Konstruksi Pustaka Genom Kakao (Theobroma cacao L.) untuk Sekuensing Genom Total Menggunakan Next Generation Sequencing HiSeq2000

I Made Tasma, Dani Satyawan, Habib Rijzaani, Rubiyo Rubiyo

Abstract


Pemuliaan kakao secara konvensional memerlukan waktu panjang (10-15 tahun). Pemanfaatan marka DNA akan memperpendek siklus pemuliaan kakao. Tujuan penelitian ini adalah mengkonstruksi pustaka genom tiga genotipe kakao yang dapat digunakan untuk sekuensing genom total kakao menggunakan NGS HiSeq2000 dan mendapatkan data resekuen genom total tiga genotipe kakao.  Bahan tanaman terdiri dari tiga klon unggul kakao (ICCR02, ICCR04, dan SUL02) diperoleh dari Balittri, Pakuwon.  DNA genomik diisolasi dari daun muda sebagai bahan konstruksi pustaka genom total. Sekuensing pustaka dilakukan pada mesin HiSeq2000 mengikuti protokol dari Illumina. Pustaka genom yang telah berhasil dikonstruksi berukuran 300 pasang basa (bp) masing-masing dengan konsentrasi 14,70 ng/µL (ICCR02), 15,20 ng/µL (ICCR04), dan 12,90 ng/µL (SUL02). Ukuran dan konsentrasi pustaka genom yang dihasilkan sangat ideal untuk sekuensing menggunakan HiSeq2000. Sekuensing ketiga genom menghasilkan data sekuen 52,9 x 109 bp.  Klaster DNA pustaka genom memiliki nilai Q scores>30 (75,0%) dengan tingkat kesalahan pembacaan basa rendah (1,47%).  Nilai densitas klaster, persen klaster PF, intensitas basa, persen phasing, dan persen prephasing menunjukkan kualitas klaster pustaka genom ketiga genotipe kakao termasuk kategori pustaka ideal. Data sekuen yang dihasilkan juga sangat ideal untuk identifikasi marka SNP genom kakao. Koleksi marka SNP digunakan untuk identifikasi gen pengendali karakter penting kakao dan pemuliaan berbasis marka DNA untuk memperpendek siklus pemuliaan kakao.

Genomic Library Construction Of Cocoa (Theobroma Cacao L.) For Whole Genome Sequensing Using A Next Generation Sequencer Hiseq2000

Conventional cocoa breeding is slow and takes about 10-15 years to complete a breeding cycle. Applying genomic technology using DNA markers will significantly decrease cocoa breeding cycle. The objectives of this study were to construct cocoa whole genome genomic libraries to be used for resequencing the whole genome of cocoa and obtain whole genome resequence data of three cocoa genotypes. Three Indonesian cocoa genotypes (ICCR02, ICRR04, and SUL02) were used. DNA genomic was isolated from young leaf and used to construct genomic DNA libraries and generate DNA clusters. DNA clusters were sequenced using a HiSeq2000 platform. The whole genome libraries of the cocoa genotypes were successfully constructed. The library size was 300 bp with concentrations of 14.70 ng/µL (ICCR02), 15.20 ng/µL (ICCR04), and 12.90 ng/µL (SUL02), respectively. The genomic library size and concentrations are suitable for sequencing study using the NGS HiSeq2000. Total sequencing output obtained was 52.9 x 109 bp. The genomic library clusters resulted during the sequencing process demonstrated the Q scores > 30 of 75.0% with low error sequencing rate of 1.47%. Cluster densities, percentage of cluster PF, base intensity, and percentage of phasing and prephasing indicated the cluster quality of the genomic libraries is classified as an ideal one to be used for resequencing study using NGS HiSeq2000. The resequence data were ideal for SNP marker discovery. SNP markers are used to identify economically important genes of cocoa and marker-aided cocoa breeding to decrase the cocoa breeding cycle.


Keywords


Genomic library; whole genome; sequencing; HiSeq2000; cocoa

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DOI: http://dx.doi.org/10.21082/jtidp.v3n2.2012.p99-108

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This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.


 Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.


 

P-ISSN: 2356-1297
E-ISSN: 2528-7222
Accredited No.699/AU2/P2MI-LIPI/10/2015 on Oktober 30, 2015 by Indonesian Institute of Science

                    


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