| Project/Area Number |
11660005
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Breeding science
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| Research Institution | Okayama University |
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Principal Investigator |
MURATA Minoru Okayama Univ., Res.Inst.Bioresour., Prof., 資源生物科学研究所, 教授 (20166292)
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| Co-Investigator(Kenkyū-buntansha) |
OGURA Yutaka Okayama Univ., Res.Inst.Bioresour., Assist.Prof., 資源生物科学研究所, 助手 (60224193)
SAKAMOTO Wataru Okayama Univ., Res.Inst.Bioresour., Assoc.Prof., 資源生物科学研究所, 助教授 (20222002)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | wheat / chromosome-specific DNA libraries / ditelosomics / microdissection / DOP-PCR / repetitive sequences / retrotrans / FISH / ダイテロソーミックス / グルテニン遺伝子 |
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| Research Abstract |
Important cereals such as wheat and barley have considerably large amounts of DNA in the genome. This makes it difficult to isolate the genes by using the methods, which are applicable to plants with small genome such as Arabidopsis. In this study, chromosome-, arm- and region-specific DNA libraries were attempted to be constructed from wheat by laser-microdissection. Telocentric chromosomes in ditelosomic lines and the midget chromosome in the rye-cytoplasm substitution line of wheat (Triticum aestivum cv. Chinese Spring) were chosen as targets, scratched off and picked up with fine glass needles adjusted to a micromanipulator under microscopic observation. The microdissected chromosomes were then harvested into a PCR tube, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). In order to evaluate the efficiency of our microdissection procedure, we attempted to amplify DNA from only one single chromosome of wheat, and compared with the results from two, four and six microdissected fragments. In almost all cases, DNA amplification could be observed even from a single chromosome. Sequencing analysis revealed that a relatively high proportion of low-copy sequences are involved in the PCR fragments. This suggests the present microdissection procedure is effective in creating painting probes and in generating region-specific DNA markers. Chromosome-specificity was confirmed in the case of the midget chromosome, because it has originated from rye. However, it was not clearly revealed in the microdissection of wheat chromosomes. It may be due to the large amount of retroposon-like repeat sequences in the amplified DNA used as probes.
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