PMID- 27275186
OWN - NLM
STAT- PubMed-not-MEDLINE
DCOM- 20160609
LR  - 20200929
IS  - 1755-8166 (Print)
IS  - 1755-8166 (Electronic)
IS  - 1755-8166 (Linking)
VI  - 9
DP  - 2016
TI  - Application of a modified drop method for high-resolution pachytene chromosome 
      spreads in two Phalaenopsis species.
PG  - 44
LID - 10.1186/s13039-016-0254-8 [doi]
LID - 44
AB  - BACKGROUND: Preparation of good chromosome spreads without cytoplasmic 
      contamination is the crucial step in cytogenetic mapping. To date, cytogenetic 
      research in the Orchidaceae family has been carried out solely on mitotic 
      metaphase chromosomes. Well-spread meiotic pachytene chromosomes can provide 
      higher resolution and fine detail for analysis of chromosomal structure and are 
      also beneficial for chromosomal FISH (fluorescence in situ hybridization) 
      mapping. However, an adequate method for the preparation of meiotic pachytene 
      chromosomes in orchid species has not yet been reported. RESULTS: Two Taiwanese 
      native Phalaenopsis species were selected to test the modified drop method for 
      preparation of meiotic pachytene chromosomes from pollinia. In this modified 
      method, pollinia were ground and treated with an enzyme mixture to completely 
      remove cell walls. Protoplasts were resuspended in ethanol/glacial acetic acid 
      and dropped onto a wet inclined slide of 30 degrees  from a height of 0.5 m. The sample 
      was then flowed down the inclined plane to spread the chromosomes. Hundreds of 
      pachytene chromosomes with little to no cytoplasmic contamination were well 
      spread on each slide. We also showed that the resolution of 45S rDNA-containing 
      chromosomes at the pachytene stage was up to 20 times higher than that at 
      metaphase. Slides prepared following this modified drop method were amenable to 
      FISH mapping of both 45S and 5S rDNA on pachytene chromosomes and, after FISH, 
      the chromosomal structure remained intact for further analysis. CONCLUSION: This 
      modified drop method is suitable for pachytene spreads from pollinia of 
      Phalaenopsis orchids. The large number and high-resolution pachytene spreads, 
      with little or no cytoplasmic contamination, prepared by the modified drop method 
      could be used for FISH mapping of DNA fragments to accelerate the integration of 
      cytogenetic and molecular research in Phalaenopsis orchids.
FAU - Kuo, Yi-Tzu
AU  - Kuo YT
AD  - Department of Life Sciences, National Cheng Kung University, No.1, University 
      Road, Tainan City, 701 Taiwan.
FAU - Hsu, Hui-Lan
AU  - Hsu HL
AD  - Department of Life Sciences, National Cheng Kung University, No.1, University 
      Road, Tainan City, 701 Taiwan.
FAU - Yeh, Chih-Hsin
AU  - Yeh CH
AD  - Taoyuan District Agricultural Research and Extension Station, Council of 
      Agriculture, Executive Yuan, 139, Sec. 2, Dongfu Rd., Houzhuang, Sinwu Dist., 
      Taoyuan City, 32745 Taiwan.
FAU - Chang, Song-Bin
AU  - Chang SB
AD  - Department of Life Sciences, National Cheng Kung University, No.1, University 
      Road, Tainan City, 701 Taiwan.
LA  - eng
PT  - Journal Article
DEP - 20160606
PL  - England
TA  - Mol Cytogenet
JT  - Molecular cytogenetics
JID - 101317942
PMC - PMC4893830
OTO - NOTNLM
OT  - Drop method
OT  - FISH mapping
OT  - Fluorescence in situ hybridization (FISH)
OT  - Orchid
OT  - Pachytene chromosome
OT  - Phalaenopsis
EDAT- 2016/06/09 06:00
MHDA- 2016/06/09 06:01
PMCR- 2016/06/06
CRDT- 2016/06/09 06:00
PHST- 2016/04/21 00:00 [received]
PHST- 2016/06/02 00:00 [accepted]
PHST- 2016/06/09 06:00 [entrez]
PHST- 2016/06/09 06:00 [pubmed]
PHST- 2016/06/09 06:01 [medline]
PHST- 2016/06/06 00:00 [pmc-release]
AID - 254 [pii]
AID - 10.1186/s13039-016-0254-8 [doi]
PST - epublish
SO  - Mol Cytogenet. 2016 Jun 6;9:44. doi: 10.1186/s13039-016-0254-8. eCollection 2016.