Dissecting a Hidden Gene Duplication: The Arabidopsis thaliana SEC10 Locus

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation

Performance and Molière radius measurements using a compact prototype of LumiCal in an electron test beam

A new design of a detector plane of sub-millimetre thickness for an electromagnetic sampling calorimeter is presented. It is intended to be used in the luminometers LumiCal and BeamCal in future linear e+e- collider experiments. The detector planes were produced utilising novel connectivity scheme technologies. They were installed in a compact prototype of the calorimeter and tested at DESY with an electron beam of energy 1–5 GeV. The performance of a prototype of a compact LumiCal comprising eight detector planes was studied. The effective Molière radius at 5 GeV was determined to be (8.1 ± 0.1 (stat) ± 0.3 (syst)) mm, a value well reproduced by the Monte Carlo (MC) simulation (8.4 ± 0.1) mm. The dependence of the effective Molière radius on the electron energy in the range 1–5 GeV was also studied. Good agreement was obtained between data and MC simulation. © 2019, The Author(s)

ColeRexBotanyPlantPathologyDissectingHiddenGene_SupportingInformation.zip

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation

ColeRexBotanyPlantPathologyDissectingHiddenGene.pdf

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation

Dissecting a Hidden Gene Duplication: The <i>Arabidopsis thaliana SEC10</i> Locus

<div><p>Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in <i>Arabidopsis thaliana</i> mutants in <i>SEC10</i>, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the <i>Arabidopsis thaliana SEC10</i> (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, <i>SEC10a</i> and <i>SEC10b</i>, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both <i>SEC10</i> genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of <i>A. thaliana</i>. Moreover, we show that the use of the existing <i>A. thaliana</i> reference genome sequence as a guide for sequence assembly of new <i>Arabidopsis</i> accessions or related species has at least in some cases led to error propagation.</p></div

The revisited structure of the <i>SEC10</i> locus in <i>Arabidopsis thaliana</i>.

<p>The revisited arrangement of the <i>SEC10</i> locus (At5g12370) depicts <i>SEC10a</i>, <i>SEC10b</i>, and parts of two neighboring genes (At5g12360, At5g12380). Coding exons are shown as black boxed, 5′UTR as gray boxes, and 3′ UTR as white boxes. Arrows indicate the position and orientation of primers used for cloning of the <i>SEC10</i> locus in four overlapping parts (a-I, a-II, b-I and b-II; lines at the bottom represent the ranges of the cloned PCR products). The orange strip marks the region omitted from the reference sequence of the <i>A. thaliana</i> genome.</p

Analysis of T-DNA insertional mutants in <i>SEC10</i> genes of <i>A. thaliana.</i>

<p>(<b>A</b>) Positions of T-DNA insertions and primers used for genotyping (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094077#pone-0094077-t001" target="_blank">Table 1</a>) are indicated by triangles or arrows, respectively. Numbers below genes indicate the exact position of each insertion (in bp counted from the start codon) and long arrows show the gene orientation. (<b>B</b>) Expression levels of <i>SEC10a</i> and <i>SEC10b</i> in young seedlings of mutant lines as analyzed by semi-quantitative RT-PCR. The expression level of the ACT7 gene was used as a control.</p

Insertional mutations in the <i>A. thaliana SEC10</i> locus do not affect viability.

<p>Insertional mutations in the <i>A. thaliana SEC10</i> locus do not affect viability.</p