Supplementary material
The new wheat vernalization response allele Vrn-D1s is caused by DNA transposon insertion in the first intron
Alexandr Muterko a, Irina Balashova a, James Cockram b , Ruslan Kalendar c, Yuri Sivolap a
a Plant Breeding and Genetics Institute – National Center of Seed and Cultivar Investigation, Department of Genomics and Biotechnology, Ovidiopolskaya Road 3, Odessa, 65036, Ukraine,
E-mail:
b National Institute of Agricultural Botany (NIAB), Huntington Road, Cambridge, CB3 0LE, UK.
c University of Helsinki, Institute of Biotechnology, MTT Plant Genomics Laboratory, Biocentre 3, P.O. Box 65, Viikinkaari 1, Helsinki, 00014, Finland.
Address for correspondence:
Alexandr Muterko, Plant Breeding and Genetics Institute – National Center of Seed and Cultivar Investigation, department of genomics and biotechnology, Ovidiopolskaya road 3, Odessa, 65036, Ukraine, E-mail:
T-tract analysis
Methods
Macroscopic curvature was calculated using the "DNA Curvature Analysis" web-service (http://www.lfd.uci.edu/~gohlke/dnacurve/, by Christoph Gohlke). The curvature (inverse of the radius of a circle passing through helix axis coordinates at i-10, i and i+10), curvature angle (an angle between the smoothed basepair normal vectors at i-15 and i+15) and the local bend angle (an angle between the basepair normal vectors at i-2 and i+2) was evaluated according to the "AA-Wedge" model (Ulanovsky et al. 1987). The curvature and bend angles are normalized to the curvature in a nucleosome. Structures were visualized by UCSF Chimera (Pettersen et al. 2004).
Results
It is a known that curved DNA fragments have a reduced electrophoretic mobility in polyacrylamide gels (Diekmann 1989; Marini et al. 1982; Stellwagen 2009; Stellwagen 1983). To further investigate the possible effect of the SNPs identified on electrophoretic mobility in PAA gels, global 3D structures of the DNA molecules were calculated from overlapping regions: 1-256 bp and 254-510 bp (which span the majority of the SNPs and InDels present in the 1019 bp fragments). Curvature, curvature angle and bend angle was evaluated, finding the 1-256 bp regions of Hap-8T (KF939527) and an intact sequence – Hap-7T (KF939526) to possess similar DNA molecule curvature (Fig. 1).
Fig. 1 Similar curvature of DNA molecule for 1-256 bp regions of the Vrn-D1 PCR fragments amplified with primers VRN1DF/VRN1-INT1R: Hap-8T (KF939527) and Hap-7T (KF939526).
These sequences differ by the deletion of two "C" nucleotides at positions 50 and 52 bp and one SNP ("T=>C") at 30 bp. A decrease of curvature (at 13%) was observed for T-tract 254-510 bp fragments that carry of a "T" insertion at position 346 bp (Hap-8T accessions) (Fig. 2).
Accordingly, we conclude that the "T" insertion at position 346 bp contributes more to the differences in the electrophoretic mobility between amplicons than other mutations within the Hap-8T haplotype. A "T" insertion is predicted to decrease DNA curvature, resulting in an increase in electrophoretic mobility in non-denaturing PAA gels. To confirm this, a 254-510 bp region of experimental sequence was tested that differed from KF939526 (Hap-7T) by a "TTT" insertion at position 346 bp instead one "T" insertion of Hap-8T (Fig. 2). The results indicated that the presence of additional "TT" nucleotides (in 346-site Hap-8T) is accompanied by a straightening of the DNA molecule (T-tract curvature is decreased at 36%).
Fig. 2 (a) Amplification of Vrn-D1 with primers VRN1DF/VRN1-INT1R for Hap-7T (1020 bp) and Hap-8T (1019 bp). Electrophoresis was performed in nondenaturing polyacrylamide gels at low voltage. The observed difference in the amplicon mobility cannot be explained by only 1 bp deletion. (b) Projection of 3D structure illustrates a bending of DNA molecule (256-510 bp region of the 1019-1020 bp amplicons) according to the dinucleotide wedge model: 1 - Hap-7T, 2 - Hap-8T, 3 - the experimental sequence = Hap-8T+"TT". The curvature at 96 bp (correspond to the 347 bp of the 1019-1020 bp amplicons) are indicated. Right graphics are showing values of macroscopic curvature, curvature and bend angles for each nucleotide of the sequence. Indicated curvature value at 96 bp position for T-tract with different lenght ((T)7, (T)8, (T)10). (c) 3D structure of the Hap-7T and Hap-8T DNA helix overlay. A wedge of T-tract region is observed.
References
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