DNA Genotyping of Kelampayan (Neolamarckia cadamba) Progenies (Sample Set I) Using Inter Simple Sequence Repeat (ISSR) Marker

TABLE OF CONTENT

Acknowledgement

List of Figures

List of Tables

List of Abbreviations

Abstract

Chapter 1 Introduction

Chapter 2 Literature Review
2.1 Neolamarckia cadamba (Roxb.) Bosser
2.2 DNA genotyping
2.3 ISSR markers and ISSR-PCR

Chapter 3 Materials and Methods
3.1 Sampling of plant materials
3.2 DNA extraction
3.3 ISSR-PCR optimization
3.4 AGE and PCR products visualization
3.5 Data analysis

Chapter 4 Results and Discussion
4.1 ISSR-PCR optimization
4.2 ISSR-PCR analysis
4.3 Data analysis
4.3.1 Data scoring
4.3.2 Genetic diversity
4.3.3 Genetic relatedness

Chapter 5 Conclusion and Recommendations

References

Appendix A

Appendix B

Appendix C

LIST OF FIGURES

Figure

2.1 Seedlings of Neolamarckia cadamba planted at the Forest Genomic and Informatics Lab, UNIMAS in 2011

2.2 N.cadamba growing in a tropical dipterocarp forest

2.3 The broad leaves of N.cadamba

2.4 The flower of N.cadamba

4.1 Optimization of PCR profiles (a) Annealing temperature, (b) MgCl2 concentration, and (c) Taq DNA polymerase concentration

4.2 Results of ISSR-PCR for (a) Matang 05, (b) Lawas 29 and (c) Lawas

4.3 Nei’s unbiased measures of biosystematics dendrogram of N. cadamba populations generated by POPGENE v.1.32 software

4.4 UPGMA dendogram showing relationship among N. cadamba progenies within Matang 05 population

4.5 UPGMA dendogram showing relationship among N. cadamba progenies within Lawas 24 population

4.6 UPGMA dendogram showing relationship among N. cadamba progenies within Lawas 29 population

4.7 The dendrogram for all the three populations, generated by using PowerMarker v 2.2 software

LIST OF TABLES

3.1 Conditions of PCR mixture for PCR amplifications using ISSR

3.2 Thermal cycling profile for ISSR-PCR amplification

4.1 Fragment size (bp) of detected loci of (AG)9C for N. cadamba

4.2 The genetic variation estimated based on the four stated indices

4.3 Nei’s genetic distance (below diagonal) and genetic identity. (above diagonal) among populations Matang 05 (pop 1), Lawas

24 (pop2) and Lawas 29 (pop3)

LIST OF ABBREVIATIONS

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DNA Genotyping of Kelampayan (Neolamarckia cadamba) Progenies (Sample Set I)

Using Inter Simple Sequence Repeat (ISSR) Marker

Valeria S Kabalon

Resource Biotechnology Programme

Faculty of Resource Science and Technology University Malaysia Sarawak

ABSTRACT

Neolamarckia cadamba, commonly known as Kelampayan is a fast-growing tropical timber species. It has many commercial applications such as production of plywood, pulp and paper, boxes and crates, furniture and light construction components. Molecular characterization of N.cadamba is needed so that the qualities of its progenies are maintained. In this project, DNA genotyping of Kelampayan were carried out to determine the genetic relatedness of Kelampayan progenies. One selected ISSR primers: (AG)9C was used to assess the genetic diversity and genetic relatedness of 150 N.cadamba progenies (half-sib) from Matang 05, Lawas 24 and Lawas 29. This primer had successfully amplified 17 loci ranging between 389 bp to 1,047 bp, by which Lawas 29 was the most polymorphic (100% polymorphic). The Shannon’s diversity index for Lawas 29 was the highest (0.4453) compared to Matang 05 (0.2064) and Lawas 24 (0.1731) populations while the G st value for the populations was 2.5837.The gene diversity within the population (H s) was 0.1783, whereas the total gene diversity (H t) between the populations was 0.2128.

Keywords: Neolamarckia cadamba, ISSR markers, molecular characterization, genetic relatedness, genetic diversity.

Neolamarckia cadamba, umumnya diketahui sebagai Kelampayan merupakan spesis kayu balak tropika yang cepat membesar. Ia mempunyai pelbagai aplikasi komersial seperti penghasilan papan lapis, pulpa dan kertas, kotak dan peti, komponen perabot dan pembinaan ringan. Pengkarakteran molekular N.cadamba diperlukan supaya kualiti generasinya dapat dikekalkan. Pengkarakteran DNA bagi Kelampayan telah dilaksanakan dalam projek ini untuk mengenalpasti keberkaitan genetik baka-baka Kelampayan. Satu primer ISSR iaitu (AG)9C telah dipilih untuk menentukan kepelbagaian genetik dan keberkaitan genetik bagi 150 anak benih N.cadamba yang berasal daripada Matang 05, Lawas 24 dan Lawas 29. Primer ini telah berjaya mengamplifikasikan 17 lokus-lokus, yang bernilai antara 389 pa sehingga 1,047 pa, dan populasi Lawas 29 mempunyai kadar polimorfik yang tertinggi (100% polimorfik). Indeks kepelbagaian Shannon ’ s bagi Lawas 29 merupakan yang tertinggi (0.4453) berbanding dengan populasi Matang 05 (0.2064) dan lawas 24 (0.1731), manakala nilai Gst bagi ketiga-tiga populasi adalah 2.5837. Nilai bagi kepelbagaian gen di antara populasi adalah 0.1783, manakala jumlah keseluruhan bagi kepelbagaian gen (Ht) di antara populasi adalah 0.2128.

Kata Kunci: Neolamarckia cadamba, penanda ISSR, pengkarakteran molekular, keberkaitan genetik, kepelbagaian genetik.

CHAPTER 1

Malaysian timber industry continues to be a major export benefactor for the nation as the demand for tropical timbers is always on hike. However, the country’s timber industry is now facing a serious problem as the prime sources for natural forest species are becoming limited due to deforestation activities. In order to ensure the continuous availability of timber sources in the future, commercial forest plantations of fast-growing species are practiced. Neolamarckia cadamba (Roxb.) Bosser (N. cadamba) of the Rubiaceae family is a typical pioneer, fast-growing tree species which grows up to 45 m tall, without branches for more than 25 m (Joker, 2000). N. cadamba self prunes and adapts very well in exploited areas (Ismail et al., 1995). It grows on varieties of soils and tolerates intermittent flooding (Joker, 2000). N. cadamba is widely distributed from India, Nepal, through Thailand and Indo-China, to Papua New Guinea (Willis 1973; Ridsdale 1978; Wong 1989; Joker 2000). It is commonly found within initial secondary forest areas below 1000 m altitude which receives more than 1500 mm rain per year, (Wong, 1989). Joker (2000) reported that due to its fast growing ability, N. cadamba is suitable for reforestation in watersheds and eroded areas, and also for windbreakers in agroforestry systems. According to Joker (2000), N. cadamba is a lightweight hardwood with poor durability, thus making it suitable for production of pulp for low- and medium- quality paper as well as for light indoors construction work (Joker, 2000). It is also reported that the leaves and bark of N. cadamba contains medicinal values (Patel and Kumar, 2007) and other commercial values (Joker, 2000). To date, the genetic information on N. cadamba is still limited and further research on it is of demand. Proper documentation on the genetic information of N.cadamba needs to be done for better understanding on this species itself. The aim of this study is to perform the DNA genotyping of N.cadamba using the ISSR markers. The term DNA fingerprinting refers to the method developed by Sir Alec Jeffreys (Jeffreys, 1985) and his associates in 1985 for the simultaneous detection of highly variable DNA fragments by hybridisation of specific multilocus probes to electrophoretically separated restriction fragments (Bhat, 2001). According to Bhat (2001), the DNA fingerprints which resembles barcodes, are unique to individuals and thus used in much the same way as conventional fingerprint which helps to identify individuals with supreme certainty. In this project, the ISSR markers are used to determine the genetic diversity of N. cadamba. Molecular markers which are based on DNA sequence polymorphisms are not influenced by environmental conditions and developmental stage of a particular plant and show high levels of polymorphisms (Domyati et al., 2010). ISSR markers have been developed as an anonymous, RAPDs-like approach which accesses variation in numerous microsatellites regions dispersed throughout the genome (Zietkiewicz et al., 1994). The ISSR markers are simple and have reproducibility and it requires small amount of DNA and does not require information on DNA sequence (Gupta et al., 1996). ISSR primers are designed from SSR motifs and can be undertaken for any plant species containing sufficient number and distribution of SSR motifs in the genome (Buhulikar et al., 2004). ISSR marker is based on a PCR-amplification of 100- 3000 base pair regions between inversely oriented SSRs or microsatellites (Bussell et al., 2005). Objective of this study was to determine the genetic diversity and genetic relatedness of N. cadamba progenies using ISSR marker.

The results from this research are useful for the conservation of gene and the tree improvement programme of this species.

CHAPTER 2 LITERATURE REVIEW

2.1 Neolamarckia cadamba (Roxb.) Bosser

The following is the taxonomy of Neolamarckia cadamba (Roxb.) Bosser (Joker, 2000):

Kingdom : Plantae

Subkingdom : Tracheobionta

Division : Magnoliophyta

Class : Magnoliopisida

Subclass : Asteridae

Order : Rubiales

Family : Rubiaceae

Genus : Neolamarckia

Species : Cadamba

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Figure 2.1 Seedlings of N. cadamba planted at the Sarawak Forest Tree Seed Bank nursery, SFC.

Neolamarckia cadamba (Roxb.) Bosser is a tropical rainforest timber species that grows fast and denominates the initial re-growth stage of tropical secondary forest (Meijer, 1970). N.cadamba (Roxb.) Bosser is well distributed from Nepal, India through Thailand and Indo-China to Malaysian Archipelago to Papua New Guinea. Joker (2000) also reported that it has been successfully introduced to Africa and Central America. N.cadamba (Roxb.) Bosser is commonly known as Kadam (India), Jabon (Jawa), Bangkal (Philippines), Laran (Sabah) and Kelampayan (Sarawak) (Joker, 2000). This timber species is a lightweight hardwood suitable for productions and manufactures of general utility furniture, plywood, pulps for low- and medium- quality papers. It is suitable for reforestation in watersheds and agroforestry systems, and also as shade tree for dipterocarp line planting (see Figure 2.2).

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Figure 2.2 N.cadamba growing in a tropical dipterocarp forest

Source:http://picasaweb.google.com/102561255743376862805/HanjaJabonSamama?feat=embedw ebsite

N. cadamba is a medium sized to large sized tree that can grow up to 44 m in height. It is bole straight, columnar and grows without branches for more than 25 m. The diameter of N. cadamba tree is normally up to 100 cm while the leaves (see Figure 2.3) are 13 to 32 cm long. Its crown is umbrella shaped and the branches are arranged in tiers. It has small orange flowers (see Figure 2.4) arranged in dense globose head (Joker, 2000). The fruits of N. cadamba are small capsules packed in a fleshy yellowish orange coloured infructescence that may contain up to approximately 8000 seeds. In Malaysia, the flowering season of N. cadamba is from June to September, while the fruiting season is from September to February (Joker, 2000).

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Figure 2.3 Figure 2.4

Figure 2.3. The broad leaves of N. cadamba. Figure 2.4. The flower of N. cadamba.

Source: http://en.wikipedia.org/wiki/Neolamarckia_cadamba

2.2 DNA genotyping

DNA genotyping is a taxonomic method used for the determination of similarities of selected species through screening of specific DNA sequences. It is performed by utilizing a sequence of nucleotides that aid in the specific identification of strain or sub strains of selected species (Yamamoto et al., 2006). DNA genotyping is normally used to identify cultivars, study plant evolutions and determine genetic relatedness of plants. DNA genotyping is dependable to the existence of numerous minisatellites families dispersed throughout the entire genome on hyper variable loci. Each of these minisatellites usually consists of tandem arrays of short repeat units and the DNA sequence variation exists in these repeated units (Lynch, 1988).

2.3 ISSR markers and ISSR-PCR

Knowledge of genetic diversity within and among populations is basically important for conservation management (Jian et al., 2004). Accurate estimates of genetic diversity are therefore useful for optimizing sampling strategies for conserving and managing the genetic diversity of trees (Hamrick and Godt, 1996).

To date, molecular markers have become common for measurement of diversity within plant populations (Smith and Wayne, 1996; Lalhruaitluanga and Prasad, 2009). DNA markers specifically characterize cultivars, provenances or genotypes and measure their genetic relationships. The markers are well heritable, environmentally stable and display adequate polymorphism to distinguish very closely related genotypes (Narayanan et al. 2007). The evaluation of genetic diversity and construction of linkage maps has been considered as desirable for the efficient use of genetic variations in the breeding programme (Tanksley et al., 1989).

ISSR-PCR is a genotyping technique based on variation found in the regions between microsatellites. It has been used in genetic fingerprinting (Blair et al., 1999), gene tagging (Ammiraju et al., 2001), detection of clonal variation (Leroy and Leon, 2000), cultivar identification (Wang et al., 2009), phylogenetic analysis (Gupta et al., 2008), detection of genomic instability (Anderson et al., 2001), and assessment of hybridization in many plant and animal species (Wolfe et al., 1998). The versatility of this genotyping technique makes ISSR useful for researchers interested in diverse fields such as conservation biology and cancer research.

ISSR markers are rapid and cost-effective and are broadly used for diversity analysis, mapping and genotype identification of plant species, including forest trees (Karp et al. 1998). ISSR analysis reported in the present work could be useful to select parents to be crossed for generating appropriate populations intended for both genome mapping and breeding purposes (Gupta et al., 2008). ISSR markers have been successfully used for varietal identification and assessment of genetic relationships in many plant species (Ajibade et al. 2000).

ISSR primers are able to amplify highly variable yet small segments, for example regions between two microsatellites of the genome (McGregor et al. 2000) and possibly make few loci available for amplification by ISSR primers (Zietkiewicz et al. 1994). ISSR technique also uses longer primers enabling higher annealing temperatures that result in greater reproducibility of the bands (Wolfe and Liston, 1998). In higher plants or animals, ISSR markers are of higher demand, because they are recognized to be abundant, very reproducible, highly polymorphic, highly informative and rapid in use (Zietkiewicz et al., 1994, Bornet et al., 2002). ISSRs was proposed for genetic diversity by Lalhruaitluanga and Prasad (2009) and commonly used in population genetics, taxonomy and phylogeny of many plant species (Wolf and Randle, 2001). ISSR primers can also confirm specific amplified DNA polymorphic fragments within varieties (Li and Ge, 2001).

ISSR fingerprinting is a method that combines most of the advantages of SSR and AFLP to the universality of RAPD. The main limitations of RAPD, AFLP, and SSR methods are low reproducibility of RAPD and high cost of AFLP while flanking sequences have to be known to develop species-specific primers for SSR polymorphism. ISSR overcomes most of these limitations (Reddy et al., 2002). ISSR fingerprinting was developed in a way that no sequence knowledge was required. Primers based on a repeat sequence and the resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. ISSR would be a better tool than RAPD for phylogenetic studies (Ajibade et al., 2000; Galvan et al., 2003). The main disadvantages of ISSR are the dominant nature and lower multiplex ratio (Yilmaz et al., 2009).

ISSR analysis is more economical and reliable than that of RAPD. Earlier studies also reported that ISSR technique generates large number of polymorphisms in chickpea (Collard et al., 2003). Because of the high polymorphism, ISSR have also been employed successfully in population genetic studies in many cultivated and wild plants (Huang and Sun, 2000; Esselman et al., 1999). ISSR also have proven useful in evaluating genetic diversity in the mangrove species Aegiceras corniculatum and Ceriops tagal (Ge and Sun, 1999, 2001). In a study done by Jian et al. (2004), the detection of high levels of polymorphism makes ISSR analysis a powerful tool for assessing genetic diversity in Heritiera littoralis. None of the individuals was genetically identical based on the ISSRs, indicating that the level of resolution in our study was sufficient to distinguish all genotypes. In preliminary studies, Jian et al. (2004) examined the repeatability of bands by both repeating the ISSR process in its entirety and running the same PCR product twice in separate lanes across 20 samples. It proves that patterns of ISSR are highly reproducible. The previous study by Narayanan (2007), they have reported the use of RAPD and ISSR markers for assessment of genetic diversity and DNA polymorphism among 48 plus trees of Tectona grandis (Teak) species . Results were useful for the efficient conservation of these plus trees and their sustainable use as suitable diverse parents in breeding strategy or as donors in clonal propagation of superior stock for teak improvement programmes.

Based on the research done by Phong et al. (2011), they reported that the total number of polymorphic bands (59) and average number of polymorphic bands/primer (2.19) detected by ISSR primers were much higher in Dalbergia assamica. This method is obviously advantageous in differentiating closely related cultivars and has been used for cultivar identification in numerous plant species, including rice (Joshi et al., 2000), apple (Goulaõ and Oliveira, 2001), mulberry (Zhao et al., 2006) and strawberry (Arnau et al., 2003).

CHAPTER 3 MATERIALS AND METHODS

3.1 Sampling of plant materials

A total of 150 healthy N. cadamba (half-sib) seedlings from Matang 05, Lawas 24 and Lawas 29 were chosen from the nursery of SFC as the research materials for this project. Green young leaf with good physical characteristics was taken from each of the chosen seedlings and packed into sampling bags which were then sealed carefully, numbered and labelled respectively based on their respective origins to avoid confusion. These leaves samples were then taken to the laboratory and were cleaned and sealed in clearly labelled sampling bags prior to storage at -20oC freezer until subjected to further use (DNA extraction). It is important that the amount of air in the sampling bags is minimized to ensure that the samples will last longer. It is also advised that the DNA extraction is carried out as soon as possible to get better qualities of DNA.

3.2 DNA extraction

DNA extractions of the N. cadamba samples were successfully carried out by using the fas Tip-X method described by Lai (2009). A sterile condition should be maintained on the working bench to avoid possible contaminations of the samples. 70% ethanol was used to establish the sterile condition and appropriate protective outfits was worn throughout the process. Six leaf discs were punched from each leaf sample by using 10 µL pipette tips to ensure uniform sizes of leaf discs. The leaf discs were placed in microcentrifuge tubes filled with 50µL extraction buffer. The samples were then be incubated at 95oC for 10 minutes. The tube were then be mixed intermittently by inverting and tapping of the tube. 120µL of dilution buffer were added prior to storage at -20oC freezer.

3.3 ISSR-PCR optimization

Extracted N. cadamba DNA samples were subjected to optimized PCR conditions with the selected ISSR primers. PCR reactions of a total 40 cycles were carried out using Mastercycler® Gradient PCR. PCR reaction mixture of 1X PCR buffer, 0.2 mM of each dNTPs, 2 mM MgCl2, 1 unit Taq polymerase, 1 µL template DNA and 10 pmol of primer were prepared (Chai, 2006). PCR optimizations were carried out using chosen ISSR primer,

(AG)9C in order to determine the annealing temperature, MgCl2 concentration and optimal DNA template concentration (Moh, 2006). Products of PCR were then checked using the agarose gel electrophoresis (AGE) using 1.5 % agarose gel and 6 V/cm.

Table 3.1. Conditions of PCR mixture for PCR amplifications using ISSR markers

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Table 3.2. Thermal cycling profile for ISSR-PCR amplification of (AG)9C

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