Development and Characterization of a New TILLING Population of Common Bread Wheat (Triticum aestivum L.)

Liang Chen,Martin A J Parry,Donghong Min,Andy Phillips,Shiqiang Wang,Yin-Gang Hu,Linzhou Huang,Pippa J Madgwick,Markus Grebe

doi:10.1371/journal.pone.0041570

Abstract

Mutagenesis is an important tool in crop improvement. However, the hexaploid genome of wheat (Triticum aestivum L.) presents problems in identifying desirable genetic changes based on phenotypic screening due to gene redundancy. TILLING (Targeting Induced Local Lesions IN Genomes), a powerful reverse genetic strategy that allows the detection of induced point mutations in individuals of the mutagenized populations, can address the major challenge of linking sequence information to the biological function of genes and can also identify novel variation for crop breeding. Wheat is especially well-suited for TILLING due to the high mutation densities tolerated by polyploids. However, only a few wheat TILLING populations are currently available in the world, which is far from satisfying the requirement of researchers and breeders in different growing environments. In addition, current TILLING screening protocols require costly fluorescence detection systems, limiting their use, especially in developing countries. We developed a new TILLING resource comprising 2610 M2 mutants in a common wheat cultivar ‘Jinmai 47’. Numerous phenotypes with altered morphological and agronomic traits were observed from the M2 and M3 lines in the field. To simplify the procedure and decrease costs, we use unlabeled primers and either non-denaturing polyacrylamide gels or agarose gels for mutation detection. The value of this new resource was tested using PCR with RAPD and Intron-spliced junction (ISJ) primers, and also TILLING in three selected candidate genes, in 300 and 512 mutant lines, revealing high mutation densities of 1/34 kb by RAPD/ISJ analysis and 1/47 kb by TILLING. In total, 31 novel alleles were identified in the 3 targeted genes and confirmed by sequencing. The results indicate that this mutant population represents a useful resource for the wheat research community. We hope that the use of this reverse genetics resource will provide novel allelic diversity for wheat improvement and functional genomics.

Highlights

Wheat is an important food crop world-wide
Genome sequencing programs for many plant species [3,4,5] has led to the availability of a large number of genomic sequences in public databases which subsequently has encouraged the development of reverse genetics tools [6]
The genomic DNA from five to ten individuals was pooled for mutation detection, but did not give clear digested bands by PAGE or agarose gel electrophoresis

Summary

Introduction

Wheat is an important food crop world-wide. many traits that are important for wheat production would benefit from the ability to understand and modify the function of specific genes [1,2]. Several reverse genetic tools are currently used for this purpose, such as T-DNA or transposon insertion, which have greatly assisted functional genomics in model species [7,8,9,10] These resources are still not available in wheat [11,12]. TILLING (Targeting Induced Local Lesions IN Genomes), which combines chemical mutagenesis with highthroughput genome-wide screening for point mutations in genes of interest, has been developed in response This methodology may be preferable to other reverse genetics approaches for various reasons. EMS can create random point mutations at high density in polyploid plants This allows multiple alleles of a specific gene to be obtained in a small population regardless of the genome size [18,19,20,21]

Methods

Results

Conclusion