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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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FISH of NicCL3 and NicCL7/30.Fluorescence in situ hybridisation (FISH) to metaphase chromosomes of (a, c) N. tomentosiformis (ac. NIC 479/84); (b, d) Th37-3; (e, h) N. tabacum (ac. 095-55); (i) N. kawakamii and; (j) TR1-A. The probes used were 18S rDNA (blue; a-e and h-i only), NtCL7 (green) and NicCL3 probes (red) counter stained with DAPI (grey). Inset (e) shows enlarged chromosome T3 with NicCL3 signal at the distal end of the long arm (arrow heads). (f, g) Genomic in situ hybridisation (GISH) to chromosomes of Th37-3, showing the N. tomentosiformis sub-genome (red) and N. sylvestris sub-genome (green). (i) Note that chromosome 3 of N. kawakamii (18S rDNA bearing) has a large NicCL3 signal proximal to the centromere (arrows). (j) TR1-A an S0 synthetic N. tabacum with the expected number of NicCL3 (red) signals and highly localised NtCL7 (green) signals. Scale bar is 5 µm.
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pone-0036963-g003: FISH of NicCL3 and NicCL7/30.Fluorescence in situ hybridisation (FISH) to metaphase chromosomes of (a, c) N. tomentosiformis (ac. NIC 479/84); (b, d) Th37-3; (e, h) N. tabacum (ac. 095-55); (i) N. kawakamii and; (j) TR1-A. The probes used were 18S rDNA (blue; a-e and h-i only), NtCL7 (green) and NicCL3 probes (red) counter stained with DAPI (grey). Inset (e) shows enlarged chromosome T3 with NicCL3 signal at the distal end of the long arm (arrow heads). (f, g) Genomic in situ hybridisation (GISH) to chromosomes of Th37-3, showing the N. tomentosiformis sub-genome (red) and N. sylvestris sub-genome (green). (i) Note that chromosome 3 of N. kawakamii (18S rDNA bearing) has a large NicCL3 signal proximal to the centromere (arrows). (j) TR1-A an S0 synthetic N. tabacum with the expected number of NicCL3 (red) signals and highly localised NtCL7 (green) signals. Scale bar is 5 µm.

Mentions: FISH using the NicCL3 clone 9 to metaphase spreads of N. tomentosiformis (ac. NIC 479/84 and Nee et al. 51771) reveals loci on eight of the large sub-metacentric chromosomes (Fig. 3 a, c and Table 2). The signal is highly localized and is exclusive to the distal region of the long arm of four chromosome pairs. The 18S rDNA-bearing chromosome (chromosome 3, following the nomenclature of Lim etal. [43]) lacks any detectable signal. In contrast there is NicCL3 signal at an interstitial locus on the orthologous 18S rDNA-bearing chromosome of the diploid relative N. kawakamii (Fig. 3 i). NicCL3 signal is also observed on chromosome T3 of N. tabacum, although it is restricted to the most distal regions of the long arm (boxed in Fig. 3 e). All NicCL3 loci in N. tabacum are noticeably smaller than those in the progenitor N. tomentosiformis and the diploid N. kawakamii.


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)

FISH of NicCL3 and NicCL7/30.Fluorescence in situ hybridisation (FISH) to metaphase chromosomes of (a, c) N. tomentosiformis (ac. NIC 479/84); (b, d) Th37-3; (e, h) N. tabacum (ac. 095-55); (i) N. kawakamii and; (j) TR1-A. The probes used were 18S rDNA (blue; a-e and h-i only), NtCL7 (green) and NicCL3 probes (red) counter stained with DAPI (grey). Inset (e) shows enlarged chromosome T3 with NicCL3 signal at the distal end of the long arm (arrow heads). (f, g) Genomic in situ hybridisation (GISH) to chromosomes of Th37-3, showing the N. tomentosiformis sub-genome (red) and N. sylvestris sub-genome (green). (i) Note that chromosome 3 of N. kawakamii (18S rDNA bearing) has a large NicCL3 signal proximal to the centromere (arrows). (j) TR1-A an S0 synthetic N. tabacum with the expected number of NicCL3 (red) signals and highly localised NtCL7 (green) signals. Scale bar is 5 µm.
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Related In: Results  -  Collection

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pone-0036963-g003: FISH of NicCL3 and NicCL7/30.Fluorescence in situ hybridisation (FISH) to metaphase chromosomes of (a, c) N. tomentosiformis (ac. NIC 479/84); (b, d) Th37-3; (e, h) N. tabacum (ac. 095-55); (i) N. kawakamii and; (j) TR1-A. The probes used were 18S rDNA (blue; a-e and h-i only), NtCL7 (green) and NicCL3 probes (red) counter stained with DAPI (grey). Inset (e) shows enlarged chromosome T3 with NicCL3 signal at the distal end of the long arm (arrow heads). (f, g) Genomic in situ hybridisation (GISH) to chromosomes of Th37-3, showing the N. tomentosiformis sub-genome (red) and N. sylvestris sub-genome (green). (i) Note that chromosome 3 of N. kawakamii (18S rDNA bearing) has a large NicCL3 signal proximal to the centromere (arrows). (j) TR1-A an S0 synthetic N. tabacum with the expected number of NicCL3 (red) signals and highly localised NtCL7 (green) signals. Scale bar is 5 µm.
Mentions: FISH using the NicCL3 clone 9 to metaphase spreads of N. tomentosiformis (ac. NIC 479/84 and Nee et al. 51771) reveals loci on eight of the large sub-metacentric chromosomes (Fig. 3 a, c and Table 2). The signal is highly localized and is exclusive to the distal region of the long arm of four chromosome pairs. The 18S rDNA-bearing chromosome (chromosome 3, following the nomenclature of Lim etal. [43]) lacks any detectable signal. In contrast there is NicCL3 signal at an interstitial locus on the orthologous 18S rDNA-bearing chromosome of the diploid relative N. kawakamii (Fig. 3 i). NicCL3 signal is also observed on chromosome T3 of N. tabacum, although it is restricted to the most distal regions of the long arm (boxed in Fig. 3 e). All NicCL3 loci in N. tabacum are noticeably smaller than those in the progenitor N. tomentosiformis and the diploid N. kawakamii.

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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