Kunda Rony Marsyal, Handayani Niken Satuti Nur, Wijayanto Hery, Widayanti Rini
Program Study of Veterinary Science, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.
Department of Genetics, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia.
Pak J Biol Sci. 2016;19(3):122-135. doi: 10.3923/pjbs.2016.122.135.
Cuscuses is marsupials animal (Phalangeridae) which has limited spread in eastern Indonesia (Sulawesi, Maluku, Papua and Timor islands), Australia and Papua New Guinea. The ex-situ and in-situ conservation of cuscuses under captivating condition is an alternative solution to protect from extinction. This study aimed to determine nucleotide sequences and genetic marker on cyt b gene with sequencing method of each species on two provinces. Whole genome DNA was extracted from 22 samples of cuscuses obtained from different habitats, Maluku (13 individuals) and Papua (8 individuals) according to the protocol of Qiamp DNA Blood Mini Kit (Qiagen) and then it was used as template for amplification of cyt b gene by using PCR method. The PCR product were then purified using column chromatography and were used as template for sequencing reaction. Results sequencing of cyt b gene were analyzed using MEGA program versions 6.0. The PCR product gives results nucleotides of 982 bp according to database GeneBank and sequencing product gives results nucleotides of 771 bp. Nucleotides alignment of Phalanger members was found 24 nucleotides distinguishing and Spilocuscus members was found 11 nucleotides distinguishing, which can be used as genetic marker between Phalanger and Spilocuscus members from Papua and Maluku.
袋貂是有袋类动物(袋貂科),分布范围有限,主要在印度尼西亚东部(苏拉威西岛、马鲁古群岛、巴布亚和帝汶岛)、澳大利亚以及巴布亚新几内亚。在圈养条件下对袋貂进行迁地和就地保护是防止其灭绝的一种替代解决方案。本研究旨在通过对两个省份各物种的测序方法,确定细胞色素b基因的核苷酸序列和遗传标记。根据Qiamp DNA Blood Mini试剂盒(Qiagen)的方案,从22个来自不同栖息地的袋貂样本中提取全基因组DNA,这些样本分别来自马鲁古(13只个体)和巴布亚(8只个体),然后将其用作模板,通过PCR方法扩增细胞色素b基因。PCR产物随后使用柱色谱法进行纯化,并用作测序反应的模板。使用MEGA 6.0版本程序分析细胞色素b基因的测序结果。根据基因库数据库,PCR产物得到982 bp的核苷酸结果,测序产物得到771 bp的核苷酸结果。发现袋貂属成员的核苷酸比对中有24个核苷酸差异,斑袋貂属成员中有11个核苷酸差异,这些差异可作为来自巴布亚和马鲁古的袋貂属和斑袋貂属成员之间的遗传标记。