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International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

Optimization of PCR Protocol and Primers Screening for Development of Rapid Marker Associated with Submergence Tolerance in Rice Genotypes Mohammad Chozin1*, Hesti Pujiwati1, Rustikawati1, and Sumardi1 1

Department of Agroecotechnology, Faculty of Agriculture, University of Bengkulu Jl. W.R. Supratman, KandangLimun, City of Bengkulu 38371A Indonesia * Corresponding author: [email protected]

ABSTRACT

Field screening on a large number of rice genotypes for submergence tolerance is impractical and time consuming. The selection period can be reduced and less tedious by the application of Random Amplified Polymorphic DNA on polymerase chain reaction (RAPD-PCR) method. However, reproducibility and validity of the DNA amplification resulted by the method is conditioned by a number of factors. This study was undertaken to determine the most suitable primers for identification of DNA marker associated to submergence tolerance in rice. DNA bands were amplified by PCR reaction and separated in agarose gel electrophoresis for generating images in UV imaging system. Sixty random decamer primers from three series of Operon Technology (OPE, OPH, and OPM) were evaluated for their capability of amplifying the DNA of nine rice genotypes differing in submergence tolerance. High quality of DNA samples were identified by RAPD-PCR thermocyclers involving single cycle of pre-denaturation at 94oC for 5 min, 45 cycles of denaturation at 94 oC for 5 sec., annealing at 4 oC lower than the recommended temperature on each primer for 30 sec, elongation at 72 C for 1 min, and single cycle of final phase at 72 C for 10 min. Nine primers (OPE-8, OPE-20, OPH-7, OPH-1, OPH-11, OPM-1, OPM-2, OPM-8, and OPM-20) could produce amplified polymorphic DNA of submergence tolerance in rice using RAPD-PCR method. Results of the present study would be useful for developing genetic RAPD marker to be implemented in rice breeding program to produce new varieties better adapted to flood prone environment. Key words: rice DNA, amplification, RAPD-PCR, primer selection, submergence tolerance

INTRODUCTION Submergence

stress due to flood is a yield limitingfactor for rice production on

lowland areas, including rainfed, inland swamp

and tidal swamp areas.Yield loss caused by

submergence can be ranged from 10 to 100 % depending on ﬂood duration, depth and ﬂoodwater conditions (Ismail et al. 2013). Singh et al. (2011) estimated that every year more than 16% global rice lowland is adversely affected by flood. In Indonesia, flood and submergence have resulted in the damaged 300,000 ha lowland rice every year with 60,000 ha harvest failure (Manikmas, 2008). Occurrence of flood on paddy field by monsoon can be varied from transient ﬂash ﬂoods leading to total submergence to long-term stagnant ﬂooding with partial submergence. The duration of total submergence on rice plant caused by flash flood can vary from few days to as long as two weeks, while partial submergence (25 cm to 50 cm deep) resulted by stagnant flooding can persist from few weeks to the entire growing season (Mackill et

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International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

al.1996). The complete submergence can be lethal to susceptible rice genotypes, especially during the early stages of plant development due to limited capability of retaining chlorophyll, carrying underwater photosynthesis, accumulating carbohydrate, preserving elongation, and survival (Das et al. 2009).

On the other hand, tolerant rice genotypes to complete

submergence maintain their chlorophyll and adopt a slow-growth strategy to enable of preserving sufficient carbohydrate to conserve metabolism during submergence and also to recover as the ﬂoodwater recedes (Sarkar et al., 1996; Setter and Laureles, 1996; Das et al. 2005; Fukao et al. 2006). Breeding rice varieties for submergence tolerant would help diminish the yield lost resulted from flood. However, one major drawback of developing submergence tolerant varieties is that the screening should be carried out by exposing the genetic materials to a complete inundation for determined period of time and this cannot be dependent on natural flooding. Similarly, the use of artificial flooding on a concrete pool for screening a large number of genotypes would be tedious and costly.

With the advance of molecular

technology these difficulty can be alleviated. Currently, molecular markers have been the valuable tool for rice breeders in improving the efficiency and accuracy of the selection program (Utami et al., 2010;

Ashkani et al., 2012).

Among the molecular markers, random amplified polymorphic DNA based on polymerase chain reaction (RAPD-PCR) is technically relatively simple and more easily automated than other markers. It is also offer an easy and quick identification without involvement of radioactive materials and requires small amount of DNA (Williams et al., 1990).

Having such characteristic, RAPD-PCR is considered suitable for screening a large

number of genotypes efficiently. The main disadvantage of this marker is that the resulted amplified profile is sensitive to the change in tissue samples, the implemented DNA extraction protocol, and conditions of PCR reaction (Weeden et al., 1992; Karp et al., 1996). Moreover, success in amplifying specific area (locus) of a genome is highly dependent on the primers used (Li et al., 2006). The objective of this study was to determine the most suitable primers for identification of DNA marker associated to submergence tolerance in rice

MATERIALS AND METHOD Screening of rice genotypes for complete submergence tolerant The screening of rice genotypes was conducted at the experimental orchard of Agriculture Production Department, Faculty of Agriculture, University of Bengkulu on a concrete pool made with size 200 cm length, 100 cm width, and 100 cm depth. As much as 398

International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

twenty two rice genotypes (6 improved varieties for swampy agro-ecosystem and 16 local races varieties collected from different agro-ecosystems) were pre-emerged in sequence of ten genotypes at plastics trays till 14 days old and transplanted to the pool that was previously filled with soil to 10 cm thick and at 20 plant density for each genotype.

Flooding to 50

cm in depth was implemented 4 days following transplanting to make the seedling were completely submerged for the next 14 days. Evaluation was made 5 days after the water was taken take out from the pool and plants recede from submergence conditions. The degree of Tolerance was scored as 1 = susceptible (> 80% plants were death), 2 = moderately susceptible (50 – 80% plants were death), 3 = moderately tolerant (50 – 80% plants were alive), and 4 = tolerant (> 80% plants were alive). DNA extraction and amplification The rice DNA extraction was carried out following the protocols as suggested by Saghai-Maroot et al. (1984) using genomic DNA isolated from the fresh leaves of all genotypes screened for the submergence tolerance.

The DNA amplification to produce

RAPD-PCR markers was made using the method developed by William et al. (1990) with some modifications as to obtaining high quality of rice DNA bands. The reaction for DNA amplification was performed in 25 l mixed solution, consisted of 1 x PCR bufer

(50 mM

KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100), 0.2 mM dNTP, 2.5 mM MgCl2, 1 unit Taq DNA polymerase (Promega), 50 ng DNA template dan 0.3 M random primer (10-mer kit operon). All the reactions were executed in DNA Thermal Cycler PE Gene Amp PCR system 2400. A programmed thermocycler (Table 1) was performed with varying annealing temperatures and the number of amplification cycles to determine the optimum condition for PCR.

Table 1. The programmed thermocyler for DNA amplification Phase Predenaturation

Temperature 94 C (5 min)

Number of cycle 1

Amplification 35 to 45 - Denaturation 94 C (5 sec) - Annealing* TM s/d TM – 5 C (30 sec) - Elongation 72 C (1 min) Final 1 72 C (1 min) * TM = the manufacturer recommended temperature for the primers

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International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

The PCR products were electrophoretically separated in agarose gel (0.8 w/v) in 1 x TAE buffer on a constant voltage of 100 volt for 90 min and stained using ethidium bromide (0.5 mg.L-1) for 20 sec. The band pattern was visualized using UV trans-illuminator and captured using Nikon D100 camera. Primer screening Screening was carried out on bulk of leave DNA samples taken proportianally from each genotypic group of submergence tolerance. Sixty random decamer primers from 3 series of Operon, namely Operon E (OPE), Operon H (OPH), and Operon M (OPM) were evaluated for their capability of generating the polymorphic for submergence tolerance.

RESULTS AND DISCUSSION Degree of complete submergence tolerance among genotypes Based on the scored submergence tolerance of the screened genotype in a 14 days complete submergence tests, 3 genotypes were classified as susceptible, 8 genotypes were classified as moderately susceptible, 9 genotypes were classified as moderately tolerant, and 4 genotypes were classified as tolerant (Table 2). In all cases, the plants of tolerant group tended to retain chlorophyll and culm elongation to renew contact with atmosphere. In contrast, most of the plants of susceptible group failed to survive in the first week of inundation with notable injuries as indicated by yellowing older leaves and decayed shoot base.

Such phenomena have been explained in detail by Sauter (2000) and Jackson and

Ram (2003). Tabel2. No.

400

Degree of submergence tolerance of 22 genotypesas subjected to a complete inundation test for 14 days Score Genoype Type of variety Tolerance group ofTolerance

1

Sirantau

Upland local race

1

Susceptible

2 3 4 5 6 7 8 9 10 11 12 13

Siung kancil Wai Putih Cina kelabu Bujang berenai Daku Gindul Hilir mudiak Kelinci Surya Pandan wangi Harum curup Hanafi putih

Upland local race Upland local race Swamp local race Lowland local race Lowland local race Lowland local race Swamp local race Lowland local race Swamp local race Lowland local race Lowland local race Lowland local race

1 1 3 3 3 3 4 2 4 2 2 3

Susceptible Susceptible Moderately tolerant Moderately tolerant Moderately tolerant Moderately tolerant Tolerant Moderately susceptible Tolerant Moderately susceptible Moderately susceptible Moderately tolerant

International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

14 15 16 17 18 19 20 21 22

Lekiten Tigo-tigo Batubara Inpara 2 Inpara 3 Inpara 4 Inpara 5 Inpara 6 Inpara 8

Upland local race Swamp local race Swamp local race Swamp improved variety Swamp improved variety Swamp improved variety Swamp improved variety Swamp improved variety Swamp improved variety

2 4 3 3 3 4 4 3 3

Moderately susceptible Tolerant Moderately susceptible Moderately tolerant Moderately tolerant Tolerant Tolerant Moderately tolerant Moderately tolerant

Optimum RAPD-PCR thermocyler for DNA amplification Thermocyler is the most critical stage in DNA amplification. Temperature and number of cycle should be optimized to produce large number of high quality of amplified DNA (Galvão and Lages-Silva, 2008). Consequently, the programmed thermocycle with varying temperature and number of cycle should be taken at initial protocol to establish an ideal condition for PCR. In this study, the optimum condition for amplifying rice DNA was determined by annealing at the recommended temperature – 4 oC for each primer with 45 cycles of amplification (Table 3). Under this thermocylcer conditions, both quantity and quality of the amplified DNA could be achieved. Table 3. The optimum PCR conditions for rice DNA amplification Phase

Temperature

Number of cycle

Predenaturation 94 C (5 min) Amplification - Denaturation 94 C (5 sec.) - Annealing* TM – 4 C (30 sec.) - Elongation 72 C (1 min) Final 72 C (1 min) * TM = the manufacturer recommended temperature for the primers

1 45

1

Primers screening for polymorphism of submergence tolerance in rice DNA It was revealed that 10 primers were identified as capable of amplifying rice DNAwith 9 primers produced polymorphic markers for tolerant and susceptible and one primer produced monomorphic marker (Table 4). These 9 primers were marker), OPE-20 (2 polymorphic markers), polymorphic markers),

OPE-8 (5 polomorphic

OPH-1 (3 marka polimormik), OPH-7 (5

OPH-11 (2 polymorphic markers), OPM-1(2 polymorphic markers),

OPM-2 (5 polymorphic markers), OPM-8 (2 polymorphic markers), dan OPM-20 (3 polymorphic markers). Among the 29 polymorphic markers, 10 polymorphic markers for submergence tolerantwere obtained from OPE-8, OPE-20, OPH-1, OPH-7, OPM-2, dan

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International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

OPM-20. Figure 1 depicts the markerbands produced by 10 DNA rice amplifying primers. Table 4.

No 1 2 3 4 5 6 7 8 9 10

Fig 1.

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The tabulated number ofpolymorphicRAPD-PCR markers resulted from ten rice DNA amplfying primers Primer

Base sequence

OPE-8 TCACCACGGT OPE-20 AACGGTGACC OPH-1 GGTCGGAGAA OPH-7 CTGCATCGTG OPH-8 GAAACACCCC OPH-11 CTTCCGCAGT OPM-1 GTTGGTGGCT OPM-2 ACAACGCCTC OPM-8 TCTGTTCCCC OPM-20 AGGTCTTGGG Total marker

Marker number Tolerant Susceptible DNA DNA 9 8 12 12 6 7 7 8 7 7 4 6 9 11 9 8 2 5 9 10 74 82

Number of Polimorphic Marker 5 2 3 5 0 2 2 5 3 3 30

Patern of RAPD-PCR markers generated from bulk of rice leave DNA from 4 groups genotypes differing in submergence tolerance, namely tolerant (1), moderately tolerant (2), moderately susceptible (3), and susceptible (4) using 10 operon primers.

International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

CONCLUSION 1. Rice genotypes had a varying degree of tolerance to complete submergence for 14 days. 2. PCR for rice DNA was optimized by thermocycler involving single cycle of pre-denaturation at 94 C for 5 min; 45 cycles of amplification with denaturation at 94 C for 5 sec., annealling at 4 oC lower than the recommended temperature on each primer for 30sec., elongation at 72 C for 1 min, andsingle cycle of final phaseat 72 C for 10 min.

3. The amplified polymorphic DNA for submergence tolerant in rice could be produced by RAPD-PCR method using OPE-8, OPE-20, OPH-7, OPH-1, OPH-11, OPM-1, OPM-2, OPM-8, and OPM-20 primers.

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International Seminar sustainable utilization of coastal resources in tropical zone, 19-20 October,2016, Bengkulu, Indonesia

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