Judul: Genomic DNA analysis of the Heat Shock Protein 70 as a  molecular marker candidate of Temperature-Shock Resilience in Osphronemus  goramy
Abstrak: Temperature stress often causes high mortality in giant gourami. One strategy to prevent this threat is to use giant gourami that are adaptive to temperature stress. Adaptive fish can be produced by molecular marker-based selection that utilizes genes associated with giant gourami resilience traits. However, the availability of gene sequence information and molecular markers for selection has not been widely done. This research was conducted as an initial stage for the development of molecular marker-based selection of gourami adaptive to temperature stress through gene expression analysis and gene identification. This research was carried out in three stages. The first stage aims to analyze gene expression levels related to gourami resilience. The second stage aims to identify markers in the gourami genome related to resilience. The third stage aims to select gourami using the Tetra primer amplification refractory mutation system-polymerase chain (Tetra-ARM PCR) method and evaluate its correlation with fish resilience. The study began with raising 200 fish to be conditioned in culture containers. For gene expression analysis, 20 giant gourami were transferred to a 55 cm × 45 cm × 45 cm aquarium with a water level of 35 cm. The fish were kept for fourteen days at 28 - 30°C and fed commercial feed with a protein content of 39% by mouth. Water changes of 30% of the aquarium volume were done once a day. The temperature stress test was conducted in two aquarium containers measuring 55 cm × 45 cm × 45 cm with a water height of 35 cm. Each aquarium had its water temperature regulated with the first aquarium at a hot temperature of 32-34 °C and the second aquarium at a cold temperature of 18-20 °C. The fish were transferred directly from the rearing container to the first aquarium for one hour, then the fish were transferred to the second aquarium for one hour. Furthermore, fish behavior was observed and differentiated into two groups of fish, namely adaptive fish and susceptible fish. Adaptive fish are fish that can still swim well, while vulnerable fish are fish that have collapsed at the bottom of the aquarium. A total of five adaptive fish and five vulnerable fish were taken for total RNA extraction. Expression analysis was performed on three resilience-related genes, namely heat shock protein 70 (HSP70), Cu-superoxide dismutase (CuSOD), dan Mn-superoxide dismutase (MnSOD) using the qPCR method. The results showed that adaptive fish had a higher gene expression level than the susceptible fish for all the genes tested. The results indicate that there is an important role of these genes in the response of giant gourami to temperature stress. Therefore, these genes can be used as a potential gene as a molecular marker for giant gourami selection. The second phase of the study aims to identify single nucleotide polymorphisms (SNPs) in the HSP70 genome sequence and evaluate fish survival after temperature stress testing. The preparation of adaptive and susceptible fish giant gourami samples for HSP70 genome sequence analysis was carried out in stages, with three replicates. A total of 40 giant gourami were used at each stage. Temperature
Keyword: genotype, HSP70, molecular marker, Osphronemus goramy, SNPs