Molecular Screening Of Cultivated Rice

Published Date: 02 Nov 2017

Amrita Iyer, Meghna Chakraborty, Priya Rai, Immanuel Selvaraj and Subramanian Babu

School of Bio Sciences and Technology, VIT University, Vellore- 632014, Tamil Nadu, India

email: [email protected]

Abstract:

Gene sequences that confer resistance to many bacteria, fungi and viruses are called resistance gene analogs (RGAs). These gene sequences work by the interaction between the resistance gene of the host and the avirulence gene of the pathogen. Therefore, plants have developed immunity to pathogens by expressing resistance genes (R-genes) as receptors. These R-genes recognize the invading molecules and mediate defense signaling in order to prevent the spread of the pathogen. The main objective of this project is to screen the resistant gene analogs of various cultivars of rice using amplification of isolated DNA through PCR. Primers for seven RGAs have been designed and used to screen five varieties of rice. Bioinformatic approach in screening the resistant gene analogs in indica and japonica genomes is also carried out.

Keywords: RGAs, PCR, bioinformatics

Introduction:

Plants use several mechanisms to defend themselves against microbial attack. One important defense mechanism is the plant’s ability to recognize pathogens and initiate defense responses. This recognition is mediated by resistance genes conferring resistance to the major classes of plant pathogens, including bacteria, virus, fungi and nematodes. These genes have been isolated from different unrelated plant species such as tobacco, Arabidopsis thaliana, flax and rice. Common motifs occur in resistance (R) genes of diverse origin and pathogen specificity. These structural motifs of various R genes suggest the cellular location of resistance proteins. By making use of conserved domains, it is possible to design oligonucleotide primers for amplifying similar regions from genomes of diverse plants species by polymerase chain reaction (PCR) (Aarts et al. 1998; Collins et al. 1998; Feuillet et al. 1997). Similarly, we have amplified resistance gene analogues from five different varieties of rice by PCR. There were seven rice varieties which were used. They were ADT 36, ADT 37, ADT 43, IR 50, Komal 101 and CR 1009. ADT 36 is a medium slender rice which is resistant to blast and stemborer. It is suitable for sornavari season and the yield is 5.05t/ha. Tamil Nadu and Pondicherry are the recommended places for cultivation. ADT 37 is a short bold white rice. It is resistant to green leaf hopper. It is suitable for kuruvai and late thaladi and the yield is 5.50-6.6t/ha.

ADT 43 is a medium slender rice variety which is suitable for sornavari, kar and Kuruvai. It is resistant to GLH, Stemborer and gallmidge and the yield is 7.0t/h. IR 50 is a dwarf rice variety which is resistant to blast and heminthosporium. It is tolerant to BLB, susceptible to RTV and yield is 60Q/ha. CR 1009 is a short bold white rice and the yield is 6.0t/h. The main objective of our study was to screen the resistant genes in different varieties of rice at the molecular level.

Materials and Methods:

Plant material: Six different varieties of rice, namely Komal 101, CR 1009, ADT 36, ADT 37, ADT 43 and IR 50 were collected from local seed shop in Vellore, Tamil Nadu, and were grown in black soil in the germination trays, for 15 days. When the seedlings reached the two-leaf stage, they were uprooted and the leaves were used for DNA isolation.

DNA isolation: The Byeong-Ha Lee University’s (Arizona) general protocol for DNA isolation was followed. For this, about 2g of fresh leaves, of the varieties, were taken in a chilled mortar and pestle and crushed along with CTAB isolation buffer. More CTAB buffer is added and sample is incubated at 600C, for 30minutes with occasional gentle swirling. Equal volume of Phenol:Choloroform (1:1) is added to the samples and centrifuged at 12000 RPM for 10 minutes. The yellow, aqueous phase, is removed and taken in a new tube. To this, same volume of Chloroform:Isoamylalcohol (24:1), is added and centrifuged at 12000RPM for 10 minutes, at room temperature. The upper aqueous phase is again removed and transferred to a new tube. 2/3rd volume of cold 100% isopropanol is added to the tube and mixed gently. This is done so that the nucleic acids can precipitate out. This is again centrifuged at 12000ROM for 10 minutes, at room temperature. Now the supernatant is discarded from the tubes, and to this, 70% Ethanol is added, which acts as a wash buffer to the DNA pellet. Incubate these tubes for 20 minutes and then centrifuge them at 10000RPM for 5 minutes, at 40C. Pour of the supernatant from the tubes and allow the DNA pellet to dry. This step of washing the DNA pellet with ethanol was repeated atleast 5 times so that the DNA pellet is completely free of any protein contamination. The final pellet was resuspended in TE buffer, after subsequent washes with RNase and Proteinase for removing the RNA and the unwanted proteins.

Electrophoretic analysis of DNA: The method of agarose gel electrophoresis was followed to determine whether DNA has been properly isolated, and to also check if the DNA is contaminated due to the presence of RNA or protein. 1% agarose gel was made. Before this the TAE buffer (50X) was prepared by taking 4.84 g Tris Base, 1.14 ml Glacial Acetic Acid, 2 ml 0.5M EDTA (pH 8.0). The total volume was made up to 1L with water. This served as the stock solution of TAE buffer. From this, 1X TAE buffer, 500ml, was prepared by taking 10ml of TAE (50X) and 490ml of distilled water. Now for preparing 1% of agarose gel, 3g of agarose was taken and it was dissolved in 30ml of the TAE buffer. This was then heated in the oven for 1 minute, such that the agarose completely dissolves, and the solution becomes transparent. This is kept in room temperature for some time so that it cools down to a certain extent. Then, Ethidium Bromide solution is added to the agarose, 0.2µl. This was then cast into a gel and kept for solidification. A comb was kept in the gel-casting slab, so that proper wells can be formed. Later the comb was carefully pulled out, and the tapes (that were used for sealing), were removed. The gel was then placed in the electrophoresis chamber and enough TAE Buffer was added so that about 2-3 mm of buffer existed over the gel. The sample (7µl) was then mixed with d sample loading dye (3µl), Bromophenol Blue, and loaded into the wells. This was then subjected to run at 50V for 1.5 hours.

Quantification of DNA: The DNA that was isolated from the above mentioned protocol, was then subjected to quantification, to calculate the total amount of DNA that was obtained in the isolation from the 2g of fresh leaf tissue. For this 990µl of sterile water and 10 µl of the DNA suspended in the TE buffer was taken. Along with this, a blank was also taken which contained 990 µl of sterile water and 10 µl of TE buffer. These contents were mixed thoroughly and the blank was set at 260nm in a spectrophotometer. The DNA was estimated at 260nm and also a ratio of 260/280 was taken and the optical density of the solution was calculated. The O.D of 1.0 was considered to contain 50 µg/ml of DNA in suspension.

PCR analysis of RGAs: PCR was done on these isolated DNA samples to amplify the DNA segments of our interest using standardized primers. Various types of PCR process were carried out for standardizing the condition for the primers to act. There were seven different primers (both forward and reverse primers of each of them) that were used in PCR. These primers were GST, RAb7, OSLEA7, OSLEA30, PK, RLRR, Non- lambda LRRAE. The primers were present in a stock solution of 100µmoles. These were hence diluted by taking 45µl of sterile water and 5µl of the desired primer (0.5µmol in 20µl of the total PCR volume). The first that was tried, was a normal PCR procedure by using only the first primer (GST), following the below mentioned protocol.

PCR REACTION MIXTURE

VARIETY

ADT36

ADT37

ADT43

IR50

KOMAL101

CR1009

DNA (µl)

1

1

2

1

2

1

FORWARD PRIMER (µl)

1

1

1

1

1

1

REVERSE PRIMER (µl)

1

1

1

1

1

1

PCR MASTER MIX (2X) (µl)

10

10

10

10

10

10

INJECTION WATER (µl)

7

7

7

7

7

7

TOTAL VOLUME (µl)

20

The PCR that was set in this followed the given conditions, and also, the cycles were repeated 30 times (from denaturation (2) to extension (4)).

SERIAL NO.

PCR CONDITIONS

TEMPERATURE

TIME (minutes)

1

Initial denaturation

950C

2

2

Denaturation

950C

1

3

Anneling

510C

1

4

Extension

720C

1.5

5

Final extension

720C

7

6

Storage

40C

∞

Other kinds of PCR such as Gradient PCR, Multiplex PCR and Touch down PCR were also tried, out of which proper results were obtained only in Touch down PCR in which the first 10 cycles were run at 300C and the next 20 cycles at 510C using the genes OSLEA30 and Non-lambda LRRAE. The PCR results were then tested using agarose gel electrophoresis.

Results and Discussion:

Fig. 1and 2: Genomic DNA isolated from cultivated rice varieties

QUANTIFICATION OF DNA:

SAMPLE

O.D (µg/µl)

260nm

260/280nm

ADT36

0.85

0.04

1.54

ADT37

7.20

0.14

1.5

ADT43

1.25

0.02

1.9

IR50

2.25

0.04

1.66

KOMAL101

1.20

0.02

1.4

CR1009

2.15

0.04

2.15

SAMPLE

AMOUNT OF DNA (O.D AT 260nmX50X200) (µg/µl)

QUANTITY OF DNA (ng)

ADT36

0.4

400

ADT37

1.4

1400

ADT43

0.2

200

IR50

0.4

400

KOMAL101

0.2

200

CR1009

0.4

400

Fig 3: PCR analysis of RGAs in cultivated rice varieties

In Fig 3, sample in lane 1 consists of ADT 43 with primers OSLEA 30 and Non-Lambda LRRAE. Sample in lane 2 consists of ADT 43 with primers PK and RLRR. In Fig 4, samples in all lanes have been taken with primers OSLEA 30 and Non-Lambda LRRAE. Lane 1 has ADT 36, lane 2 has ADT 37, lane 3 has ADT 43, lane 4 has IR 50, lane 5 has Komal 101, lane 6 has CR 1009 and the last lane has marker.

ADT 43 was observed to have two copies of OSLEA RGA and the primers used have resulted in amplification of a 0.5 kb and 1.2 kb fragment. Similarly CR 1009 was found to have two copies of resistant gene analogue under study.

Discussion:

The difference among the varieties in terms of amplified products for the RGAs under study indicates their possible differential reaction to diseases. Further sequencing of these amplified products and developing them as molecular markers, is likely to help in screening of the breeding lines in the resistant variety development programmes. Developing these resistant variety plants can help, in turn, in reducing the use of chemical insecticides to the plants thereby preventing soil or environmental damage.