DNA Analysis Materials and Methods

149

8.2.1. DNA Analysis

a. Plant materials. Sample used were obtained from fresh dried material kept in Silica Gell collected both from the field and plant growing at green houese of Bogor Botanic Gardens Table 8.1.. Voucher specimens were deposited in Herbarium Bogoriense BO and Herbarium of Bogor Botanic GardensBOHB. b. DNA Extraction. DNA was extracted by a modified CTAB method of Doyle and Doyle 1978 or using the Dneasy Plant Mini Kit Qiagen, Inc., Tokyo following the manufacturer’s intruction. 1 500 um CTAB was poured in the 1.5 ml tube and it was incubated on 60ºC. 2 A piece of lamina ± 4 cm2 was ground on the bowl by using liquit nitrogen until forming smooth powder . 3 The smooth powder was poured into the tube containing 500 um CTAB and shaked for 1 minute and then incubated in 60ºC for 30-60 minutes. c. Separating DNA from Protein. 1 500 um of cloroform and isoamil alcohol mixture was poured into the tube containing sample and 500 um CTAB and shaked by hand for 10 minutes. 2 The tube was centrifuged on 20ºC for 20 minutes at 15000 rpm. 3 The top layer containing water with DNA was poured into the 1.5 um tube. Step no.1 – no.3 were repeated once more. d. DNA Purification. 1 400 ul Isoamilalcohol was added into the tube containing DNA liquit and than kept in the freezer for15 minutes or longer. 2 The tubes were centrifuged at 0ºC for 15 minutes at 15000rpm. 3 The liquid was disposed of the residu was DNA. 4 300 ul 70 Ethanol was added into the tube and shaked by using vortex and centrifuged at 20ºC for 10 minutes 15000rpm. 5 The ethanol was evaporated by using aspirator. 6 Tubes containing DNA was dried in the desicator for10 minutes. 7 The DNA was dissolved in 100um TE and than shaked well. 150 e. rbcL Multiplication by Using PCR. PCR was performed using a DNA thermal cycler Perkin-Elmer 9700, Applied Bosystems, CA with Ex Taq DNA polymerase TaKaRa Biomedical, Tokyo. Some samples were amplified with Amp direct Shimazu, Kyoto, whike most others were amplified with Ex buffer. 1 10xPCR Buffer, dNTPmix, 10 pM primer x 2, MgCl2, DNA template, Taq polymerase and DW Destilate Water were poured into 0.2 ml tubes. Taq must be kept at 0ºC in the ice box before PCR was begun to run. 2 PCR was runned at 94ºC for 3 minutes and turned on 35 cycles at 94ºC for 30 seconds, at 48ºC for 40 second, at 72ºC for 90 seconds, and at 72ºC for 7 minutes and than the tubes were kept at 4ºC. f. Checking PCR Product by Using Electrophoretic 1 making gel agarose LE 1. 2 3.5 ul PCR product was mixed with dye in the parafilm and than poured into sample holes 3 Elektroforesi was runned 4 When blue colour reaching the forth line electrophoretic was stopped. Gel was moved and pu t into etidiumbromid for 20 minutes. 5 Gel was illuminated by using UV beam; if bars pictures with proportion sizes were seen it was concluded that PCR product have been multiplied well. g. PCR Product Purification PCR products were purified with GFX DNA and GEL purification Kit Amarsham Pharmacia Biotech, Piscataway, NJ or with ExoSAP-IT USB corporation, Ohio following the manufacture’s intruction. a. Autocycle. 1 3 ul premix, 0.5 ul sequence buffer, 0.5 ul MgCl2, 1.6 ul 2pm primer, and 4.4 ul DNA were put into the 0.2 ml tubes. The premix was containing of dNTP, ddNTP, buffer and taq polymerase. Taq polymerase must be kept at 0ºC before autocyle. 2 PCR was begun at 96ºC for 90 second and than runned at 30 cycles 96ºC for 10 second, at 50ºC for 15 second and at 60ºC for 4 minutes and than the tubes were kept at 4ºC. b. Purifying Autocycle Product. 1 12.5ul 100 Etanol and 0.5um 3M sodium asetat were put into 1.5 ml tubes 2 Autocycle product were added into the tubes and shaked by using vortex. 3 The tubes were kept in the ice box for 20 minutes. 4 The tubes were centrifuged for 20 minutes at 20ºC 15000rpm and than their liquit were run off. 5 250ul etanol 70 was added into 151 the tubes and than shaked by using vortex. 6 The tubes were centrifuged at 20ºC for 10 minutes at 15000rpm speed and than ethanol was evaporated by using aspirator. 7 Autocycle product in the tubes were dried in the desiccator for 15 minutes. h. Gel Squencing Preparation 1 Glass plate for gel was washed off with water and rinsed off with miliQ and isoprophanol, and than dried at room temperature. 2 preparing gel. 18g urea was mixed with 5 ml 10x TBE buffer and amount of water and than stirred by using magnetic stirrer for 10 minutes 3 The mixture was added with water till 48 ml volume. 4 The mixture was evaporated by using aspirator and than kept in the refrigerator 5 The mixture was evaporated by using dessicator for 5 minutes. 6 10 APS was made in the tube. 7 Glass plate was lay out on the flat tabel, tweezers were tightened 8 TEMED 30ul and 270ul APS 10 were poured into the mixture and shaked manually 9 The Gel was poured into the glass plate and than chomb was installed. 10 After 2 hours, manicure was smeared for preventing buffer spilled out. i. Preparing Samples for Squencing 1 Formamide and blue dextran 5:1 mixtured was made. 3ul DNA sample for every coll. number were also prpepared. All samples and blue dextran must alwasy be kept in the ice box and blue dextran. 2 The tubes containing samples were shaked by using shaker for 5 minutes. 3 The tubes cantaining were incubated at 98ºC for 2 minutes and than kept in the ice box.. j. Run Sequencer 1 Chomb was removed from gel and than changed with the chomb with 36 holes. 2 Glass plate gel was washed and rinsed off with isopropanol and mili-Q till the white line not seen. 3 Glass plate gel was installed on the chamber sequencer and than cassette recorder was also installed on the Sequencer. 4 Checking glass plate gell if the noise voice was heared from the machine we must clean again the plate. 5 Chamber was installed on the sequencer and than 1x TBE buffer was poured into the chamber. 6 All of the bubbles attaching the holes chomb must be removed by using injector. 152 Table 8.1. List of Taxa Used in This Study Species Locality Collector and Number Herbarium Holding Voucher Genebank Accession No. References Outgroup Athyrium distentifolium Unknown AB059577 Yatabe and Murakami2001 Athyrium filix-femina L. Roth ex Meretens AY818676 Skog et al 2004 Athyrium niponicum Mett. Hance Tokyo, Japan CT1005 TI AB232413 Tsutsumi and Kato 2006 Athyrium vidalii Miq. Koidz. Chiba, Japan Sano 25 CBM D43894 Sano et al 2000b Athyrium sheareri Aichi, Japan Sano 41 CBM D43892 Sano et al 2000b Athyrium yokoscense Fr. Et Sav. Christ Chiba, Japan Sano 22 CBM D43893 Sano et al 2000b Ingroup Diplazium angustipinna Holttum Near Sungai Kobet, Track to Batu Ayau, Muller Range, Central Kalimantan, Borneo. 440 m. T.Ng. Praptosuwiryo 1905 BO Present Study Diplazium asymmetricum Praptosuwiryo Sp. Nov. Petak 4 Desa Kemutuk Lor, Converted forest-Natural forest boundary, Wanawisata Baturraden, Mt. Slamet, Cetral Java. 970-1000 m. T.Ng. praptosuwiryo 1094 BO Present Study Diplazium asymmetricum Praptosuwiryo Sp. Nov. 2=1 Loop Trail, Cikaniki Forest Research, Mt. Halimun, West Java. Ca. 1000 m T.Ng. praptosuwiryo 1728 BO Present Study Diplazium cordifolium Blume ‘simple fronds’ Cangkuang Forest, Southern Slopes of Mt. Salak, West Java T.Ng. Praptosuwiryo 1202 BO Present Study 153 Table 8.1. Continued Species Locality Collector and Number Herbarium Holding Voucher Genebank Accession No. References Diplazium cordifolium Blume ‘Pinnate fronds’ Cangkuang Forest, Southern Slopes of Mt. Salak, West Java T.Ng. Praptosuwiryo 1237 BO Present Study Diplazium cordifolium Blume ‘Pinnate fronds’ Cangkuang Forest, Southern Slopes of Mt. Salak, West Java T.Ng. Praptosuwiryo 1238 BO Present Study Diplazium cf cordifolium Blume Primary forest between S. Anak Kobet- S. Kobet, track to Batu Ayau, Central Kalimantan, Borneo. T.Ng. Praptosuwiryo 1912 BO Present Study Diplazium accedens Blume Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1001 BO Present Study Diplazium accedens Blume Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1161 BO Present Study Diplazium accedens Blume Cangkuang Forest, Southern Slopes of Mt. Salak, West Java. T.Ng. Praptosuwiryo 1211 BO Present Study Diplazium bantamense Blume Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park, West Java T.Ng. Praptosuwiryo 1160 BO Present Study Diplazium batuayanense Praptosuwiryo Above Sungai Kobet, Track to Batu Ayau, Muller Range, Central Kalimantan, Borneo. 440 m. T.Ng. Praptosuwiryo 1909 BO Present Study D_cavalerianum Christ. C.Chr. Chiba, Japan Sano 11 CBM D43909 Sano et al 2000b D. chinense Bak. C.Chr. Kumamoto, Japan Ohta Takamiya 739 KUMA AB021718 Sano et al 2000b 154 Table 8.1. Continued Species Locality Collector and Number Herbarium Holding Voucher Genebank Accession No. References Diplazium dilatatum Blume Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1011 BO Present Study Diplazium simplicivenium Holttum Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1025 BO Present Study Diplazium simplicivenium Holtum Converted Forest, Petak 55, Desa Karang Mangu, Wana Wisata Baturraden, G. Slamet, Baturraden, Central Java. 860 m. T.Ng. Praptosuwiryo 1073 BO Present Study Diplazium dilatatum Blume Kagoshima, Japan Ohta Takamiya 602 KUMA AB021719 Sano et al 2000b Diplazium donianum Mett. Tard. Kagoshima, Japan Sano 29 CBM D43911 Sano et al 2000b Diplazium esculentum Retz. Sw. Situ Patengan, Mt. Patuha, Bandung, West Java. T.Ng. Praptosuwiryo 730 BO Present Study Diplazium esculentum Retz. Sw. Gede-Pangrango National Park, West Java T.Ng. Praptosuwiryo 1227 BO Present Study Diplazium esculentum Retz. Sw. Kagoshima, Japan Hasebe 276001 TI U05619 Hasebe et al 1994 Diplazium hottae Tagawa Near Sungai Anak Kobet, Track to Batu Ayau, Muller Range, Central Kalimantan, Borneo. 450 m. T.Ng. Praptosuwiryo 1911 BO Present Study Diplazium lobbianum Moore Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1007 BO Present Study 155 Table 8.1. Continued Species Locality Collector and Number Herbarium Holding Voucher Genebank Accession No. References Diplazium lobbianum Moore Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1031 BO Present Study Diplazium lobbianum Moore Cibodas Forest, Behind Cibodas Botanic Gardens, Track I Mt. Gede, Gede-Pangrango National Park, West Java. 1500 m T.Ng. Praptosuwiryo 1181 BO Present Study Diplazium lobbianum Moore Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park, West Java T.Ng. Praptosuwiryo 1008 BO Present Study Diplazium lonchophyllum Kunze Unknown U05920 Wolf et al 1994 Diplazium megasegmentum Praptosuwiryo Sp.Nov. Cangkuang Forest, Southern Slope, Mt. Salak, West Java. T.Ng. Praptosuwiryo 1450 BO Present Study Diplazium mesosorum Tochigi, Japan Sano 44 CBM D43910 Sano et al 2000 Diplazium okudairaekami Diplazium pallidum Blume Moore Near Telaga Warna, Gede-Pangrango National Park, West Java T.Ng. Praptosuwiryo 1172 BO Present Study Diplazium pallidum Blume Moore Mt. Payung, Ujung Kulon National Park, West Java T.Ng. Praptosuwiryo 1406 BO Present Study Diplazium poiense C.Chr. in C.Chr. Holttum Unknown TML1 Diplazium polypodioides Blume Cangar Nature Reserve, Mt. Welirang, Batu, East Java. 1370-1400 m. T.Ng. Praptosuwiryo 667 BO Present Study Diplazium polypodioides Blume Cibodas Forest, Mt. Gede, Gede- Pangrango National Park, West Java T.Ng. Praptosuwiryo 1185 BO Present Study 156 Table 8.1. Continued Species Locality Collector and Number Herbarium Holding Voucher Genebank Accession No. References Diplazium porphyrorachis Unknown TML2 Diplazium procumbens Holttum Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1015 BO Present Study Diplazium procumbens Holttum Cibodas Forest, Track I of Mt. Gede, Behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1047 BO Present Study Diplazium procumbens Holttum Cibodas Forest, Behind Cibodas Botanic Gardens, Track I Mt. Gede, Gede-Pangrango National Park, West Java T.Ng. Praptosuwiryo 1163 BO Present Study Diplazium procumbens Holttum Converveted Forest-Natural Forest Boundary, Petak 4 Desa Kemutuk Lor Wana Wisata Baturraden, Mt. Slamet, Central Java. 970-1000 m. T.Ng. Praptosuwiryo 1094 BO Present Study Diplazium procumbens Holttum Cimisblung, Mt. Masigit, Mt. Pangrango, Gede-Pangrango National Park. 1300 m. T.Ng. Praptosuwiryo 1281 BO Present Study Diplazium procumbens Holttum Cangkuang Forest, Souther Slopes of Mt. Salak, West Java. T.Ng. Praptosuwiryo 1216 BO Present Study Diplazium rhachidosorus Unknown TML3 Diplazium riparium Holttum Unknown TML4 157 Table 8.1. Continued Species Locality Collector and Number Herbarium Holding Voucher Genebank Accession No. References Diplazium sibiricum var. sibiricum Akita, Japan Ohta Takamiya 695 KUMA AB021720 Sano et al 2000b Diplazium silvaticum Bory Sw. Wild fern of Bogor Botanic Gardens, Java T.Ng. Praptosuwiryo 1300 BO Present Study Diplazium silvaticum Bory Sw. Wild fern of Bogor Botanic Gardens, Java T.Ng. Praptosuwiryo 1302 BO Present Study Diplazium silvaticum Bory Sw. Wild fern of Bogor Botanic Gardens, Java T.Ng. Praptosuwiryo 1303 BO Present Study Diplazium simplicivenium Holttum Track to Cibeureum, Mt. Gede, Gede- Pangrango National Park, West Java T.Ng. Praptosuwiryo 1141 BO Present Study Diplazium simplicivenium Holttum Rawa Denok I, Gede-Pangrango National Park, West Java T.Ng. Praptosuwiryo 1171 BO Present Study Diplazium simplicivenium Holttum Near Batu Kukusan I, Gede-Pangrango National Park, West Java T.Ng. Praptosuwiryo 1175 BO Present Study Diplazium simplicivenium Holttum Cibodas Forest, Mt. Gede, Gede- Pangrango National Park, West Java T.Ng. Praptosuwiryo 1179 BO Present Study Diplazium sorzogonense C. Presl. Loop Trail , Cikaniki Research Center, Mt. Halimun, West Java. Ca. 1000 m. T.Ng. Praptosuwiryo 1720 BO Present Study Diplazium sorzogonense C. Presl. Loop Trail , Cikaniki Research Center, Mt. Halimun, Halimun National Park, West Java. Ca. 1000 m. T.Ng. Praptosuwiryo 1725 BO Present Study Diplazium sorzogonense C. Presl. Loop Trail, Cikaniki Forest Research Center, Mt. Halimun, Halimun National Park, West Java. Ca. 950 m. T.Ng. Praptosuwiryo 1731 BO Present Study Diplazium sorzogonense C. Presl. Jalur Owa, Cikaniki Reseacrh Center, Mt. Halimun, West Java. Ca. 975 m. T.Ng. Praptosuwiryo 1744 BO Present Study 158 Table 8.1. Continued Species Locality Collector and Number Herbarium Holding Voucher Genebank Accession No. References Diplazium speciosum Blume Geger Bentang, Mt. Pangrango, Gede- Pangrango National Park, West Java. 1960 m. T.Ng. Praptosuwiryo 1243 BO Present Study Diplazium squamigerum Mett. Matsum. Kumamoto, Japan Ohta Takamiya 893 KUMA Sano et al 2000b Diplazium subpolypodioides Alderw. Alderw. Cibodas Forest, Mt. Gede, Gede- Pangrango National Park, West Java. T.Ng. Praptosuwiryo 1184 BO Present Study Diplazium subserratum Blume Moore. Converted Forest, Petak 55, Desa Karang Mangu, Wana Wisata Baturraden, G. Slamet, Baturraden, Central Java. 860 m. T.Ng. Praptosuwiryo 1070 BO Present Study Diplazium subserratum Blume Moore. Converted Forest, Petak 55, Desa Karang Mangu, Wana Wisata Baturraden, G. Slamet, Baturraden, Central Java. 860 m. T.Ng. Praptosuwiryo 1072 BO Present Study Diplazium subvirescens Praptosuwiryo Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park 1500 m. T.Ng. Praptosuwiryo 1178 BO Present Study Diplazium subvirescens Praptosuwiryo Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National T.Ng. Praptosuwiryo 1012 BO Present Study Diplazium tomentosum Blume Loop Trail, Cikaniki Forest Research, Mt. Halimun, West Java. Ca. 1000 m T.Ng. Praptosuwiryo 1722 BO Present Study Diplazium umbrosum Smith Bedd. Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1002 BO Present Study 159 Table 8.1. Continued Species Locality Collector and Number Herbarium Holding Voucher Genebank Accession No. References Diplazium umbrosum Smith Bedd. Cibodas Forest, Track I of Mt. Gede, behind Cibodas Botanic Gardens Guest House, Gede-Pangrango National Park T.Ng. Praptosuwiryo 1050 BO Present Study Diplazium wichurae Mett. Diels Shizuoka, Japan Sano 13 CBM D43915 Sano et al 2000b Diplazium xiphophyllum Baker C.Chr. Eas Kalimantan, Borneo TD 902 Living Coll. at Nuserry of BBG Present Study Diplazium xiphophyllum Baker C.Chr. Loop Trail, Cikaniki Forest Research, Mt. Halimun, West Java. Ca. 950 m. T.Ng. Praptosuwiryo 1717 BO Present Study Diplazium xiphophyllum Baker C.Chr. Loop Trail, Cikaniki Forest Research, Mt. Halimun, West Java. Ca. 950 m. T.Ng. Praptosuwiryo 1719 BO Present Study Diplazium xiphophyllum Baker C.Chr. HM 39 Cikuda Paeh, Cikaniki Forest Research, Mt. Halimun, West Java. T.Ng. Praptosuwiryo 1791 BO Present Study 160 7 Prerun for 5 minutes. 8 Stopping for a moment and than samples with odd number collectons were injected into the odd holes. 9 Resume for 5 minutes. 10 Stop prerun and than the sample with the even collentions number were injected into the even holes and than the chambercover was installed 11 Running. k. Gene rbcL sequence alignment. The PCR product were sequenced with the Big Dye Terminator Cycle Squencing Kit Ver. II or Ver. III Applied Biosystems, CA following manufacturer’s intructions with amplification primers of Hasebe et al Tabel 1.. l. Homology searching on the data base by internet using a reprsentative database of nucleotide sequence, DNA DataBase of Japan DDBJ, http:www.ddbj.nig.ac.jp to compare predicted gene sequence with the registered sequence in the database. Table 8.2. Primers Used for Amplifying and Sequencing DNA from Diplazium Hasebe et al, 1994 Primer Sequence 5’ to 3’ Position rbcL -aF rbcL-bF rbcL-sR rbcL -aR rbcL -cF rbcL-sF rbcL -cR rbcL-bR ATGTCACCACAAACAGAGACTAAAGC TATCCCCTGGATTTATTTGAGGAAGGTTC GAACCTTCCTCAAATAAATCCAGGGGATA CTTCTGCTACAAATAAGAATCGATCTCTCCA TGAAAACGTCGTGAATTCCCAACCGTTTATGCG ACTGTAGTGGGCAAATTGGAAGGCGAACG GCAGCAGCTAGTTCCGGGCTCCA CGTTCGCCTTCCAATTTGCCCACTACAGT 1-26 307-335 335-307 670-640 609-638 988-1016 1373-1351 1016-988 161

8.2.2. Phylogenetic analysis