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

Molecular Marker-Assisted Selection of Rice Grain Quality on Rice (Oryza Sativa L.) Lines Tolerant to Fe Toxicity Stress

The elite rice has been produced, including iron (Fe) tolerant varieties. To get the appropriate rice lines which superior not only Fe tolerant but also have good grain quality needs to be developed selection system, especially in the use of molecular markers. This study was aimed to develop molecular markers for selection the rice grain quality characters of selected rice lines Fe tolerant. A total of 30 selected Fe tolerant rice lines and 5 parents as control lines were used in this research. Characterization of grain quality were quantitatively using the standard. While for genotyping analysis used 19 molecular markers of STS, SSR, Indel and SNP. This study showed that 14 of 19 markers result polymorphic DNA band (DNA markers). Association analysis of genotype and phenotype showed that 10 of 14 markers were significantly (p < 0.05) related to high quality of rice grain. Among four types of markers used in this study, STS was the most widely associated significantly with four characters of rice quality. The phenotyping analysis showed that the physical grain and palatability quality which obtained from the total mean of 30 rice lines tested tend to nearly with the parent\u27s value as controls lines. The most of these lines were included in the group IV of National Rice Grain Quality Standard (SNI). The amylose content (AC) showed that the texture was varied from firm and dry (high AC) to soft and sticky (low AC). The association results showed that there were significant (p ≤ 0.05) markers related with the biosynthesis starch genes, i.e: SBE1, SS1, SSIIa, GPA, PUL and S3cl which contributed on the character of rice palatability. These selected significant markers could be useful for screening of other population with Fe tolerant and/ or other desired morpho-agronomical traits in support of rice breeding program 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

Keragaman Genetika Empat Belas Aksesi Kentang (Solanum Tuberosum L.) Berdasarkan Marka SSR Dan STS (Genetic Diversity of Fourteen Potato Accessions Based on SSR and STS Markers)

Potato is one of high economically horticultural plant. The increasing of national consumption of potato becomes a challenge forpotato breeders. The success of breeding programs is depending on availability of genetic diversity. The aim of this research wasto analyze the genetic diversity of fourteen accessions of potato by using SSR and STS markers. PCR analysis was scored as binerdata and the collected data was analyzed using NTSYS and PowerMarker. The result showed that there were 63% polymorphic(12 markers) of total markers. As many as 60 alleles with the size of 200–500 bp were identified by a range of 2–9 alleles perlocus. The polymorphism level was 0.59 (0.36–0.74). Result also showed the average of major allele frequency was 49.42%(35.71–63.64%). Nine markers which have polymorphism level more than 0.5 could be used to detect genetic diversity of potato.The average of genetic diversity index was 0.65. Cluster analysis showed that 14 accessions of potato were split in two groups(coefficient 0.70). The first groups consisted of Atlantik, GM 05, Granola Kembang Merbabu 17, and the second groups consist ofRepita, Maglia, Medians, CIP397078.7, CIP392781.1, Margahayu, Granola, CIP394613.139, Amabile, and Tenggo. The informationof genetic diversity of this germplasm could be used as a preliminary basis for choosing crossing parents in potato breeding inIndonesia

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

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

Keragaman Genetika Empat Belas Aksesi Kentang (Solanum tuberosum L.) Berdasarkan Marka SSR dan STS (Genetic Diversity of Fourteen Potato Accessions Based on SSR and STS Markers)

Potato is one of high economically horticultural plant. The increasing of national consumption of potato becomes a challenge forpotato breeders. The success of breeding programs is depending on availability of genetic diversity. The aim of this research wasto analyze the genetic diversity of fourteen accessions of potato by using SSR and STS markers. PCR analysis was scored as binerdata and the collected data was analyzed using NTSYS and PowerMarker. The result showed that there were 63% polymorphic(12 markers) of total markers. As many as 60 alleles with the size of 200–500 bp were identified by a range of 2–9 alleles perlocus. The polymorphism level was 0.59 (0.36–0.74). Result also showed the average of major allele frequency was 49.42%(35.71–63.64%). Nine markers which have polymorphism level more than 0.5 could be used to detect genetic diversity of potato.The average of genetic diversity index was 0.65. Cluster analysis showed that 14 accessions of potato were split in two groups(coefficient 0.70). The first groups consisted of Atlantik, GM 05, Granola Kembang Merbabu 17, and the second groups consist ofRepita, Maglia, Medians, CIP397078.7, CIP392781.1, Margahayu, Granola, CIP394613.139, Amabile, and Tenggo. The informationof genetic diversity of this germplasm could be used as a preliminary basis for choosing crossing parents in potato breeding inIndonesia