Document Type : Original Article

Authors

1 Molecular marker lab, Registration and identification of plant varieties department, Seed and plant certification and registration institute (SPCRI), Agricultural Research, Education, and Extension Organization (AREEO), 31535-1516 Karaj, Iran

2 Seed qualification lab, Seeds registration and certification department, Seed and plant certification and registration institute (SPCRI), Agricultural Research, Education, and Extension Organization (AREEO), 31535-1516 Karaj, Iran

10.22034/jpbb.2022.355919.1027

Abstract

Introduction: Reaching an appropriate DNA isolation protocol is a great challenge in chickpea seeds due to its richness in protein and carbohydrates. In this research, a chickpea seed collection from various seed classes of candidate varieties was tested for genetic purity. Some off-type seeds could not germinate properly and using seedlings are not available for DNA isolation. Thus, seven experiments were designed to find a proper protocol for chickpea seed DNA extraction.
Methods: SDS based seed extraction buffer along with protein kinase K, various levels of DTT, and PVP including, 2% PVP with 0.2% , 0.4%, and 0.8% DTT as well as applying organic reagents including phenol: chloroform (25:24, v/v) and chloroform: isoamyl alcohol (24:1, v/v) were compared with a DNA extraction kit. Machine learning techniques contain regression linear modeling and discriminant analysis (DA) employed to evaluate the protocols.
Results: The results indicated that seed extraction buffer with 0.8% DTT showed the highest efficiency in DNA concentration, but the lowest in purity. Thus, using DTT can increase DNA yield in chickpea seeds but not great impact on diminishing protein residuals. Both 0.4% DTT and protein kinase K presented similar purity results. DTT 0.4% can be the proper alternatives of protein kinase K to remove protein residuals from chickpea seeds in DNA isolation. However, the best DNA purity achieved by using 0.4% DTT and organic reagents that was similar to the kit.  Both 260/280 and 260/230 ratios were above 2 and the DNA yield was higher than the kit. Furthermore, Discriminant analysis (DL) demonstrated that applying 0.4% DDT with organic reagents and kit categorized in the same group.
Conclusions: This method of DNA isolation is the reliable and efficient protocol can be employed for seeds that are rich in protein. 

Graphical Abstract

Optimization of an Efficient DNA Isolation Method from Protein-Rich Seeds of Chickpea (Cicer arietinum L.) for Seeds Certification Program

Keywords

Main Subjects

  1. Introduction

Healthy, original, and high quality seeds are important fundamentals of agricultural production and development that lead to increase efficiency of production resources and achieving the food security. Morphological evaluations in field observations are not sufficient and unreliable. High genetic purity in crop cultivars is mandatory for achieving the certification presenting reasonable agronomic performance in the field and attracting farmers and investors. DNA-based technologies were developed to provide a better discrimination of varieties for breeders‎ (Smith and Register Iii, 2008). ‎

Crops contain various levels of nutrients, including protein, carbohydrates, and polyphenols that make DNA isolation challenging due to attachment with nucleic acids ‎(Aboul-Maaty and Oraby, 2019; Sabriu-Haxhijaha, Ilievska et al., 2020). ‎The chickpea seed is the main source of carbohydrates and proteins, which comprises  80% of the total dry seed weight‎ (Vural and Akcin, 2010). Therefore, employing an efficient and reliable protocol for high purity DNA ‎isolation results in an accurate DNA amplification (Xia, Chen et al., 2019) ‎.

An efficient isolation protocol should enhance DNA yield and quality along with diminishing DNA degradation. The protocol would be cost-effective, time, and labor saving. Two basic methods are used frequently in various plant species, including sodium dodecyl Sulfate (SDS) ‎(Edwards, Johnstone et al., 1991) and cetyltrimethyl ammonium bromide (CTAB) (Porebski, Bailey et al., 1997); (Chen, Rangasamy et al., 2010).‎

In this project, several suspected off-types of candidate varieties of chickpea seeds from their different seed classes were investigated in terms of the genetic purity. The off-types germination and vigor test were not promising. Therefore, seeds were used to evaluate genetic purity. SDS-base DNA isolation method with some modifications was developed in these seeds collection to use in SSR marker to discriminate the candidate cultivars from off-types. Modifications accomplished regarding the use of protein kinase K, PVP, DTT, and organic reagents to improve DNA yield and quality. Machine learning methods were exploited to asses protocols compared with the kit.

  1. Materials and Methods

2.1. Plant materials

A chickpea seeds collection comprised 150 seed samples from various candidate varieties and their off-types in every seed class, from breeders to certified seeds. The primary seed physical screening and germination test were carried out in seed quality lab, and then transmit to molecular marker lab for genetic purity qualification in seed and plant certification and registration institute.

2.2. DNA isolation

Plant genomic DNA extraction kit (ADDBIO, cat no: 10023, South Korea) and SDS-based DNA extraction method with some modifications including 2% PVP and 0.2%, 0.4%, and 0.8% DTT (Table 1) were employed to isolate DNA from the chickpea seeds. Extraction buffer was applied in this research was based on Edward protocol ‎(Edwards, Johnstone et al., 1991).‎

2.2.1. Protocol no.1, DNA extraction kit

This protocol was prepared by ADDBIO company (cat no: 10023) including protein kinase K, extraction, precipitation, binding, and 2 washing buffers applied for DNA isolation.

2.2.2. Protocol No.2

Protein kinase K (20 µL) was added to the grounded sample and followed by 1 mL of SDS-based extraction buffer (without PVP and DTT) that were mixed well by a vortex. The samples were left in the water bath for 45 min at 65 °C and inverted 2-3 times gently. The samples were centrifuged for 5-10 min at 13000 rpm and the supernatants were transferred to a fresh tube. One-tenth (0.1 V) 5 M potassium acetate was added and incubated on ice for 10-15 min. The mixture was centrifuged for 10 minutes at 13000 rpm and the supernatants were transferred to a fresh tube.

Two volumes of pre-chilled 96% ethanol and 0.1 volume sodium acetate were added to precipitate the DNA. The samples were left at -20 °C for at least one hour. The tubes were centrifuged for 7 min at 7000 rpm and discarded the supernatants. The resulting pellets were washed with 500 µL cold 70% ethanol and centrifuged at 7000 rpm for 2 min and air-dried. The pellets were dissolved in 100 µl of TE or ultrapure water and stored at -20 °C.

2.2.3. Protocols No.3, 4, 5, and 6

In protocol no.3, PVP 2% and DTT 0.2% were added to Edward’s 0.5% SDS extraction buffer, while PVP 2% and DTT 0.4% applied in protocol no.4 and DTT 0.8% in protocol no.5. The DNA extraction procedure was the same as protocol no.2, whereas protein kinase K was not included in the samples. In protocol no.6, both DTT 0.4% and protein kinase K were used to decrease protein residuals in chickpea seed samples. The DNA extraction procedure was the same as protocol no.2.

2.2.4. Protocol No.7

1 mL SDS-based extraction buffer with 0.4% DTT and 2% PVP was mixed well by vortex (the first extraction). The supernatant was extracted twice with phenol/chloroform (25: 24, v/v, the second extraction) and chloroform/isoamyl alcohol (24: 1, v/v, third extraction). Then, the upper aqueous phase was added with 0.1 volume potassium acetate solution (5M, pH=5.5) and double volume of ethanol 96% and left them at -20 °C for 20 min followed by the gentle inversion and centrifuged 10 min at 13000 rpm to pellet DNA. Next, the pellets were washed with cold 70% ethanol twice and air-dried.

2.3. Qualitative and Quantitative Analysis of Extracted DNA

The DNA products were measured by NanoDrop (ND 1000) at 260 nm. DNA purity was evaluated by computing the absorbance ratio A260/280 and A260/230. Carbohydrate and polyphenol residuals in samples were measured by the absorbance ratio A260/280, while the absorbance ratio A260/230 offers protein residuals in samples ‎(Wilson and Walker, 2010).‎

2.4. Data analysis

The data from NanoDrop evaluated by SPSS software version 22 (IBM Analytics) in form of One-way ANOVA analysis to discover the significant differences amongst DNA yields and purity. Furthermore, ANCOVA analysis was employed to assess covariates effect (Protein and carbohydrate residuals) on DNA yield. Regression linear modeling and discriminant analysis (DL) were carried out through SPSS Software version 22 (IBM Analytics) to classify 7 protocols.

  1. Results

In this project, various amounts of DTT were tested in protocols no. 3, 4, 5, 6, and 7 besides protein kinase K in protocol no.1, 2, and 6 and organic reagents in protocol no.7 to improve DNA concentration and purity (Table 1).

 

Table 1. Concentrations of components in-7 protocols for DNA extraction

 

Protocol

Lysis buffer

Protein kinase K

DTT

PVP

Organic reagents

1

Kit

20 µL

Kit

Kit

Kit

2

SDS 0.5%, 0.5 M Tris, 0.35 M NaCl

20 µL

-

-

-

3

SDS 0.5%, 0.5 M Tris, 0.35 M NaCl

-

0.2%

2%

-

4

SDS 0.5%, 0.5 M Tris, 0.35 M NaCl

-

0.4%

2%

-

5

SDS 0.5%, 0.5 M Tris, 0.35 M NaCl

-

0.8%

2%

-

6

SDS 0.5%, 0.5 M Tris, 0.35 M NaCl

20 µL

0.4%

2%

-

7

SDS 0.5%, 0.5 M Tris, 0.35 M NaCl

-

0.4%

2%

Phenol/chloroform (25: 24) and chloroform/isoamyl alcohol (24: 1, v/v)

The results from 7 protocols for DNA isolation showed that protocol no.5 by using DTT 0.8% had the highest DNA concentration (612 ng µL-1 in average), while kit DNA extraction and protocol no.2 through protein kinase presented the lowest DNA yield, 230 ng µL-1 and 226 ng µL-1 in average, respectively (Figure 1). Protocol no.4 by using 0.4% DTT illustrated a much higher DNA concentration (526 ng µL -1 in average) after protocol no.5 followed by protocol no. 3 with 0.2% DTT (366 ng µL-1 in average). Furthermore, protocol no. 6 with the 0.4% of DTT plus protein kinase K revealed 446.86 ng µL–1 DNA yield   in average that was higher than protocol no.7 through DTT 0.4% and organic reagents (373 ng µL-1 in average )(Figure 1).

Figure 1. DNA concentrations in 260 nm from 7 protocols

When it comes to DNA quality, the data presented that the kit extraction showed the highest quality that 260/280 absorbance ratio was higher than 1.8 (in average 2.16), while the 260/230 absorbance ratio was 2.2 (Figures 2 and 3), while the lowest DNA quality achieved in protocol no.5 through using 0.8% DTT (260/280 and 260/230 absorbance ratios were 1.44 and 0.59, respectively). Applying organic reagents in protocol no. 7 comprising phenol/chloroform (25:24, v/v) and chloroform/isoamyl alcohol (24: 1, v/v) remarkably decreases residuals in chickpea seeds and significantly boost DNA purity. The 260/280 absorbance ratio was 2.1 that was the same as the kit and considered the highest compared with the other procedures. Likewise, the average figure of 260/230 absorbance ratio was 2 that was lower than kit (2.2), but higher than others (Figures 2 and 3).


Figures 2. 260/280 absorbance ratio that indicates carbohydrates residuals in 7 protocols

In the protocol no.2, protein kinase K was used to decrease protein residuals. The data from this protocol indicated that 260/280 absorbance was higher than 1.8 (in average 1.92) whereas the 260/230 absorbance ratio was low (0.98). Furthermore, the pellet gathered from protocol no.2 extraction made extract unclear along with visible discoloration. Similarly, both absorbance ratios of protocol no.4 were the same as protocol no.2 and they categorized in the same group. Applying protein kinase K along with DTT 0.4% in protocol no.6 showed no significant changes in DNA residuals that 260/280 ratio was 1.8, but 260/230 ratio was 0.86. Using small amount of DTT (0.2%) in protocol no.3 illustrated the lower 260/280 absorbance ratio that was 1.73 and 260/230 ratio was 0.76 that were lowest after protocol no. 5 (Figures 2 and 3).

Figures 3. 260/230 absorbance ratio presents protein residuals in 7 protocols

Regression linear modeling with boosting technique suggested that among 7 protocols, the 260/230 absorbance ratio with 63% importance is the more important than 260/280 absorbance ratio (37%), while DNA yield was not involved in the model due to no regression with the other variables. The accuracy of modeling was 82%.

Discriminant analysis (DA) showed that 3 canonical discriminant functions obtained from this analysis that function 1 with 92.6% variance could discriminate the most independent variables and function 2 could discriminate 6.3%, while function 3 with 1.1% was the least discriminant function (Table 2).  

 

Table2. Eigenvalues in independent variables

Eigenvalues

Function

Eigenvalue

% of Variance

Cumulative %

Canonical Correlation

1

19.24a

92.6

92.6

.975

2

1.31a

6.3

98.9

.753

3

.230a

1.1

100.0

.432

Standardized Canonical Discriminant Function Coefficients data indicated that absorbance ratio of 260/230 had the greatest coefficient (1.07) in function 1 that had the highest discriminant function and absorbance ratio of 260/280 allocated the greatest coefficient (1.18) in function 2 (Table 3). 

Table 3. The standardized canonical discriminant function coefficients in independent variables

 

Function

1

2

3

DNA Yield

-.32

-.27

.93

A260/280

-.09

1.18

.27

A260/230

1.07

-.61

-.04

 

Therefore, protocols no. 1 and 7 were categorized in the same group while protocols no. 2, 4, 6, and 3 characterized in another group and protocol no.5 considered in a separate group (Figure 4).

  1. Discussion

Seeds of legume and cereal that are known as the major food are the storage tissue, rich in protein, starch, and oil ‎(Bewley and Black, 1994). The majority of DNA isolation protocols in chickpea employed the CTAB ‎method mostly in leaves (Vural and Akcin, 2010); (Tapan Kumar, C. Bharadwaj  et al., 2013); (Talebi ‎‎2008); (Neha Joshi, Anamika Rawat et al., 2010); (EMMA S. MACE, BUHARIWALLA et al., 2003); ‎‎(Dipankar Chakraborti, Anindya Sarkar et al., 2006), but few in seeds (ARUN DEV SHARMA, GILL et al., 2012). CTAB is a frequently used surfactant in DNA extraction published by Doyle and Doyle ‎‎(1987), while Edward protocol (Edwards, Johnstone et al., 1991) used SDS as detergent. The SDS-‎based protocol was frequently used in seeds DNA isolation in several seed varieties including corn ‎and roasted soybean (Sabriu-Haxhijaha, Ilievska et al., 2020) and raw soybean (Xia, Chen et al., ‎‎2019).‎ In this project, we used SDS 0.5% with Protein kinase K (20 µL), PVP 2% along with DTT 0.2%, 0.4%, and 0.8% and organic reagents in the forms of 7 protocols. One of the main factors for high quality DNA would be to use of a higher concentration of low molecular weight PVP (2.5‎ %) (Zhang and Stewart, 2000). The role of DTT is to inhibit polyphenol oxidation (Sa, Pereira et al., ‎‎2011)‎. Our data showed that using just only protein kinase K is not satisfactory for removing protein from chickpea seeds and resulted in gelatinous and discolored DNA extract (Figures 2,3, and 5).

Figure 4. Classification chart of 7 protocols through canonical discriminant functions

This gelatinous material are hard to isolate from organelles and make the DNA inappropriate for PCR and restriction enzyme digestion analyses ‎(Porebski, Bailey et al., 1997) and classical 2-primer PCR amplification (Demeke, Adams et al., ‎‎1992) by inhibiting Taq DNA polymerase activity (Fang, Hammar et al., 1992). ‎On the other hand, our results demonstrated that adding PVP 2% and DTT  (0.2%, 0.4%, and 0.8%) removed this viscosity and made the DNA extract clear (Figure 5). This result may indicated that using small amount of DTT (0.2% an 0.4%) could improve DNA quality partially, whereas a higher amount of DTT (0.8%) could increase DNA yield dramatically, but led to more residuals (Figures 2, 3, and  5).

Similarly, the other research indicated that DNA isolation was successfully done by using high level of β-mercaptoethanol (DTT is the safe alternative of βME) that led to high concentration white pellets with no visible discoloration with the higher DNA yield  in chickpea seeds (ARUN DEV SHARMA, GILL et al., 2012). Our results are in accordance with the research ‎‎(Moreira and Oliveira, 2011) ‎that emphasized on using DTT (substitute of βME) was effective for achieving high-quality DNA with no visible discoloration. Our data indicated that applying protein kinase, PVP, and DTT was effective in higher DNA yield and eliminating polyphenolics/polysaccharide residuals (260/280 absorbance ratio) (Figures 1 and 2), while, it was not sufficient for removing protein residuals completely (260/230 absorbance ratio). Even, adding both DTT 0.4% and protein kinase K simultaneously could not diminish protein residuals (260/230 absorbance ratio) in protocol no.6 (Figures 2 and 3). The other reports represented that DNA quality was boosted in soybean seeds through application of organic reagents ‎(Xia, Chen et al., 2019); (Sabriu-Haxhijaha, Ilievska et al., 2020). ‎Subsequently, the organic detergents containing phenol/chloroform (25: 24, v/v) and chloroform/isoamyl alcohol (24: 1, v/v) as the second and the third extractions were applied to achieve high DNA yield and quality. It has been verified that phenol can separate protein from DNA and mixture with chloroform (1:1, v/v) was formed in two phases, DNA in the aqueous supernatant separated from the pro- tein-chloroform gel.

                                                                                 

Figure 5. Schematic view of application various amount of DTT, protein kinase K, and organic reagents on DNA extraction quality

Furthermore, the other residuals separated by chloroform and isoamyl alcohol to obtain a high purity extraction ‎(Green and Sambrook, 2017). ‎In protocol no. 7, organic compounds were used to remove all endured residuals that led to high DNA purity computable with the kit along with reasonable DNA yield higher than kit (Figure 1, 2, 3, and 5). Using organic detergents was reported in transgenic soybean that is rich in protein that resulted in improving DNA yield and purity ‎(Xia, Chen et al., 2019). ‎Besides, the linear regression with boosting method revealed higher importance of 260/230 absorbance ratio (63%) than 260/280 absorbance ratio (37%). Thus, increasing 260/230 absorbance ratio along with 260/280 absorbance ratio was vital to obtain a proper protocol for further molecular studies. Furthermore, discriminant analysis (DL) confirmed 260/230 absorbance ratio had the highest proportions to discriminate protocols (Tables 2 and 3). Classification of 7 protocols through canonical discriminant discovered that protocols no. 1 and 7 with the highest 260/230 absorbance ratio (over 2) and high in 260/280 absorbance ratio (over 1.8) were classified in a distinct group whereas protocols no. 2, 5, and 3 that were low in 260/230 absorbance ratio, but high in 260/280 absorbance ratio (over 1.8) considered in one group. However, protocol no.5 that was low in both 260/230 and 260/280 absorbance ratios was categorized in a single group (Figure 4). Discriminant analysis (DA) is a flexible classifier employed to classify observations into two or more groups or classes and it assesses procedures and the contribution degrees of variables to group partitioning ‎(Pourreza, Pourreza et al., 2012).‎

  1. Conclusion

A simple, safe, reliable, and cost-efficient SDS-based DNA extraction method was described to provide high-quality DNA from chickpea seeds and other crops containing elevated concentrations of protein and polysaccharide compounds. The resulting optimized SDS-based protocol enables the isolation of high quality genomic DNA amenable to ISSR and SSR markers.

Data Availability

All the data presented in this study are available from the corresponding author.

Competing Interests

The author declares that there are no competing interests associated with the manuscript.

Author contributions

The experiments design and data gathering were carried out by Akram Ghaffari, chickpea samples were provided by Bita Oskouei, and the manuscript was revised by Mohammad Reza Jazayeri.

Funding

This project was funded by seed and plant certification and registration institute (SPCRI), Karaj, Iran.

Ethics approval and consent to participate

The authors declare that they do not use human or animals in their research.

Orcid

Akram Ghaffari: https://orcid.org/0000-0001-5456-9911

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