Materials and Reagents

1. Consumables
   1. Microfuge tubes (Corning, Axygen^®, catalog number: MCT-150-C )
      
   2. Pipette tips (Corning, Axygen^®, catalog number: T-1000-C , T-200-C , T-300 )
      
      
2. Plant material
   
   1. 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.
      
      
3. Chemicals
   1. Taq PCR mastermix (Tiangen, catalog number: KT201 )
      
   2. Liquid nitrogen
      
   3. Absolute ethanol (Sinopharm Chemical Reagent, catalog number: 10009228 ) (ice cold)
      
   4. 70% ethanol (ice cold)
      
   5. 7.5 M ammonium acetate (Sigma-Aldrich, catalog number: A1542-250G )
      
   6. Chloroform: isoamyl alcohol (24:1) (Sinopharm Chemical Reagent, catalog number: chloroform, 10006818 ; isoamyl alcohol, 10003218 )
      
   7. ddH₂O (sterile)
      
   8. 5x Loading buffer with GelRed (Shanghai Generay Biotech, catalog number: GR0205-500 )
      
   9. Cetyltrimethylammonium bromide/Hexadecyl trimethyl-ammonium bromide (CTAB) (Thermo Fisher Scientific, catalog number: ICN19400480 )
      
   10. Ethylenediaminetetraacetic acid (0.5M solution/pH 8.0) (EDTA) (Thermo Fisher Scientific, catalog number: BP2482-500 )
       
   11. Polyvinyl pyrrolidone, MW 40,000 (PVP 40)
       
   12. NaCl (Sigma-Aldrich, catalog number: S5886-500G )
       
   13. HCl (Sigma-Aldrich, catalog number: 258148 )
       
   14. Tris base (Thermo Fisher Scientific, catalog number: BP152-1 )
       
   15. Boric acid (Sigma-Aldrich, catalog number: B0394-100G )
       
   16. Agarose (Biowest, catalog number: 111860 )
       
   17. Hi-Di^TM formamide (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4311320 )
       
   18. GeneScan 500Liz (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4322682 )
       
   19. CTAB buffer (see Recipes)
       
   20. 1 M Tris (pH 8.0) (see Recipes)
       
   21. TE buffer (see Recipes)
       
   22. 5x TBE (Tris-Borate-EDTA) buffer (Stock) (see Recipes)
   23. 1% agarose gel (see Recipes)
       

Equipment

1. Mortar and pestle
   
2. Microcentrifuge (Eppendorf, model: 5427R )
   
3. Water bath (IKA, model: HB10 )
   
4. PCR thermal cycler (EASTWIN, model: ETC-811 )
   
5. Agarose gel electrophoresis system [include PowerPac^TM Basic Power Supply for electrophoresis (Bio-Rad Laboratories, catalog number: 1645050 )]
   
6. Genetic Analyzer (Invitrogen, model: ABI PRISM 3100 )
   

Software

1. GeneMarker version 2.4.0 (http://www.softgenetics.com/GeneMarker.html)
   
2. FSTAT version 2.9.3 (http://www2.unil.ch/popgen/softwares/fstat.htm)
   
3. GenAlEx version 6.2 (http://biology-assets.anu.edu.au/GenAlEx/Download.html)
   
4. STRUCTURE version 2.2 (http://pritchardlab.stanford.edu/software/structure2_2.html)
   
5. NewHybrids version 1.1 beta (http://ib.berkeley.edu/labs/slatkin/eriq/software/software.htm#NewHybs)
   

Procedure