Natural genetic variation in zinc (Zn) accumulation in Brassicaceae

Zinc (Zn) is an essential plant nutrient. Most plant species have a shoot Zn concentration ([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

Natural genetic variation in zinc (Zn) accumulation in Brassicaceae

Zinc (Zn) is an essential plant nutrient. Most plant species have a shoot Zn concentration ([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

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

Background: Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results: We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions: Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service

Genetical and comparative genomics of Brassica under altered Ca supply identifies Arabidopsis Ca-transporter orthologs

Although Ca transport in plants is highly complex, the overexpression of vacuolar Ca2+ transporters in crops is a promising new technology to improve dietary Ca supplies through biofortification. Here, we sought to identify novel targets for increasing plant Ca accumulation using genetical and comparative genomics. Expression quantitative trait locus (eQTL) mapping to 1895 cis- and 8015 trans-loci were identified in shoots of an inbred mapping population of Brassica rapa (IMB211 x R500); 23 cis- and 948 trans-eQTLs responded specifically to altered Ca supply. eQTLs were screened for functional significance using a large database of shoot Ca concentration phenotypes of Arabidopsis thaliana. From 31 Arabidopsis gene identifiers tagged to robust shoot Ca concentration phenotypes, 21 mapped to 27 B. rapa eQTLs, including orthologs of the Ca2+ transporters At-CAX1 and At-ACA8. Two of three independent missense mutants of BraA.cax1a, isolated previously by targeting induced local lesions in genomes, have allele-specific shoot Ca concentration phenotypes compared with their segregating wild types. BraA.CAX1a is a promising target for altering the Ca composition of Brassica, consistent with prior knowledge from Arabidopsis. We conclude that multiple-environment eQTL analysis of complex crop genomes combined with comparative genomics is a powerful technique for novel gene identification/prioritization

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea. A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOSTM clones with insert sizes ~20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs. Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter b-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue. This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae

Genetical and comparative genomics of Brassica under altered Ca supply identifies Arabidopsis Ca-transporter orthologs

Although Ca transport in plants is highly complex, the overexpression of vacuolar Ca2+ transporters in crops is a promising new technology to improve dietary Ca supplies through biofortification. Here, we sought to identify novel targets for increasing plant Ca accumulation using genetical and comparative genomics. Expression quantitative trait locus (eQTL) mapping to 1895 cis- and 8015 trans-loci were identified in shoots of an inbred mapping population of Brassica rapa (IMB211 x R500); 23 cis- and 948 trans-eQTLs responded specifically to altered Ca supply. eQTLs were screened for functional significance using a large database of shoot Ca concentration phenotypes of Arabidopsis thaliana. From 31 Arabidopsis gene identifiers tagged to robust shoot Ca concentration phenotypes, 21 mapped to 27 B. rapa eQTLs, including orthologs of the Ca2+ transporters At-CAX1 and At-ACA8. Two of three independent missense mutants of BraA.cax1a, isolated previously by targeting induced local lesions in genomes, have allele-specific shoot Ca concentration phenotypes compared with their segregating wild types. BraA.CAX1a is a promising target for altering the Ca composition of Brassica, consistent with prior knowledge from Arabidopsis. We conclude that multiple-environment eQTL analysis of complex crop genomes combined with comparative genomics is a powerful technique for novel gene identification/prioritization

Magnesium and calcium overaccumulate in the leaves of a schengen3 mutant of Brassica rapa

Magnesium (Mg) and calcium (Ca) are essential mineral nutrients poorly supplied in many human food systems. In grazing livestock, Mg and Ca deficiencies are costly welfare issues. Here, we report a Brassica rapa loss-of-function schengen3 (sgn3) mutant, braA.sgn3.a-1, which accumulates twice as much Mg and a third more Ca in its leaves. We mapped braA.sgn3.a to a single recessive locus using a forward ionomic screen of chemically mutagenized lines with subsequent backcrossing and linked-read sequencing of second back-crossed, second filial generation (BC2F2) segregants. Confocal imaging revealed a disrupted root endodermal diffusion barrier, consistent with SGN3 encoding a receptor-like kinase required for normal formation of Casparian strips, as reported in thale cress (Arabidopsis thaliana). Analysis of the spatial distribution of elements showed elevated extracellular Mg concentrations in leaves of braA.sgn3.a-1, hypothesized to result from preferential export of excessive Mg from cells to ensure suitable cellular concentrations. This work confirms a conserved role of SGN3 in controlling nutrient homeostasis in B. rapa, and reveals mechanisms by which plants are able to deal with perturbed shoot element concentrations resulting from a “leaky” root endodermal barrier. Characterization of variation in leaf Mg and Ca accumulation across a mutagenized population of B. rapa shows promise for using such populations in breeding programs to increase edible concentrations of essential human and animal nutrients

Natural genetic variation in zinc (Zn) accumulation in Brassicaceae

Zinc (Zn) is an essential plant nutrient. Most plant species have a shoot Zn concentration ([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.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Biofortifying Brassica with calcium (Ca) and magnesium (Mg)

Billions of people worldwide consume insufficient calcium (Ca) or magnesium (Mg) for adequate health. Dietary Ca and Mg intakes can potentially be increased through crop biofortification. Recently, we reported sufficient natural genetic variation and heritability in a leafy crop plant (Brassica oleracea; C-genome, 1n=9; cabbage, cauliflower, kale etc.) to indicate that genetic biofortification is feasible in vegetable Brassica. We also reported loci affecting shoot Ca and Mg concentration (shoot-Ca and Mg). Here, we extend the previous study to explore the closely related species B. rapa (A-genome, 1n=10; Chinese cabbage, pak choi, a more tractable species genetically, and the amphidiploid species B. napus(AC-genome, 1n=19; canola/oilseed rape etc.). Wide variation in shoot/leaf-Ca and Mg occurs among all three species. Shoot/leaf-Ca and Mg is significantly and highly heritable. Quantitative trait loci (QTL) affecting shoot/leaf Ca and Mg concentration occur in potentially paralogous regions of B. oleracea and B. rapa. If confirmed, allelic variation at such loci could be used in biofortification breeding programs for vegetable Brassica. As genome sequencing and marker generation improves, it will be possible to resolve these (and other) putative loci to the gene level. Further studies on the regulation, interaction and function of these genes will enable us to understand Ca and Mg dynamics in plants

Biofortifying Brassica with calcium (Ca) and magnesium (Mg)

Billions of people worldwide consume insufficient calcium (Ca) or magnesium (Mg) for adequate health. Dietary Ca and Mg intakes can potentially be increased through crop biofortification. Recently, we reported sufficient natural genetic variation and heritability in a leafy crop plant (Brassica oleracea; C-genome, 1n=9; cabbage, cauliflower, kale etc.) to indicate that genetic biofortification is feasible in vegetable Brassica. We also reported loci affecting shoot Ca and Mg concentration (shoot-Ca and Mg). Here, we extend the previous study to explore the closely related species B. rapa (A-genome, 1n=10; Chinese cabbage, pak choi, a more tractable species genetically, and the amphidiploid species B. napus(AC-genome, 1n=19; canola/oilseed rape etc.). Wide variation in shoot/leaf-Ca and Mg occurs among all three species. Shoot/leaf-Ca and Mg is significantly and highly heritable. Quantitative trait loci (QTL) affecting shoot/leaf Ca and Mg concentration occur in potentially paralogous regions of B. oleracea and B. rapa. If confirmed, allelic variation at such loci could be used in biofortification breeding programs for vegetable Brassica. As genome sequencing and marker generation improves, it will be possible to resolve these (and other) putative loci to the gene level. Further studies on the regulation, interaction and function of these genes will enable us to understand Ca and Mg dynamics in plants