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GENETIC DIVERSITY OF KELAMPAYAN USING DOMINANT DNA MARKERS BASED ON INTER-SIMPLE

SEQUENCE REPEATS IN SARAWAKW.S. Ho1, K.S. Liew1, A.B. Elias1, F.B. Fedrick1, M.A. Mohamad Khairil1,

S.L. Phui2 & A. Julaihi2

1Forest Genomics and Informatics Laboratory (fGiL)Department of Molecular Biology

Faculty of Resource Science and TechnologyUniversiti Malaysia Sarawak

94300 Kota Samarahan, Sarawak

2Applied Forest Science and Industry Development (AFSID)Sarawak Forestry Corporation

93250 Kuching, SarawakEmail: wsho@frst.unimas.my

Abstract

Neolamarckia cadamba (Roxb.) Bosser, or locally known as kelampayan, is a fast-growing timber species which produces one of the best sources of raw material for the plywood industry and also for the pulp and paper industry. It has been selected as one of the promising plantation tree species for large-scale planted forest development in Sarawak. Therefore, the molecular characterization of this indigenous tropical tree species is needed to maintain its high quality. Inter-simple sequence repeats (ISSR) markers were used in this study to determine the genetic diversity of kelampayan in three progeny trial blocks at the Landeh Nature Reserve, Semengok, Sarawak. The seeds were collected from the selected mother trees located at the Pasai Bon, Niah and Lawas seed production areas (SPAs) in Sarawak. Three ISSR primers, namely (GTG)6, (AG)10 and (AC)10, that yielded reproducible, informative and scorable fragments were chosen for ISSR analysis. A total of 64 loci were generated of which 45.3–74.6% of the loci were identified as polymorphic bands with the size ranging from 500 bp to 2 kb among 247 kelampayan progenies selected in the present study. Molecular diversity based on Shannon’s diversity indices (I) among 247 trees ranged from 0.268 to 0.350. In general, the kelampayan trees in the three progeny trial blocks exhibited a high level of molecular diversity and DNA polymorphism compared with its natural populations. This preliminary information will form the base for kelampayan tree improvement and conservation programmes.

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INTRODUCTION

The Sarawak state government has set a target to establish one million hectares of planted forest by the year 2020. The state government has issued 43 licenses for planted forests (LPFs) and about 250 000 ha of planted forests have been established to date. Planted forests are a viable and sustainable method to produce high-value commercial timber to meet the current increase in global demand for wood. Datuk Len Talif Salleh (State Forestry Department Director) stated that planted forests can reduce dependency on natural forest and generate about 10 to 20 times more wood volume than the natural forests (Borneo Post Online 2010). Besides, planted forests also can provide a number of social and environmental services such as rehabilitation of degraded lands, soil and water protection, sequestering and storing carbon, conservation of biological diversity, creating rural employment, helping communities raise their standard of living and contributing to sustainable development (FAO 2010).

In order to achieve this goal, approximately 30 million high quality planting materials are required annually for planted forest development in Sarawak. In this regard, forest genetics and tree improvement research will help respond to the need to develop adequate tools for producing good quality seedlings that are of faster growth, high yield and high wood quality in the shortest of time at a reasonable cost. With the advent in DNA sequencing, data analysis and PCR technology, a diverse array of DNA marker systems have been described in the literature such as SSR, RAPD, ISSR, RFLP and AFLP (Semagn et al. 2006). These DNA markers will greatly facilitate the selection of quality planting materials for planted forest development in Sarawak. Besides, such DNA markers also have proven their utility in the analysis of phylogenetic relationships, population structure, mating system, gene flow, parental assignment, introgressive hybridization, marker-assisted selection and genetic linkage (Kumar et al. 2009).

ISSR technique is a PCR-based method to amplify DNA fragments between two closely spaced and oppositely oriented SSRs (Moreno et al. 1998). Compared to other DNA fingerprinting methods, ISSR markers offer several advantages such as: a) fast, inexpensive and no radioactive handling facilities required, b) does not require the knowledge of flanking sequence, c) highly dispersed throughout the genome, and d) highly polymorphic (Galvan et al. 2003). A study carried out by Moreno et al. (1998) demonstrated that the reproducibility of ISSR (91.8%) was higher than RAPD (85.8%) due to the use of longer ISSR primers (16–25 mers) which permits much more stringent annealing conditions. ISSR also generates a large number of markers by amplifying multiple microsatellite loci, thus allowing screening of a large number of samples in a single gel (Nagaraju et al. 2001). Therefore, these markers are useful in studies on genetic diversity (Balasaravanan et al. 2005, Okun et al. 2007, Chezhian et al. 2010), gene tagging (Ammiraju et al. 2001), cultivar identification (Wong et al. 2009), genome mapping (Zietkiewicz et al. 1994, Godwin et al. 1997) and phylogenetic analysis (Dogan et al. 2007).

Neolarmarckia cadamba (Roxb.) Bosser, or locally known as kelampayan, is one of the selected indigenous species for planted forest development in Sarawak (Figure 1). Kelampayan is characterized as a large, deciduous and fast-growing tree that gives economic returns within 8 to 10 y (Joker 2000). The tree can grow

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up to 40–45 m tall and 100–160 cm in diameter. Kelampayan is one of the best sources of raw material for the plywood industry, besides pulp and paper production (Joker 2000). It also can be used for other purposes such as picture frame, disposable chopstick, wooden sandal, general utility furniture and plywood. Moreover, the leaves and bark of kelampayan are reported to possess various medicinal uses (Patel & Kumar 2008, Mondal et al. 2009).

Figure 1 Morphological characteristics of N. cadamba. (a) Mature tree; (b) Twig with inflorescence; (c) Fruit

Despite kelampayan being an economically and pharmaceutically important timber species, genetic information of this species is still scanty until now. Thus, we applied ISSR markers to determine the genetic diversity of kelampayan in three progeny trial blocks at the Landeh Nature Reserve, Semengok, Sarawak.

MATERIALS AND METHODS

A total of 247 kelampayan samples (83, 74 and 90 samples from Blocks 1, 2 and 3; respectively) were collected from the Kelampayan Provenance Trial Plot at Landeh Natural Reserve, Sarawak (Figure 1, Table 1). Total genomic DNA was extracted from the fresh leaf samples using a modified CTAB method (Doyle & Doyle 1990). The quality and quantity of the extracted DNA were estimated spectrophotometrically and verified using a 0.8% agarose gel. The DNA was then subjected to ISSR-PCR amplifications.

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Table 1 Origins of kelampayan tree according to lines in three different blocks

LineTree origin

Block 1 Block 2 Block 3

1 Pasai Bon Niah Lawas

2 Niah Pasai Bon Pasai Bon

3 Pasai Bon Pasai Bon Lawas

4 Niah Niah Niah

5 Lawas Lawas Pasai Bon

6 Niah Pasai Bon Lawas

7 Lawas Lawas Niah

8 Lawas Niah Niah

9 Pasai Bon Lawas Pasai Bon

Figure 1 Kelampayan progeny trial plot at Landeh Nature Reserve, Sarawak

Three microsatellite primers, namely (AC)10, (AG)10 and (GTG)6, were used in this study to amplify the ISSR region. PCR was carried out using a Mastercycler Gradient PCR (Eppendorf, Germany). Amplification was carried out in 25-µl reaction volume containing 1X PCR buffer (10 mM Tris-HCl at pH 8.8 and 50 mM KCl), 2.5-mM MgCl2, 0.2 mM of each dNTPs (dATP, dCTP, dTTP and dGTP), 0.5 unit of Taq DNA polymerase (Promega, USA), 10.0 pmol µl-1 of primer and 2 ng µl-1 of genomic DNA. The thermal cycling profile was programmed at 94 °C for 2 min as the initial denaturation step, 39 cycles of 30 s at 94 °C, 30 s at 59.1 °C for (GTG)6, at 57.8 °C for

Block 1

Block 2

Block 3

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(AC)10 and at 56.6 °C for (AG)10, 1 min at 72 °C and final extension step at 72 °C for 10 min. The PCR products were then examined on 1.5% agarose gel and 1 kb DNA ladder (Promega, USA) was ran simultaneously. The gel was documented using the Geliance 200 Imaging System (Perkin Elmer, USA).

The DNA bands produced at different loci were determined and named for each DNA sample. Banding profiles generated were converted into binary data matrices on the basis of present (1) or absent (0) of bands. Data scoring was based on several criteria: (1) locus was assumed as independent or non-allelic, (2) there was no bias in scoring monomorphic fragments versus polymorphic fragments, (3) amplified loci were expected to be in the range of 500 bp to 2000 bp, and (4) the similarity of fragment size was assumed to be the indicator of homology. POPGENE Version 1.32 software was used to estimate the genetic diversity of N. cadamba using Shannon’s diversity Index, H’=-∑piln pi-[(S-1/(2N)] where pi stands for frequency of the ISSR fragment and the second fraction of the formula is the correction factor (Lewinton 1972). Clustering of all kelampayan samples was also performed based on shared allele distance, DSA (Chakraborty & Jin 1993) using PowerMarker Version 3.25 and neighbour-joining tree was constructed using MEGA Version 4 (Tamura et al. 2007).

RESULTS AND DISCUSSION

The genetic diversity of kelampayan was successfully determined using dominant DNA markers based on inter-simple sequence repeats. Three ISSR primers produced 64 loci across all the 247 individuals ranging from 500 bp to 2.0 kb in size. Out of 64 loci, 29 loci were generated by (GTG)6 primer, 16 loci by (AC)10 and 19 loci by (AG)10. The genetic diversity of kelampayan is summarized in Table 2. The percentages of polymorphic loci were in the range of 45.3–74.6% with an average of 58.2%. Shannon’s diversity indices (I) ranged 0.268–0.350. It was found that the genetic diversity of kelampayan in the present study was higher compared with its natural populations, with Shannon’s diversity indices ranging 0.154–0.235 and polymorphic loci ranging 41.3–59.4% (Tiong et al. 2010).

Kelampayan progenies from Block 1 (0.350) were found to be more diverse compared with Blocks 2 and 3. Low percentages of polymorphic loci indicate that the genetic diversity of kelampayan is low when compared with other plant species such as Tectona grandis plus tree with 95.5% polymorphic loci (Narayanan et al. 2007), 100% in Asparagus acutifolius (Sica et al. 2005), 85.7% in Swertia chirayita (Joshi & Dhawan 2007) and Glycyrrhiza uralensis with 92.2% polymorphic loci (Yao et al. 2008) amplified by ISSR primers.

Table 2 Summary of mean Shannon’s diversity indeices and percentage of polymorphic loci in three different blocks

Block Shannon’s diversity index (I) Percentage of polymorphic loci (%)

Block 1 0.350 74.6

Block 2 0.270 45.3

Block 3 0.268 54.7

Mean 0.296 58.2

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Kelampayan progenies from the three different blocks were grouped into 6, 4 and 5 clusters for Blocks 1, 2 and 3 respectively (Figure 2). Each cluster consists of kelampayan progenies that originated from the selected mother trees in Pasai Bon, Niah and Lawas seed production areas (SPAs). In other words, all kelampayan progenies were genetically closely related. A study carried out by Tiong et al. (2010) demonstrated that the coefficient of population differentiation of six natural forests of kelampayan was low (Gst = 0.2013) compared with other species such as Ceriops tagal (Gst = 0.529) (Ge & Sun 2001), C. decandra (0.882) (Tan et al. 2005), Hagenia abyssinica (Gst = 0.25), (Feyissa et al. 2007) and Taxus fauna (Gst = 0.5842) (Shah et al. 2008). The low genetic differentiation value was also observed in other species such as Shorea leprosula, Gst = 0.085 (Lee et al. 2000), Larix potaninii, Gst = 0.116 (Yu et al. 2006) and Calocedrus macrolepis, Gst = 0.042 (Wang et al. 2003). Furthermore, relatively low genetic diversity or high level of genetic differentiation was also recorded in several conifers (Wang et al. 2003).

Figure 2 Neighbour-joining trees showing relationship among kelampayan trees by using 64 ISSR loci (a) Block 1, (b) Block 2 and (c) Block 3

L2T7

L1T6

L2T3

L1T7

L1T2

L8T7

L9T1

1L6

T2

L2T1

0

L6T4

L2T5

L5T8

L4T8

L2T9

L2T1

6

L1T9

L3T9

L2T6

L2T4

L2T13 L5T16

L3T3

L6T6 L9T10

L7T14 L2T1L7T6

L6T14L4T9

L4T3

L9T12

L6T3 L3T12

L2T11

L5T4L5T12L3T5

L4T13

L3T1

L5T11

L1T4

L8T13

L4T6

L3T14

L2T8L4

T10

L3T1

5

L1T1

2L4T1

2

L5T6L4

T16L5

T9L5T1

5

L2T15

L9T3

L1T8

L4T15

L1T5

L8T12L8T1

L6T1

L7T5

L7T11

L9T6L8T8L6T8

L1T11L9T15

L7T3 L3T13 L7T10

L3T4

L8T6L7T13

L8T2

L8T5

L7T9

L5T3

L6T12

L3T16L3T15

L3T14L1T12

L2T2L2T1

L2T3L2T5

L2T4

L2T10 L2T16

L2T13

L2T11 L2T8

L2T7

L2T6

L1T6

L1T4L1T9

L1T16

L1T8L1T5

L1T3L1T2

L1T7

L6T8

L6T1

1L6

T9 L6T7

L6T1

4L6

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3

L6T12

L6T5

L6T3L5T2

L5T10L6T2L5T7

L4T16 L4T13

L4T3L5T4

L4T14L5T6

L3T11L3T10L3T12

L3T9L3T7L3T3

L3T1

L3T13L4T10

L4T1

L4T11

L4T2L4T12L7T2

L7T1L7T3

L8T4L8T15

L8T8

L8T3L8T7L8T11

L8T9

L8T2L7T15

L7T13

L7T16

L7T6L7T11

0.02

L4T12

L2T1

5

L5T1

5

L6T5

L6T1

0L9

T7

L4T9

L4T2

L6T9

L6T8

L4T1

L7T1

3L3

T1L1

T7L8

T2L1

T2L3

T9

L6T4

L4T1

0

L6T1

1

L1T6

L4T1

4L6

T7

L3T2

L9T9

L5T13 L5T5

L3T12

L3T4L2T1

L9T10L4T8L8T1

L1T12L7T1L8T13L6T16

L2T14

L1T5

L3T6

L9T1

L1T11

L9T6L7T4L7

T12

L8T1

2

L8T1

6L6

T15

L8T5

L7T7

L9T4

L7T1

1L9

T5L8

T14

L9T2

L8T7

L3T8

L2T1

1L9

T8L7

T14

L7T5

L3T1

4L2

T10

L8T3

L7T2

L9T1

4

L1T4

L8T6L5

T10

L3T15L9T13

L5T4

L7T9L7T16

L5T16L8T4L5T7L5T14

L3T7L2T13L2T3

L1T8L7T3L1T10

L3T5 L1T9

L4T6

L3T11 L9T12

L4T4

0.02

(a)

(c)(b)

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The low level of genetic differentiation among populations of kelampayan may be due to long-distance gene flow that has occurred over fairly large geographical areas (224–519 km) (Table 3). As explained by Hamrick et al. (1992), long-lived woody species with large geographic ranges, outcrossing breeding systems, and wind- or animal-dispersed seeds typically display less variation among populations. Further studies are needed because there is lack of evidence regarding the reproductive biology as well as pollen and seed dispersal mechanisms of kelampayan.

Table 3 Geographical distances between three different kelampayan seed production areas in Sarawak

Pasai Bon Niah Lawas

Pasai Bon 295 km 519 km

Niah 295 km 224 km

Lawas 519 km 224 km

CONCLUSION

In this study, ISSR analysis was proven as a powerful tool for assessing genetic diversity of kelampayan progenies collected from the three progeny trial blocks at the Landeh Nature Reserve, Semengok, Sarawak. This preliminary information will form the base for kelampayan tree improvement and conservation programmes.

ACKNOWLEDGMENTS

The authors would like to thank all the laboratory assistants and foresters involved in this research programme for their excellent field assistance in sample collection. This work was part of the joint Industry-University Partnership Programme, a research programme funded by the Sarawak Forestry Corporation (SFC) and Universiti Malaysia Sarawak (UNIMAS).

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