Post on 20-Jul-2015
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Rekayasa Tanaman III
Pemanfaatan Marka Molekuler
dalam Pemuliaan Tanaman
Tim Rektan, Lab. Pemuliaan tanaman
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Kompetensi
Mahasiswa akan dapat:
Menjelaskan aplikasi marka molekuler dalam pemuliaan
tanaman
Menjelaskan karakteristik marka molekuler
Menjelaskan perkembangan & teknik marka molekuler
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Apakah Marka Molekuler ?:
Aplikasi Marka Molekuler
Jenis – jenis marka/ penanda
1.Marka morfologi dan fisiologi: tinggi tanaman, warnadaun, kand. alkaloid, kand. klorofil.
2. Marka Biokimia: marka berbasis protein (isozym)
3. Marka Molekuler: berbasis molekul DNA
Molekul DNA yang digunakan untuk menandai sekuensDNA spesifik/gen/segmen genom suatu individu yang kita
inginkan.
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What are molecular markers?
• Genetic information resides in the genome
• A molecular marker is based on DNA sequence
• Polymorphisms arise by mutation
ATP5L2 ATP synthase
C22orf11 chromosome 22 ORF
PANX2 pannexin 2
…gtataagtgaaccactcagggtcctggccgggcacagtggctcacgcctgt
aatcccagcctttgggaggccgaggtgggtggatcatgaggtcaggagttcaa
gaccagcctggccaaatggcgaaacattgtctctactaaaagtacaaaaattag
ccagacgtggtggtgctcactgtaatcccagctactcgggaggctgaggcagg
aaaatcacttgaacccgggaggcggaggtcacagttagccaagatcacaccac
tgtactccag …
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Perfect DNA marker
(Linkage Gene)
Flanking marker
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Aplikasi Marka Molekuler:
Keragaman genetik (Genetic diversity) dan fingerprinting
Hubungan kekerabatan antar plasma nutfah
Genetic distances vs. heterosis Pemurnian varietas dan identifikasi
Hubungan filogenetik (Phylogenetic relationships)
Pewarisan dan pemetaan
Pewarisan karakter yang sederhana
Karakter Quantitative (dissection into QuantitativeTraits Loci - QTL)
Aplikasi Marka Molekuler
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Aplikasi Marka Molekuler:
Sebagai alat seleksi: Marker-Assisted Selection (MAS)
Karakter sederhana: konversi galur (faster backcross)
Transfer segment multiple genomic (QTL) Pyramidisasi gen-gen: mengumpulkan gen-gen baik
dalam satu individu
Introgresi suatu karakter dari plasma nutfah liar
Seleksi dan manipulasi alil-alil resesif Seleksi awal
Identifikasi dan isolasi gen dengan bantuan marka DNA
Aplikasi Marka Molekuler
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Hal yang mendasari aplikasi Marka molekuler:
Adanya variasi dalam DNA sequence
Yang disebabkan oleh adanya:
Single base mutation
Insertion / deletion
Rearrangement (perpindahan DNA
sequence dr satu posisi ke posisi yanglain)
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Tidak dipengaruhi lingkungan
Tidak dipengaruhi oleh tingkat
perkembangan tanaman (vegetatif-generatif: sama)
Tidak bersifat destruktif
Tidak ada interaksi interlocus
Kelebihan marka molekuler:
Properti Marka Molekuler
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Perkembangan Marka Molekuler
C A P A
C I T Y
1985 1990 1995 2000
DNA Chips
Multi-SSRs
AFLPsSSRs
RAPDsRFLPs
SNP
???
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Karakteristik marka yang baik
Sangat Polymorphic: ada perbedaan pola pitaDNA
Reproducible (dapat diulang dg hasil yg sama)
Kodominan (mampu membedakan antarahomosigot dan heterosigot)
Terdistribusi dalam genom
Murah dan akurat
Mudah dilaksanakan (dapat diotomatisasi)
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Polymorphism- yang mana?
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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Teknik-teknik Marka Molekuler:
RFLP (Restriction Fragment length Polymorphism)
RAPD (Random Amplified Polymorphism DNA)
Microsatellite/SSRs (Simple Sequence Repeats) AFLP (Amplified Fragment Length Polymorphism)
Teknik- teknik Marka Molekuler
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Pertimbangan dalam memilih marka DNA:
- Tujuan: diversity, isolasi gen, gen yang terpautdengan marka, seleksi, skrining, mapping,deteksi lokus karakter kuantitatif (QTL),
- Karakter yg susah/mahal diamati,
- Tingkat polimorfisme tinggi
- Pengembangan metode sudah established
- Ketersediaan peralatan pendukung- Ketersediaan SDM dan dana
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RFLP (Restriction Fragment Length
Polymorphism)Genomic DNA
Separation of fragments
Hybridisation
Autoradiography
1 2 3 4 5 6
Dipotong oleh E. restriksi
Southern Blot
Teknik- teknik Marka Molekuler
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RAPD’s (Random Amplified
Polymorphism DNA’s)
Genomic DNA
PCR-amplification
1 2 3 4 5 6short unspec. primer
Gel Electrophoresis
Teknik- teknik Marka Molekuler
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AFLP (Amplified Fragment Length
Polymorphisme)
Genomic DNA
Frequent + rare cutter digest
Adapter ligation
Amplification with
preselective primers
Amplification with
selective primers
AFLP-pattern
Gel electrophoretic analysis
Teknik- teknik Marka Molekuler
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SSR’s (Simple Sequence Repeats)
CGT CGT CGT CGT CGT CGT CGT
GCA GCA GCA GCA GCA GCA GCA
CGT CGT CGT CGT CGT CGT
GCA GCA GCA GCA GCA GCA
Genomic DNA
spec. primer
PCR-Amplification
Gel electrophoretic analysis
Teknik- teknik Marka Molekuler
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Molecular Markers - summary
• Many types of molecular markers available• Type(s) chosen for use will depend on many
factors
• Dominant or co-dominant, co-dominant
preferable in most cases
• Polymorphism is necessary for geneticmapping
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PERTANYAAN
????????
Marker Assisted Selection (MAS)
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Marker Assisted Selection (MAS)
Kegunaan Introgresi Alil pada QTL
Pengembangan galur tertentu yang digunakan secaraluas dalam program pemuliaan
Introgresi alil target yang terdapat pada plasma nutfahyang memiliki penampilan agronomi yang tidakdikehendaki
Tidak ada keragaman diantara plasma nutfah elit
untuk digunakan pada lingkungan yang dikehendaki
Introgresi Alil pada Gen yg sdh diidentifikasi Piramidisasi alil-alil elit dari suatu gen yang sudah
teridentifikasikan
Marker Assisted Selection (MAS)
Marker Assisted Selection (MAS)
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Pemulihan secara cepat melalui BC-MAS
Donor genome
Recurrent genome
F1 Backcross 1 Backcross 2 Backcross 3 Backcross 20 Backcross 100
Traditionalbackcrossbreeding
Years 0.5 1 1.5 2 10 50
Marker-assistedbackcrossbreeding
Years 0.5 1 1.5
Marker Assisted Selection (MAS)
Marker Assisted Selection (MAS) Kelemahan
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Kelemahan MAS
Keterbatasan teoritis
Kompleksitas genetik karakter yang
ingin dikembangkanAkurasi evaluasi fenotipik
Evaluasi interaksi QxE
Evaluasi interaksi epistasis
Marker Assisted Selection (MAS)-Kelemahan
Marker Assisted Selection (MAS) Kelemahan
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Kelemahan MAS
Keterbatasan secara Praktikal
Jml. Sampel yang dapat dianalisis terbatas
Keyakinan bahwa identifikasi QTL dibutuhkanbilamana dilakukan penambahan plasmanutfah
Jml. Genotip yang dpt dikembangkan dalamwaktu tertentu terbatas
Marker Assisted Selection (MAS)- Kelemahan
MAS untuk QPM
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MAS Untuk Quality Protein of Maize
Tier 1
Tier 2
Tier 3
Tier 4
SSR Phi 57
MAS untuk QPM
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