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Structural characterization of copia-type retrotransposons leads to insights into the marker development in a biofuel crop, Jatropha curcas L.

Alipour A, Tsuchimoto S, Sakai H, Ohmido N, Fukui K - Biotechnol Biofuels (2013)

Bottom Line: Fluorescence in situ hybridization (FISH) to metaphase chromosomes reveals that copia-type retrotransposons are scattered across chromosomes mainly located in the distal part regions.This is the first report on genome-wide analysis and the cytogenetic mapping of copia-type retrotransposons of jatropha, leading to the discovery of families bearing high potential as DNA markers.Distinct dynamics of individual copia-type families, feasibility of a retrotransposon-based insertion polymorphism marker system in examining genetic variability, and approaches for the development of breeding strategies in jatropha using copia-type retrotransposons are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan. kfukui@bio.eng.osaka-u.ac.jp.

ABSTRACT

Background: Recently, Jatropha curcas L. has attracted worldwide attention for its potential as a source of biodiesel. However, most DNA markers have demonstrated high levels of genetic similarity among and within jatropha populations around the globe. Despite promising features of copia-type retrotransposons as ideal genetic tools for gene tagging, mutagenesis, and marker-assisted selection, they have not been characterized in the jatropha genome yet. Here, we examined the diversity, evolution, and genome-wide organization of copia-type retrotransposons in the Asian, African, and Mesoamerican accessions of jatropha, then introduced a retrotransposon-based marker for this biofuel crop.

Results: In total, 157 PCR fragments that were amplified using the degenerate primers for the reverse transcriptase (RT) domain of copia-type retroelements were sequenced and aligned to construct the neighbor-joining tree. Phylogenetic analysis demonstrated that isolated copia RT sequences were classified into ten families, which were then grouped into three lineages. An in-depth study of the jatropha genome for the RT sequences of each family led to the characterization of full consensus sequences of the jatropha copia-type families. Estimated copy numbers of target sequences were largely different among families, as was presence of genes within 5 kb flanking regions for each family. Five copia-type families were as appealing candidates for the development of DNA marker systems. A candidate marker from family Jc7 was particularly capable of detecting genetic variation among different jatropha accessions. Fluorescence in situ hybridization (FISH) to metaphase chromosomes reveals that copia-type retrotransposons are scattered across chromosomes mainly located in the distal part regions.

Conclusion: This is the first report on genome-wide analysis and the cytogenetic mapping of copia-type retrotransposons of jatropha, leading to the discovery of families bearing high potential as DNA markers. Distinct dynamics of individual copia-type families, feasibility of a retrotransposon-based insertion polymorphism marker system in examining genetic variability, and approaches for the development of breeding strategies in jatropha using copia-type retrotransposons are discussed.

No MeSH data available.


Related in: MedlinePlus

Chromosomal distribution of jatropha copia-type retrotransposons. Mitotic metaphase spread of J. curcas (2n = 22) after fluorescence in situ hybridization with RT element had high, moderate and low copy number as a probe. The chromosomes were counterstained light blue with DAPI. Green and red signal represent RT sequences labeled with DIG and biotin, respectively. (a) RT sequence belongs to family Jc1 (high copy), (b)Jc5 (high copy), (c)Jc3 (moderate copy) and (d)Jc8 (low copy) were used as probes. (e) Double fluorescence in situ hybridization with the 5S rRNA gene sequence and the RT sequence of Jc5. Bars= 3 μm.
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Figure 8: Chromosomal distribution of jatropha copia-type retrotransposons. Mitotic metaphase spread of J. curcas (2n = 22) after fluorescence in situ hybridization with RT element had high, moderate and low copy number as a probe. The chromosomes were counterstained light blue with DAPI. Green and red signal represent RT sequences labeled with DIG and biotin, respectively. (a) RT sequence belongs to family Jc1 (high copy), (b)Jc5 (high copy), (c)Jc3 (moderate copy) and (d)Jc8 (low copy) were used as probes. (e) Double fluorescence in situ hybridization with the 5S rRNA gene sequence and the RT sequence of Jc5. Bars= 3 μm.

Mentions: Determining the chromosomal locations of copia-type retrotransposons could contribute to the better understanding of the role and the dynamics of the repetitive elements in the genome and karyotype of jatropha, as well as facilitate the selection of families for informative markers. In order to gain insight into the chromosomal distribution of copia-type retrotransposons in the jatropha genome, FISH analysis was carried out using biotin- or digoxigenin (DIG)-labeled RT sequences as probes, which were selected from families of the three lineages that showed high (Jc1 and Jc5), moderate (Jc3), and low (Jc8) copy numbers (Figures 8a-d). The copia-type retrotransposons of the all four families dispersed throughout the chromosomes but were predominantly located in the distal regions of chromosome arms. These results demonstrate the similar distribution patterns of copia-type retrotransposons in jatropha chromosomes among families of various lineages with different copy numbers. Interestingly, the intensities of FISH signals were obviously different among the chromosomes. This might be due to difference in copy numbers of the family members among the chromosomes. It was previously indicated that the genes for 5S rRNA were mapped at the terminal heterochromatin regions in two of the jatropha chromosomes [36]. Employing a double-label FISH assay to detect the physical distribution patterns of 5S rRNA genes and Jc5 RT demonstrated that they are not overlapped, despite the presence in the distal part of chromosomes and intense hybridization signals of RT (Figure 8e). There is a good evidence for the similar distribution patterns of copia-type retrotransposons in jatropha chromosomes among families of various lineages with different copy numbers.


Structural characterization of copia-type retrotransposons leads to insights into the marker development in a biofuel crop, Jatropha curcas L.

Alipour A, Tsuchimoto S, Sakai H, Ohmido N, Fukui K - Biotechnol Biofuels (2013)

Chromosomal distribution of jatropha copia-type retrotransposons. Mitotic metaphase spread of J. curcas (2n = 22) after fluorescence in situ hybridization with RT element had high, moderate and low copy number as a probe. The chromosomes were counterstained light blue with DAPI. Green and red signal represent RT sequences labeled with DIG and biotin, respectively. (a) RT sequence belongs to family Jc1 (high copy), (b)Jc5 (high copy), (c)Jc3 (moderate copy) and (d)Jc8 (low copy) were used as probes. (e) Double fluorescence in situ hybridization with the 5S rRNA gene sequence and the RT sequence of Jc5. Bars= 3 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852365&req=5

Figure 8: Chromosomal distribution of jatropha copia-type retrotransposons. Mitotic metaphase spread of J. curcas (2n = 22) after fluorescence in situ hybridization with RT element had high, moderate and low copy number as a probe. The chromosomes were counterstained light blue with DAPI. Green and red signal represent RT sequences labeled with DIG and biotin, respectively. (a) RT sequence belongs to family Jc1 (high copy), (b)Jc5 (high copy), (c)Jc3 (moderate copy) and (d)Jc8 (low copy) were used as probes. (e) Double fluorescence in situ hybridization with the 5S rRNA gene sequence and the RT sequence of Jc5. Bars= 3 μm.
Mentions: Determining the chromosomal locations of copia-type retrotransposons could contribute to the better understanding of the role and the dynamics of the repetitive elements in the genome and karyotype of jatropha, as well as facilitate the selection of families for informative markers. In order to gain insight into the chromosomal distribution of copia-type retrotransposons in the jatropha genome, FISH analysis was carried out using biotin- or digoxigenin (DIG)-labeled RT sequences as probes, which were selected from families of the three lineages that showed high (Jc1 and Jc5), moderate (Jc3), and low (Jc8) copy numbers (Figures 8a-d). The copia-type retrotransposons of the all four families dispersed throughout the chromosomes but were predominantly located in the distal regions of chromosome arms. These results demonstrate the similar distribution patterns of copia-type retrotransposons in jatropha chromosomes among families of various lineages with different copy numbers. Interestingly, the intensities of FISH signals were obviously different among the chromosomes. This might be due to difference in copy numbers of the family members among the chromosomes. It was previously indicated that the genes for 5S rRNA were mapped at the terminal heterochromatin regions in two of the jatropha chromosomes [36]. Employing a double-label FISH assay to detect the physical distribution patterns of 5S rRNA genes and Jc5 RT demonstrated that they are not overlapped, despite the presence in the distal part of chromosomes and intense hybridization signals of RT (Figure 8e). There is a good evidence for the similar distribution patterns of copia-type retrotransposons in jatropha chromosomes among families of various lineages with different copy numbers.

Bottom Line: Fluorescence in situ hybridization (FISH) to metaphase chromosomes reveals that copia-type retrotransposons are scattered across chromosomes mainly located in the distal part regions.This is the first report on genome-wide analysis and the cytogenetic mapping of copia-type retrotransposons of jatropha, leading to the discovery of families bearing high potential as DNA markers.Distinct dynamics of individual copia-type families, feasibility of a retrotransposon-based insertion polymorphism marker system in examining genetic variability, and approaches for the development of breeding strategies in jatropha using copia-type retrotransposons are discussed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan. kfukui@bio.eng.osaka-u.ac.jp.

ABSTRACT

Background: Recently, Jatropha curcas L. has attracted worldwide attention for its potential as a source of biodiesel. However, most DNA markers have demonstrated high levels of genetic similarity among and within jatropha populations around the globe. Despite promising features of copia-type retrotransposons as ideal genetic tools for gene tagging, mutagenesis, and marker-assisted selection, they have not been characterized in the jatropha genome yet. Here, we examined the diversity, evolution, and genome-wide organization of copia-type retrotransposons in the Asian, African, and Mesoamerican accessions of jatropha, then introduced a retrotransposon-based marker for this biofuel crop.

Results: In total, 157 PCR fragments that were amplified using the degenerate primers for the reverse transcriptase (RT) domain of copia-type retroelements were sequenced and aligned to construct the neighbor-joining tree. Phylogenetic analysis demonstrated that isolated copia RT sequences were classified into ten families, which were then grouped into three lineages. An in-depth study of the jatropha genome for the RT sequences of each family led to the characterization of full consensus sequences of the jatropha copia-type families. Estimated copy numbers of target sequences were largely different among families, as was presence of genes within 5 kb flanking regions for each family. Five copia-type families were as appealing candidates for the development of DNA marker systems. A candidate marker from family Jc7 was particularly capable of detecting genetic variation among different jatropha accessions. Fluorescence in situ hybridization (FISH) to metaphase chromosomes reveals that copia-type retrotransposons are scattered across chromosomes mainly located in the distal part regions.

Conclusion: This is the first report on genome-wide analysis and the cytogenetic mapping of copia-type retrotransposons of jatropha, leading to the discovery of families bearing high potential as DNA markers. Distinct dynamics of individual copia-type families, feasibility of a retrotransposon-based insertion polymorphism marker system in examining genetic variability, and approaches for the development of breeding strategies in jatropha using copia-type retrotransposons are discussed.

No MeSH data available.


Related in: MedlinePlus