Seed Germination and DNA genotyping of Neolamarckia cadamba (Roxb.) Progenies (half-sib family).

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2 Seed Germination and DNA genotyping of Neolamarckia cadamba (Roxb.) Progenies (half-sib family). Nurfaizah Matra 1, Petrus Bulan 2, Ho Wei Seng 1 1 Forest Genomic and Informatics laboratory (fgil), Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak. 2 Department of Plant Science and Environmental Ecology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak. Abstract Neolamarckia cadamba or locally known as Kelampayan is an economically and ecologically important forest tree species. The species is an indigenous fast growing tree that gives early economic returns, within eight to ten years. Kelampayan has been planted in plantation for commercial purposes due to its wood characteristics that are suitable to produce different products, such as plywood and paper. In the present study, germination test was used to determine the seed viability and to gain more information with respect to field planting value of the Kelampayan seeds. Germination test also can provide results which can be used to compare the value of different seed lots. Fruits of ten mother trees from the Ravens Court, Lawas, Sarawak, were processed and air-dried to collect the seeds. The germination test was conducted on the filter paper under optimum condition and the growth rates between progenies of different mother trees were compared. Progenies from mother tree number 00059, showed the highest germination rate (88%) while progenies from mother tree number showed the lowest germination rate (14%). The information on germination test can be used to predict the rate of germinating progenies from different mother trees and to determine the planting value of seed lots to provide standardized marketing seed. For DNA genotyping, a total of 139 progenies from the 7 selected mother trees earlier were genotyped using four SSR primers, namely AC03, AC11, AC15 and GTG11. All 4 loci were successfully amplified and a total of 17 alleles were detected. The value for H e was highest for NCAC11 (0.7190) and lowest for NCGTG11 (0.3832). Introduction Kelampayan (Neolamarkcia cadamba) is a fast growing tree species that grows naturally in Australia, China, India, Indonesia, Malaysia, Papua New Guinea, Philippines, Singapore and Vietnam. It is a favoured plantation species inside and outside its native range. It has been planted as an ornamental and plantation tree and has been successfully introduced into Costa Rica, Puerto Rico, South Africa, Surinam, Taiwan, Venezuela and other tropical and subtropical countries (Orwa et al. 2009). N. cadamba is lightweight hardwood. The wood is mainly suitable for multiple end uses, such as plywood, light construction materials, flooring, beams and rafters, boxes and crates, teachests, packing cases, shuttering, ceiling boards, toys, wooden shoes, bobbins, yokes, carvings, matches, chopsticks and pencils (Soerianegara and Lemmens 1993). The dried bark is used to relieve fever and as a tonic. A yellow dye, obtained from the bark of the roots, can serve as tannin or dyestuff (Soerianegara and Lemmens 1993). N. cadamba is considered as one of the best raw material for plywood industries and expected to become increasingly important for wood industries. Since most of the plywood industries are facing acute shortage of raw material, plywood manufacturers will be glad to purchase the trees at remunerative prices. The pulp is sometimes mixed with other, generally long-fibred material to produce medium quality paper. The tree is also suitable for ornamental use and shade along roadsides and villages as well as for shelter for other crops in agroforestry systems. It is also used in reforestation and afforestation programmes (Orwa et al. 2009). Germination testing is designed to estimate the maximum number of seeds that will produce a normal seedling and to give results that are as repeatable as possible. Without uniform procedures, there would be no standard on which to base the value of seedlots for commercial transactions and the seed trade would be chaotic and filled with dispute. Germination also tells a grower about a seedlots potential (Karrfalt, 2004). A seed lot is composed of a population of individual seed units; each possessing its own distinct capability to produce a mature plant. A seed vigour test is an analytical procedure to evaluate seed vigour under standardized conditions. It enables a seed producer to determine and compare the vigour of a seed lot before it is marketed (McDonald, 1988). Seed testing played an important role in guaranteeing good quality seed to the farmer (Gassim, 1988). A normal seedling has all the essential plant structures necessary for the plant to continue to grow normally under favourable conditions (AOSA 1996; ISTA 1996).

3 Microsatelite markers have been used for genetic diversity studies, because of their large number, distribution throughout the genome, high level polymorphism, co-dominant inheritance, neutrally with respect to selection and easy automation of analytical procedures (Canon et al, 2001). SSRs are composed of a 1- to 6-base pair (bp) DNA sequence that is repeated a variable number of times. SSRs are amplified by PCR with primers that are complementary to the conserved sequences that flank an SSR locus. Polymorphic fragments (alleles) resulting from variations in SSR repeat length are separated electrophoretically to display genetic profiles of individuals. SSR alleles typically show monogenic-codominant inheritance that enables classification of homozygotes and heterozygotes in a segregating population (Narvel et. al., 2000) The existence of large gene pool is important for the potential future mating system and for the development of sustainable timber tree production system. The ability of N. cadamba to grow very fast on a variety of soils, its favourable silvicultural characteristics and the absence of serious pests and diseases, has brought N. cadamba for industrial plantations as well as for reforestation and afforestation. This species is also expected to become increasingly important for wood-based industries, particularly when the supplies for plywood from natural forests decrease. Hence, studying the marketing potential of N. cadamba and its plantation investment opportunities are of considerable interest. In this study, seed germination test was conducted to compare the value of different seed lots from ten different mother trees, which will be used to predict the rate of germinating progenies from different mother trees and to determine the planting value of seed lot to evaluate seed vigour under standardized conditions. For DNA genotyping, SSR primers were used to determine genetic diversity and genetic relationship among seven mother trees and their progenies. Materials and Methods Sample Collection N. cadamba seeds were collected from the Ravens Courts, Lawas, Sarawak. The ripe fruits were soaked in water for a week until rotted, ground by hand, and air dried to collect the seeds. Once the seeds were totally dried, the seeds were stored in an air-tight glass jar and kept in -20 C until used. Germination Test Germination tests should be run in cabinets or rooms that meet exacting requirements for temperature and light control in order to make accurate and repeatable estimates (Karrfalt, 2004). Seeds require certain conditions for normal germination. The most important requirements are substrata, moisture, temperature and light (Chauhan et. al, 2009). The germination test was conducted on the filter paper under optimum condition. Plant Growth Chamber was used to provide controlled and normal condition for seed germination test. For each mother tree seedlots, seeds were germinated on Petri dishes with each treatment was replicated four times at a rate of 100 seeds per Petri dish. Germination counts were made from the 3 rd days after sowing up to 51 days consecutively. DNA Extraction The total DNA was isolated from 7 mother trees and 139 seedlings following the modified protocol of the CTAB method by Doyle and Doyle (1990). Subsequently, the purified total DNA was quantified by gel electrophoresis, the DNA quality was verified by spectrophotometer. The DNA concentration of each sample was diluted to 50ng/µl. Data Analysis For DNA genotyping, four SSR primers, namely AC03, AC11, AC15 and GTG11 were used in this study. Table 1 lists the sequences of the primers. The observed heterozygosity (H o ), expected heterozygosity (H e ), F IS, F IT and F ST and genetic distance among populations were calculated with Population Genetics Analysis 3.2 (POPGENE) software. Polymorphism information content (PIC), gene diversity was computed using Powermarker 3.25 software. A phylogenetic tree and radiation tree was constructed by UPGMA method based on pairwise Nei s standard genetics distances using the same software. Results and Discussion Germination Test: In this study, progenies from mother tree number showed the highest germination (88%), while progenies from mother tree number showed the lowest germination (14%) (Table 2). Progenies from mother tree might have the possibility to have higher germination rate on the field compared to other mother trees in this study.

4 Table 2: Number of seed germination for each day observed and total mean number for ten different mother trees Sample Day/ mean no. of seed germination Total germination Germination Percentage (%) Genetic Variability at microsatellite loci All 4 loci were successfully amplified and a total of 17 alleles were detected; NCAC03 and NCA11 showed the highest number of alleles per locus (5) while NCGTG11 the lowest (3) (Table 3). The four primers used were found to have high polymorphism in all populations, with PIC ranging from (NCGTG11) to (NCAC11). The mean gene diversity across loci was indicating high level of information of the chosen microsatellite set (Table 4). Overall, the mean H e across loci was approximately , while the H o was The value for H e was highest for NCAC11 (0.7190) and lowest for NCGTG11 (0.3832). Table 3: Number of alleles per locus Locus Total No. of Alleles NCAC03 5 NCAC11 5 NCAC15 4 NCGTG11 3 Total 17 Table 4: Polymorphic information content (PIC), genetic diversity, observed heterozygosity (H o ), expected heterozygosity (H e ), seven N. cadamba populations. Locus PIC Gene Diversity H o H e NCAC NCAC NCAC NCGTG Mean Conclusion The information on germination test and SSR data analysis in this study will be very useful for genetic diversity study and also in determining the mating system parameters. Thus, very important in determining the genetic structure and evolutionary inherent of natural populations.

5 Acknowledgement We gratefully acknowledge the Universiti Malaysia Sarawak (UNIMAS) and Sarawak Forestry Corporatiom (SFC) for providing financial support for these studies. References AOSA Rules for testing seeds. Journal of Seed Technology 16(3): 1B113. Doyle, JJ and Doyle JL Isolation of plant DNA from fresh tissue. Focus 12: ISTA [International Seed Testing Association] International Rules for Seed Testing, Seed Science and Technology 21(Suppl.): 1B288. J.S. Chauhan, Y.K. Tomar, N. Indrakumar Singh, Seema Ali, Anoop Badoni, Debarati and A. Rana Assessment of Compatible Substratum for Andrographis paniculata Standard Seed Germination Testing. Journal of American Science,;5(6): Canon, J., Alexandrino, P., Bessa, I., Carleos, C., Carretoro, Y., Dunner, S., Ferran, N., Garcia, D., Jordana, J., Laloe, D., Pereira, A., Sanchez, A., Moazarmigouudarzi, K., Genetic Diversity of Local European Beef Cattle Breeds for Conservation purposes. Genet. Sel. Evol. (33), (2001). Gassim, AB. Seed quality and vigour. In: Quality Seed Production. Van Gastel, A.J.G. and Kerley, J. (Eds.), 1988; pp ICARDA, Syria. James M. Narvel, Walter R. Fehr, Wen-Chy Chu, David Gran c and Randy C. Shoemaker Simple Sequence Repeat Diversity among Soybean Plant Introductions and Elite Genotypes. CELL BIOLOGY & MOLECULAR GENETICS. Crop Science: 40: McDonald, M.B Challenges in seed technology. pp In Proceedings 10th Seed Technology Conference. ed. J.S. Burris. Ames, IA. Karrfalt, R. P Seed Testing. USDA Forest Services National Tree Seed Laboratory, Dry Branch, Georgia. Nei, M Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, 89, Orwa, C., Mutua, A., Kindt, R., Jamnadass, R. And Anthony, S Agroforestry tree database: a tree reference and selection guide version AFTPDFS/Anthocephalus_cadamba.pdf [7 December 2010]. Richter, H.G. & Dallwitz, M.J Commercial Woods < -intkey.com%2fwood%2fde%2fwww%2frubanchi.htm --> Accessed on 20 August Soerianegara, I. and Lemmens, R.H.M.J Plant resources of South-east Asia 5 (1): Timber trees: Major commercial timbers. Pudoc Scientific Publishers, Wageningen, Netherlands. Weir, B. S., Cockerham, C.C., Estimating F-statistics for the analysis of population structure. Evolution (38), (1984).