73 inbred lines, and transcripts were associated with a specific parental allele when possible. In the "transcript_counts.csv" file, each sample is given 4 columns to separate the allele calls: '_g1' contains counts matching the B73 allele, '_g2' contains counts matching the A188 allele, 'ua' means unassigned (no SNPs were present in the transcript), and '_cf' means conflicting (the transcript had SNPs matching both alleles -- this is most likely sequencing error). Code to reproduce all figures from the main text of Nelms and Walbot (2022) can be found at https://doi.org/10.5281/zenodo.6620997

ORGANISM(S): Zea mays

PROVIDER: GSE175505 | GEO | 2021/10/26

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study are to identify Mutator insertion sites and determine the affect of these insertions on nearby genes Methods: Wiseq-based Mu element profiling was performed as described previously using B73/Mo17 F1 hybrid progeny seedlings. The B73 parent was carried Mutator activity that had been introgressed into the B73 genetic background. The Mo17 parent lacked active Mu elements. Thus, all new insertions were into the B73 genome. Genomic DNA was extracted from 6-day-old seedlings of B73/Mo17 hybrid plants. Amplicon-based enrichment of Mu flanking DNA was then performed. The purified PCR products were subject to Miseq-based Wideseq pipeline at Purdue Genomics Core Facility (https://www.purdue.edu/hla/sites/genomics/wideseq-2/). Wideseq reads were mapped to the B73 reference genome as described previously. By identifying the Mu target site duplications (TSDs), a set of genes targeted by Mu insertions that segregated in hybrid progeny was obtained and those containing B73/Mo17 SNPs in their mRNA sequences were used for allele-specific expression analysis. Because new insertions were into the B73 genome, the effect of these insertions would be expected to be specific in all cases to the B73 allele. To quantify the allele frequency, we performed RT-PCR followed by Wideseq from the identical shoot tissues of the hybrid seedlings mentioned above. Total RNA was extracted using the RNA Extraction Kit (Zymo) and cDNAs were synthesized using Promega M-MLV Reverse Transcriptase. For for a subset of 16 genes that carried SNPs, RNA fragments containing B73/Mo17 SNPs were amplified by RT-PCR. Libraries were prepared using an mRNA preparation kit (Illumina) following a standard protocol (https://www.purdue.edu/hla/sites/genomics/wi-deseq-2/). The RT-PCR products were then sequenced by the “WideSeq” pipeline. Results: A knockdown index was deduced by normalizing the observed ratio to that observed in genes that lacked Mu insertions in both genetic backgrounds for each insertion. We found that none of the four promoter insertions changed the expression of nearby genes. A quarter of 5’ UTR insertions (5 out of 20) caused knockout or strong knockdown effects and one of seven intronic insertions resulted in a knockout effect. Collectively, of a total of 33 Mu insertions, all of which were within 200 bp of genes, only 11 significantly reduced gene expression, and only two knocked it out completely. Conclusions: These results indicate that that Mu element insertions near TSSs of host genes are often associated with quantitative and in many cases neglectable functional consequences on nearby gene expression.

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Allele-specific RNA-seq of single pollen precursors

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