PMID- 22442728
OWN - NLM
STAT- MEDLINE
DCOM- 20120723
LR  - 20211021
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 7
IP  - 3
DP  - 2012
TI  - Preparations of meiotic pachytene chromosomes and extended DNA fibers from cotton 
      suitable for fluorescence in situ hybridization.
PG  - e33847
LID - 10.1371/journal.pone.0033847 [doi]
LID - e33847
AB  - Fluorescence in situ hybridization (FISH) has become one of the most important 
      techniques applied in plant molecular cytogenetics. However, the application of 
      this technique in cotton has lagged behind because of difficulties in chromosome 
      preparation. The focus of this article was FISH performed not only on cotton 
      pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen 
      mother cells (PMCs) instead of buds or anthers were directly digested in enzyme 
      to completely breakdown the cell wall. Before the routine acetic acid treatment, 
      PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and 
      clear the background. The method of ice-cold Carnoy's solution spreading 
      chromosome was adopted instead of nitrogen removed method to avoid chromosomes 
      losing and fully stretch chromosome. With the above-improved steps, the 
      high-quality well-differentiated pachytene chromosomes with clear background were 
      obtained. FISH results demonstrated that a mature protocol of cotton pachytene 
      chromosomes preparation was presented. Intact and no debris cotton nuclei were 
      obtained by chopping from etiolation cotyledons instead of the conventional 
      liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis 
      buffer on slide, the parallel and clear background DNA fibers were acquired along 
      the slide. This method overcomes the twist, accumulation and fracture of DNA 
      fibers compared with other methods. The entire process of DNA fibers preparation 
      requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding 
      method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by 
      nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent 
      oxidation. The probability of success in isolating nuclei for DNA fiber 
      preparation is almost 100% tested with this method in cotton. So a rapid, safe, 
      and efficient method for the preparation of cotton extended DNA fibers suitable 
      for FISH was established.
FAU - Peng, Renhai
AU  - Peng R
AD  - State Key Laboratory of Cotton Biology, China and Cotton Research Institute of 
      Chinese Academy of Agricultural Science, Anyang, Henan, China.
FAU - Zhang, Tao
AU  - Zhang T
FAU - Liu, Fang
AU  - Liu F
FAU - Ling, Jian
AU  - Ling J
FAU - Wang, Chunying
AU  - Wang C
FAU - Li, Shaohui
AU  - Li S
FAU - Zhang, Xiangdi
AU  - Zhang X
FAU - Wang, Yuhong
AU  - Wang Y
FAU - Wang, Kunbo
AU  - Wang K
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20120319
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (DNA, Plant)
SB  - IM
MH  - Chromosomes, Plant/*chemistry/genetics
MH  - DNA, Plant/chemistry/genetics/*isolation & purification
MH  - Gossypium/*chemistry/genetics
MH  - In Situ Hybridization, Fluorescence/methods
MH  - *Pachytene Stage
PMC - PMC3307766
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2012/03/24 06:00
MHDA- 2012/07/24 06:00
PMCR- 2012/03/19
CRDT- 2012/03/24 06:00
PHST- 2011/10/20 00:00 [received]
PHST- 2012/02/18 00:00 [accepted]
PHST- 2012/03/24 06:00 [entrez]
PHST- 2012/03/24 06:00 [pubmed]
PHST- 2012/07/24 06:00 [medline]
PHST- 2012/03/19 00:00 [pmc-release]
AID - PONE-D-11-20663 [pii]
AID - 10.1371/journal.pone.0033847 [doi]
PST - ppublish
SO  - PLoS One. 2012;7(3):e33847. doi: 10.1371/journal.pone.0033847. Epub 2012 Mar 19.