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Isolation and characterization of simple sequence repeat (SSR) DNA marker from marine herbivorous fish
|Authors: ||Ibnu Sahidhir|
|Contributors: ||NTOU:Department of Aquaculture|
Chanos chanos;Siganus canaliculatus;SSR;Isolation
|Issue Date: ||2013-10-07T02:42:39Z
|Abstract: ||在台灣、菲律賓和印尼，虱目魚 (Chanos chanos, milkfish) 與臭肚魚 (Siganus canaliculatus, rabbitfish) 為受大眾喜愛的草食性魚類且已可完全養殖。即使在未來可能面臨魚粉和魚油短缺的問題，虱目魚的產量仍可以穩定成長。目前尚無研究指出虱目魚和臭肚魚的簡單重複序列 (simple sequence repeat, SSR) 分子標誌，因此本研究目的在於分離與鑑定虱目魚與臭肚魚的簡單重複序列標誌。 本研究中以磁珠分離技術進行虱目魚與臭肚魚之SSR 標誌。菌落PCR結果得知，虱目魚與臭肚魚分別有400個與360個克隆。再分別挑選出31個虱目魚與29個臭肚魚的SSR克隆，在這些克隆中我們感興趣的SSR均大於500 bp。定序結果得知在虱目魚中只有1個克隆沒有SSR，有13個dinucleotide motif 克隆，分別為12個 (AC)n與1個 (AG)n motif。 其餘的17個SSR 為trinucleotide motif，分別為4個(CGT)n、4個 (TGC)n、8個 (TCC)n motif、與其他1個 (AGA)n motif。 臭肚魚中則有16個克隆有SSR ， 13個克隆無SSR 。有3 個 (T)n mononucleotide 與1 個 (CA)n dinucleotide motif。其他12個trinucleotide motif 分別為4 個 (GAG)n、4 個 (CAG)n、2 個 (CTG)n 與 2 個 (TCC)n motif。 接著設計17 組虱目魚與9 組臭肚魚的引子對SSR 進行多態性的評估，所有引子皆可使用PCR 增幅，但在虱目魚與臭肚魚中並沒有顯示出多態性。|
Milkfish (Chanos chanos) and white-spotted spine-foot rabbitfish (Siganus canaliculatus) are very popular as table fish, especially in Taiwan, Philippines, and Indonesia and have been successfully domesticated for whole lifecycle. Milkfish production increases steadily without altered by fish meal and fish oil shortage, so that has the opportunity for future development. No researches have been conducted to reveal simple sequence repeat DNA marker in these species. The purpose of this study is to isolate and characterize simple sequence repeat DNA in milkfish and white-spotted spinefoot rabbitfish and to search polymorphism. In this study, SSR DNA markers of the fish were developed by magnetic bead separation technique. Colony PCR had been conducted for 400 clones of milkfish and 360 clones of rabbitfish respectively. Afterward, it was selected 31 and 29 positive clones having inserted DNA size more than 500bp from milkfish and rabbitfish samples for sequencing the DNA, respectively. The DNA sequence analysis of milkfish revealed that only one clone had no SSR. There were 13 clones contained dinucleotide motif, which 12 loci contained (AC)n motif and one locus contained (AG)n motif. The remaining 17 SSR loci showed trinucleotide motif; four loci had (CGT)n motif; four loci contained (TGC)n motif; eight loci showed (TCC)n motif; and one locus showed (AGA)n motif. In rabbitfish, the DNA sequencing revealed that there were 16 clones contained SSR DNA sequences; 13 clones did not contain SSR sequence. Three loci revealed mononucleotide motif of (T)n. One locus revealed dinucleotide motif of (CA)n. Twelve loci showed trinucleotide motif, which four loci presented (GAG)n motif, four loci exhibited (CAG)n motif, two loci showed (CTG)n motif, and two loci presented (TCC)n. Subsequently, 17 primer pairs of milkfish and nine primer pairs of rabbitfish were designed for evaluating polymorphism. All the primers could be amplified in PCR but there were no polymorphism revealed from milkfish and rabbitfish primers.
|Appears in Collections:||[水產養殖學系] 博碩士論文|
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