Ó Lochlainn, Seosamh
(2011)
Natural genetic variation in zinc (Zn) accumulation in Brassicaceae.
PhD thesis, University of Nottingham.
Abstract
Zinc (Zn) is an essential plant nutrient. Most plant species have a shoot Zn concentration ([Zn]shoot) <0.1 mg Zn g-1 dry weight (DW), but extensive natural genetic variation occurs. For example, within the Brassicaceae, some Noccaea (Thlaspi) and Arabidopsis species hyperaccumulate [Zn]shoot >10 mg Zn g-1 DW. There is compelling evidence that orthologues of the Arabidopsis thaliana PIB-type Heavy-Metal-Associated domain-containing ATPase 4 (AtHMA4), which transport Zn2+ and other cations, have a major involvement in the Zn hyperaccumulation trait. The aim of this thesis was to study aspects of genetic variation in the Brassicaceae using a comparative genomic approach, focussing primarily on orthologues of AtHMA4 in Noccaea and Brassica.
The first major objective was to clone the full genomic sequence of NcHMA4. This locus was successfully sequenced in Noccaea caerulescens Saint Laurent Le Minier. First, a new genomic fosmid library was generated comprising 36,864 clones with 40 kb inserts, giving ~5-fold genomic coverage. Through DNA fingerprinting, Genome Sequencer (GS) FLX 454 sequencing and contig assembly, a single region collinear with AtHMA4 flanking genes was identified. Unlike A. thaliana, four novel tandem HMA4 gene repeats with highly conserved coding regions, but substantially divergent promoter regions, were present. Preliminary evidence indicates cis-regulated high expression, supporting previous expression data for N. caerulescens. Notably, this observation is remarkably consistant with recent findings in A. halleri.
In planta analysis of NcHMA4 remains challenging in N. caerulescens due to a vernal obligate lengthy life cycle (7–9 months) and lack of a robust transformation system. To facilitate future analyses, genetically-stable faster cycling M4 lines were therefore created using fast neutron (FN) mutagenesis. Two non vernal obligate lines have been characterised bearing fruit as soon as 92 days after sowing (DAS) and showing no perturbed [Zn]shoot or obvious pleiotropic effects. Future efforts should focus on their efficient transformation to improve future in planta biological understanding.
In Brassica, data from previously reported glasshouse and field studies on B. oleracea L. [Zn]shoot were further analysed to test for the presence of HMA4 orthologues in QTL regions. However, large QTL and multiple paralogues have hindered progress. A more efficient Targeting Induced Local Lesions In Genomes (TILLing)-based approach has therefore been pursued in B. rapa during the latter stages of this study. Locus specific allelic variants in a candidate metal transporter gene BraA.CAX1.a have been identified and methods for rapid downstream genotyping (High Resolution Melt (HRM)-based efficient SNP detection technology) and characterisation have been developed successfully. These approaches are now underway for BraA.HMA4 and an additional candidate metal transporter BraA.ESB1. Since A. thaliana knock-outs of ESB1, CAX1 and HMA genes have altered nutritional phenotypes, future studies will focus on their characterisation under contrasting mineral environments.
This thesis has pursued a comparative genomics approach. A previously unreported quadruplication and cis-regulation probably contributes to high HMA4 expression in N. caerulescens. Fast cycling Noccaea lines and a robust Brassica genotyping platform were developed. These will become valuable tools for downstream molecular genetic approaches for in planta functional analysis of HMA4 and other transporters to determine their role in regulating mineral accumulation in Brassicaceae. Ultimately, a greater understanding of genetic variation in [Zn]shoot may have downstream application in genetic biofortification or phytoremediation strategies.
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