Improvement in Extraction Methods of Genomic DNA from Pitaya Stems

doi:10.11983/CBB18134

Abstract

Abstract:

Pitaya (Hylocereus undulatus) is a tropical fruit developed in recent years. Because its stem is full of polysaccharides, polyphenols and other secondary metabolites, extracting high-quality DNA from the stem is difficult, especially from stems more than 1 year old. At present, there is no good method for DNA extraction. We examined three methods to improve DNA extraction based on the CTAB+Tris-HCl washing method for better and easier DNA extraction. The third method obtained the best-quality DNA without sticky substances from pitaya stem more than 1 year old and was not restricted by sampling data and sampling site. The extracted DNA can be used in enzyme digestion and inter-simple sequence repeat amplification experiments. Our study provides an ideal method for extracting DNA from pitaya stem, which is worth popularizing.

Key words: pitaya, stem, DNA extraction, improved CTAB+Tris-HCl washing method

Quandong Nong,Mingyong Zhang,Mei Zhang,Shuguang Jian,Hongfang Lu,Kuaifei Xia,Heming Wen. Improvement in Extraction Methods of Genomic DNA from Pitaya Stems[J]. Chinese Bulletin of Botany, 2019, 54(3): 371-377.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB18134

https://www.chinbullbotany.com/EN/Y2019/V54/I3/371

Figures/Tables 3

Figure 1 The washed samples of pitaya mixed with CTAB lysate 1-3: Represented the precipitation can not be mixed with CTAB lysate; 4-9: Represented the precipitation can be mixed with CTAB lysate

Table 1 The OD260/OD280 and OD260/OD230 of DNA extracted from pitaya by 3 different improved CTAB+Tris-HCl washing methods

No.	Improved 1		Improved 2		Improved 3
No.	OD₂₆₀/OD₂₈₀	OD₂₆₀/OD₂₃₀	OD₂₆₀/OD₂₈₀	OD₂₆₀/OD₂₃₀	OD₂₆₀/OD₂₈₀	OD₂₆₀/OD₂₃₀
1	1.99	1.38	3.83	0.48	2.06	1.51
2	1.90	1.16	2.12	0.92	2.04	1.79
3	2.03	1.30	2.69	0.63	2.05	1.65

Figure 2 Electrophoretograms of pitaya DNA, enzyme digestion and ISSR amplification (A) Electrophoretogram of DNA extracted by 3 different improved methods (M: DNA marker; Lane 1-3: DNA extracted by improved 1 method; Lane 4-6: DNA extracted by improved 2 method; Lane 7-9: DNA extracted by improved 3 method). (B) Electrophoretogram of pitaya DNA extracted by improved 3 method and enzyme digestion (M: DNA marker; Lane 1-7: DNA; Lane 8-14: DNA digested by HindIII; Lane 15-21: DNA digested by HindIII and EcoRI; The DNA template of 8-14 and 15-21 is corresponding to the DNA in 1-7 lanes, respectively). (C) Electrophoretogram of ISSR amplification (M: DNA marker; Lane 1-7: Products of 7 different DNA templates ISSR amplification).

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