Progress 08/15/06 to 08/14/09
Outputs
OUTPUTS: The complication of linkage group 2 was resolved by high density genetic mapping of the papaya genome using sequence-tagged co-dominant microsatellite markers. The current high density genetic map consists of nine major linkage groups and three minor linkage groups. Our revised objective 2 is to assign the three minor linkages to chromosomes defined by major linkage groups. Our molecular cytogenetic mapping results placed the three minor linkage groups 10, 11, and 12 to chromosomes defined by major linkage groups 8, 9, and 7 respectively. The papaya chromosome 1 contains the male-specific Y chromosome region (MSY), which controls sex in papaya. We selected 12 SSR markers along the papaya chromosome 1, spanning 121cM with average distance between neighbor markers at 10cM. The relative order of markers on the chromosome 1 was tested by fluorescence in situ hybridization (FISH) and their physical locations were measured based on at least 10 meiotic pachytene chromosomes. The result is showed in Table 1. Compared with genetic map, 10 out of the 12 markers showed consistent order. The 2 exemptions are P3K2981 and P6K1460, which flanked the MSY region. The locations of P3K2981 and P6K1460 were inverted compared with the genetic map that separated the two markers by only 2 cM. The cytogenetic map is consistent with our physical map result (Yu et al., 2009) and the enhanced AFLP and SSR genetic map (Blas et al., 2009). All the 12 markers we have tested are located at euchromatin regions, and confirmed that SSR markers are enriched in genic region. We also investigated the relationships between physical distance and cytological distance. Our estimated relative physical size in euchromatin region was 145-174 kb/faction length (FL). Thus, euchromatin region of the chromosome 1 might be 17.81-21.34 Mb. The papaya MSY is located between 46 and 48cM on the chromosome 1. The genetic distance between these 2 markers were only 2cM. However, it occupied 12.2% of the chromosome (or 16.97 fraction length). The physical size of this region is 8.5-fold higher than the size indicated on the genetic map, due to the suppression of recombination in the MSY region. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
An accurate chromosome map is an essential resource for genome sequence assembly, genetic and physical mapping of targeted genes, and papaya improvement via genetic engineering and gene manipulation. With the completion of the papaya genome sequencing project, genomic resources are available to construct a sequence-tagged genetic map and linking the genetic map to chromosomes by florescent in situ hybridization of strategically selected DNA markers. The integrated genetic, physical, and chromosome map will be an enormously useful tool for the papaya research community and ultimately benefit the papaya growers with improved papaya cultivars and products. We reported here the 1st molecular cytogenetic map of the papaya chromosome 1. This map revealed the relationships between physical, cytological and genetic distances along papaya chromosome which was unknown before. An accurate and integrated map is necessary for mapping QTLs. Our result indicated that 96% of the papaya chromosome 1 was covered by the SSR genetic map. The remaining 4% uncovered regions are telomeric regions. By labeling BAC clones with genes of interest on pachytene chromosome, the genetic position of unknown BAC clones can be estimated through its cytological position and estimating the physical distance between BAC clones. This will facilitate marker-assisted selection of agronomically important traits and cloning of target genes.
Publications
- Ming R., S. Hou, Y. Feng, Q. Yu, A. Dionne-Laporte, J.H. Saw, P. Senin, W. Wang, B.V. Ly, K.L.T. Lewis, S.L. Salzberg, L. Feng, M.R. Jones, R.L. Skelton, J.E. Murray, C. Chen, W. Qian, J. Shen, P. Du, M. Eustice, E. Tong, H. Tang, E. Lyons, R.E. Paull, T.P. Michael, K. Wall, D. Rice, H. Albert, M.-L. Wang, Y.J. Zhu, M. Schatz, N. Nagarajan, R. Agbayani, P. Guan, A. Blas, C. M. Wai, C.M. Ackerman, Y. Ren, C. Liu, J. Wang, J. Wang, J.-K. Na, E.V. Shakirov, B. Haas, J. Thimmapuram, D. Nelson, X. Wang, J.E. Bowers, A.R. Gschwend, A.L. Delcher, R. Singh, J.Y. Suzuki, S. Tripathi, K. Neupane, H. Wei, B. Irikura, M. Paidi, N. Jiang, W. Zhang, G. Presting, A. Windsor, R. Navajas-Perez, M.J. Torres, F. Alex Feltus, B. Porter, Y. Li, A.M. Burroughs, M.-C. Luo, L. Liu, D.A. Christopher, S.M. Mount, P.H. Moore, T. Sugimura, J. Jiang, M.A. Schuler, V. Friedman, T. Mitchell-Olds, D.E. Shippen, C.W. dePamphilis, J.D. Palmer, M. Freeling, A.H. Paterson, D. Gonsalves, L. Wang, M. Alam. 2008. The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus). Nature 452:991-996
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Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: To assign each linkage group of the papaya high density map to individual chromosomes we selected markers and labeled papaya BAC clones containing these markers on papaya chromosomes. In total, 57 BAC clones showed specific signals on papaya pachytene chromosomes and 21 BAC clones showed specific signals on papaya metaphase chromosomes. For linkage 1 to 9, at least two BAC clones at two distal ends were mapped with specific FISH signals and will be used for papaya chromosome characterization. To characterize structural features of papaya chromosomes, the DIG-labeled PCR fragments containing papaya 45S rDNA and 5S rDNA were used as probes to label papaya chromosomes. Our FISH result showed that only one pair of papaya chromosomes contain 45S rDNA and it is located in interstitial position of the chromosome. The signals were also interacted with nucleolus. Multicolor FISH simultaneously detecting 45S rDNA and papaya chromosome-specific markers revealed the 45S rDNA was located on Linkage group 2. Fine-mapping of 45S rDNA on Linkage group 2 showed that 45S rDNA was located between CPM1759 (68cM) and CPM1868 (101cM). FISH result showed at least 2 pairs of papaya chromosomes contained 5S rDNA. These 2 pairs of papaya chromosomes were identified as LG 4 and LG 5 using papaya chromosome-specific markers. The position of 5S rDNA was located between SSR marker P3K398 (31cM) and CPM1111 (91cM) on LG 4 and between SSR marker CPM988 (44cM) and CPM1655 (66cM) on LG 5. The complication of linkage group 2 was resolved by high density genetic mapping of the papaya genome using sequence-tagged co-dominant microsatellite markers. The current high density genetic map consists of nine major linkage groups (linkage group 1 to 9) and three minor linkage groups (linkage group 10 to 12). DNA markers were chosen from each of the three minor linkage groups (LG 10-12) and hybridized with chromosome-specific markers. LG 10 was assigned to LG8 and this cytogenetic result was coincident with result from the papaya whole genome shotgun sequence. Also, LG11 was assigned to LG9 and the relative order of DNA markers on this chromosome revealed that the flip LG11 was located on the top of LG9. Lastly, LG12 was located at the same chromosome with LG 7. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
An accurate chromosome map is an essential resource for genome sequence assembly, genetic and physical mapping of targeted genes, and papaya improvement via genetic engineering and gene manipulation. With the completion of the papaya genome sequencing project, genomic resources are available to construct a sequence-tagged genetic map and linking the genetic map to chromosomes by florescent in situ hybridization of strategically selected DNA markers. The integrated genetic, physical, and chromosome map will be an enormously useful tool for the papaya research community and ultimately benefit the papaya growers with improved papaya cultivars and products. In the past years, scientists at UH, HARC, and the University of Illinois have prepared papaya chromosome slides, selected target DNA markers from individual linkage groups, and isolated DNA from BACs containing these markers. The carefully prepared papaya chromosome spread revealed morphological features of papaya chromosomes that were unknown before. Molecular cytogenetic mapping placed two minor linkage groups 10, 11, and 12 to chromosomes defined by major linkage groups 8, 9, and 7 respectively. Construction of a molecular cytogenetic map will help us understand the structure and organization of the papaya genome and expedite the process to improve papaya quality and productivity.
Publications
- Ming R., S. Hou, Y. Feng, Q. Yu, A. Dionne-Laporte, J.H. Saw, P. Senin, W. Wang, B.V. Ly, K.L.T. Lewis, S.L. Salzberg, L. Feng, M.R. Jones, R.L. Skelton, J.E. Murray, C. Chen, W. Qian, J. Shen, P. Du, M. Eustice, E. Tong, H. Tang, E. Lyons, R.E. Paull, T.P. Michael, K. Wall, D. Rice, H. Albert, M.-L. Wang, Y.J. Zhu, M. Schatz, N. Nagarajan, R. Agbayani, P. Guan, A. Blas, C. M. Wai, C.M. Ackerman, Y. Ren, C. Liu, J. Wang, J. Wang, J.-K. Na, E.V. Shakirov, B. Haas, J. Thimmapuram, D. Nelson, X. Wang, J.E. Bowers, A.R. Gschwend, A.L. Delcher, R. Singh, J.Y. Suzuki, S. Tripathi, K. Neupane, H. Wei, B. Irikura, M. Paidi, N. Jiang, W. Zhang, G. Presting, A. Windsor, R. Navajas-Perez, M.J. Torres, F. Alex Feltus, B. Porter, Y. Li, A.M. Burroughs, M.-C. Luo, L. Liu, D.A. Christopher, S.M. Mount, P.H. Moore, T. Sugimura, J. Jiang, M.A. Schuler, V. Friedman, T. Mitchell-Olds, D.E. Shippen, C.W. dePamphilis, J.D. Palmer, M. Freeling, A.H. Paterson, D. Gonsalves, L. Wang, M. Alam. 2008. The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus). Nature 452:991-996
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Progress 08/15/06 to 08/14/07
Outputs
Objective 1: Assign each linkage group of the papaya high density map to individual chromosomes by in situ hybridization of selected DNA markers mapped to each linkage group. Papaya chromosomes are relatively small compared with other plant species. It is still not possible to identify individual chromosomes based on their morphological characteristics, although they are variable in sizes. To assign papaya linkage groups to individual chromosomes, we developed one set of papaya chromosome-specific markers based on a sequence tagged high density papaya genetic map. We selected markers from each linkage group and label papaya BAC clones containing these markers on papaya chromosomes. Some BAC clones showed high specificity, while others non-specific hybridization due to repetitive sequences. In total, 57 BAC clones was examined for specificity. All BAC clones were labeled by either biotin or digoxigenin, and florescence in situ hybridization (FISH) analysis was performed
using papaya pachytene chromosomes, which has higher resolution than metaphase chromosomes. For linkage 1 to 9, at least two BAC clones at two distal ends were mapped with specific FISH signals and will be used for papaya chromosome characterization. Objective 2: Define the breakpoint of linkage group 2 and clarify the orders of linkage groups on chromosomes. The complication of linkage group 2 was resolved by high density genetic mapping of the papaya genome using sequence-tagged co-dominant microsatellite markers. The current high density genetic map consists of nine major linkage groups (linkage group 1 to 9) and three minor linkage groups (linkage group 10 to 12). Our hypothesis is that each of the 9 major linkage groups represents one chromosome and specific chromosomal features such as the nucleolar organizer might have contributed to the break down of linkage groups. Our revised objective 2 is to assign the three minor linkages to chromosomes defined by major linkage groups.
Assembly of whole genome shotgun sequences provided molecular evidence that linkage groups 8 and 10 are on the same chromosomes as a contiguous genome sequences contains microsatellite markers on both linkage groups 8 and 10. Our FISH mapping results demonstrated that linkage group 9 and 11 are on the same chromosome. The linkage group 11 is at one end of the chromosome. We are now focusing on placing linkage group 12 to a chromosome defined by a major linkage group.
Impacts
An accurate chromosome map is an essential resource for genome sequence assembly, genetic and physical mapping of targeted genes, and papaya improvement via genetic engineering and gene manipulation. With the completion of the papaya genome sequencing project, genomic resources are available to construct a sequence-tagged genetic map and linking the genetic map to chromosomes by florescent in situ hybridization of strategically selected DNA markers. The integrated genetic, physical, and chromosome map will be an enormously useful tool for the papaya research community and ultimately benefit the papaya growers with improved papaya cultivars and products. In the past year, scientists at UH, HARC, and the University of Illinois have prepared papaya chromosome slides, selected target DNA markers from individual linkage groups, and isolated DNA from BACs containing these markers. The carefully prepared papaya chromosome spread revealed morphological features of papaya
chromosomes that were unknown before. Molecular cytogenetic mapping placed two minor linkage groups 10 and 11 to chromosomes defined by major linkage groups 8 and 9, respectively. Construction of a molecular cytogenetic map will help us understand the structure and organization of the papaya genome and expedite the process to improve papaya quality and productivity.
Publications
- No publications reported this period
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