|Abstract: ||尖吻鱸為台灣及東南亞地區重要之養殖魚類，因其成長快、抗病能力強、肉質細膩，所以普遍受養殖戶及消費者喜愛。早期鱸魚被認為是手術後傷口癒合的補品藥膳。由於價格比一般養殖魚類高，所以僅在餐廳或宴客場所才能看到以鱸魚烹飪材料的菜餚，近年來產量逐漸上升，價格降低到一般民眾有能力負擔。在全球暖化所造成氣候變遷的趨勢下，近年來極端氣候發生率大幅提高，每年12月至2月間寒流來襲造成低溫環境緊迫，使得台灣重要養殖經濟魚種如虱目魚、鱸魚、石斑魚、白蝦等在寒流期間活動力與攝食降低、生長緩慢，甚至大規模的凍傷與凍斃。嚴重衝擊養殖魚業的產量及周遭環境與生態，造成大量的經濟損失。本文研究目的為開發尖吻鱸耐低溫相關之分子標記，本研究將尖吻鱸進行低溫試驗後，分別採取耐低溫組（SPT）與不耐低溫（SPS）兩組之鰓、肝臟、腦、頭腎、肌肉組織建立轉錄體資料庫。利用轉錄體次世代定序之數位化基因表現分析，以基因層面來探討並開發尖吻鱸耐低溫相關之分子標記，供未來育種耐低溫尖吻鱸之用。轉錄體資料庫共產出44,183,427,900 nt，組裝後總共獲得142,824個Unigene，平均長度為1320個鹼基。共同註釋到NR、NT、SwissPort、KEGG、COG、GO資料庫的Unigene有19,370個。利用MicroSAtellite（MISA）分析軟體共搜尋到62,034個包含單、雙、三、四、五及六鹼基重複的微衛星標記，將各組織間上調和下調的基因進行Venny線上軟體（2.1）交叉比對分析，找到共同差異性表達基因有308個，從中搜尋出20個與耐低溫性狀相關的功能性微衛星標記。而後進一步利用20組分子標記對尖吻鱸試驗族群進行多型性分析，結果得出有14個微衛星標記在尖吻鱸為多型性標記，其中CL12362_4標記使用判別分析法進行辨別後，耐低溫組的正確率達75%，不耐低溫組的正確率為69%，且在耐低溫組EE基因型頻率為0.5，在不耐低溫組中為0.19。本試驗所開發之抗寒相關微衛星標記與尖吻鱸低溫轉錄組資料庫，期望在未來可以輔助業者選育抗寒品系，減低寒害所造成養殖漁業經濟上的損失。|
Asian seabass (Lates calcarifer) is important commercial aquaculture species in Taiwan and Southeast Asia. Asian seabass has advantages of fast-growing, resistance to disease and tender meat, so is popular in farmers and consumer. In early days, Asian seabass been known as good for recover after surgery. Due to it’s price is higher than normal aquaculture species, only in the restaurant have meal made by Asian seabass. In recent years, production has gradually increased, price reduced to the general public have the ability to afford. Global warming caused the trend of climate change, the incidence of extreme climate increased significantly in recent years. Because of the cold spell from December to February every year cause sudden drops in water temperatures, important economic species such as milkfish, bass, grouper and white shrimp freeze to death, cause a great losses in economic. The purpose of this study is to develop putative molecular markers of cold tolerance-related genes in Asian seabass. Afier Asian seabass were subjected to cold stress, we sampled five tissues: brain, gill, brain, liver, head kidney and muscle from two groups, the cold- tolerance group (SPT), which survived under the cold stress; the cold-sensitive (SPS) group, which couldn’t survived at the low temperature. Five tissues sample were performed using the Illumina HiSeq 2000 platform for transcriptome sequencing. Transcriptome data base produce 44,183,427,900 nt, we obtained 142,824 unigenes with an average length of approximately 1320 base pairs. Total 19,370 unigenes were co-annotated in public protein databases, including the NCBI non-redundant (NR), nucleotide (NT), SwissPort, Kyoto Encyclopedia of Genes and Genome (KEGG), Clusters of Orthologous Groups (COG) and Gene Ontology (GO) database. In addition, 62,034 SSRs were identified using molecular marker loci detection software MicroSAtellite (MISA). We use Venny(2.1) to analysis Up-regulated and down-regulate genes between tissues, founds 308 co-differential expressed genes. Cold related gene sequences were established out of 20 functionality SSR markers. The result shown that 14 of 20 SSR markers were polymorphism . Using discriminant analysis to discriminate 14 SSR markers, founds the correct rate of CL12362_4 marker in SPT and SPS group were 75% & 69%, respectively.Genotype EE frequency in SPT and SPS were 50%& 18.75%, respectively. These polymorphism results among and within the strains can be further use to identify the strain-specific markers for identification of cold-tolerance and improve the genetic stability of breeding programs for L. calcarifer.