Targeted parallel sequencing of large genetically-defined genomic regions for identifying mutations in Arabidopsis

Matthew Mccormack,Kun-Hsiang Liu,Jen Sheen

doi:10.1186/1746-4811-8-12

Matthew Mccormack, Kun-Hsiang Liu + Show 1 more

Open Access

https://doi.org/10.1186/1746-4811-8-12

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Abstract

Large-scale genetic screens in Arabidopsis are a powerful approach for molecular dissection of complex signaling networks. However, map-based cloning can be time-consuming or even hampered due to low chromosomal recombination. Current strategies using next generation sequencing for molecular identification of mutations require whole genome sequencing and advanced computational devises and skills, which are not readily accessible or affordable to every laboratory. We have developed a streamlined method using parallel massive sequencing for mutant identification in which only targeted regions are sequenced. This targeted parallel sequencing (TPSeq) method is more cost-effective, straightforward enough to be easily done without specialized bioinformatics expertise, and reliable for identifying multiple mutations simultaneously. Here, we demonstrate its use by identifying three novel nitrate-signaling mutants in Arabidopsis.

Highlights

Genetic screens are a powerful approach for studying diverse processes by isolating mutants showing phenotypes directly or indirectly involved in biological pathways
We designed a dual genetic screen strategy to isolate mutants involved in nitrate signaling
We selected nitrite reductase (NIR) as our nitrate response marker gene because NIR plays a critical role in the nitrate assimilation pathway, it is encoded by a single gene, and NIR expression can be rapidly and consistently induced by nitrate [14]

Summary

Introduction

Genetic screens are a powerful approach for studying diverse processes by isolating mutants showing phenotypes directly or indirectly involved in biological pathways. Identifying the molecular lesion underlying these phenotypes is crucial towards understanding the mechanism of the process it is involved in. In order to reveal the molecular identity of the mutant, positional cloning is commonly employed to identify the mutations [1]. Despite the availability of the Arabidopsis genome sequence, positional cloning from diverse mutant screens can be time-consuming or even hampered due to low chromosomal recombination in megabase-sized regions surrounding the mutation [1,2,3,4]. The copious numbers of mutations generated during the mutagenesis processes become a single mutant is described in one report [7]. Detection of the known mutations were found only in some cases using one-lane sequencing due to variable and low coverage of the genome [7]

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Conclusion