Genetic Diversity Analysis of Jatropha Curcas Provenances Using Randomly Amplified Polymorphic DNA Markers

Genetic Diversity Analysis of Jatropha CurcasProvenances Using Randomly Amplified PolymorphicDNA Markers. Dani Satyawan and I Made Tasma.Jatropha curcas nuts are rich in oil that is higly suitable forHak Cipta © 2011, BB-Biogenthe production of bio-diesel or to be used directly inmodified diesel engines. The objective of this study was toassess the extent of genetic diversity among 50 J. curcasprovenances and one accession of J. integerrima usingRAPD markers. The fifty J. curcas provenances werecollected from ecologically diverse regions of Indonesia, andplanted in the Pakuwon Experimental Station (Sukabumi,West Java). Fourteen RAPD primers with 60-80% G+Ccontent were used in this genetic diversity analysis andproduced 64 bands with 95.7% polymorphism level. ThePolymerase Chain Reactions used to generate the RAPDbands sometimes produced inconsistent and nonreproducibleresults, necessitating the duplication of eachreaction to prevent scoring errors. Sixty one validated bandswere subsequently used for genetic diversity analysis usingUnweighted Pair Group Method Arithmetic (UPGMA)method and Dice coefficients. It was shown that thesimilarity coefficients among the provenances ranged from0.2 to 0.98 with an average similarity of 0.75. Dendrogramanalysis produced two major groups of provenances, withone outlier from South Lampung. There was no tendency forprovenances originated from nearby regions to clustertogether in each group, and several provenances showedmore similarities with provenances originated from distantregions. This pattern lent credence to reports that Jatrophawas introduced to Indonesia around four centuries ago andwas mainly spread by humans. Based on the meansimilarities among the accessions and their clusteringpattern, the genetic diversity of the Jatropha collectionappeared to be fairly low. Future additions of geneticmaterials from more diverse genetic background will benecessary to maintain the current progress of Jatrophaimprovement program

Pembentukan Pustaka Genom, Resekuensing, Dan Identifikasi SNP Berdasarkan Sekuen Genom Total Genotipe Kedelai Indonesia

Resequencing of the soybean genome facilitates SNP marker discoveries useful for supporting the national soybean breedingprograms. The objectives of the present study were to construct soybean genomic libraries, to resequence the whole genome offive Indonesian soybean genotypes, and to identify SNPs based on the resequence data. The studies consisted of genomiclibrary construction and quality analysis, resequencing the whole-genome of five soybean genotypes, and genome-wide SNPidentification based on alignment of the resequence data with reference sequence, Williams 82. The five Indonesian soybeangenotypes were Tambora, Grobogan, B3293, Malabar, and Davros. The results showed that soybean genomic library wassuccessfully constructed having the size of 400 bp with library concentrations range from 21.2–64.5 ng/μl. Resequencing of thelibraries resulted in 50.1 x 109 bp total genomic sequence. The quality of genomic library and sequence data resulted from thisstudy was high as indicated by Q score of 88.6% with low sequencing error of only 0.97%. Bioinformatic analysis resulted in atotal of 2,597,286 SNPs, 257,598 insertions, and 202,157 deletions. Of the total SNPs identified, only 95,207 SNPs (2.15%) werelocated within exons. Among those, 49,926 SNPs caused missense mutation and 1,535 SNPs caused nonsense mutation. SNPsresulted from this study upon verification will be very useful for genome-wide SNP chip development of the soybean genome toaccelerate breeding program of the soybean

Phylogenetic and Maturity Analyses of Sixty Soybean Genotypes Used for DNA Marker Development of Early Maturity Quantitative Trait Loci in Soybean

Phylogenetic and Maturity Analyses of Sixty SoybeanGenotypes Used for DNA Marker Development of EarlyMaturity Quantitative Trait Loci in Soybean. I MadeTasma, Dani Satyawan, Ahmad Warsun, MuhamadYunus, and Budi Santosa. The Indonesian soybeanproductivity is still very low with the national average of 1.3t/ha. One means to improve national soybean productivity isby manipulating harvest index by cultivating very earlymaturing soybean cultivars. Development of early maturingsoybean cultivars can be expedited by using marker-aidedselection. The objective of this study was to select parentallines having contrasted maturity traits and selected parentsmust be genetically distance. The parents then were used todevelop F2 populations for detecting early maturity QTL insoybean. Maturity tests of 60 soybean genotypes wereconducted at two locations, Cikeumeuh (Bogor) and Pacet(Cianjur) using a randomized block design with threereplications. Genomic DNA of the 60 genotypes wereanalyzed using 18 SSR markers and genetic relationship wasconstructed using the Unweighted Pair-Group MethodArithmatic through Numerical Taxonomy and MultivariateSystem program version 2.1-pc. Results showed that the 60genotypes demonstrated normal distribution in bothlocations for days to R1 (32-48d), days to R3 (35-55d), days toR7 (75-92d), and days to R8 (78-99d). Four early maturinggenotypes and three late genotypes were obtained. TotalSSR alleles observed were 237 with average allele per locusof 12.6 (3-29), and average PIC value of 0.78 (0.55-0.89).Genetic similarity among genotypes ranges from 74.8-95%.At similarity level 77% divided the genotypes into six clusters(the four selected early maturing genotypes located inclusters III and IV, while the three late genotypes located incluster II). Based on maturity data, pubescent color, andphygenetic analysis seven parents were selected (four earlymaturing genotypes B1430, B2973, B3611, B4433 and threelate genotypes B1635, B1658, and B3570). Twelve F2populations were developed with the aid of SSR markersSatt300 dan Satt516. Two of the populations will be used todevelop DNA markers for earliness in soybean

Genome-Edited Plants and the Challenges of Regulating Their Biosafety in Indonesia

Genome editing is a precise breeding technique to improve plant properties by editing specific genes that regulate desired trait. Genome editing techniques can be designed so that the resulting plant does not contain foreign genes and the resulting changes in DNA sequences cannot be distinguished from products obtained by conventional gene mutations which have been considered as safe and therefore unregulated. Thus, genome editing products in some countries are also not specifically regulated as GM products even though their assembly process uses recombinant DNA and genetic transformation. Brazil, likeIndonesia ratified the Cartagena Protocol, but it issued a special  regulation that provides dispensation for several types of genome editing products and exempts them from regulations that apply to transgenic plants. The steps taken by other countries in regulating genome editing products can be taken into consideration in drafting regulations in Indonesia, in order to create a conducive environment that supports the use of this potential technology while at the same time provides assurance regarding its safety to human health and the environment. The purpose of this review was to provide information onthe development of genome editing technologies in plant breeding, analyze its risks compared to that of conventional breeding, and compare its biosafety regulation in various countries to provide some considerations for drafting regulations on the risk assessment of genome editing products in Indonesia, as a ratifying country of the Cartagena Protocol

Pembentukan Pustaka Genom, Resekuensing, dan Identifikasi SNP Berdasarkan Sekuen Genom Total Genotipe Kedelai Indonesia

Resequencing of the soybean genome facilitates SNP marker discoveries useful for supporting the national soybean breedingprograms. The objectives of the present study were to construct soybean genomic libraries, to resequence the whole genome offive Indonesian soybean genotypes, and to identify SNPs based on the resequence data. The studies consisted of genomiclibrary construction and quality analysis, resequencing the whole-genome of five soybean genotypes, and genome-wide SNPidentification based on alignment of the resequence data with reference sequence, Williams 82. The five Indonesian soybeangenotypes were Tambora, Grobogan, B3293, Malabar, and Davros. The results showed that soybean genomic library wassuccessfully constructed having the size of 400 bp with library concentrations range from 21.2–64.5 ng/μl. Resequencing of thelibraries resulted in 50.1 x 109 bp total genomic sequence. The quality of genomic library and sequence data resulted from thisstudy was high as indicated by Q score of 88.6% with low sequencing error of only 0.97%. Bioinformatic analysis resulted in atotal of 2,597,286 SNPs, 257,598 insertions, and 202,157 deletions. Of the total SNPs identified, only 95,207 SNPs (2.15%) werelocated within exons. Among those, 49,926 SNPs caused missense mutation and 1,535 SNPs caused nonsense mutation. SNPsresulted from this study upon verification will be very useful for genome-wide SNP chip development of the soybean genome toaccelerate breeding program of the soybean

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

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 Hiseq2000Conventional 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

GENOMIC VARIATION OF FIVE INDONESIAN CACAO (Theobroma cacao L.) VARIETIES BASED ON ANALYSIS USING NEXT GENERATION SEQUENCING

Indonesian cacao productivity is still low mainly due to the lack availability of superior cacao planting materials. A new breeding method is necessary to expedite cacao yield improvement programs. To date, no study has yet been done to characterize Indonesian cacao varieties at the whole genome level. The objective of this study was to characterize genomic variation of five superior Indonesian cacao varieties using next-generation sequencing. Genetic materials used were five Indonesian cacao varieties, i.e. ICCRI2, ICCRI3, ICCRI4, SUL2 and ICS13. Genome sequences were mapped to the cacao reference genome sequence of Criollo variety. Sequence alignment and genomic variation discovery were done using Bowtie2 and mpileup software of Samtools, respectively. A total of 2,326,088 single nucleotide polymorphisms (SNPs) and 362,081 insertions and deletions (Indels) were obtained from this study. In average, a DNA variant was identified in every 121 nucleotides of the genome sequence. Most of the DNA variants were located outside the genes. Only 347,907 SNPs and Indels (13.18%) were located within protein coding region (exon).  Among the DNA variations within exon, 188,949 SNPs caused missense mutation and 1,535 SNPs induced nonsense mutation.  Unique gene-based SNPs were also discovered from this study that can be used as fingerprints for the particular cacao variety. The DNA variants obtained were excellent DNA marker resources to support cacao breeding programs. The SNPs discovered are useful as materials for genome-wide SNP chip development to be used for gene and QTL tagging of important traits for expediting national cacao breeding program

IDENTIFICATION OF SINGLE NUCLEOTIDE POLYMORPHISMS ON CATTLE BREEDS IN INDONESIA USING BOVINE 50K

Single nucleotide polymorphisms (SNPs) abundant in bovine genome influence genetic variation in biological mechanism. The study aimed to identify SNPs on Indonesian cattle breeds and analyze their genetic diversity using Bovine 50K SNP chip. Twenty eight "Ongole Grade" (OG) beef cattle and 20 "Holstein Friesian" (HF) dairy cattle were used for the Infinium II assay test. This assay included amplification of genomic DNA, fragmenta-tion, precipitation, resuspension, hybridization, processing bead chip for single-base extension, and imaging at iScan. Data and clusters were analyzed using GenomeStudio software. The Bovine 50K SNP chip containing 54,609 SNPs was observed spanning all chromosomes of bovine genome. Genotyping for the total SNPs was successfull based on Call Rate, GeneCall and GeneTrain scores. Most SNP markers had alleles that shared among the individuals or breeds, or had specific alleles at distinctive frequencies. Minor allele frequency (MAF) spreads equally with intervals of 0-0.5. The breeds of OG and HF tended to be separated in different clusters without considering their genetic history and twin or normal. This result suggests that most individuals are closely related to one another, regardless of the same breed. Some genes identified on chromosomes 3, 4, 5, 7, 13, 17 and 18 were located in the loci/regions that contained SNPs with specific alleles of either HF or OG breed. These SNPs were more powerful for differentiation of beef cattle and dairy cattle than among individuals in the same breed. These SNP variations and genetic relatedness among individuals and breeds serve basic information for cattle breeding in Indonesia

Genomic Variation of Five Indonesian Cacao (Theobroma Cacao L.) Varieties Based on Analysis Using Next Generation Sequencing

Indonesian cacao productivity is still low mainly due to the lack availability of superior cacao planting materials. A new breeding method is necessary to expedite cacao yield improvement programs. To date, no study has yet been done to characterize Indonesian cacao varieties at the whole genome level. The objective of this study was to characterize genomic variation of five superior Indonesian cacao varieties using next-generation sequencing. Genetic materials used were five Indonesian cacao varieties, i.e. ICCRI2, ICCRI3, ICCRI4, SUL2 and ICS13. Genome sequences were mapped to the cacao reference genome sequence of Criollo variety. Sequence alignment and genomic variation discovery were done using Bowtie2 and mpileup software of Samtools, respectively. A total of 2,326,088 single nucleotide polymorphisms (SNPs) and 362,081 insertions and deletions (Indels) were obtained from this study. In average, a DNA variant was identified in every 121 nucleotides of the genome sequence. Most of the DNA variants were located outside the genes. Only 347,907 SNPs and Indels (13.18%) were located within protein coding region (exon). Among the DNA variations within exon, 188,949 SNPs caused missense mutation and 1,535 SNPs induced nonsense mutation. Unique gene-based SNPs were also discovered from this study that can be used as fingerprints for the particular cacao variety. The DNA variants obtained were excellent DNA marker resources to support cacao breeding programs. The SNPs discovered are useful as materials for genome-wide SNP chip development to be used for gene and QTL tagging of important traits for expediting national cacao breeding program