INDONESIAN JOURNAL OF AGRICULTURAL SCIENCE

Number 2, 2015

51-58
GENETIC STABILITY ANALYSIS OF RB GENE IN GENETICALLY MODIFIED POTATO LINES TOLERANT TO Phytophthora infestans

Authors: Edy Listanto, Eny Ida Riyanti, Tri Joko Santoso, Toto Hadiarto and A. Dinar Ambarwati

Abstract

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Development of potato cultivars with high levels of broad spectrum resistance is a key long-term management strategy against late blight disease caused by Phytophthora infestans. Six progeny lines of hybridization between transgenic potato Katahdin SP951 with non-transgenic Granola and Atlantic were selected based on agronomical characteristics and resistance to late blight disease. The study aimed to analyze the number of insertions and stability of inserted RB gene in the transgenic potato lines. The research was carried out through plant DNA extraction, southern blot analysis and polymerase chain reaction (PCR). Southern blot analysis was used to detect the number of inserts integrated into potato genome, while PCR analysis was used to detect stability of RB gene from generation to generation. The results showed that the progenies obtained from hybridization between Atlantic and transgenic Katahdin SP951 (lines No. 20 and 27) and between Granola and transgenic Katahdin SP951 (line No. 69) contained one copy number of RB gene, according to the probing of nptII. The result is similar to that of inserted RB gene found in the parental transgenic Katahdin SP951. The presence of RB gene in four different generations (G0, G1, G2 and G3) showed stable integration of the gene into the plant genome. The single copy number of RB gene will repress the occurrence of silencing gene expression. The stability analysis of RB gene can determine that the gene is still present in plant genome after several generations.

59-70
IDENTIFICATION OF SINGLE NUCLEOTIDE POLYMORPHISMS ON CATTLE BREEDS IN INDONESIA USING BOVINE 50K

Authors: Puji Lestari, Habib Rijzaani, Dani Satyawan, Anneke Anggraeni, Dwinita W. Utami, Ida Rosdianti, Muchamad Lutfi and I Made Tasma

Abstract

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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.

71-78
EVALUATION OF GENETIC DIVERSITY IN CACAO COLLECTED FROM KOLAKA, SOUTHEAST SULAWESI, USING SSR MARKERS

Authors: Rubiyo, Nur Kholilatul Izzah, Indah Sulistiyorini and Cici Tresniawati

Abstract

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Kolaka, which is located in Southeast Sulawesi, has long been known as one of cacao production centers in Indonesia. Therefore, many different cacao germplasms can be found in this region. The study aimed to evaluate genetic diversity and relationships of 12 cacao genotypes collected from Kolaka. Genomic DNA was extracted by using a modified CTAB method. Meanwhile, genetic diversity was analyzed based on 16 SSR markers, which then separated by 6% non-denaturing polyacryl-amide gel electrophoresis. The result showed that all of those markers, 14 markers exhibited polymorphism and subsequently used for data analysis using NTSYS and PowerMarker program. About 70 different alleles were generated from 12 cacao genotypes analyzed with an average of 5 alleles per locus. Average value of polymorphism information content (PIC) resulted in this study was 0.59. The cluster analysis using UPGMA method based on the genetic similarity coefficient revealed that all cacao genotypes were separated into three major groups. The first group consisted of five cacao genotypes, the second one held four cacao genotypes, whereas the third group contained three genotypes. This result indicates that three genotypes that clustered separately from the others could be used as a good clonal candidate for cacao breeding program. The information resulted from this present study would be useful for future cacao breeding program, especially in efforts to release a new variety.

79-86
GENETIC DIVERSITY OF S3 MAIZE GENOTYPES RESISTANT TO DOWNY MILDEW BASED ON SSR MARKERS

Authors: Amran Muis, Nurnina Nonci and Marcia B. Pabendon

Abstract

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The compulsory requirement for releasing new high yielding maize varieties is resistance to downy mildew. The study aimed to determine the level of homozygosity, genetic diversity, and genetic distance of 30 S3 genotypes of maize. Number of primers to be used were 30 polymorphic SSR loci which are distributed over the entire maize genomes. The S3 genotypes used were resistant to downy mildew with homozygosity level of >80%, genetic distance between the test and tester strains >0.7, and anthesis silking interval (ASI) between inbred lines and tester lines was maximum 3 days. The results showed that 30 SSR primers used were spread evenly across the maize genomes which were manifested in the representation of SSR loci on each chromosome of a total of 10 chromosomes. The levels of polymorphism ranged from 0.13 to 0.78, an average of 0.51, and the number of alleles ranged from 2 to 8 alleles per SSR locus, an average of 4 alleles per SSR locus. The size of nucleotides in each locus also varied from 70 to 553 bp. Cophenetic correlation value (r) at 0.67 indicated that the Unweighted Pair-Group Method Using Arithmetic Averages (UPGMA) was less reliable for differentiating genotypes in five groups. Of the total of 30 genotypes analyzed, 17 genotypes had homozygosity level of >80% so it can be included in the hybrid assembly program.

87-96
HOST-RESTRICTED RANGE OF H5N1 AVIAN INFLUENZA VIRUSES ASSOCIATED WITH CHARACTERS OF POLYMERASE COMPLEX OF PB2 AND PB1-F2 PROTEINS

Authors: NLP Indi Dharmayanti and Risza Hartawan

Abstract

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Epidemiological studies on H5N1 avian influenza viruses indi-cated that the viruses do not transmit efficiently from human to human. Transmissibility of viruses among human population is very complex and polygenic. Studies on molecular determinants facilitating interspecies transmission of the viruses suggested that two polymerase complex proteins such as PB2 and PB1-F2 are important. PB2 is critical in determining the host specificity, whereas mutations in PB1-F2 increase the viral virulence. The study aimed to characterize the polymerase complex of PB2 and PB1-F2 proteins of H5N1 avian influenza viruses isolated from Indonesia. The DNA samples encoding the PB2 and PB1-F2 complex proteins of several H5N1 isolates were sequenced and analyzed. Pathogenicity of the viruses was studied in both avian and mammal models. The sequencing results showed that there was no mutation in both proteins of PB2 and PB1-F2 of the avian influenza virus isolates. The molecular character for host specificity was consistent with the animal experiment results. The H5N1 virus isolates were only infectious and pathogenic in chickens, but not in BALB/C mice as the mammal model. The study suggests that host range of H5N1 virus isolates of Indonesia is restricted to poultry and not transmisible to mammal model used in this study.