Progress 10/01/04 to 09/30/08
Outputs
OUTPUTS: Centromeres and telomeres are the two most important landmarks of eukaryotic chromosomes. Telomeres serve as inert caps, preventing chromosomal rearrangements stemming from the reactive and recombinogenic behavior of broken chromosome ends. Centromeres are responsible for chromosome movement in both mitosis and meiosis by serving as an attachment site for spindle fibers. Cloning and characterization of DNA sequences associated with telomeres and centromeres from plant species is an essential step to construct plant artificial chromosomes (PACs). With an ultimate goal to develop PACs using potato as a model species we launched an effort to characterize the DNA sequences associated with the telomeres and centromeres of potato chromosomes. We developed a special procedure to isolate highly repetitive satellite DNA from the potato genome. Our approach was to search the most dominant tandem repeats using a bacterial artificial chromosome (BAC) library-based screening method. A large set of potato BAC clones containing satellite repeats were isolated and confirmed by fluorescence in situ hybridization (FISH) mapping. Several satellite repeats specific to the telomeric and centromeric regions of potato chromosomes were isolated and characterized from these BAC clones. Most interestingly we found that the potato centromeres contain megabase-sized satellite DNA arrays amplified from sequences similar to telomeric DNA. Such long arrays of telomere-similar sequences in centromeres are unique in multicellular eukaryotes. We also characterized several repetitive DNA sequences that are located in the pericentromeric regions of potato chromosomes, including the 4.7-kb Sobo repeat. This repeat was found to be derived from the long terminal repeats (LTRs) of a retrotransposon. Characterization of the Sobo repeat has provided significant implications on origin of tandemly repeated DNA sequences in complex eukaryotic genomes. The centromere-specific histone H3 variant, referred to as CENH3, is an universal marker for centromeric chromatin, with homologues having been characterized in species ranging from yeast to insects, vertebrates, and plants. We have successfully developed an antibody against potato CENH3. Our initial immunoassays using this antibody failed because it binds to some unknown proteins in the cytoplasm. We tried various methods to fix the potato root tip tissues and to prepare the chromosome spreads. We have developed a technique that allows us to remove the cytoplasm but retain the nuclei and chromosomes with reasonable quality. We have confirmed that this antibody stained only the centromeric regions on potato chromosomes. This antibody will provide an important tool for our future research to confirm the functional role of the centromere-related DNA sequences isolated through this project. PARTICIPANTS: Jiming Jiang: Principal Investigator; Ahmet L. Tek: Ph. D. Student (Graduated in 2003); Robert M. Stupar: Ph. D. Student (Graduated in 2005); Pudota B. Bhaskar: Ph. D. Student. TARGET AUDIENCES: Potato genetics and genomics research community, plant biotechnology industry. PROJECT MODIFICATIONS: No major changes were implanted.
Impacts
Centromeres and telomeres govern the stability and proper segregation and transmission of chromosomes during cell division. Cloning and characterization of plant centromeric and telomeric DNA is essential to understand the structure and organization of plant chromosomes and is a critical step to develop plant artificial chromosomes (PACs). Potato is an optimal model plant species for PAC construction because it is asexually propagated, tolerant to aneuploidy, and is highly transformable. We have identified a number of DNA elements located in the telomeric, centromeric, and pericentromeric regions of potato chromosomes. The isolation of potato BAC clones containing large centromeric DNA fragments is the first step toward our goal to develop a PAC in potato. Our technique for centromeric DNA isolation can potentially be applied in other eukaryotic species. Successful applications of PACs will rely on techniques to deliver large DNA fragments into plant cells. We have also exploited the limit and potential of the currently available binary vector systems. We have demonstrated that the currently available Agrobacterium strains are not ideal to hold large insert plasmids. We need to continue to engineer these stains to increase the stability of large insert constructs. We have developed an antibody against potato CENH3, the most critical centromeric protein. This antibody will be a critical tool to confirm if the centromeric DNA elements are located in the functional domain of potato centromeres and to assess the function of centromeres associated with rearranged chromosomes or artificially constructed chromosomes. Future work will lead us to characterize the DNA components of the functional centromeres as well as the pericentromeric heterochromatin in the potato genome, which will be the key foundation for potato PAC development.
Publications
- Tek, A.L. and Jiang, J. (2004) The centromeric regions of potato chromosomes contain megabase-sized tandem arrays of telomere-similar sequence. Chromosoma 113: 77-83.
- Dong, F., Tek, A.L., Frasca, A.B.L., McGrath, J.M., Wielgus, S.M., Helgeson, J.P., and J. Jiang (2005) Development and characterization of potato-Solanum brevidens chromosomal addition/substitution lines. Cytogenet. Genome Res. 109: 368-372.
- Tek, A.L., Stevenson, W.R., Helgeson, J.P., and Jiang, J. (2004) Introgression of tuber soft rot and early blight resistances from Solanum brevidens into cultivated potato. Theor. Appl. Genet. 109: 249-254.
- Tek, A.L., Song, J., Macas, J. and Jiang, J. (2005) Sobo, a recently amplified satellite repeat of potato, and its implications for the origin of tandemly repeated sequences. Genetics 170: 1231-1238.
- Stupar, R.M., Beaubien, K.A., Jin, W., Song, J., Lee, M.-K., Wu, C., Zhang, H.-B., Han, B. and Jiang, J. (2006) Structural diversity and differential transcription of the patatin multicopy gene family during potato tuber development. Genetics 172: 1263-1275.
- Stupar, R.M., Bhaskar, P.B., Yandell, B.S., Rensink, W.A., Hart, A.L., Ouyang, S., Veilleux, R.E., Busse, J.S., Erhardt, R.J., Buell, C.R., and Jiang, J. (2007) Phenotypic and transcriptomic changes associated with potato autopolyploidization. Genetics 176: 2055-2067.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We have continued our effort to isolate and characterize the centromeric DNA sequences from the potato genome. In a collaboration with Dr. Robin Buell at the Michigan State University, we searched the 155,130 sequences derived from the ends of potato bacterial artificial chromosome (BAC) clones. Retrotransposon sequences are the most abundant repetitive DNA component and account for ~12% of the sequences. We found a significant number of sequences (~1.31%) show homology to plant telomeric repeats. We selected two Potato BACs in which both end sequences contained the telomeric repeats and six clones in which only one of the two ends contained the telomeric sequences. These BACs were used in fluorescent in situ hybridization (FISH) studies to assess where on the chromosomes these repetitive sequences are localized. We discovered that these clones did not generate unambiguous signals at the telomeres of potato chromosomes but produced major signals in the centromeric and
pericentromeric regions of several potato chromosomes. These results showed that these potato BACs were most likely derived from centromeric rather than telomeric regions of potato chromosomes. We have mapped the chromosomal locations of more than 30 potato BAC clones, including several BACs located in the pericentromeric regions of potato chromosome 6. We have identified BACs that contains the most dominant repetitive DNA in potato pericentromeres. Cloning and characterization of these DNA elements are underway.
PARTICIPANTS: Jiming Jiang: Principal Investigator; Pudota B. Bhaskar: Ph. D. Student; Mallory Choudoir: Undergraduate Student
TARGET AUDIENCES: Potato Genetics, Genomics Research Community and Plant Biotechnology Industry.
PROJECT MODIFICATIONS: No major changes were implanted.
Impacts
Plant artificial chromosomes (PACs) are expected to become the next-generation technology for plant genetic engineering. The genetic material stably contained in a PAC will be in the order of multi-millions of DNA base pairs and will be able to carry the genetic information of hundreds of genes. Thus PACs can be used to design transgenic constructs including multiple genes or genes covering an entire pathway. The long term goal of this project is to use cloned potato centromeric DNA to develop potato PACs. We have identified a number of DNA elements located in the centromeres and pericentromeric regions of potato chromosomes. We have also developed an antibody against potato CENH3, the most critical centromeric protein. Future work will lead us to characterize the DNA components of the functional centromeres as well as the pericentromeric heterochromatin in the potato genome, which will be the key foundation for potato PAC development.
Publications
- Stupar, R.M., Bhaskar, P.B., Yandell, B.S., Rensink, W.A., Hart, A.L., Ouyang, S., Veilleux, R.E., Busse, J.S., Erhardt, R.J., Buell, C.R., and Jiang, J. (2007) Phenotypic and transcriptomic changes associated with potato autopolyploidization. Genetics 176: 2055-2067.
- Groza, H.I., Bowen, B.D., Bussan, A.J., Stevenson, W.R., Navarro, F., Kichefski, D., Peloquin, S.J., Palta, J., and Jiang, J. (2007) MegaChip - A new potato variety for chipping. Am. J. Potato Res. 84: 343-350.
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Progress 01/01/06 to 12/31/06
Outputs
The centromere is the most characteristic landmark of chromosomes in higher eukaryotic species and appears cytologically as a distinct primary constriction on condensed metaphase chromosomes. The centromere is responsible for sister chromatid cohesion and is the site for kinetochore assembly and spindle fiber attachment, thereby allowing for faithful pairing and segregation of sister chromatids during cell division. Cloning of a functional centromere is also an essential step to construct artificial chromosomes, which have attracted tremendous interests from both academia and the biotech industry to develop plant artificial chromosomes (PACs). Several proteins associated with centromeres are highly conserved among eukaryotes. In particular, a centromere-specific histone H3 variant, referred to as CENH3, appears to be a universal marker for centromeric chromatin, with homologues having been characterized in species ranging from yeast to insects, vertebrates and plants.
CENH3s replace the regular histone H3 in centromeric chromatin. There are numerous lines of evidence that CENH3 plays the key role in the establishment and function of kinetochores in various organisms. We have successfully developed an antibody against potato CENH3. Our initial immunoassays using this antibody failed because it binds some unknown proteins in the cytoplasm. We tried various methods to fix the potato root tip tissues and to prepare the chromosome spreads. We have developed a technique that allows us to remove the cytoplasm but retain the nuclei and chromosomes with reasonable quality. We have confirmed that this antibody stained only the centromeric regions on potato chromosomes. We have also developed several transgenic potato lines using a construct containing the maize CenH3 gene. Preliminary results have shown that the maize CENH3 were incorporated into the potato centromeres. Further characterization of these transgenic lines is underway.
Impacts
Plant artificial chromosomes (PACs) are expect to be one of the next-generation tools for genetic engineering. PACs will have the capacity to hold large amounts of DNA. Thus PACs can carry large genes and can be used to design transgenic constructs including multiple genes or genes covering an entire pathway. The long term goal of this project is to develop PACs using potato as a model. We have developed an antibody against potato CENH3, the most critical centromeric protein. This antibody will be a critical tool to confirm if the centromeric repetitive DNA elements are located in the functional domain of potato centromeres and to assess the function of centromeres on any rearranged chromosomes or artificially developed chromosomes in the future.
Publications
- Groza, H.I., Bowen, B.D., Stevenson, W.R., Sowokinos, J.R., Glynn, M.T., Thill, C., Peloquin, S.J., Bussan, A.J., and Jiang, J. (2006) White Pearl - A chipping potato variety with high level of resistance to cold sweetening. Am. J. Potato Res. 83: 259-267.
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Progress 01/01/05 to 12/31/05
Outputs
The centromere is the most characteristic landmark of chromosomes in higher eukaryotic species and appears cytologically as a distinct primary constriction on condensed metaphase chromosomes. The centromere is responsible for sister chromatid cohesion and is the site for kinetochore assembly and spindle fiber attachment, thereby allowing for faithful pairing and segregation of sister chromatids during cell division. Cloning of a functional centromere is also an essential step to construct artificial chromosomes, which have attracted tremendous interests from both academia and the biotech industry to develop plant artificial chromosomes (PACs). We successfully developed a special strategy to isolate centromeric DNA repeats in potato. Our approach was to search the most dominant tandem repeats in the potato genome using a bacterial artificial chromosome (BAC) library-based screening method. A BAC clone, 28G12, was identified to be a potential candidate centromeric
clone. Fluorescence in situ hybridization analysis using BAC 28G12 as a probe revealed that this BAC hybridizes to only one of the 24 Solanum bulbocastanum (a diploid wild potato species) chromosomes. This hemizygous locus was mapped to the pericentromeric region of potato chromosome 7. A 4.7-kb tandem repeat, named as Sobo, was isolated from this BAC clone. The Sobo locus spans 360 kb of a 4.7-kb monomer. Sequence analysis revealed that the major part of the Sobo monomer shares significant sequence similarity with the long terminal repeats (LTRs) of a retrotransposon. The Sobo repeat was not detected in other Solanum species and is absent in some S. bulbocastanum accessions. Sobo monomers are highly homogenized and share more than 99% sequence identity. These results suggest that the Sobo repeat is a recently emerged satellite and possibly originated by a sudden amplification of a genomic region including the LTR of a retrotransposon and its flanking genomic sequences.
Characterization of the Sobo repeat has provided significant implications on origin of tandemly repeated DNA sequences in complex eukaryotic genomes. We have also developed an antibody against potato CENH3, the centromeric H3 histone variant. Similar antibodies have been developed in several other plant species, including Arabidopsis thaliana, rice and maize, and have been proved to be a powerful tool in centromere biology research. The study of the centromere specificity of the potato anti-CENH3 antibody is underway.
Impacts
Centromeres govern the proper segregation and transmission of chromosomes during cell division. Cloning and characterization of plant centromeric DNA is essential to understand the structure and organization of plant chromosomes and is a critical step to develop plant artificial chromosomes (PACs). Potato is the optimal plant model for PAC construction because it is asexually propagated, tolerant to aneuploidy, and is highly transformable. We have isolated several repetitive DNA elements that are located in the centromeric and pericentromeric regions of potato chromosomes. These DNA elements are valuable probes to isolate large centromeric DNA fragments. The antibody against potato CENH3 will be a critical tool to confirm if the centromeric repetitive DNA elements are located in the functional domain of potato centromeres.
Publications
- Dong, F., Tek, A.L., Frasca, A.B.L., McGrath, J.M., Wielgus, S.M., Helgeson, J.P., and J. Jiang (2005) Development and characterization of potato-Solanum brevidens chromosomal addition/substitution lines. Cytogenet. Genome Res. 109: 368-372.
- Tek, A.L., Song, J., Macas, J. and Jiang, J. (2005) Sobo, a recently amplified satellite repeat of potato, and its implications for the origin of tandemly repeated sequences. Genetics 170: 1231-1238.
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