Abstract
【Objective】The study aims to optimize chromosome sorting and construction of BAC library from specific chromosomes and present technical support for accelerating wheat genome research.【Method】Using double-block method to synchronize root tip meristem cells of common wheat ditelosomic line 5DL, the chromosomes were released for flow karyotypic analysis after fixed in 2% polyformaldehyde in Tris-HCl buffer followed by mechanical homogenization. Chromosome 5DL was subjected to flow sorting and PCR analysis to optimize chromosome sorting in wheat. Chromosomes from composite peaks were digested with 1U BamHI to prepare HMW (high-molecular-weight, HMW) DNA. The ratio of 10:1 (HMW DNA:BIBAC vector, w/w) was applied and then 1:20 (ligation:electro-competent cell, v/v) performed in electroporation. To determine the insert size,random 100 recombinators were selected for full digestion with NotI to optimize the technique for chromosome-specific library.【Result】The mitotic index can reach over 60% after the following treatments with 1.25 mmol·L-1 hydroxyurea for 18 h, 6 h in hydroxyurea (HU)-free Hoagland’s solution for recovery and 2.5 h 1 μmol·L-1 trifluralin on wheat meristem cells. Based on fluorescence microscope and PCR detection, histograms of fluorescence intensity acquired after the analysis of DAPI-stained common wheat ditelosomic line 5DL chromosomes comprised three composite peaks and two single peaks representing chromosome 5DL and 3B, respectively. Partial enzyme digestion of high-molecular-weight (HMW) DNA demonstrated that 50-300 kb fragments can be acquired with an average insert size of 100 kb or so. According to the fluorescence channel, the size of chromosome 5DL was 482 Mb. Therefore, the library of chromosome 5DL with 19 000 clones could achieve four fold coverage.【Conclusion】The two above optimized techniques present a useful method in constructing chromosome- and chromosome arm-specific library from sorted chromosomes in wheat.
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Claims 6
Claim · model-written summary
The synchronization treatment raised the wheat meristem mitotic index above 60%.
- Taxon
- wheat
Triticum aestivume0 - Organ / stage
- meristem cellse0
- Treatment
- 1.25 mmol·L-1 hydroxyurea for 18 h, 6 h in hydroxyurea (HU)-free Hoagland’s solution for recovery and 2.5 h 1 μmol·L-1 trifluraline0
- Outcome
- mitotic indexe0
- Direction
- reach overe0
- Value
- over 60%e0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The mitotic index can reach over 60% after the following treatments with 1.25 mmol·L-1 hydroxyurea for 18 h, 6 h in hydroxyurea (HU)-free Hoagland’s solution for recovery and 2.5 h 1 μmol·L-1 trifluralin on wheat meristem cells”
Claim · model-written summary
Fluorescence profiles showed three composite peaks and two single peaks for chromosomes 5DL and 3B.
- Taxon
- wheatTriticum aestivume0
- Material
- ditelosomic line 5DLe0
- Outcome
- three composite peaks and two single peaks representing chromosome 5DL and 3Be0
- Value
- three composite peaks and two single peakse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“DAPI-stained common wheat ditelosomic line 5DL chromosomes comprised three composite peaks and two single peaks representing chromosome 5DL and 3B, respectively”
Claim · model-written summary
Partial digestion produced DNA fragments of 50-300 kb.
- Taxon
- wheatTriticum aestivume1
- Outcome
- fragmentse0
- Value
- 50-300 kbe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Partial enzyme digestion of high-molecular-weight (HMW) DNA demonstrated that 50-300 kb fragments can be acquired”
e1“wheat meristem cells”
Claim · model-written summary
The resulting average insert size was about 100 kb.
- Taxon
- wheatTriticum aestivume1
- Outcome
- average insert sizee0
- Value
- 100 kbe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“with an average insert size of 100 kb or so”
e1“wheat meristem cells”
Claim · model-written summary
Fluorescence channel analysis estimated chromosome 5DL size at 482 Mb.
- Taxon
- wheatTriticum aestivume1
- Outcome
- size of chromosome 5DLe0
- Value
- 482 Mbe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“According to the fluorescence channel, the size of chromosome 5DL was 482 Mb”
e1“wheat meristem cells”
Claim · model-written summary
The chromosome 5DL library contained 19 000 clones and achieved four fold coverage.
- Taxon
- wheatTriticum aestivume1
- Outcome
- library of chromosome 5DLe0
- Value
- 19 000 clonese0
- Qualifier
- four fold coveragee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the library of chromosome 5DL with 19 000 clones could achieve four fold coverage”
e1“wheat meristem cells”
The same questions in other papers
rewatering · cytology · Triticum aestivum (wheat)Results agree
1 paper in the same direction as this paper
- positive
Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.