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Independent, rapid and targeted loss of highly repetitive DNA in natural and synthetic allopolyploids of Nicotiana tabacum.

Renny-Byfield S, Kovařík A, Chester M, Nichols RA, Macas J, Novák P, Leitch AR - PLoS ONE (2012)

Bottom Line: Allopolyploidy (interspecific hybridisation and polyploidy) has played a significant role in the evolutionary history of angiosperms and can result in genomic, epigenetic and transcriptomic perturbations.We examine the immediate effects of allopolyploidy on repetitive DNA by comparing the genomes of synthetic and natural Nicotiana tabacum with diploid progenitors N. tomentosiformis (paternal progenitor) and N. sylvestris (maternal progenitor).Abundance estimates, based on sequencing depth, indicate NicCL3 is almost absent in N. sylvestris and has been dramatically reduced in copy number in the allopolyploid N. tabacum.

View Article: PubMed Central - PubMed

Affiliation: School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom.

ABSTRACT
Allopolyploidy (interspecific hybridisation and polyploidy) has played a significant role in the evolutionary history of angiosperms and can result in genomic, epigenetic and transcriptomic perturbations. We examine the immediate effects of allopolyploidy on repetitive DNA by comparing the genomes of synthetic and natural Nicotiana tabacum with diploid progenitors N. tomentosiformis (paternal progenitor) and N. sylvestris (maternal progenitor). Using next generation sequencing, a recently developed graph-based repeat identification pipeline, Southern blot and fluorescence in situ hybridisation (FISH) we characterise two highly repetitive DNA sequences (NicCL3 and NicCL7/30). Analysis of two independent high-throughput DNA sequencing datasets indicates NicCL3 forms 1.6-1.9% of the genome in N. tomentosiformis, sequences that occur in multiple, discontinuous tandem arrays scattered over several chromosomes. Abundance estimates, based on sequencing depth, indicate NicCL3 is almost absent in N. sylvestris and has been dramatically reduced in copy number in the allopolyploid N. tabacum. Surprisingly elimination of NicCL3 is repeated in some synthetic lines of N. tabacum in their forth generation. The retroelement NicCL7/30, which occurs interspersed with NicCL3, is also under-represented but to a much lesser degree, revealing targeted elimination of the latter. Analysis of paired-end sequencing data indicates the tandem component of NicCL3 has been preferentially removed in natural N. tabacum, increasing the proportion of the dispersed component. This occurs across multiple blocks of discontinuous repeats and based on the distribution of nucleotide similarity among NicCL3 units, was concurrent with rounds of sequence homogenisation.

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Tandem arrangement of NicCL3.Southern blot hybridisation of genomic DNA from (TH) synthetic tobacco Th37-5, (TO) N. tomentosiformis ac. NIC 479/84 and (TA) N. tabacum ac. SR1 digested with SpeI, BamHI and HaeII (a methylation sensitive isoschizomer of BamHI) and probed with NicCL3. Size indicators on the left are in kb. Digestion with BamHI and SpeI results in a ladder like pattern, typical of tandem repeats. Digestion is inhibited when using HaeII, indicating extensive CG methylation of tandem units.
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pone-0036963-g004: Tandem arrangement of NicCL3.Southern blot hybridisation of genomic DNA from (TH) synthetic tobacco Th37-5, (TO) N. tomentosiformis ac. NIC 479/84 and (TA) N. tabacum ac. SR1 digested with SpeI, BamHI and HaeII (a methylation sensitive isoschizomer of BamHI) and probed with NicCL3. Size indicators on the left are in kb. Digestion with BamHI and SpeI results in a ladder like pattern, typical of tandem repeats. Digestion is inhibited when using HaeII, indicating extensive CG methylation of tandem units.

Mentions: Southern blot hybridisation was carried out using NicCL3 as a probe. For each species 1–2 µg of genomic DNA was digested with BamHI and SpeI enzymes (Fig. 4, Table 2), which have a single restriction site within NicCL3. A ladder pattern of bands was evident in N. kawakamii, N. tomentosiformis (TW142 and NIC 479/84), natural N. tabacum (095-55 and SR1), synthetic N. tabacum, Th37-3, 5, 6, 7 and 8. The bands are indicative of tandemly arranged satellite repeats arranged head to tail. The fastest migrating band corresponded to the satellite monomer (2.2 kb), contained within the 2.9 kb in silico reconstruction (Fig. 1 b). There was no signal detected in Th37-1, N. sylvestris N. glutinosa or N. otophora. Other species (N. setchellii and N. tomentosa) have trace amounts of background signal but lack any detectable ladder pattern (Table 2).


Independent, rapid and targeted loss of highly repetitive DNA in natural and synthetic allopolyploids of Nicotiana tabacum.

Renny-Byfield S, Kovařík A, Chester M, Nichols RA, Macas J, Novák P, Leitch AR - PLoS ONE (2012)

Tandem arrangement of NicCL3.Southern blot hybridisation of genomic DNA from (TH) synthetic tobacco Th37-5, (TO) N. tomentosiformis ac. NIC 479/84 and (TA) N. tabacum ac. SR1 digested with SpeI, BamHI and HaeII (a methylation sensitive isoschizomer of BamHI) and probed with NicCL3. Size indicators on the left are in kb. Digestion with BamHI and SpeI results in a ladder like pattern, typical of tandem repeats. Digestion is inhibited when using HaeII, indicating extensive CG methylation of tandem units.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351487&req=5

pone-0036963-g004: Tandem arrangement of NicCL3.Southern blot hybridisation of genomic DNA from (TH) synthetic tobacco Th37-5, (TO) N. tomentosiformis ac. NIC 479/84 and (TA) N. tabacum ac. SR1 digested with SpeI, BamHI and HaeII (a methylation sensitive isoschizomer of BamHI) and probed with NicCL3. Size indicators on the left are in kb. Digestion with BamHI and SpeI results in a ladder like pattern, typical of tandem repeats. Digestion is inhibited when using HaeII, indicating extensive CG methylation of tandem units.
Mentions: Southern blot hybridisation was carried out using NicCL3 as a probe. For each species 1–2 µg of genomic DNA was digested with BamHI and SpeI enzymes (Fig. 4, Table 2), which have a single restriction site within NicCL3. A ladder pattern of bands was evident in N. kawakamii, N. tomentosiformis (TW142 and NIC 479/84), natural N. tabacum (095-55 and SR1), synthetic N. tabacum, Th37-3, 5, 6, 7 and 8. The bands are indicative of tandemly arranged satellite repeats arranged head to tail. The fastest migrating band corresponded to the satellite monomer (2.2 kb), contained within the 2.9 kb in silico reconstruction (Fig. 1 b). There was no signal detected in Th37-1, N. sylvestris N. glutinosa or N. otophora. Other species (N. setchellii and N. tomentosa) have trace amounts of background signal but lack any detectable ladder pattern (Table 2).

Bottom Line: Allopolyploidy (interspecific hybridisation and polyploidy) has played a significant role in the evolutionary history of angiosperms and can result in genomic, epigenetic and transcriptomic perturbations.We examine the immediate effects of allopolyploidy on repetitive DNA by comparing the genomes of synthetic and natural Nicotiana tabacum with diploid progenitors N. tomentosiformis (paternal progenitor) and N. sylvestris (maternal progenitor).Abundance estimates, based on sequencing depth, indicate NicCL3 is almost absent in N. sylvestris and has been dramatically reduced in copy number in the allopolyploid N. tabacum.

View Article: PubMed Central - PubMed

Affiliation: School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom.

ABSTRACT
Allopolyploidy (interspecific hybridisation and polyploidy) has played a significant role in the evolutionary history of angiosperms and can result in genomic, epigenetic and transcriptomic perturbations. We examine the immediate effects of allopolyploidy on repetitive DNA by comparing the genomes of synthetic and natural Nicotiana tabacum with diploid progenitors N. tomentosiformis (paternal progenitor) and N. sylvestris (maternal progenitor). Using next generation sequencing, a recently developed graph-based repeat identification pipeline, Southern blot and fluorescence in situ hybridisation (FISH) we characterise two highly repetitive DNA sequences (NicCL3 and NicCL7/30). Analysis of two independent high-throughput DNA sequencing datasets indicates NicCL3 forms 1.6-1.9% of the genome in N. tomentosiformis, sequences that occur in multiple, discontinuous tandem arrays scattered over several chromosomes. Abundance estimates, based on sequencing depth, indicate NicCL3 is almost absent in N. sylvestris and has been dramatically reduced in copy number in the allopolyploid N. tabacum. Surprisingly elimination of NicCL3 is repeated in some synthetic lines of N. tabacum in their forth generation. The retroelement NicCL7/30, which occurs interspersed with NicCL3, is also under-represented but to a much lesser degree, revealing targeted elimination of the latter. Analysis of paired-end sequencing data indicates the tandem component of NicCL3 has been preferentially removed in natural N. tabacum, increasing the proportion of the dispersed component. This occurs across multiple blocks of discontinuous repeats and based on the distribution of nucleotide similarity among NicCL3 units, was concurrent with rounds of sequence homogenisation.

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