Novel Informatic Tools to Support Functional Annotation of the Durum Wheat Genome.

Mario Fruzangohar,Priyanka Kalambettu,Elena Kalashyan,Curtis J Pozniak,Krysta Wiebe,Penny J Tricker,Ute Baumann,Jennifer Ens

doi:10.3389/fpls.2019.01244

Abstract

Seed mutagenesis is one strategy to create a population with thousands of useful mutations for the direct selection of desirable traits, to introduce diversity into varietal improvement programs, or to generate a mutant collection to support gene functional analysis. However, phenotyping such large collections, where each individual may carry many mutations, is a bottleneck for downstream analysis. Targeting Induced Local Lesions in Genomes (TILLinG), when coupled with next-generation sequencing allows high-throughput mutation discovery and selection by genotyping. We mutagenized an advanced durum breeding line, UAD0951096_F2:5 and performed short-read (2x125 bp) Illumina sequencing of the exome of 100 lines using an available exome capture platform. To improve variant calling, we generated a consolidated exome reference using the recently available genome sequences of the cultivars Svevo and Kronos to facilitate the alignment of reads from the UAD0951096_F2:5 derived mutants. The resulting exome reference was 484.4 Mbp. We also developed a user-friendly, searchable database and bioinformatic analysis pipeline that allowed us to predict zygosity of the mutations discovered and extracts flanking sequences for rapid marker development. Here, we present these tools with the aim of allowing researchers fast and accurate downstream selection of mutations discovered by TILLinG by sequencing to support functional annotation of the durum wheat genome.

Highlights

Mutants are valuable tools for the identification and functional analysis of genes
We mapped reads from the unmutagenized control line to the two publically available durum wheat genome assemblies [Svevo (Maccaferri et al, 2019) and Kronos (‘Kronos EI v1’)]
By combining these aligned read data with contigs assembled from unmapped reads, we constructed a new durum exome reference, DECaR, which consists of 220,114 contigs with a total length of 484,479,862 bp covering ca. 4% of the estimated 11-Gbp durum wheat genome

Summary

Introduction

Mutants are valuable tools for the identification and functional analysis of genes. Mutations can arise spontaneously or can be induced physically (e.g., radiation), chemically (e.g., alkylating agents), and by transposon insertions or through gene editing, such as with the CRISPR/Cas system for specific modifications of target genes (Adli, 2018).The use of chemical mutagenesis has had a renaissance with the development of Targeting Induced Local Lesions in Genomes (TILLinG) method in Arabidopsis (McCallum et al, 2000). Mutants are valuable tools for the identification and functional analysis of genes. Mutations can arise spontaneously or can be induced physically (e.g., radiation), chemically (e.g., alkylating agents), and by transposon insertions or through gene editing, such as with the CRISPR/Cas system for specific modifications of target genes (Adli, 2018). The use of chemical mutagenesis has had a renaissance with the development of Targeting Induced Local Lesions in Genomes (TILLinG) method in Arabidopsis (McCallum et al, 2000). TILLinG is a high-throughput method of inducing and identifying genetic variations in target genes. Its main advantage is that it can be employed as a functional genomics platform for virtually any species, independent of genome size and ploidy. It is not surprising that TILLinG populations have been generated for various animal and plant species as described in (Kurowska et al, 2011)

Methods

Results

Discussion

Conclusion