Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2017, Vol. 43 ›› Issue (07): 947-953.doi: 10.3724/SP.J.1006.2017.00947

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

dsDNA Fluorescent Quantification and Genotyping in Common Wheat by FLUOstar System

XIAO Yong-Gui1,Susanne DREISIGACKER2,Claudia NUÑEZ-RíOS2,HU Wei-Guo3,XIA Xian-Chun1,HE Zhong-Hu1,4,*   

  1. 1 Institute of Crop Science, Chinese Academy of Agricultural Sciences (CAAS) /National Wheat Improvement Center, Beijing 100081, China; 2 International Maize and Wheat Improvement Center (CIMMYT), 06600 México, DF México; 3 Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China; 4 CIMMYT China Office, c/o CAAS, Beijing 100081, China
  • Received:2016-09-08 Revised:2017-03-02 Online:2017-07-12 Published:2017-03-30
  • Contact: He Zhonghu, E-mail: zhhecaas@163.com E-mail:xiaoyonggui@caas.cn
  • Supported by:

    This study was supported by the Key Project of the National Research and Development Program (2016YFD0101804-6), the National Natural Science Foundation of China (31671691), the National Key Technology R&D Program of China (2014BAD01B05), and the International Science & Technology Cooperation Program of Ministry of Science and Technology (2013DFG30530).

Abstract:

Quantitative analysis on double-stranded DNA (dsDNA) lays a foundation in molecular biology research in plants, particularly important for genotyping in molecular breeding. The objective of this study was to establish standard curve for fluorescence quantitative analysis by lambda DNA, to compare the difference between dsDNA value in fluorescence system and ultraviolet spectrophotometry, and to identify the allelic variations of rust resistance genes in wheat. The fluorescent dye could be efficiently performed in the quantitative analysis with micro dsDNA concentration (< 1.1 ng ?L?1). However, the fluorescent dye could lead to uncertainty of original concentrations of wheat leaf and grain genome DNA, due to more fold serial dilutions for higher DNA concentration. A downward tendency was happened in fluorescent intensity when fluorescent reaction volume was tapered, which influenced the accuracy of DNA concentration. The volume of reaction system mixed nucleic acid and fluorescent dye should be more than 200 ?L for accurate determination of micro dsDNA. For genotyping on PCR products, the volume of fluorescent reaction system should be more than 40 ?L. FLUOstar could be used for identifying the dominant marker, for instance csSr32#1 (Sr32) and IB-267 (Sr50), its accuracy was 100% in correspondence with that from agarose gel electrophoresis. Co-dominant marker with the characteristic of peculiarity and major difference in amplified fragment length ( ≥100 bp), such as We173 (Yr26), could also be identified by fluorescent analysis. Compared with agarose gel electrophoresis method, fluorescent method have a simple, convenient, and rapid oparetion with high repeatability, and can be used for segregating generations in marker-assisted breeding.

Key words: Common wheat, dsDNA, Fluorescent quantitative analysis, Dominant marker, Molecular breeding

[1] Bhat S, Curach N, Mostyn T, Bains G S, Griffiths K R, Emslie K R. Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units. Anal Chem, 2010, 82: 7185–7192 [2] 聂晓静, 赵筱萍, 王毅. 基于荧光图像的抗肿瘤细胞迁移药物筛选方法. 药学学报, 2011, 46: 793–797 Nie X J, Zhao X P, Wang Y. Development of fluorescence imaging based assay for screening compounds with anti-migration activity. Acta Pharm Sin, 2011, 46: 793?797 (in Chinese with English abstract) [3] 王宇, 刘景晶. 核酸的定量技术研究进展. 药学进展, 2006, 30: 385?390 Wang Y, Liu J J. Recent advances in research on quantification of nucleic acids. Prog Pharmac Sci, 2006, 30: 385?390 (in Chinese with English abstract) [4] Lyu Z Z, Liu J C, Zhou Y, Guan Z, Yang S M, Li C, Chen A L. Highly sensitive fluorescent detection of small molecules, ions, and proteins using a universal label-free aptasensor. Chem Commun (Camb), 2013, 49: 5465–5467 [5] Rengarajan K, Cristol S M, Mehta M, Nickerson J M. Quantifying DNA concentrations using fluorometry: a comparison of fluorophores. Mol Vis, 2002, 8: 416–421 [6] Lucena-Aguilar G, Sánchez-López A M, Barberán-Aceituno C, Carrillo-ávila J A, López-Guerrero J A, Aguilar-Quesada R. DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreserv Biobank, 2016, 14: 264–270 [7] Haque K A, Pfeiffer R M, Beerman M B, Struewing J P, Chanock S J, Bergen A W. Performance of high-throughput DNA quantification methods. BMC Biotechnol, 2003, 3: 20 [8] Georgiou C D, Papapostolou I. Assay for the quantification of intact/fragmented genomic DNA. Anal Biochem, 2006, 358: 247–256 [9] Marie D, Vaulot D, Partensky F. Application of the novel nucleic acid dyes YOYO-1, YO-PRO-1 and PicoGreen for flow cytometric analysis of marine prokaryotes. Appl Environ Microb, 1996, 62: 1649?1655 [10] 桂海娈, 金庆日, 张亚军, 王晓杜, 杨永春, 邵春艳, 程昌勇, 卫芳芳, 杨扬, 杨梦华, 宋厚辉. 基于荧光染料PicoGreen 和核酸适配体的伏马毒素B1检测方法. 生物工程学报, 2015, 31: 1393–1400 Gui H L, Jin Q R, Zhang Y Z, Wang X D, Yang Y C, Shao C Y, Cheng C Y, Wei F F, Yang Y, Yang M H, Song H H. Development of an aptamer/fluorescence dye PicoGreen-based method for detection of fumonisin B1. Chin J Biotechnol, 2015, 31: 1393–1400 (in Chinese with English abstract) [11] 曾国平, 向东山, 何治柯. 基于Hoechst33258荧光染料检测单链DNA的方法研究. 化学学报, 2011, 69: 1450?1456 Zeng G P, Xiang D S, He Z K. Fluorimetric method for the determination of sequence-specific DNA with the fluorescent dye Hoechst 33258. Acta Chim Sin, 2011, 69: 1450–1456 (in Chinese with English abstract) [12] Mago R, Verlin D, Zhang P, Bansal U, Bariana H, Jin Y, Ellis J, Hoxha S, Dundas I. Development of wheat–Aegilops speltoides recombinants and simple PCR-based markers for Sr32 and a new stem rust resistance gene on the 2S#1 chromosome. Theor Appl Genet, 2013, 126: 2943–2955 [13] Mago R, Bariana H S, Dundas I S, Spielmeyer W, Lawrence G L, Prior A J, Ellis J G. Development of PCR markers for the selection of wheat stem rust resistance genes Sr24 and Sr26 in diverse wheat germplasm. Theor Appl Genet, 2005, 111: 496–504 [14] Zhang X, Han D, Zeng Q, Duan Y, Yuan F, Shi J, Wang Q, Wu J, Huang L, Kang Z. Fine mapping of wheat stripe rust resistance gene Yr26 based on collinearity of wheat with Brachypodium distachyon and rice. PLoS One, 2013, 8: e57885 [15] Huang H, Shi S, Gao X, Gao R, Zhu Y, Wu X, Zang R, Yao T. A universal label-free fluorescent aptasensor based on Ru complex and quantum dots for adenosine, dopamine and 17 β-estradiol detection. Biosens Bioelectron, 2016, 79: 198?204 [16] Leung K, He B, Yang C, Leung C H, Wang H D, Ma D. Development of an aptamer-based sensing platform for metal ions, proteins, and small molecules through terminal deoxynucleotidyl transferase induced G-Quadruplex formation. ACS Appl Mater Interfaces, 2015, 7: 24046–24052 [17] Lisanti S, Omar W A W, Tomaszewski B, De Prins S, Jacobs G, Koppen G, Mathers J C, Langie S A S. Comparison of methods for quantification of global DNA methylation in human cells and tissues. PLoS One, 2013, 8: e79044 [18] Fraga M F, Uriol E, Borja D L, Berdasco M, Esteller M, Ca?al M J, Rodriguez R. High-performance capillary electrophoretic method for the quantification of 5-methyl 2’-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues. Electrophoresis, 2002, 23: 1677–1681 [19] Karimi M, Johansson S, Stach D, Corcoran M, Grander D, Schalling M, Bakalkin G, Lyko F, Larsson C, Ekstr?m T J. LUMA (LUminometric Methylation Assay): a high throughput method to the analysis of genomic DNA methylation. Exp Cell Res, 2006, 312: 1989–1995 [20] Kuo K C, McCune R A, Gehrke C W, Midgett R, Ehrlich M. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA. Nucl Acids Res, 1980, 8: 4763–4776 [21] Burns M J, Nixon G J, Foy C A, Harris N. Standardization of data from real-time quantitative PCR methods-evaluation of outliers and comparison of calibration curves. BMC Biotechnol, 2005, 5: 31 [22] Wilfinger W W, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques, 1997, 22: 474–481 [23] Sedlackova T, Repiska G, Celec P, Szemes T, Minank G. Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods. Biol Proced Online, 2013, 15: 5 [24] Hollegaard M V, Grove J, Grauholm J, Kreiner-M?ller E, B?nnelykke K, N?rgaard M, Benfield T L, N?rgaard-Pedersen B, Mortensen P B, Mors O, S?rensen H T, Harboe Z B, B?rglum A D, Demontis D, ?rntoft T F, Bisgaard H, Hougaard D M. Robustness of genome-wide scanning using archived dried blood spot samples as a DNA source. BMC Genet, 2011, 12: 58

[1] JIN Yi-Rong, LIU Jin-Dong, LIU Cai-Yun, JIA De-Xin, LIU Peng, WANG Ya-Mei. Genome-wide association study of nitrogen use efficiency related traits in common wheat (Triticum aestivum L.) [J]. Acta Agronomica Sinica, 2021, 47(3): 394-404.
[2] ZHANG Ping-Ping,YAO Jin-Bao,WANG Hua-Dun,SONG Gui-Cheng,JIANG Peng,ZHANG Peng,MA Hong-Xiang. Soft wheat quality traits in Jiangsu province and their relationship with cookie making quality [J]. Acta Agronomica Sinica, 2020, 46(4): 491-502.
[3] Di JIN,Dong-Zhi WANG,Huan-Xue WANG,Run-Zhi LI,Shu-Lin CHEN,Wen-Long YANG,Ai-Min ZHANG,Dong-Cheng LIU,Ke-Hui ZHAN. Fine mapping and candidate gene analysis of awn inhibiting gene B2 in common wheat [J]. Acta Agronomica Sinica, 2019, 45(6): 807-817.
[4] Fang-Ping YANG,Jin-Dong LIU,Ying GUO,Ao-Lin JIA,Wei-E WEN,Kai-Xiang CHAO,Ling WU,Wei-Yun YUE,Ya-Chao DONG,Xian-Chun XIA. QTL mapping of adult-plant resistance to stripe rust in wheat variety holdfast [J]. Acta Agronomica Sinica, 2019, 45(12): 1832-1840.
[5] Yong-Jie MIAO, Jun YAN, De-Hui ZHAO, Yu-Bing TIAN, Jun-Liang YAN, Xian-Chun XIA, Yong ZHANG, Zhong-Hu HE. Relationship between Grain Filling Parameters and Grain Weight in Leading Wheat Cultivars in the Yellow and Huai Rivers Valley [J]. Acta Agronomica Sinica, 2018, 44(02): 260-267.
[6] DONG Xue,LIU Meng,ZHAO Xian-Lin,FENG Yu-Mei,YANG Yan. Isolation and Characterization of LMW-GS Glu-A3 in Common Wheat Related Species [J]. Acta Agron Sin, 2017, 43(06): 829-838.
[7] LIU Kai,DENG Zhi-Ying,ZHANG Ying,WANG Fang-Fang,LIU Tong-Tong,LI Qing-Fang,SHAO Wen,ZHAO Bin,TIAN Ji-Chun*,CHEN Jian-Sheng*. Linkage Analysis and Genome-Wide Association Study of QTLs Controlling Stem-Breaking-Strength-Related Traits in Wheat [J]. Acta Agron Sin, 2017, 43(04): 483-495.
[8] GONG Xi,JIANG Yun-Feng,XU Bin-Jie,QIAO Yuan-Yuan,HUA Shi-Yu,WU Wang,MA Jian,ZHOU Xiao-Hong,QI Peng-Fei,LAN Xiu-Jin. Mapping QTLs for Awn Length in Recombinant Inbred Line Population Derived from the Cross between Common Wheat and Tibetan Semi-wild Wheat [J]. Acta Agron Sin, 2017, 43(04): 496-500.
[9] XU Wen,SHEN Hao,GUO Jun,YU Xiao-Cong,LI Xiang,YANG Yan-Hui,MA Xiao,ZHAO Shi-Jie,SONG Jian-Min. Drought Resistance of Wheat NILs with Different Cuticular Wax Contents in Flag Leaf [J]. Acta Agron Sin, 2016, 42(11): 1700-1707.
[10] WANG Xin,MA Ying-Xue,YANG Yang,WANG Dan-Feng,YIN Hui-Juan,WANG Hong-Gang. Identification of Dwarfing Wheat Germplasm SN224 and Analysis of QTLs for Its Agronomic Characters [J]. Acta Agron Sin, 2016, 42(08): 1134-1142.
[11] LIU Kai,DENG Zhi-Ying,LI Qing-Fang,ZHANG Ying,SUN Cai-Ling,TIAN Ji-Chun*,CHEN Jian-Sheng*. Mapping QTLs For Wheat Panicle Traits with High Density SNP Genetic Map [J]. Acta Agron Sin, 2016, 42(06): 820-831.
[12] ZHAO De-Hui,YAN Jun,HUANG Yu-Lian,XIA Xian-Chun,ZHANG Yan,TIAN Yu-Bing,HE Zhong-Hu,ZHANG Yong. Effect of 1BL/1RS Translocation on Gluten Protein Fraction Quantities and Dough Rheological Properties [J]. Acta Agron Sin, 2015, 41(11): 1648-1656.
[13] XIAO Yong-Gui, LI Si-Min, LI Fa-Ji, ZHANG Hong-Yan, CHEN Xin-Min, WANG De-Sen, XIA Xian-Chun, HE Zhong-Hu. Genetic Analysis of Yield and Physiological Traits in Elite Parent Jing 411 and Its Derivatives under Two Fertilization Environments [J]. Acta Agron Sin, 2015, 41(09): 1333-1342.
[14] ANG Li,HUANG Yu-Lian,CHANG Ping,YAN Jun,ZHANG Ye-Lun,XIA Xian-Chun,TIAN Yu-Bing,HE Zhong-Hu,ZHANG Yong. QTL Mapping for Arabinoxylans Content and Its Relationship with Processing Quality in Common Wheat [J]. Acta Agron Sin, 2014, 40(09): 1695-1701.
[15] JIN Xiu-Feng,WANG Xian-Guo,REN Wan-Jie,ZHANG Xiao-Ke,XIE Hui-Min,FAN Feng-Gui. Relationship between a Water Stress Responsive Protein and Drought Resistance and Molecular Mapping of the Target Gene in Common Wheat [J]. Acta Agron Sin, 2014, 40(02): 198-204.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!