Crops ›› 2020, Vol. 36 ›› Issue (5): 103-109.doi: 10.16035/j.issn.1001-7283.2020.05.016

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Extraction of High-Quality Chromosome DNA in Yunnan Wild Rice Oryza officinalis

Li Exian(), Yin Fuyou, Zhang Dunyu, Chen Yue, Yu Tengqiong, Lei Yongtao, Xiao Suqin, Cheng Zaiquan, Ke Xue()   

  1. Institute of Biotechnology and Germplasm Resources, Yunnan Academy of Agricultural Sciences/Key Laboratory of Southwestern Crop Gene Resources and Germplasm Innovation, Ministry of Agriculture and Rural Affairs/Rice Materials Engineering Technology Research Center of Yunnan Province, Kunming 650205, Yunnan, China
  • Received:2020-03-05 Revised:2020-05-16 Online:2020-10-15 Published:2020-10-12
  • Contact: Ke Xue E-mail:xiaogaogao4850@126.com;kexue7788@163.com

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Abstract:

Oryza officinalis is one of the three wild species found in China. Although riched in excellent genes, however, due to the large difference between the CC genome of O.officinalis and the AA genome of cultivated rice, distant hybridization is not compatible, and excellent genes are difficult to use. Genomic library construction with large fragment DNA is an important method to study and to use its genes. This kind of genomic library, such as BIBAC library requires a high-quality genomic DNA. But it is not easy to extract chromosome DNA with the size of Mb from O.officinalis by convention methods, so the extraction system need to be optimized. In this paper, the experimental steps such as sampling and grinding, nucleus separation and embedding, histone removal and DNA release, and enzymatic hydrolysis and recovery of DNA were optimized. High quality chromosomal DNA of 1.9Mb was successfully extracted from O.officinalis, and the enzymatic digestion of DNA was also explored. The isolated DNA fragment can used to construct a large-fragment genomic library.

Key words: Oryza officinalis, Chromosome DNA, DNA extraction

Fig.1

Nucleus separation from leaves of O.officinalis A. O.officinalis; B. Leaves homogenate; C. Nuclear centrifugation; D. Nuclear centrifugation and cleaning; E. Nucleus"

Fig.2

Nucleus embedding of O. officinalis by low-melting point agarose A. Nuclear resuspension; B. Agarose solidified on the ice; C. Embedding block is solidified in the medical syringe; D. Cut into a circular piece; E. Embedded block washing; F. Embedded block after washing"

Fig.3

Effects of different concentrations of BamH I on genomic DNA digestion of O.officinalis under eight minutes treatment M. Yeast chromosome PFG marker; Non. DNA without enzyme treatment. The same below"

Fig.4

Effects of digestion time on genomic DNA of O. officinalis under 0.6U BamH I treatment"

Fig.5

Chromosome DNA of O. officinalis without or with BamHⅠdigestion A. Optimum conditions for BamHⅠ, B and C. Extraction of target DNA region after enzymatic digestion"

Fig.6

BIBAC vector and large-fragment DNA recovery of O.officinalis A. BIBAC and digested by BamHⅠ, B. Target DNA recovery for BamHⅠ;M. Yeast chromosome PFG marker (A) and D2000 marker (B) "

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