Chin Bull Bot ›› 2019, Vol. 54 ›› Issue (3): 371-377.doi: 10.11983/CBB18134

• TECHNIQUES AND METHODS • Previous Articles     Next Articles

Improvement in Extraction Methods of Genomic DNA from Pitaya Stems

Nong Quandong1,2,3,Zhang Mingyong1,2,Zhang Mei2,Jian Shuguang2,Lu Hongfang2,Xia Kuaifei1,2,*,Wen Heming3,*   

  1. 1. Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South China Botanical Garden, CAS, Guangzhou 510650, China
    2. Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, CAS, Guangzhou 510650, China
    3. Wenshan Academy of Agricultural Sciences, Wenshan 663099, China
  • Received:2018-06-11 Accepted:2018-10-14 Online:2019-11-24 Published:2019-05-01
  • Contact: Xia Kuaifei,Wen Heming

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

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 1Improved 2Improved 3
OD260/OD280OD260/OD230OD260/OD280OD260/OD230OD260/OD280OD260/OD230
11.991.383.830.482.061.51
21.901.162.120.922.041.79
32.031.302.690.632.051.65

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)."

[1] 陈明贤 (2012). 福建省火龙果种质资源ISSR分析及耐盐生理研究. 硕士论文. 福州: 福建农林大学. pp. 11-26.
[2] 程文, 夏正俊, 冯献忠, 杨素欣 (2016). 一种快速、无损大豆种子DNA提取方法的建立和应用. 植物学报 51, 68-73.
doi: 10.11983/CBB15057
[3] 邓仁菊, 范建新, 蔡永强 (2011). 国内外火龙果研究进展及产业发展现状. 贵州农业科学 39(6), 188-192.
doi: 10.3969/j.issn.1001-3601.2011.06.052
[4] 董美超, 岳建强, 李进学, 高俊燕 (2013). 火龙果育种研究进展. 热带农业科学 33(5), 56-59.
doi: 10.3969/j.issn.1009-2196.2013.05.014
[5] 冯建成 (2006). 分子标记辅助选择技术在水稻育种上的应用. 中国农学通报 22(2), 43-47.
doi: 10.3969/j.issn.1000-6850.2006.03.011
[6] 傅建敏, 梁玉琴, 孙鹏, 姬燕晓, 谭晓风 (2015). 柿属植物叶绿体DNA提取方法优化. 中南林业科技大学学报 35(12), 25-28.
doi: 10.14067/j.cnki.1673-923x.2015.12.004
[7] 韩静丹, 秦萌, 王献 (2012). 铁线蕨总DNA提取方法比较分析. 河南农业大学学报 46, 182-187.
doi: 10.3969/j.issn.1000-2340.2012.02.015
[8] 李会, 任志莹, 王颖, 王建忠 (2013). 不同DNA提取试剂盒提取作物种子基因组DNA效果的比较. 湖北农业科学 52, 1956-1958.
doi: 10.3969/j.issn.0439-8114.2013.08.059
[9] 李建生 (2007). 玉米分子育种研究进展. 中国农业科技导报 9(2), 10-13.
doi: 10.3969/j.issn.1008-0864.2007.02.002
[10] 李金璐, 王硕, 于婧, 王玲, 周世良 (2013). 一种改良的植物DNA提取方法. 植物学报 48, 72-78.
doi: 10.3724/SP.J.1259.2013.00072
[11] 梁雪莲, 郑奕雄, 陈晓玲, 曾锦姬 (2007). 花生DNA提取方法比较. 生物技术 17, 41-44.
doi: 10.3969/j.issn.1004-311X.2007.01.014
[12] 罗焜, 马培, 姚辉, 宋经元, 陈科力, 刘义梅 (2012). 中药DNA条形码鉴定中的DNA提取方法研究. 世界科学技术——中医药现代化 14, 1433-1439.
doi: 10.11842/wst.2012.2.[sequence]
[13] 罗小艳, 郭璇华 (2007). 火龙果的研究现状及发展前景. 食品与发酵工业 33(9), 142-145.
[14] 马小军, 莫长明 (2017). 药用植物分子育种展望. 中国中药杂志 42, 2021-2031.
[15] 邱丽娟, 王昌陵, 周国安, 陈受宜, 常汝镇 (2007). 大豆分子育种研究进展. 中国农业科学 40, 2418-2436.
doi: 10.3321/j.issn:0578-1752.2007.11.005
[16] 申世辉, 马玉华, 蔡永强 (2015). 火龙果研究进展. 中国热带农业 (1), 48-52.
[17] 田新民, 李洪立, 何云, 洪青梅, 胡文斌, 李琼 (2015). 火龙果研究现状. 北方园艺 (18), 188-193.
[18] 吴丽圆 (2004). 4种思茅松总DNA提取方法的比较. 福建林学院学报 24, 237-240.
doi: 10.3969/j.issn.1001-389X.2004.03.011
[19] 吴琳 (2016). 火龙果种质资源评价及繁育研究. 硕士论文. 南宁: 广西大学. pp. 11-61.
[20] 吴敏娜, 武亚琦, 屈艳, 魏纪东, 钟根深 (2015). 四种小鼠肠道微生物DNA提取方法比较. 生态学杂志 34, 1183-1188.
[21] 余志雄, 欧高政, 陈清西, 袁亚芳 (2010). 火龙果总DNA提取方法比较研究. 中国农学通报 26, 300-303.
[22] 詹少华, 尹艺林 (2008). 大豆基因组DNA提取纯化方法研究. 安徽农业科学 36, 9871-9872, 9928.
doi: 10.3969/j.issn.0517-6611.2008.23.029
[23] 张冰雪, 范付华, 乔光, 宋莎, 文晓鹏, 刘涛 (2013). 贵州地方火龙果芽变种质DNA指纹图谱及遗传多样性的ISSR分析. 果树学报 30, 573-577.
[24] 张芬, 李达, 吕长平, 张力, 冯岳东, 许威, 张宏志 (2011). 兰花SRAP指纹图谱的构建. 湖南农业科学 (3), 129-132.
doi: 10.3969/j.issn.1006-060X.2011.03.039
[25] 张桂春, 刘玉静, 李延敏, 牟萍, 曲明娟, 李清, 周菊华 (2017). 火龙果果皮中可溶性膳食纤维的提取方法. 植物学报 52, 622-630.
doi: 10.11983/CBB16116
[26] 张玉晶, 李牡丹, 石旭, 关萍 (2011). 珙桐基因组DNA的提取及ISSR-PCR体系的优化. 山地农业生物学报 30(3), 211-214.
doi: 10.3969/j.issn.1008-0457.2011.03.005
[27] 郑育声, 李东栋, 王哲魁 (2007). 椰子不同组织基因组DNA的提取及质量分析. 海南大学学报自然科学版 25, 51-56.
doi: 10.3969/j.issn.1004-1729.2007.01.013
[28] Kaczyńska A, ?o? M, W?grzyn G (2013). An improved method for efficient isolation and purification of genomic DNA from filamentous cyanobacteria belonging to generaAnabaena, Nodularia and Nostoc. Oceanol Hydrobiol Stud 42, 8-13.
doi: 10.2478/s13545-013-0058-y
[29] Murray MG, Thompson WF (1980). Rapid isolation of high molecular weight plant DNA.Nucleic Acids Res 8, 4321-4325.
doi: 10.1093/nar/8.19.4321
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