Development of SCAR Markers for Rapid Identification of Resistance to Phytophthora in Durian using Inter Simple Sequence Repeat Markers

Noor Camellia, N. A.1*, Salma, I.2, & Mohd Norfaizal, G.2

1 Industrial Crop Research Center, Malaysian Agricultural Research and Development Institute, Serdang, MALAYSIA

2 Agrobiodiversity and Environment Research Center, Malaysian Agricultural Research and Development Institute, Serdang, MALAYSIA

* Correspondence E-mail: camellia@mardi.gov.my

DOI: http://dx.doi.org/10.33980/ajabs.2019.v07i01.005

(Received 21 May, 2019; Accepted 27 Jun, 2019; Published 30 Jun, 2019)

ABSTRACT: Eighty inter simple sequence repeat (ISSR) primers were applied to detect polymorphisms among eight durian clones. From eighty ISSR primers used, only six primers UBC824, UBC860, UBC886, UBC888, UBC889 and UBC891 generated distinct polymorphic fragments. Out of the total of 42 bands produced from six primers, eight were polymorphic. Only 5 fragments successfully cloned to convert into sequence characterized amplified regions (SCAR) markers. Based on the sequences of the cloned ISSR fragments, six ISSR-SCAR primer sets were produced. Three SCAR markers designed from this study were examined using genomic DNA extracted from 4 accessions of Durio lowianus andDurio kutejensis and 6 accessions of hybrids and clones of Durio zibethinus. Resistance to Phytophthora infection is an important objective for cultivar improvement programmes of Durian. Molecular markers linked to such key traits can be used to screen resistant individuals for future breeding. ISSR -SCAR marker 886-2 was identified to separate the resistant and susceptible Durio species towards Phytophthora infections.

Keywords: Durio; Canker; Resistance; Susceptible; Disease and Sequencing.

INTRODUCTION: Durian is one of the important fruit in Malaysia. The country is the worlds second largest producer and exporter of durian after Thailand (Vanijajiva, 2012). There are several problems in maintaining durian orchard. One of the most important problems in durian cultivation is the susceptibility to stem canker caused by Phytophthora palmivora (Muryati et al., 2009). The disease of Phytophthora is one of the most serious problems attacking durian plants occurring everywhere in cultivated area including Malaysian Agricultural Research and Development Institute (MARDI) genebank, which represent the world largest collections of durian.

This disease can attack any stage of the plant from seedlings, matured and old trees. The fungus can attack the trunk, twigs, branches, leaves, flowers, fruit and the underground parts of the stem and roots. Various cultivation methods and chemicals have been applied and proven to be somewhat effective (Muryati et al., 2009). However, their effectiveness is only for short period of time. Using resistant genetic resources is considered to be the most effective approach against this disease for long-term cultivation (Mohd Shamsudin et al., 2000). However, information on the genetic backgrounds of durian genetic resources is still lacking and there is a need to evaluate and utilize the genetic diversity of durian in breeding for resistant varieties.

Breeding of durian for resistance to Phytophthora has become an important objective of durian crop improvement program. Conventionally, durian plants were selected based on the visible or measurable traits called the phenotype. Phenotypic reaction to infection of Phytophthora palmivora has been previously conducted for MARDI germplasm. Wounded barks of durian trees were inoculated with Phytophthora palmivora isolates to observe the reaction (Nik Masdek, 2008). Unfortunately, this conventional method can be difficult due to the influence by the environmental factors and costly. Identification of the disease resistance trait based on molecular markers is a practical method to be approached. Identified region of DNA that showed resistance to Phytophthora would be used as a marker to track and develop the trait. These markers will help researchers especially breeders to make selection for resistant plant at the stage of seedlings. The objectives of this study were to develop ISSR-SCAR markers of durian and to identify the potential ISSR-SCAR markers associated with Phytophthora resistance in Durio species.

MATERIALS AND METHODS:

PCR amplification with ISSR primers: Approximately 80 ISSR primers were tested on 8 durian clones. The reaction volume was 20l containing 20ng genomic DNA, 1X PCR buffer, (various=1/2) mM MgCl2, 0.2mM dNTPs, 0.6M primer and 1U Taq polymerase (vivantis). The thermal cycler conditions were 2 min at 94C; followed by 40 cycles of 94C for 1 min, annealing temp (various C) for 1 min, and 72C for 2min; followed by a final elongation at 72C for 8 min. A total of 8 distinct polymorphic fragments were selected from 6 ISSR Primers (Table 1).

Cloning of polymorphic fragments and designing of SCAR primers: These fragments were excised and purified using QIA quick Gel Extraction kit (QIAGEN). Then, these fragments were cloned using TOPO TA Cloning Kit for sequencing (Invitrogen). DNA fragments were ligated to vector pCR-TOPO. Then, these ligated products were transformed chemically into E. coli competent cells (One Shot TOP10) through heat shock process (42C for 1 min). The transformed cells were added with SOC medium and incubated at 37C and lastly spread onto selective LB agar with X-gal and 100g/ml Ampicillin. The agar plates were incubated 37C overnight. Single colonies were selected and cultured in 10l selective LB broth (100g/ml Ampicillin) at 37C for 1hour. After that, colony PCR was carried out to identify clones carrying the desired DNA fragment. The right clones were incubated in 8ml selective LB broth (100g/ml Ampicillin) at 37C overnight. Plasmids were extracted using QIAprep Spin Miniprep Kit (QIAGEN). The extracted plasmids were digested with enzyme EcoRI (37C, 5hours) to confirm the presence of the desired insert within the plasmid. The right clones (in plasmid form) were then sent for sequencing. From those fragments, only 5 fragments were successfully cloned and sent for sequencing. Based on these sequences, ISSR-SCAR primers had been designed using primer premier 5 software.

Detection of SCAR markers associate to Phytophthora resistance : DNA samples of 2 Durio species, Durio lowianus and Durio kutejensis, 3 hybrid of clones (MDUR78, MDUR79, MDUR88) and 1 clone (D24) of Durio zibethinus were collected at MARDI field genebank in Serdang, Selangor. Samples have been earlier identified for their phenotypic reaction to Phytophthora palmivora infection (Table 2). Only plants that showed extreme reaction of resistant and susceptible to Phytophthora infection were used in this experiment (Table 2). Sample of young leaves from each species were extracted using i-genomic Plant DNA Mini Kit from iNtRON Biotechnology. DNAs obtained were quantified by electrophoresis using 1 % agarose gel and 100bp DNA ladder. DNAs concentrations were quantified using spectrophotometer.

A total of three SCAR primers that have been converted from ISSR polymorphisms were used in this experiment which are ISSR-SCAR 888, ISSR-SCAR 886-1 and ISSR-SCAR 886-2 (Table 3). DNA amplification was performed in a 20l reaction volume containing 20ng genomic DNA, 1x PCR Buffer, 1 or 2 mM MgCl2, 0.2mM dNTPs, 0.6M primer, and 1U Taq Polymerase. Amplification was performed in a thermo-cycler. The PCR assay was performed in 41 cycles: 1 cycle of 2 minutes at 94C, followed by 40 cycles of 1 minute at 94C, 1 minute at annealing temperature (various C), 2 minutes at 72C and finishing with an extension step of 8 minutes at 72C. The amplification products were separated in 1.4% agarose gel. The gel was stained with Syber Safe and allele size was estimated by comparing with 100bp DNA ladder. DNA fragments were photographed using Gel documentation system and stored as digital pictures.

RESULTS:

Development of SCAR markers: From eighty ISSR primers used, only six primers UBC824, UBC860, UBC886, UBC888, UBC889 and UBC891 generated distinct polymorphic fragments. From these six primers, number of amplicons per primers varied from 4 (UBC860) to 12 (UBC889) and the amplicon sizes varied from 750bp to 1400bp (Table 1). Out of the total of 42 bands, 8 bands (18.6%) were polymorphic with an average of 1.3 polymorphic bands per primer (Table 1). Figure 1 showed the polymorphic banding profile generated using six primers UBC824, UBC860, UBC886, UBC888, UBC889 and UBC891. From those eight polymorphic fragments, only 5 fragments successfully cloned to convert into sequence characterized amplified regions (SCAR) markers. Based on the sequences of the cloned ISSR fragments, six ISSR-SCAR primer sets were produced (Table 3).

Detection of SCAR markers associate to Phytophthora resistance: Three SCAR markers designed for this study were examined using genomic DNA extracted from 4 accessions of Durio lowianus andDurio kutejensis and 6 accessions of hybrids and clones of Durio zibethinus. All of the primers yielded clear banding profiles with the size of amplicon varied from 1 to 3 and the amplicon size varied from 200bp to 1250bp (Figure 2, 3 & 4). All of the bands, which are 5 in total, were polymorphic (Figure 2, 3 & 4). From these 3 SCAR primers, Primer 886-2 separated the resistance and susceptible Durio species (Figure 4). Five known resistant individual amplified the SCAR markers 886-2. They were 2 accesions of Durio lowianus, 1 accession of Durio kutejensis and 2 hybrids of Durio zibethinus MDUR78 and MDUR79.

Table 1: List of Inter Simple Sequence Repeat (ISSR) primers showing genetic polymorphisms among eight durian clones.

ISSR primer

(UBC)

Primer sequence*

PCR condition [MgCl2] & Annealing Temp.

Fragment size

Total Bands

No. of polymorphic bands

824

(TC)8G

2mM;45C

1.4kb

6

1

860

(TG)8RA

2mM;45C

800bp

4

1

886

VDV(CT)7

2mM;45C

1-800bp

2-780bp

7

2

888

BDB(CA)7

1mM;40C

850bp

8

1

889

DBD(AC)7

1mM;45C

1-950bp

2-1.0kb

12

2

891

HVH(TG)7

1mM;45C

750bp

6

1

*Single letter abbreviations for mixed-base positions: Y = (C, T), R = (A, G), B = (non A), D = (non C), V = (non T), H = (non G).

Table 2: The plant materials collected from MARDI field gene bank, Serdang and their reactions to phytophthora.

No.

Samples Name

Reactions to Phytophtora

Samples ID

1.

Durio lowianus

(Wild species)

Highly Resistant

(Nik Masdek, 2008; Mohd Shamsudin et al., 2000)

D1

2.

Durio lowianus

(Wild species)

Resistant

(Nik Masdek, 2008)

D2

3.

Durio kutejensis

(Wild species)

Resistant

(Nik Masdek, 2008)

D3

4.

Durio kutejensis

(Wild species)

Susceptible

(Nik Masdek, 2008)

D4

5.

Durio zibethinus (Hybrid : MDUR88 D24x D10)

Intermediate

(Zainab & Abidin, 2008; Perak Agriculture Department, 2013 )

D5

6.

Durio zibethinus (Hybrid : MDUR88- D24x D10)

Intermediate

(Zainab & Abidin, 2008; Perak Agriculture Department, 2013)

D6

7.

Durio zibethinus (Hybrid : MDUR79- D24x D10)

Resistant

(Zainab & Abidin, 2008; Perak Agriculture Department, 2013 )

D7

8.

Durio zibethinus (Hybrid : MDUR78 D10xD24)

Resistant

(Zainab & Abidin, 2008; Perak Agriculture Department, 2013)

D8

9.

Durio zibethinus (Clone : D24)

Very susceptible (Tai, 1971)

D9

10.

Durio zibethinus (Clone : D24)

Very susceptible (Tai, 1971)

D10

Table 3: Characteristics of the SCAR loci derived from ISSR markers selected from previous study.

No.

Oligo Name

Primer sequence

(5-3)

No. Bases

Tm value

GC (%)

1.

886-1.F

CCC TTG TAT TTT TTC CTA TC

20

45.0

35.0

886-1.R

CTC TCT CAT GCT TCT CCC AAC

21

52.7

52.4

2.

888.F

CAC ACA GCA GCA ACA CAA C

17

49.9

52.6

888.R

GTT GGT GGT GGA GGC AGT C

18

54.7

63.2

3.

889-2.F1

GGG AAC ACG GCA AGG GGA GT

19

61.7

65.0

889-2.R1

GCT ACG GCG GAG GGC AAA GT

19

62.1

65.0

4.

886-2.F

CTC TCT TAT CCA GGG GG

20

45.8

58.8

886-2.R

CTT TGC TGT CAT CTG GGT

20

47.3

50.0

5.

891.F

GTG TGT GAT GGA CAG TGA A

19

45.5

47.4

891.R

CTT TTG CTG TTT CAC ATT CA

19

48.4

35.0

6.

889-2.F2

CAC ACA AGA ATT TAG ATG G

19

41.9

36.8

889-2.R2

CAC ACA CAC ATA TAT ACA T

20

33.3

31.6

img2

Figure 1: Polymorphic banding patterns of eight durian clones generated using ISSR primers. a) UBC824 b) UBC888 c) UBC860 d) UBC886 e) UBC889 and f) UBC891. Abbreviations ; red box , Failed to clone; blue box, Successfully cloned.

img2

Figure 2: Polymorphic banding patterns of Durio species generated using ISSR-SCAR primers 886-1.

img2

Figure 3: Polymorphic banding patterns of Durio species generated using ISSR-SCAR primers 888.

img2

Figure 4: Polymorphic banding patterns of Durio sp. generated using ISSR-SCAR primers 886-2.

DISCUSSION : The SCAR markers have some advantages over ISSR markers to distinguish Durio plants. Due to ISSR dominant nature, the amplification of multiple loci and their sensitivity to reaction conditions restricted their further use. SCARs are more independent to reaction conditions and are usually codominant markers that amplify a single locus, making them robust and more reproducible assay than could be obtained with short primers used for ISSR analysis (Bautista et al., 2002). Since SCAR markers can identify a single or a few bands instead of a complex pattern, SCAR markers are more straightforward to be analyzed than other molecular techniques such as RAPD, ISSR, SSR and AFLP (Luqua et al., 2013). The SCAR marker technique has been successfully applied in many crops for the marking of resistance genes and marker-assisted selection (Evans and James, 2003; Ardiel et al., 2002; Jei-Wan et al., 2011; Dong Hyeon et al., 2013). In this study, clear and bright, amplified DNA bands from ISSR markers were selected for SCAR marker development. In SCAR, pairs of 17-21bp oligonucleotide primers specific to the sequence of polymorphic bands can be used to amplify the characterized regions from genomic DNA under stringent conditions, which makes this marker more specific and dependable as compared to ISSR marker.

Durio lowianus (D1) reaction towards Phytophthora palmivora was the highest resistance among all the durian clones and species used in this study. It can be the best benchmark for choosing the desirable primers which is resistant to Phytophthora. MDUR78 and MDUR 79 hybrid were also both resistant to Phytophthora infections and also claimed by Zainab & Abidin (2008) and Perak Agriculture Department (2013) to be resistant towards Phytophthora. Durio zibethinus D24 clone was known to be susceptible to Phytophthora disease. The samples were taken from trees that were severely infected by Phytophthora palmivora. The study shown that SCAR primer 886-2 detected the Durio lowianus (D1 & D2), Durio kutejensis (D3), MDUR78 (D8) and MDUR 79 (D7) hybrid only. The D24 clones (D9 & D10) were not amplified with the primer 886-2. This preliminary work to detect Phytophthora resistance, has shown that SCAR primer 886-2 can be used to screen for durian trees or seedlings which have the resistance towards Phytophthora infections for future breeding or as rootstocks.

Arguably, this markers need to be further tested on the F1 progenies of the resistance and susceptible clones before it can be used. Further study on marker assisted selection (MAS) need to be done. Seggregation of the full progeny for Phytophthora resistance must be tested with this marker. SCAR markers by directly sequencing from ISSR products were useful for the MAS and suitable for further applications such as cultivar identification and mapping (Busconi et al., 2006).

CONCLUSION: Six ISSR-SCAR primer sets were produced from three successfully cloned polymorphic ISSR primers which are ISSR-SCAR 886-1, ISSR-SCAR 888, ISSR-SCAR 886-2, ISSR-SCAR 889-2.1, ISSR-SCAR 889-2.2 and ISSR-SCAR 891. ISSR-SCAR marker 886-2 was identified to separate the resistant and susceptible Durio species towards Phytophthora infections.

ACKNOWLEDGEMENTS: The researchers are grateful to those who have directly and indirectly contributed to this study that was funded by the Malaysian Agricultural Research and Development Institute (MARDI) Short Term Research Grant Project (JP-RS-0377).

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