back-cross (BC1) between A. halleri, and the non-hyperaccumu- lator, A. lyrata ssp. petraea, co-segregated with the A. halleri HMA4 allele and with Cd tolerance [18]. Using RNA interference (RNAi), it was demonstrated that Zn and Cd hypertolerance were associated with HMA4 expression in A. halleri [19]. These plants were sensitive to increased exogenous Zn and Cd treatments, translocated less Zn from the root to the shoot, and were phenotypically more similar to A. thaliana [19]. Conversely, expression of AhHMA4 cDNA under its endogenous promoter in A. thaliana resulted in increased Zn concentrations in xylem parenchyma cells, resembling Zn distribution in A. halleri roots [19]. Subsequent sequencing and functional analyses of AhHMA4 revealed that enhanced HMA4 expression was the result of both tandem gene triplication and altered cis regulation [19]. For N. caerulescens, expression of a NcHMA4 cDNA in yeast (Saccharomyces cerevisae) associated with enhanced Zn tolerance and increased Zn transport out of cells which supported a role for Zn efflux across plasma membranes in planta [20]. In general, P1B-type ATPases are more highly expressed in the shoots of N. caerulescens than non-hyperaccumulating Thlaspi arvense [21] and Arabidopsis thaliana [22,23]. Further studies characterising N. caerulescens HMA4 transcripts found increased expression as exogenous Zn was applied at levels which were either deficient or toxic to non- hyperaccumulating species [20,21]. Despite circumstantial evi- dence for similar roles in Zn hyperaccumulation, genomic sequence data has not been published for HMA4 in Noccaea caerulescens. The aim of this study was to test the hypothesis that tandem duplication and deregulation of HMA4 expression, which occurs in A. halleri [19], also occurs in N. caerulescens. Results and Discussion To test for tandem duplications of the HMA4 locus in N. caerulescens required de novo sequence. To achieve this goal, the creation of a single copy genomic fosmid library coupled with high-throughput pyrosequencing were selected as appropriate strategies. Fosmid libraries yield large insert sizes, have high stability and reduced susceptibility to aberrent recombination, thereby ensuring maximum genomic sequence representation [24,25]. By randomly shearing DNA fragments, these libraries also retain a wider selection of sequences than those based on traditional restriction digestion [26]. Sequences were generated via Next Generation Genome Sequencer (NextGen GS) FLX 454 technology as it offered the greatest read length (350–450 bp) of current pyrosequencing technologies, and is routinely employed for de novo sequencing [27–29]. Construction and characterisation of a Noccaea caerulescens fosmid library The genomic fosmid library was constructed for the self- compatible Zn and Cd hyperaccumulator Noccaea caerulescens (J.&C. Presl) F.K. Mey., from a first generation accession from a geographically isolated population in Saint Laurent Le Minier, southern France (supplied by Guy Delmot, Saint Laurent le Minier, France, 43u559480 N, 3u409120 E) [30]. Such populations are self- compatible and highly inbred [31–34], and demonstrate low levels of heterozygosity and high inbreeding coefficients [34–37]. The creation of a laboratory inbred line was not pursued, since this could result in an accumulation of mutations [38] leading to increased genetic load [39] and reduced fitness, as well as gene copy number variation [40–42] and perturbed sequencing results. To further prevent potential allelic perturbations in sequencing results, the library was constructed using leaf genomic DNA from a single plant (250 Mb), and cloned into 36,864 Escherichia coli HMA4 Quadruplication in Noccaea caerulescens EPI300TM-T1R host cells containing highly stable, randomly sheered, ,40 kb genomic inserts, representing ,5.9 fold genomic coverage, while 454 sequencing reads returned .20 fold coverage. Such sequencing strategies compare favourably with those adopted by [19] to robustly identify tandem triplication of HMA4 in the self-incompatible Arabidopsis halleri. To elucidate the genomic sequence of HMA4 in N. caerulescens, the library was probed with a radiolabelled NcHMA4 specific sequence. Seven clones, N18P80, P6P46, N12P82, H2P47, B3P40, B22P20 and J12P81, were identified as containing NcHMA4 sequences following PCR amplification using primers specific for the NcHMA4 probe. Six of these fosmids demonstrated unique evidence of multiple copies of the NcHMA4 locus following restriction digest fingerprinting (Figure 1). Initial pyrosequencing [27] of a pool containing all seven fosmids returned 3 Mbp of sequence at 5-fold coverage per fosmid. Sequences were assembled into contigs and aligned to syntenic regions in the A. thaliana genome to confirm the presence of multiple NcHMA4 copies. Individual copies were assigned to unique clones through PCR analyses using locus specific primers (Figure 2). Fosmids were then sequenced individually to improve the specificity and efficiency of prior pooled sequence assemblies, and returned 2.4 Mbp at .20 fold coverage per fosmid. Two independent HMA4 copies were identified in fosmids B3P40 (27,978 bp; NcHMA4-1 and NcHMA4-2) and P6P46 (31,521 bp; NcHMA4-3 and NcHMA4-4) (Figures S1 & S2, Data S1 & S2). Fosmid J12P81 (31,218 bp) contained NcHMA4-2 as well as two genes downstream to its 39 end, whose sequences were homologous to the A. thaliana genes At2g19160 and At2g19170, and so demonstrated synteny with Arabidopsis thaliana (Figure S3, Data S3). Fosmid N18P80 (20,090 bp) contained 941 bp of the 59 region of NcHMA4-3 in addition to four orthologues to At2g19060, At2g19070, At2g19080 and At2g19090, which were syntenic to this region in A. thaliana (Figure S4, Data S4). As indicated through locus specific PCR analysis (Figure 2), sequence data from fosmid H2P47 (20,258 bp) showed homology to NcHMA4-4 and its 59 intergenic region, as well as the 59 intergenic region of NcHMA4-1 (Figure S5, Data S5). Fosmid inserts, containing homologous sequences which demonstrated .99% sequence identity along 59 and 39 ends of between 425 and 14,866 bp, were assembled into unique contiguous sequences. Consequently, fosmid H2P47 assembled both fosmids P6P46 (containing HMA4-3 and HMA4-4) and B3P40 (containing HMA4-1 and HMA4-2) into a unique locus (Data S7,S8), flanked to its 59 by N18P80, and to its 39 by J12P81 (Figure 3). In support of this HMA4 quadruplication, a genomic Southern illustrated hybridisation intensities for HindIII fragments, which were indicative of a 3:1 (1040–1050 bp fragment (represent- ing HMA4-1, HMA4-3 and HMA4-4): 1.9 kb fragment (representing HMA4-2)) genomic ratio (Figure 1). All five overlapping N. caerulescens fosmids spanned a single 101,480 bp locus in N. caerulescens and contained four HMA4 tandem repeats (corresponding to At2g19110 in A. thaliana), compared to syntenic regions in A. thaliana and A. halleri, containing one and three copies respectively (Figure 3, Data S6). Sequences flanking NcHMA4 tandem repeats remained essentially syntenic with A. thaliana. Analysis of NcHMA4 sequences Within the deduced coding sequences, all four NcHMA4 gene copies share between 87 and 99% nucleotide sequence identity, whilst introns demonstrated between 81 and 100% identity to consensus NcHMA4 sequences (Figures 4 & S6). The deduced coding sequences showed lower sequence identities with those of AtHMA4 (between 76–78%) and of all three AhHMA4 copies (between 62–66%), which may indicate that quadruplication was a PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17814