Arabidopsis mutants may represent recombinant introgression lines
It is a common practice in Arabidopsis to transfer a mutation generated in one genetic background to other genetic background via crossing. However, the drawback of this methodology is unavoidable presence of genomic fragments from the donor parent being often replacing desirable genomic fragments of the recurrent parent. Here, we highlighted problem of Arabidopsis mutants being recombinant introgression lines that can lead to unreliable and misinterpreted results.
We studied the regulation of low copy number transposable elements Tag1 and Evelknievel (EK), located at the end of the bottom arm of chromosome 1 and both are present in the Arabidopsis Landsberg erecta (Ler) but not in Columbia (Col) ecotype. Using various epigenetic mutants (cmt3, ddm1, kyp2, ago4, rdr2 hen1 etc.), we found that certain mutants in the Ler background are deficient of Tag1 or EK or both and represent recombinant introgression lines whereby chromosomal regions from Col have been recombined into the Ler genome. Our data support a recent proposal calling for formulating standards for authentication of plant lines that are used in plant research. Most important is to verify that a given trait or genomic locus under study is correctly identified, particularly when using mutants generated by crossing.
KeywordsArabidopsis thaliana Ler ecotype Columbia ecotype Evelknievel retroelement Tag1 transposable element cmt3 kyp2 ago4 ddm1 Recombinant introgression lines Backcrossing
decrease in DNA methylation1
kryptonite/suppressor of variegation homolog4
RNA dependent DNA methylation
Contamination and misidentification of cell lines is a common, long-standing problem in medical research calling for establishing proper controls and standards for cell culture authentication . Obviously, studies that are conducted with misidentified cell lines are deceptive, misconceived by the scientific community adding disinformation to the literature that might affect future studies . In a recent letter, Bergelson et al.  raised a concern regarding the identity of the plant genetic material used by plant biologists including transgenic lines, mutants, or accessions claiming that plant lines “may not be what they are supposed to be”. The authors suggested formulating standards for validation of genetic stocks to avoid contamination and misidentification of genetic material used in plant research. Indeed, a recent report demonstrated SNP match as an efficient tool for genotyping Arabidopsis stock collections . Here we highlight the necessity for developing standards for genotyping and identification of plant material by describing a special case whereby mutant lines in the Arabidopsis thaliana Landsberg erecta (Ler) genetic background appear to be recombinant introgression lines between Ler and Columbia (Col) ecotypes where desirable genomic regions of Ler were replaced by the corresponding, yet undesirable genomic regions of Col ecotype.
Materials and methods
We studied wild type Col and Ler, as well as mutants in the Ler background, namely, ddm1 (Ler background CSHL-GT24941), cmt3–7 (CS6365, provided by Autran) and kyp2 (CS6367, provided by Autran), hen1 (provided by Mlotshwa, V. Vance lab) and rdr2 hen1 double mutant (Bin Yu lab). In addition, five ago4-1 lines (Ler background) obtained from various labs were analyzed including ago4-1a (Zilberman lab, University of California, Berkeley, USA; ABRC CS6364), ago4-1b and ago4-1c (Daphne Autran lab, IRD, University of Montpllier, France; ABRC CS6364), ago4-1d (Judith Bender lab, Brown University, USA; ABRC CS6364) and ago4-1e (Caroline Dean, John Innes Centre, UK). All Arabidopsis thaliana lines, were grown in a controlled growth room under long day photoperiod (16 h light and 8 h dark, light intensity 200 μmol photons m−2 s−1) at 22 °C ± 2 and 70% humidity.
DNA isolation and PCR analysis
DNA was extracted from wild type and mutant leaves using Genomic DNA Mini kit (Cat. No. GP100, Geneaid, Taiwan). This DNA was subjected to PCR to amplify the Tag1, Evelknievel (EK), indel-1, indel-7, indel-9 and nga225 (for primer sequences see Additional file 1). PCR conditions were 95 °C, 5 min; 30–40 cycles of 95 °C, 30 s; 60 °C, 30 s; 72 °C, 30 s; followed by 72 °C, 5 min. PCR products were resolved on 1.5% agarose (SeaKem LE AGAROSE Cat. No. 50004, Lonza, USA) gel stained with ethidium bromide. The PCR analysis repeated at least three times.
Results and discussion
Our study is limited to the genomic locus containing the transposable elements Tag1 and Evelknievel, which are located at the end of the bottom arm of chromosome 1 of Arabidopsis thaliana WT Ler ecotype; these TEs are not present in the WT Col ecotype. Thus our study is limited to the analysis of only these TEs; other TEs in other chromosomal regions were not studied. In this study, we have verified various epigenetic mutants for introgression only at the bottom arm of chromosome 1, but we didn’t test the possibility for introgression in other chromosomal loci. We assembled a practical tool of various polymorphic markers covering large part of the Arabidopsis genome that can be used for the assessment of introgression between Arabidopsis ecotypes. The work is also limited to only mutant lines used in this study and derived from crosses between Ler and Col ecotypes.
NSY and JK carried out all assays; NSY, JK and GG design experiments, analyzed and conceived the data, wrote the draft. All authors read and approved the final manuscript.
We thank Daphne Autran (IRD, University of Montpllier, France), Caroline Dean (John Innes Centre, UK), Assaf Zemach (Zilberman lab, University of California, Berkeley, USA), Judith Bender (Brown University, USA), Bin Yu (University of Nebraska-Lincoln, USA), Sizolwenkosi Mlotshwa and Vicki Vance (University of South Carolina, USA) and the ABRC for providing mutant lines. We also thank A. Zemach for discussing the results.
The authors declare that they have no competing interests.
Availability of data and materials
All data is given in the main body of the manuscript; materials are available from the authors.
Consent for publication
Ethics approval and consent to participate
This work was supported by the Israel Science Foundation [175/12 to GG]; the Blaustein Center for Scientific Cooperation post-doctoral fellowship to NSY; PBC Program of Israeli Council for Higher Education for post-doctoral fellowship to NSY.
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