**2.2 Equipments and solution preparation**

Mortars, pestles, glassware and plasticware was autoclaved prior to use. The CTAB extraction buffer was composed of 2.0% CTAB (High Purity grade, Amresco), 100 mM Tris-HCl (pH 8.0)) (Ultra pure grade, Amresco), 2 M NaCl (Biotechnology grade, Amresco), 25 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) (High purity Grade Amresco) in 0.1% ultra pure water. The components in the extraction buffer were mixed and autoclaved. The 5% PVPP (Sigma P-6755) was added when the material was grounded and the 5% beta-mercaptoethanol (Biotechnology grade, Amresco) was added before DNA extraction. The final solution was warmed in a water bath to 65°C for use in DNA extraction. TE buffer was prepared with 10 mM Tris-HCl (pH 8,0) and 1.0 mM EDTA (pH 8.0). A phenol (pH 5.0)/chloroform /isoamyl alcohol mixture (25/24/1) (Biotechnology grade, Amresco) and a chloroform /isoamyl alcohol mixture (24/1) were prepared before use, and all other solutions including 3 M sodium acetate (NaAc) (pH 5.2) (Biotechnology grade, Amresco), 1 M NaCl, pre-cooled 75% ethanol were prepared with ultra pure water and autoclaved. DNase-Free RNase (Ultra pure grade) was purchased from Amresco Corporation.

#### **2.3 Grinding**

The frozen fresh samples were transferred into a mortar with liquid nitrogen and the ceramic pestle was pre-chilled for grinding. By providing liquid nitrogen as a cooling jacket, the samples were sprinkled with PVPP and ground vigorously to fine powder using the ceramic pestle. This powder was used for the following extraction protocols of the CTAB methods. And the commercial DNA isolation kit was ground in liquid nitrogen free of adding to PVPP.

### **2.4 DNA extraction**

For our modified CTAB method, the steps of this protocol was carried out as follows:


Tender *R. soongorica* leaves were collected from Ejina in Mogo, China and snap-frozen in liquid nitrogen. The frozen leaves were transported in liquid nitrogen and stored at -80ºC

Mortars, pestles, glassware and plasticware was autoclaved prior to use. The CTAB extraction buffer was composed of 2.0% CTAB (High Purity grade, Amresco), 100 mM Tris-HCl (pH 8.0)) (Ultra pure grade, Amresco), 2 M NaCl (Biotechnology grade, Amresco), 25 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) (High purity Grade Amresco) in 0.1% ultra pure water. The components in the extraction buffer were mixed and autoclaved. The 5% PVPP (Sigma P-6755) was added when the material was grounded and the 5% beta-mercaptoethanol (Biotechnology grade, Amresco) was added before DNA extraction. The final solution was warmed in a water bath to 65°C for use in DNA extraction. TE buffer was prepared with 10 mM Tris-HCl (pH 8,0) and 1.0 mM EDTA (pH 8.0). A phenol (pH 5.0)/chloroform /isoamyl alcohol mixture (25/24/1) (Biotechnology grade, Amresco) and a chloroform /isoamyl alcohol mixture (24/1) were prepared before use, and all other solutions including 3 M sodium acetate (NaAc) (pH 5.2) (Biotechnology grade, Amresco), 1 M NaCl, pre-cooled 75% ethanol were prepared with ultra pure water and autoclaved. DNase-Free RNase (Ultra pure grade) was purchased

The frozen fresh samples were transferred into a mortar with liquid nitrogen and the ceramic pestle was pre-chilled for grinding. By providing liquid nitrogen as a cooling jacket, the samples were sprinkled with PVPP and ground vigorously to fine powder using the ceramic pestle. This powder was used for the following extraction protocols of the CTAB methods. And the commercial DNA isolation kit was ground in liquid nitrogen free of

For our modified CTAB method, the steps of this protocol was carried out as follows:

mixed gently by inversion and incubated on ice for 30 min.

1. The ground powder sample (100 mg) was transferred to 2 ml micro-centrifuge tubes filled with 700 µl of pre-warmed CTAB extraction buffer containing 35 µl βmercaptoethanol, following by incubation at 65°C for 30 min in a warm water bath. The mixture was regularly mixed three to four times by gently inversion during the

2. 200 μl 3 mol/L sodium acetate (NaAc) (pH 5.2) was added to the incubated mixture and

3. An equal volume of chloroform/isoamyl alcohol (24/1) was added to the homogenate and mixed thoroughly for 2 min, following by centrifugation at 12,000×g for 10 min at

**2. Materials and methods** 

upon reaching the laboratory.

from Amresco Corporation.

**2.3 Grinding** 

adding to PVPP.

**2.4 DNA extraction** 

incubation..

**2.2 Equipments and solution preparation** 

**2.1 Plant materials** 

room temperature. The upper aqueous phase was carefully collected from each sample without disturbing the interface. This step was repeated twice.


Initial tests for DNA isolation from the leaves of *R. soongorica* were carried out with the modified CTAB-A method and the Plant Genomic DNA Kit (TIANGEN Biotech Co., Ltd., Beijing). The modified CTAB-A method was modified based on the classical Doyle and Doyle (1987) method. The steps of the modified CTAB-A are similar to those of the CTAB-B method before step 4 (the precipitate of crude nucleic pellet). The main difference is that the crude nucleic pellet in CTAB-A method was solved in TE and extracted by chloroform/isoamyl alcohol (24/1) again instead of being treated with DNase-free RNase. Briefly, the crude nuclei pellet was dissolved in 500 µl of TE buffer, followed by the steps 3 and 4 of the CTAB-B method repeatedly. The protocols for the commercial DNA isolation kit was performed according to the manufacturer' procedures on their website:http://www.tiangen.com/newEbiz1/EbizPortalFG/portal/html/ProductInfoExhi bit.html?ProductInfoExhibit\_ProductID=c373e923ec4bc4d68f7efc2e13bcb309&ProductInfoE xhibit\_isRefreshParent=false. The protocol of the modified TianGen Plant Genomic DNA Kit was based on those of the TianGen Plant Genomic DNA Kit with some slight modifications. The modifications were listed as follows: (1) The plant materials were ground free of liquid nitrogen, but were added to the cooled sterile mortar and ground with Gp1 buffer poured into the mortar. (2) The ground tissue was transferred to 2 ml micro-centrifuge tubes prepared a warm (65°C) Gp1extraction buffer, and then the 5% beta-mercaptoethanol and 10 μl DNase-free RNase were added to the mixture immediately and mixed gently by inversion. The other steps are carried out by the instructions of the kit. For each method, three independent experiments were done, and three samples were prepared in each independent experiment.

#### **2.5 Testing the quality of the genomic DNA**

Three microliters of each genomic DNA sample is examined by electrophoresis and remnant DNA sample is stored at −70°C. Mixing 1 μl of 5× DNA loading buffer (TIANGEN Biotech (Beijing) Co. Ltd.) with 3 μl of genomic DNA at room temperature for 1 min. Then the sample was loaded on 0.8% agarose formaldehyde denaturing gels stained with ethidium bromide (EtBr) (Biotechnology grade, Amresco), and run on gels in the 1× formaldehyde electrophoresis buffer at 5-7 V/cm.

### **2.6 Assessment of the purity and the yield of the genomic DNA**

Two microliters of each genomic DNA sample was diluted into 200 μl of sterilized ultra pure water (pH 7.0). The absorbance of each diluted genomic DNA sample was evaluated at 260 and 280 nm using a ND-2000C (Thermo, America). The yield of genomic DNA was calculated according to the formula: DNA yield = 50 × OD260 × dilution factor × volume of sample in milliliters/material weight (g). Measured the values at the wavelengths of 260, 280 nm and 230 nm and calculated the ratios of A260/A280 and the A260/A230.

#### **2.7 ISSR amplification**

The DNAs isolation from different *R. soongorica* populations by our promoted CTAB-B protocol were used as template for inter simple repeat sequence primers (ISSR) amplification (Gajera et al., 2010). ISSR amplification reactions were performed in 20 μl reaction volume containing 1 μl gDNA template, 0.25 mmol/L of each dNTPs, 2.5 mmol/L MgCl2, 1×PCR buffer (10mmol/L Tris-HCl pH8.3, 5mmol/L KCl), 1 U Taq DNA polymerase and 0.5 mmol/L of UBC-807 primer (AGA GAG AGA GAG AGA GT). The amplification reaction were carried out on a thermocycler (Biometra) and programmed for an initial pre-denaturing at 94°C for 5 min, followed by 35 cycles of 1 min at 94°C (denaturation), 1 min at 48°C (annealing temperature), and 1.5 min at 72°C (extension) followed by a final extension step at 72°C for 10 min. Amplification products (5 μl) were electrophoresed in 1.5% agarose in 1× TBE buffer and stained with ethidium bromide.
