Lampiran 1. Urutan basa dari 4 primer SSR. Nama Primer Sekuen (5 3 )
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1 45 Lampiran 1. Urutan basa dari 4 primer SSR Nama Primer Sekuen (5 3 ) 1 FR FR FR FR 3745 F: 5 - GAATGTGGCTGTAAATGCTGAGTG -3 R: 5 - AAGCCGCATGGACAACTCTAGTAA -3 F: 5 - AATGCAGACCAAGCTAATCATATAC -3 R: 5 - GTTCAGGTGATGGTGACTCAGATAG -3 F: 5 -AAGCAATATAGGTCAGTG-3 R: 5 -TCATTTCTAATTCAATAC-3 F: 5 - GGAAGTCTTGATGTTGAAAG -3 R: 5 - ATCAAGCAGTCGCATAATAC -3
2 46 Lampiran 2. Pembuatan Larutan Stok CTAB 5 % (100 ml): Timbang NaCl sebanyak 2.0 gram dan CTAB sebanyak 5.0 gram. Masukkan bahan kimia ke dalam erlenmeyer dan ditambahkan 100 ml aquades. Aduk campuran larutan dengan menggunakan stirrer kemudian diletakkan diatas hote plate. Tris HCl 1 M ph 8.0 (100 ml): Timbang Tris sebanyak gram. Masukkan Tris ke dalam erlenmeyer dan ditambahkan 80 ml aquades. Aduk campuran larutan dengan menggunakan stirrer kemudian diletakkan diatas hote plate. Selanjutnya, ditambahkan 4.2 ml HCl pekat sedikit demi sedikit sampai ph mencapai 8. Masukkan ke dalam gelas ukur, kemudian volume ditepatkan dengan aquades hingga volume larutan menjadi 100 ml. Tris HCl 1 M ph 7.4 (50 m): Timbang Tris sebanyak gram. Masukkan Tris ke dalam erlenmeyer dan ditambahkan 30 ml aquades. Aduk campuran larutan dengan menggunakan stirrer kemudian diletakkan diatas hote plate. Selanjutnya, ditambahkan NaOH 2.5 M sedikit demi sedikit sampai ph mencapai 7.4. Masukkan ke dalam gelas ukur, kemudian volume ditepatkan dengan aquades hingga volume larutan menjadi 50 ml. EDTA O.5 M ph 8.0 (100 ml): Timbang EDTA sebanyak gram dan NaOH 2.0 gram. Masukkan bahan kimia ke dalam erlenmeyer dan ditambahkan 80 ml aquades. Aduk campuran larutan dengan menggunakan stirrer kemudian diletakkan diatas hote plate. Selanjutnya, ditambahkan HCl pekat sedikit demi sedikit sampai ph mencapai 8. Masukkan ke dalam gelas ukur, kemudian volume ditepatkan dengan aquades hingga volume larutan menjadi 100 ml.
3 47 NaCl 5 M (l00 ml): Timbang NaCl sebanyak gram. Masukkan ke dalam erlenmeyer dan ditambahkan 80 ml aquades. Aduk campuran larutan dengan menggunakan stirrer kemudian diletakkan diatas hote plate. Masukkan ke dalam gelas ukur, kemudian volume ditepatkan dengan aquades hingga volume larutan menjadi 100 ml. **Semua bahan di atas disterilkan dengan menggunakan autoklaf kecuali CTAB 5%.
4 48 Lampiran 3. Pembuatan Larutan Buffer Buffer Ekstraksi/CTAB (100 ml): Campurkan 40 ml CTAB 5%, 25.1 ml NaCl 5 M, 4 ml EDTA 0.5 M ph 8.0, 10 ml Tris HCl 1 M ph 8.0 dan 20.8 ml aquades. Buffer TAE 50 X (100 ml): Campurkan 24.2 ml Tris HCl 1 M ph 7.4, 5.7 ml Asam Asetat Glasial, 10 ml EDTA 0.5 M PH 8.0, dan aquades hingga volume larutan menjadi 100 ml. Buffer TAE 1X (500 ml): Campurkan 10 ml Buffer TAE 50 X dan 490 ml aquades. Buffer TE (50 ml): Campurkan 0.5 ml Tris HCl 1 M PH 8.0, 0.1 ml EDTA 0.5 M PH 8.0 dan 49.4 ml aquades. Kloroform Isoamilalkohol 24 : 1 (50 ml): Campurkan 48 ml Kloroform dan 2 ml Isoamilalkohol. Etanol 70 % (100 ml): Campurkan 70 ml Etanol dan 30 ml aquades.
5 49 Lampiran 4. Alur Penelitian Pengambilan Sampel DNA Klon Kelapa Sawit Isolasi DNA Uji Kualitas Uji Kuantitas Amplifikasi PCR-SSR Analisis Hasil Amplifikasi PCR-SSR Elektroforesis
6 50 Lampiran 5. Kode sampel klon BTC 60 PT Socfindo No. Kode Sampel Klon
7 51 Lampiran 6. Uji kuantitas klon kelapa sawit BTC 60 Kode Sampel Absorbansi ( Å260 /280) Konsentrasi (μl/mg) 1 2,03 41,5 2 1,16 12,8 3 2,53 7,9 4 1,60 134,1 5 1,34 116,4 6 2,40 46,9 7 1,66 52,2 8 1,36 105,5 9 1,33 34,4 10 2,01 19,2 11 1,98 31,2 12 1,12 3,1 13 1,48 41,1 14 2,50 16,0 15 1,48 40,4 16 3,15 27,0 17 1,37 86,4 18 1,57 45,7 19 1,05 69,7 20 1,98 58,7 21 2,03 37,1 22 1,26 41,7 23 1,80 12,4 24 1,58 15,2 25 1,62 104,7 26 1,37 40,6 27 1,67 32,5 28 2,23 25,6 29 1,62 33,3 30 3,32 23,7 x ± Sd 1.79 ± ± x ± 2Sd 1.79 ± ± 66.79
8 52 Lampiran 7. Gambar sampel klon Kelapa Sawit BTC 60 PT. Socfindo sampel 1 sampel 2 sampel 3 sampel 4 sampel 5 sampel 6 sampel 7 sampel 8 sampel 9 sampel 10
9 53 sampel 11 sampel 12 sampel 13 sampel 14 sampel 15 sampel 16 sampel 17 sampel 18 sampel 19 sampel 20
10 54 sampel 21 sampel 22 sampel 23 sampel 24 sampel 25 sampel 26 sampel 27 sampel 28 sampel 29 sampel 30
11 55 Lampiran 8. Analisis faktorial dengan menggunakan DARwin 6.0 Factorial analysis on dissimilarity Dissimilarity file: KLON BTC 60.dis Dissimilarity calculated from data file: KLON BTC 60.var (type:'allelic') User selection Units: 30/30 and Loci: 4/4 Dissimilarity index: Simple matching Missing data options: No missing data 1000 bootstraps Imported Allelic data Ploidy = selected units on 30 Selected unit list: Number Unit No. Sampel Warning: At least one negative eigenvalue. (smallest eigenvalue: ) Non Euclidean dissimilarity Axis Eigenvalue Inertia% Axis Eigenvalue Inertia%
12 56 There is only 2 axes with positive eigenvalue (user selection was 5 axes) Unit coordinates on factorial axes WARNING: negative eigenvalue => cosinus² may be negative or greater than one! 1st column = coordinate 2nd column = cosinus² x 1000 Axis 1 Axis 2 Coord. Cos² Coord. Cos²
13 57 Lampiran 9. Analisis Kluster Metode UnWeighted Neighbor-Joining dengan Menggunakan DARwin 6.0 Tree construction Dissimilarity file: KLON BTC 60.dis Tree file : KLON BTC 60.arb Method: UnWeighted Neighbor-Joining Dissimilarity min value = Dissimilarity max value = selected units on 30 Selected unit list: Number Unit No. Sampel Iteration: 1 - Maximum level = Iteration: 2 - Maximum level = Iteration: 3 - Maximum level = Iteration: 4 - Maximum level = Iteration: 5 - Maximum level =
14 Iteration: 6 - Maximum level = Iteration: 7 - Maximum level = Iteration: 8 - Maximum level = Iteration: 9 - Maximum level = Iteration: 10 - Maximum level = Iteration: 11 - Maximum level = Iteration: 12 - Maximum level = Iteration: 13 - Maximum level = Iteration: 14 - Maximum level = Iteration: 15 - Maximum level = Iteration: 16 - Maximum level = Iteration: 17 - Maximum level = Iteration: 18 - Maximum level = Iteration: 19 - Maximum level = Iteration: 20 - Maximum level = Iteration: 21 - Maximum level =
15 Iteration: 22 - Maximum level = Iteration: 23 - Maximum level = Iteration: 24 - Maximum level = Iteration: 25 - Maximum level = Iteration: 26 - Maximum level = Iteration: 27 - Maximum level = Last grouping Edges and lengths : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : 0.062
16 : : : : : : : : : : : : : : : : : : : : : : : : : : 0 Edge length sum = Edge bootstrap values (%) : 68 Average 'edge' distance between initial tree and bootstrapped trees: percentile: percentile: 1
17 61 Lampiran 10. Jarak genetik klon BTC 60 berdasarkan 4 marka SSR Units
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