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Characterization of repetitive DNA landscape in wheat homeologous group 4 chromosomes.

Garbus I, Romero JR, Valarik M, Vanžurová H, Karafiátová M, Cáccamo M, Doležel J, Tranquilli G, Helguera M, Echenique V - BMC Genomics (2015)

Bottom Line: SSR frequency was found one per 24 to 27 kb for all chromosome assemblies except 4DLI, where it was three times higher.Their physical distribution in wheat genome was analyzed by fluorescent in situ hybridization (FISH) and one of them, the Carmen retrotransposon, was found specific for centromeric regions of all wheat chromosomes.The presented work is the first deep report of wheat repetitive sequences analyzed at the chromosome arm level, revealing the first insight into the repeatome of T. aestivum chromosomes of homeologous group 4.

View Article: PubMed Central - PubMed

Affiliation: CERZOS (CCT - CONICET Bahía Blanca) and Universidad Nacional del Sur, Bahía Blanca, Argentina. igarbus@criba.edu.ar.

ABSTRACT

Background: The number and complexity of repetitive elements varies between species, being in general most represented in those with larger genomes. Combining the flow-sorted chromosome arms approach to genome analysis with second generation DNA sequencing technologies provides a unique opportunity to study the repetitive portion of each chromosome, enabling comparisons among them. Additionally, different sequencing approaches may produce different depth of insight to repeatome content and structure. In this work we analyze and characterize the repetitive sequences of Triticum aestivum cv. Chinese Spring homeologous group 4 chromosome arms, obtained through Roche 454 and Illumina sequencing technologies, hereinafter marked by subscripts 454 and I, respectively. Repetitive sequences were identified with the RepeatMasker software using the interspersed repeat database mips-REdat_v9.0p. The input sequences consisted of our 4DS454 and 4DL454 scaffolds and 4ASI, 4ALI, 4BSI, 4BLI, 4DSI and 4DLI contigs, downloaded from the International Wheat Genome Sequencing Consortium (IWGSC).

Results: Repetitive sequences content varied from 55% to 63% for all chromosome arm assemblies except for 4DLI, in which the repeat content was 38%. Transposable elements, small RNA, satellites, simple repeats and low complexity sequences were analyzed. SSR frequency was found one per 24 to 27 kb for all chromosome assemblies except 4DLI, where it was three times higher. Dinucleotides and trinucleotides were the most abundant SSR repeat units. (GA)n/(TC)n was the most abundant SSR except for 4DLI where the most frequently identified SSR was (CCG/CGG)n. Retrotransposons followed by DNA transposons were the most highly represented sequence repeats, mainly composed of CACTA/En-Spm and Gypsy superfamilies, respectively. This whole chromosome sequence analysis allowed identification of three new LTR retrotransposon families belonging to the Copia superfamily, one belonging to the Gypsy superfamily and two TRIM retrotransposon families. Their physical distribution in wheat genome was analyzed by fluorescent in situ hybridization (FISH) and one of them, the Carmen retrotransposon, was found specific for centromeric regions of all wheat chromosomes.

Conclusion: The presented work is the first deep report of wheat repetitive sequences analyzed at the chromosome arm level, revealing the first insight into the repeatome of T. aestivum chromosomes of homeologous group 4.

No MeSH data available.


Physical localization of newly identified transposons. The identified retrotransposons were localized on metaphase spreads of vc. Chinese Spring using in situ hybridization with fluorescent labeled probes. Red color was used for the repetitive probes, Green color was used for Afa probe and Blue was stained chromosome DNA using DAPI. Arrows are pointing at 4D chromosome. a) Probes of all retrotransposons besides retrotransposon Carmen produced weak disperse signals on all chromosomes. The red signals represent distribution of retrotransposon Victoria. b) The red channel of figure A to demonstrate distribution of weak signals of Victoria probe on whole chromosomes. c) All probes derived from retrotransposon Carmen gave similar hybridisation pattern on metaphase chromosomes. Surprisingly, the retrotransposon was preferentially localized in centromeric region of all chromosomes with varying intensity. On few chromosomes were observed weak and dispersed signals on distal parts of chromosome arms too. d) The red channel of figure C to demonstrate distribution of weak signals of Carmen probe on whole chromosomes. Scale bars represent 10 μm.
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Fig5: Physical localization of newly identified transposons. The identified retrotransposons were localized on metaphase spreads of vc. Chinese Spring using in situ hybridization with fluorescent labeled probes. Red color was used for the repetitive probes, Green color was used for Afa probe and Blue was stained chromosome DNA using DAPI. Arrows are pointing at 4D chromosome. a) Probes of all retrotransposons besides retrotransposon Carmen produced weak disperse signals on all chromosomes. The red signals represent distribution of retrotransposon Victoria. b) The red channel of figure A to demonstrate distribution of weak signals of Victoria probe on whole chromosomes. c) All probes derived from retrotransposon Carmen gave similar hybridisation pattern on metaphase chromosomes. Surprisingly, the retrotransposon was preferentially localized in centromeric region of all chromosomes with varying intensity. On few chromosomes were observed weak and dispersed signals on distal parts of chromosome arms too. d) The red channel of figure C to demonstrate distribution of weak signals of Carmen probe on whole chromosomes. Scale bars represent 10 μm.

Mentions: The transposon insertion site based markers are specific and highly abundant, especially in large genomes where repetitive sequences represent major portions of genomic sequence, and became popular in plant genetic, physical mapping and diversity assessments. Several approaches were developed to visualize polymorphisms in the insertion sites and the most widely used in wheat are the RJM [20] or ISBP markers [18,19]. In light of this, identification and characterization of any new transposon adds to the pool of possible markers. After identification of six new LTR retrotransposons (Table 6) their DNA was amplified and labeled with fluorescent dye. The resulting probes were hybridized on metaphase chromosomes. Most of the probes (Additional file 7: Table S6) provided weak and mostly randomly distributed unreliable signals on several chromosomes (similar to Figure 5a, data not shown). An exception was probe from LTR retrotransposon Carmen which provided signal in centromeric region of all chromosomes (Figure 5b) with highly varying intensity. Unfortunately, FISH analysis could not provide quantitative data which limits assessment of abundance of the retrotransposon for centromeres of particular chromosomes. Surprisingly, chromosome 4D showed very weak signal for this probe in all metaphase figures analyzed (Figure 5c and d). These findings support previously identified facts that repetitive elements and particularly transposons can, besides their selfish multiplication, play also an important role in evolution of genomes in moderating gene expression and creating new genes by exon reshuffling [52] or are part of important genome structures as centromeres and have ability to specifically target such structures [53,54].Figure 5


Characterization of repetitive DNA landscape in wheat homeologous group 4 chromosomes.

Garbus I, Romero JR, Valarik M, Vanžurová H, Karafiátová M, Cáccamo M, Doležel J, Tranquilli G, Helguera M, Echenique V - BMC Genomics (2015)

Physical localization of newly identified transposons. The identified retrotransposons were localized on metaphase spreads of vc. Chinese Spring using in situ hybridization with fluorescent labeled probes. Red color was used for the repetitive probes, Green color was used for Afa probe and Blue was stained chromosome DNA using DAPI. Arrows are pointing at 4D chromosome. a) Probes of all retrotransposons besides retrotransposon Carmen produced weak disperse signals on all chromosomes. The red signals represent distribution of retrotransposon Victoria. b) The red channel of figure A to demonstrate distribution of weak signals of Victoria probe on whole chromosomes. c) All probes derived from retrotransposon Carmen gave similar hybridisation pattern on metaphase chromosomes. Surprisingly, the retrotransposon was preferentially localized in centromeric region of all chromosomes with varying intensity. On few chromosomes were observed weak and dispersed signals on distal parts of chromosome arms too. d) The red channel of figure C to demonstrate distribution of weak signals of Carmen probe on whole chromosomes. Scale bars represent 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4440537&req=5

Fig5: Physical localization of newly identified transposons. The identified retrotransposons were localized on metaphase spreads of vc. Chinese Spring using in situ hybridization with fluorescent labeled probes. Red color was used for the repetitive probes, Green color was used for Afa probe and Blue was stained chromosome DNA using DAPI. Arrows are pointing at 4D chromosome. a) Probes of all retrotransposons besides retrotransposon Carmen produced weak disperse signals on all chromosomes. The red signals represent distribution of retrotransposon Victoria. b) The red channel of figure A to demonstrate distribution of weak signals of Victoria probe on whole chromosomes. c) All probes derived from retrotransposon Carmen gave similar hybridisation pattern on metaphase chromosomes. Surprisingly, the retrotransposon was preferentially localized in centromeric region of all chromosomes with varying intensity. On few chromosomes were observed weak and dispersed signals on distal parts of chromosome arms too. d) The red channel of figure C to demonstrate distribution of weak signals of Carmen probe on whole chromosomes. Scale bars represent 10 μm.
Mentions: The transposon insertion site based markers are specific and highly abundant, especially in large genomes where repetitive sequences represent major portions of genomic sequence, and became popular in plant genetic, physical mapping and diversity assessments. Several approaches were developed to visualize polymorphisms in the insertion sites and the most widely used in wheat are the RJM [20] or ISBP markers [18,19]. In light of this, identification and characterization of any new transposon adds to the pool of possible markers. After identification of six new LTR retrotransposons (Table 6) their DNA was amplified and labeled with fluorescent dye. The resulting probes were hybridized on metaphase chromosomes. Most of the probes (Additional file 7: Table S6) provided weak and mostly randomly distributed unreliable signals on several chromosomes (similar to Figure 5a, data not shown). An exception was probe from LTR retrotransposon Carmen which provided signal in centromeric region of all chromosomes (Figure 5b) with highly varying intensity. Unfortunately, FISH analysis could not provide quantitative data which limits assessment of abundance of the retrotransposon for centromeres of particular chromosomes. Surprisingly, chromosome 4D showed very weak signal for this probe in all metaphase figures analyzed (Figure 5c and d). These findings support previously identified facts that repetitive elements and particularly transposons can, besides their selfish multiplication, play also an important role in evolution of genomes in moderating gene expression and creating new genes by exon reshuffling [52] or are part of important genome structures as centromeres and have ability to specifically target such structures [53,54].Figure 5

Bottom Line: SSR frequency was found one per 24 to 27 kb for all chromosome assemblies except 4DLI, where it was three times higher.Their physical distribution in wheat genome was analyzed by fluorescent in situ hybridization (FISH) and one of them, the Carmen retrotransposon, was found specific for centromeric regions of all wheat chromosomes.The presented work is the first deep report of wheat repetitive sequences analyzed at the chromosome arm level, revealing the first insight into the repeatome of T. aestivum chromosomes of homeologous group 4.

View Article: PubMed Central - PubMed

Affiliation: CERZOS (CCT - CONICET Bahía Blanca) and Universidad Nacional del Sur, Bahía Blanca, Argentina. igarbus@criba.edu.ar.

ABSTRACT

Background: The number and complexity of repetitive elements varies between species, being in general most represented in those with larger genomes. Combining the flow-sorted chromosome arms approach to genome analysis with second generation DNA sequencing technologies provides a unique opportunity to study the repetitive portion of each chromosome, enabling comparisons among them. Additionally, different sequencing approaches may produce different depth of insight to repeatome content and structure. In this work we analyze and characterize the repetitive sequences of Triticum aestivum cv. Chinese Spring homeologous group 4 chromosome arms, obtained through Roche 454 and Illumina sequencing technologies, hereinafter marked by subscripts 454 and I, respectively. Repetitive sequences were identified with the RepeatMasker software using the interspersed repeat database mips-REdat_v9.0p. The input sequences consisted of our 4DS454 and 4DL454 scaffolds and 4ASI, 4ALI, 4BSI, 4BLI, 4DSI and 4DLI contigs, downloaded from the International Wheat Genome Sequencing Consortium (IWGSC).

Results: Repetitive sequences content varied from 55% to 63% for all chromosome arm assemblies except for 4DLI, in which the repeat content was 38%. Transposable elements, small RNA, satellites, simple repeats and low complexity sequences were analyzed. SSR frequency was found one per 24 to 27 kb for all chromosome assemblies except 4DLI, where it was three times higher. Dinucleotides and trinucleotides were the most abundant SSR repeat units. (GA)n/(TC)n was the most abundant SSR except for 4DLI where the most frequently identified SSR was (CCG/CGG)n. Retrotransposons followed by DNA transposons were the most highly represented sequence repeats, mainly composed of CACTA/En-Spm and Gypsy superfamilies, respectively. This whole chromosome sequence analysis allowed identification of three new LTR retrotransposon families belonging to the Copia superfamily, one belonging to the Gypsy superfamily and two TRIM retrotransposon families. Their physical distribution in wheat genome was analyzed by fluorescent in situ hybridization (FISH) and one of them, the Carmen retrotransposon, was found specific for centromeric regions of all wheat chromosomes.

Conclusion: The presented work is the first deep report of wheat repetitive sequences analyzed at the chromosome arm level, revealing the first insight into the repeatome of T. aestivum chromosomes of homeologous group 4.

No MeSH data available.