Minjia LU, Xiufang MO, Qin WANG, Guoquan LU, Yurong JIANG

College of Agriculture and Food Science, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China

Chenopodium quinoa Willd, also known as the South American quinoa, is an annual Chenopodiaceous herb. It originated in the area with an altitude of 2 800-4 200 m in the Andes of South America. Chenopodium quinoa Willd is distributed in the range of 12° N -39° S,and has been cultivated in the Andes for more than 5 000 years. It is called as the “mother grain” by Incas[1].Chenopodium quinoa Willd contains high contents of proteins, calcium,phosphorus and iron, and has nearly perfect amino acid composition. It is the only perfect nutritional food identified by the FAO,and is known as “super grain in future” and “nutritional gold”[2-3]. Studies have shown that the long-term consumption of Chenopodium quinoa Willd plays a good role of adjuvant therapy against heart disease, high blood pressure, high blood sugar and high cholesterol.In addition,Chenopodium quinoa Willd also has functions of strengthening immunity,restoring energy, supplementing nutri tion and reducing weight, et al[4-5].The United Nations named 2013 as the“International year of quinoa”, aiming to make the world pay more attention to the role of quinoa’s biological diversity and nutritional value in food supply and nutrition security. In recent decades, the nutrition and utilization value of quinoa has gotten people’s widely recognization and attention. Currently, the quinoa’s researches mainly focus on biological properties[6-7],chemical composition[8-9],stress resistance and other physiological characteristics[10-11].Compared withthose in other countries,the researches on quinoa,especially on the molecular biology of quinoa,are less and almost empty in China. The preparation of high-quality DNA samples is the basis of plant molecular biological researches.The DNA extraction method and treatment method differ among different plants and different parts of the same plants[12-16].There have been currently no reports on genomic DNA extraction method for quinoa. In this study, the genomic DNA in leaves,stems and roots of quinoa was extracted respectively by modified CTAB method, SDS method and high-salt low-pH method. At the same time, the detection using SSR, SSCP and other molecular markers was also performed so as to screen out a rapid,simple and high-quality DNA available extraction method,thereby providing a reference for further molecular biological researches on quinoa.

Material and Methods

Material

The test material was Chenopodium quinoa Willd PI596293,provided by the College of Agronomy,Zhejiang Agriculture and Forestry University.The leaf samples were collected from Chenopodium quinoa Willd plants cultivated in fields. The PI596293 seeds were cultured on germination paper at 25 ℃for 12 d, and then the stems and roots were sampled for DNA extraction.

Main reagents

The main reagents used in this study included EDTA, Tris, CTAB,PVP 40, SDS, β-mercaptoethanol(Sigma Corporation), ethanol, chloroform, isopropanol, sodium chloride,sodium acetate, potassium acetate (of analytical grade, made in China),TaqDNA polymerase, dNTP and DL2000 DNA Marker (TaKaRa Biotechnology (Dalian) Co., Ltd). The primers were synthesized by the Beijing Genomics Institute.

DNA extraction methods

Certain amounts(0.15 g for each)of Chenopodium quinoa Willd young leaves, stems and roots were placed in 2 ml centrifuge tubes. Total three replicates were arranged for each tissue part. The centrifuge tubes were frozen in liquid nitrogen for 5 s by holding tube caps with long-handled tweezers. Subsequently, the samples in the centrifuge tubes were fully ground with a glass rod driven by electric drill.

CTAB method A certain amount(600 μl) of pre-heated CTAB extract[containing 0.1 mol/L Tris-HCl (pH 8.0), 0.02 mol/L EDTA (pH 8.0), 2%CTAB, 2% PVP40, 1 mol/L NaCl,0.2% β-mercaptoethanol]was added to each of the centrifuge tubes. The centrifuge tubes were bathed in water at 65 ℃for 50-60 min,and during this duration, the solutions in centrifuge tubes were mixed several times. Subsequently, the solutions in centrifuge tubes were cooled to room temperature, and then equal-volume chloroform-isoamyl alcohol-ethanol(76∶4∶20)was added and mixed.The tubes were centrifuged at 10 000 r/min for 10 min.The supernatants were removed to other new centrifuge tubes,added with two-fold volume of ice-ethanol and mixed by reversion. The fluffy DNA was singled out and washed with 70%ethanol twice. After dried, the DNA was dissolved in 80 μl ddH2O and preserved at 4 ℃.

SDS method A certain amount (800 μl) of pre-heated extraction buffer[containing 0.5 mol/L NaCl, 0.1 mol/L Tris-HCl (pH 8.0), 0.05 mol/L EDTA(pH 8.0), 0.01 mol/L β-mercaptoethanol]was first added to each of the centrifuge tubes, and then a certain amount (80 μl) of 20% SDS was added(final concentration of 2%).After mixed by shaking, the mixtures in centrifuge tubes were bathed at 65 °C for 30 min, and during this duration,the mixtures were also mixed by reversion. Subsequently, the centrifuge tubes were removed and the mixtures inside were cooled to room temperature. A certain amount (0.3 ml) of 5 mol/L potassium acetate was added to each of the tubes and mixed. After bathed in iced water for 20 min, the centrifuge tubes were centrifuged at 10 000 r/min for 10 min.Subsequently,equal volume of chloroform-isoamyl alcohol (24∶1)was added to each of the tubes. After mixed, the centrifuge tubes were centrifuged at 12 000 r/min for 10 min. The supernatants were removed to other new tubes. Subsequently, 0.7-fold volume of pre-cooled ice isopropanol was added to each tube and mixed gently by reversion.After placed at -20 ℃for 20 min, the centrifuge tubes were centrifuged at 12 000 r/min for 10 min at room temperature. The precipitates were collected and washed with 70% ethanol twice. Subsequently, they were dried,dissolved in 60 μl of ddH2O and preserved at 4 ℃.

High-salt low-pH method A certain amount(1 ml)of pre-heated extraction buffer (containing 0.1 mol/L NaAc,0.05 mol/L EDTA, 0.5 mol/L NaCl,2.5% PVP, 3% SDS, 1% β-mercaptoethanol) was added to each of the centrifuge tubes. The centrifuge tubes were bathed in water at 65 ℃for 30 min,and then cooled to room temperature.They were centrifuged at 10 000 r/min for 10 min. The supernatants were removed to other new tubes.Subsequently, 2/3-fold volume of 25 mol/L of KAC (pH 4.8) was added to each tube. After placed on rice for 30 min, the centrifuge tubes were centrifuged at 10 000 r/min for 10 min.The supernatants were removed to other new tubes.Subsequently,equalvolume chloroform-isoamyl alcohol(24 ∶1) was added to each tube and mixed repeatedly. The tubes were then centrifuged at 10 000 r/min for 10 min. The supernatants were removed to other new tubes. Subsequently,0.6-fold volume of isopropanol (-20℃)was added to each tube and mixed gently. After placed at -20 ℃for 20 min, the tubes were centrifuged at 8 000 r/min for 10 min. The obtained precipitates were washed with 70%ethanol twice. After dried, the DNA was dissolved in 60 μl of ddH2O and preserved at 4 ℃.

DNA purity,concentration and yield detection

The DNA concentrations and D260nm/D280nmvalues were determined using NanoDrop/ND1000 nucleic acid protein analyzer.If D260nm/D280nmvalue≈1.8, the extracted DNA was pure; If D260nm/D280nmvalue ＞1.9, the extracted DNA was contaminated;If D260nm/D280nmvalue ＜1.6, the extracted DNA was contaminated by proteins or phenol.Then the DNA yields were calculated according to following formula:

DNA yield(μg/g)=Extracted DNA amount(μg)/Sample amount(g).

A certain amount (3 μl) of DNA solution of each sample was examined by 1% agarose gel electrophoresis.The graphing was performed using gel imaging system.

PCR of genomic DNA

Referring to study results of Jarvis et al[17],the SSR primers KCAA003 and KAAT009 were designed. The upstream sequence(5’-3’)of KCAA003 was ACCTTTCGGCTGCTCAGATA,and the downstream sequence(5’-3’)was TGCT-GATGTTGTTGCAGATG;the upstream sequence (5’-3’) of KAAT009 was AGTTGCCAACATGCAGAGC, and the downstream sequence (5’-3’) was CGACGACGCAAGACATTAGA. The PCR reaction system was as follows:1.5 μl 10×PCR buffer, 1 μl template DNA, 0.3 μl SSR primers, 0.25 μl dNTPs (10 mmol/L),0.15 μl Taq enzyme and 11.5 μl ddH2O.The amplification program was as follows:pre-denaturation at 94 ℃for 5 min; denaturation at 94 ℃for 30 s,annealing at 55 ℃for 30 s, extending at 72 ℃for 30 s, total 30 cycles; extending at 72 ℃for 10 min, preserved at 4 ℃. The amplification products were examined by 1.5% agarose gel electrophoresis, and the graphing was performed using gel imaging system.

SSR molecular marker detection

A certain amount (1 μl) of PCR amplified product of each sample was examined by 12% non-denaturing polyacrylamide gel electrophoresis(constant power of 80 W, 30 min). After the electrophoresis ended, the gel was immersed in fixative (10%ethanol, 0.05% acetic acid) and fixed on a shaker for 10 min. Subsequently,the gel was washed with distilled water two times, and stained with 0.2%AgNO3for 20 min. And then the gel was washed with distilled water 1-2 times(less than 30 s).Finally,the coloration was completed with color solution(containing 30 g/L NaOH and 13.6 ml/L formaldehyde), and ended by eluting with distilled water three times.

SSCP molecular marker detection

The PCR amplified product and denaturing loading buffer [containing 98% deionized formamide, 10 mmol/L EDTA(pH 8.0),0.025%xylene cyanide,0.025% bromophenol blue]were mixed together(v/v,1∶5)and added to 0.5 ml micro-centrifuge tube.After denatured at 98 ℃for 10 min, the samples were rapidly bathed in iced water for 10 min.About 5 μl of each denaturing sample was taken and examined by 12% non-denaturing polyacrylamide gel electrophoresis at 4 ℃(constant power of 80 W, 12 h). Subsequently, the fixation, silver staining and coloration were performed as described above.

Results and Analysis

DNA quality detection by gel electrophoresis

Agarose gel electrophoresis is a common mean to detect DNA quality[18].As shown in Fig.1, the genomic DNA extracted from different tissues of Chenopodium quinoa Willd all could be amplified to obtain obvious strips, and DNA concentration in leaves was significantly higher than those in stems and roots.The genomic DNA concentrations, purities and yields by three different extraction methods were shown in Table 1. The comprehensive analysis showed that the DNA yield by modified CTAB method was highest, and the DNA yields in leaves, stems and roots were 824.5, 209.3 and 254.1 μg/g, respectively; the DNA yield by modified SDS method ranked second, and the DNA yields in leaves, stems and roots were 496.0, 207.2 and 231.4 μg/g, respectively; the DNA yield by high-salt lowpH was lowest, and the yields in leaves, stems and roots were 322.6,112.0 and 156.2 μg/g, respectively.The DNA yields in leaves by the three extraction methods were all more than twice the yields in stems and roots.The gel test results were consistent with the results of the spectrophotometer. Table 1 also showed that the D260nm/D280nmvalues of genomic DNA extracted by modified CTAB method ranged from 1.7 and 1.8, indicating that the proteins, phenols, polysaccharides and other impurities were removed completely; the D260nm/D280nmvalues of genomic DNA extracted by modified SDS method indicated that the quality of extracted DNA was relatively good, and RNA and proteins were almost removed; the D260nm/D280nmvalues of genomic DNA extracted by high-salt low-pH method indicated that the leaf genomic DNA might be contaminated by RNA or decomposed.

Table 1 Concentrations and purities of genomic DNA extracted from different tissues of quinoa by different methods

Molecular detection

PCR-SSR detection The DNA con-centrations were all adjusted to approximately 300 ng/μl. Then the extracted DNA was amplified by 2 SSR primers. The results (Fig.2) showed that the genomic DNA extracted from different Chenopodium quinoa Willd tissues by different methods all could be amplified to obtain apparent strips,and it could be used for polyacrylamide gel electrophoresis. Fig.3 showed that the obtained strips were clear, and there were no impure strips or non-specific strips.It suggested that the selected 2 SSR primers had good specificity; the extracted DNA met the requirements by SSR markers, and could be used in molecular markerbased molecular assisted breeding.

SSCP analysis SSCP(single strand conformation polymorphism) is a conformation-based method for detecting single nucleotide polymorphism (SNP)in genomic DNA. It is a rapid, simple and sensitive detection method.SSCP can detect, at the single strand level,small differences in DNA, which cannot be detected at the double-strand level[19]. As shown in Fig.4, the genomic DNA extracted from different Chenopodium quinoa Willd tissues by different methods all could be amplified by PCR, and apparent strips were shown on 12% agarose gel after electrophoresis.

Conclusions and Discussion

There have been currently CTAB,SDS, high-salt low-pH method, urea method and sodium citrate method for extracting genomic DNA from plants[20].The establishment of simple and fast extraction method to obtain high-quality DNA is very important for studying and revealing genetic diversity and genetic characteristics of quinoa germplasm resources by SSR and SSCP molecular markers. Quinoa contains polysaccharides,polyphenols and other secondary metabolites[21-23].These metabolites can combine with nucleic acids,forming compounds during the extraction of DNA, affecting DNA yield and quality. The results of this study showed that the genomic DNA extracted from different tissues of quinoa by modified CTAB, SDS and high-salt low-pH methods all met the requirements by molecular biological analysis in future, such as PCR-SSR,SSCP, etc. The yields of genomic DNA extracted from leaf tissue by different extraction methods were all higher than those from stems and roots. So quinoa leaves are more suitable to be used for genomic DNA extraction.

The results of this study also showed that the genomic DNA yield by modified CTAB method was highest,and the proteins, polysaccharides and other impurities were removed more effectively, so high-purity DNA was obtained. These study results were consistent with the comparison results of DNA extraction methods in plants in other studies[20,24-26].In this study,in the extracts of modified CTAB and high-salt low-pH methods, PVP-40 and PVP (polyvinylpyrrolidone) were added. They are all complexes of phenol, which can combine with polyphenols to form an insoluble complex, removing polyphenols more effectively and reducing DNA pollution by phenol. These complexes can also combine with polysaccharides. So polysaccharides in genomic DNA can be effectively removed by centrifugation, thereby improving the purity of extracted DNA[27].However,the yield of genomic DNA extracted by high-salt low-pH method is relatively low.

In the three extraction methods,electric-drill crushing method, as a replacement for hand grinding, was adopted in this study,saving labor and costs. The comparison of extraction methods showed the yield of genomic DNA extracted from quinoa leaves by modified CTAB method was higher,and the consumed time by modified CATB method was 20-30 min shorter than those by the other two methods,saving a lot of research time. The modified CTAB method has a good supporting role for map construction and QTL analysis of quinoa in future.In this study, the young tissues of quinoa are used,and in future studies,optimum extraction method for old leaves of quinoa will be explored.

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