Identification of Natural Hybrids by SSR Markers in Mussaenda
用SSR标记物识别玉叶金花属中的自然杂交品种
发布: 2016年07月05日第6卷第13期 DOI: 10.21769/BioProtoc.1853 浏览次数: 9519
评审: Samik BhattacharyaGazala AmeenAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Oct 2015
Abstract
Detection of natural hybrids is of great significance for plant taxonomy, reproductive biology, and population genetic studies. Compared with methods depending on morphological characters, molecular markers provide reliable and much more accurate results. This protocol describes approaches employing microsatellite (SSR) markers to identify inter-specific hybrids in Mussaenda (Rubiaceae).
Keywords: Gene flow (基因流)
Natural hybridization (自然杂交)
SSR (SSR)
Materials and Reagents
- Consumables
- Plant material
- Fresh leaves of the putative hybrid individuals, as well as the parental species, were collected and dried in silica gel. It usually takes 3-5 days for complete drying of the leaves.
- Fresh leaves of the putative hybrid individuals, as well as the parental species, were collected and dried in silica gel. It usually takes 3-5 days for complete drying of the leaves.
- Chemicals
- Taq PCR mastermix (Tiangen, catalog number: KT201 )
- Liquid nitrogen
- Absolute ethanol (Sinopharm Chemical Reagent, catalog number: 10009228 ) (ice cold)
- 70% ethanol (ice cold)
- 7.5 M ammonium acetate (Sigma-Aldrich, catalog number: A1542-250G )
- Chloroform: isoamyl alcohol (24:1) (Sinopharm Chemical Reagent, catalog number: chloroform, 10006818 ; isoamyl alcohol, 10003218 )
- ddH2O (sterile)
- 5x Loading buffer with GelRed (Shanghai Generay Biotech, catalog number: GR0205-500 )
- Cetyltrimethylammonium bromide/Hexadecyl trimethyl-ammonium bromide (CTAB) (Thermo Fisher Scientific, catalog number: ICN19400480 )
- Ethylenediaminetetraacetic acid (0.5M solution/pH 8.0) (EDTA) (Thermo Fisher Scientific, catalog number: BP2482-500 )
- Polyvinyl pyrrolidone, MW 40,000 (PVP 40)
- NaCl (Sigma-Aldrich, catalog number: S5886-500G )
- HCl (Sigma-Aldrich, catalog number: 258148 )
- Tris base (Thermo Fisher Scientific, catalog number: BP152-1 )
- Boric acid (Sigma-Aldrich, catalog number: B0394-100G )
- Agarose (Biowest, catalog number: 111860 )
- Hi-DiTM formamide (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4311320 )
- GeneScan 500Liz (Thermo Fisher Scientific, Applied BiosystemsTM, catalog number: 4322682 )
- CTAB buffer (see Recipes)
- 1 M Tris (pH 8.0) (see Recipes)
- TE buffer (see Recipes)
- 5x TBE (Tris-Borate-EDTA) buffer (Stock) (see Recipes)
- 1% agarose gel (see Recipes)
- Taq PCR mastermix (Tiangen, catalog number: KT201 )
Equipment
- Mortar and pestle
- Microcentrifuge (Eppendorf, model: 5427R )
- Water bath (IKA, model: HB10 )
- PCR thermal cycler (EASTWIN, model: ETC-811 )
- Agarose gel electrophoresis system [include PowerPacTM Basic Power Supply for electrophoresis (Bio-Rad Laboratories, catalog number: 1645050 )]
- Genetic Analyzer (Invitrogen, model: ABI PRISM 3100 )
Software
- GeneMarker version 2.4.0 (http://www.softgenetics.com/GeneMarker.html)
- FSTAT version 2.9.3 (http://www2.unil.ch/popgen/softwares/fstat.htm)
- GenAlEx version 6.2 (http://biology-assets.anu.edu.au/GenAlEx/Download.html)
- STRUCTURE version 2.2 (http://pritchardlab.stanford.edu/software/structure2_2.html)
- NewHybrids version 1.1 beta (http://ib.berkeley.edu/labs/slatkin/eriq/software/software.htm#NewHybs)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Luo, Z., Duan, T., Yuan, S., Chen, S., Bai, X. and Zhang, D. (2016). Identification of Natural Hybrids by SSR Markers in Mussaenda. Bio-protocol 6(13): e1853. DOI: 10.21769/BioProtoc.1853.
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分类
植物科学 > 植物分子生物学 > DNA
分子生物学 > DNA > 基因分型
分子生物学 > DNA > PCR
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