Abstract
基因體標記輔助臺灣鯛抗逆良種選拔與品系培育技術開發應用
優質臺灣鯛選拔育種改良計畫為台灣現今刻不容緩的首要任務，以基因體和分子標記輔助種原庫選拔與優質種苗培育之遺傳管理策略，為提升臺灣鯛水產品質之重要關鍵。多年來種苗業者在品系選種與開發效率之遺傳改良過程面臨許多急待克服之問題。為能實質解決產業之瓶頸，本計畫共分2年執行，於本年度之目標為(1)蒐集與建立耐寒/鹽/熱等抗逆境特性臺灣鯛種原庫之系譜追踨系統以及體表型與基因型之遺傳相關資訊。(2)建立臺灣鯛抗逆境特性次世代轉錄體定序生物資訊分析所開發之功能性基因標記資訊庫，整合重要經濟性狀相關之功能性標記與基因型資訊，開發可輔助優質臺灣鯛選育之微衛星或單一核苷酸多型性分子標記。透過本計畫所開發之分子標記，可提供臺灣鯛種苗產業縮短台灣鯛育種時程之整合式分子標記，達真正解決優質臺灣鯛品種篩選所需耗費之養殖空間與時間及遺傳穩定度等問題。研究成果兼具基礎研究與產業應用價值，未來可提供業者建構完整產銷履歷及品牌認證之科學化管理，為水產養殖產業注入新元素。
建構台灣鯛基因體育種分析整合平台以應用於抗病台灣鯛分子標誌輔助選育
本計畫目標為發展台灣鯛之基因體選育平台以進行高抗病台灣鯛品系之分子標誌輔助選育。利用已建立之尼羅吳郭魚全基因體及轉錄體資料庫，建立台灣鯛參與免疫調節、抗病基因相關之微衛星DNA分子標誌資料庫，挑選具多態性微衛星DNA分子標誌。以多態性微衛星DNA分子標誌分析對鏈球菌感染不同抗性之海大尼羅吳郭魚純系NT1、NT2、NT1 X NT2雜交NTH品系及商業台灣鯛品系 (YH與SD品系) 之基因型多樣性，挑選與抗病力高度相關之微衛星分子標誌。以高致死劑量致病性海豚或無乳鏈球菌處理海大抗病力佳尼羅吳郭魚純系NT1品系、NTH雜交品系及商業台灣鯛品系，以抗病基因相關分子標誌輔助建立高抗病力之台灣鯛品系。為能使台灣鯛養殖產業邁向永續發展經營，本研究將整合台灣鯛基因體、轉錄體與其他體學研究資源，發展產業導向之台灣鯛基因體資料庫平台。結合巨量分子序列數據與雲端服務，透過持續加值並維護台灣鯛線上基因體與多維體線上分析資料庫，將可讓研究團隊快速獲取基因標誌、種原資訊等情報，並進一步整合強化與蒐集種原庫之抗逆境(抗病性、耐熱性、耐寒性及耐鹽性)、成長優勢等性狀之功能特徵，有效提升遺傳管理、病原檢驗及病害防治等資訊分析，並在日後提供相關研究社群使用，將成為其他高經濟水產生物基因資訊應用平台的雛形，提供相關數位服務，建構便捷之分子鑑定支援體系，強化分子育種與病原防治之能力，掌握並解讀基因型與表現型的關鍵數據，將基因資料庫訊息落實到水產生物基因體選種、育種、保種及新品種開發之實際應用面，促進農業優質種苗之運用與營運效率。並瞭解特定飼料添加物，對台灣鯛健康與肉質及其腸道菌相的互動關係，減少養殖過程中不必要的藥物使用，提昇整個台灣鯛養殖產業的質與量，進一步提高我養殖產業之收益，並藉此高品質產品來積極拓展外銷市場，推動臺灣成為亞太種苗繁殖中心及種苗培育與供應中心。
建立臺灣鯛之無抗養殖技術模式
本計畫擬提出臺灣鯛綠色無抗養殖方法。根據臺灣鯛的各養殖階段進行調節，綜合動態調節臺灣鯛的生長環境。並通過水質管理、高溫抗病品系、綠色的飼料與營養管理與疾病防治，來提高臺灣鯛的存活率，長程目標是建立吳郭魚無抗養殖模式，為達到此一長程目標；設定下列短程目標以生物技術與智能化物聯網建立吳郭魚環境安全、生物安全與食品安全之養殖體系。在未來2年建立下列實施項目，包括：一、建立臺灣鯛環境安全養殖體系─將應用複合微生物制劑與生物絮團技術(Biofloc technology)等進行水質管理，改良水體環境。二、建立臺灣鯛生物安全養殖體系─以分子選育技術（Marker assist selection, MAS）選育高溫抗病台灣鯛品系；開發臺灣鯛魚苗用鏈球菌與湖泊病毒疫苗和快篩鏈球菌與湖泊病毒檢測試劑組，可在養殖現場迅速檢驗出病原菌與即時對症下藥。三、建立臺灣鯛食品安全養殖體系─採用抗菌肽、植物免疫刺激物、微生物發酵豆粕、魚小肽和複合微生態製劑的無抗飼料，提供一種綠色無抗的飼養方法，亦可促進臺灣鯛成長與降低發病率。四、構建智能化臺灣鯛翻譯養殖系統─養殖全程將應用水產物聯網系統實時監控養殖場的水質環境、線上疾病診斷與生長表現實踐臺灣鯛養殖上的環境安全、生物安全與食品安全。本計畫預期效益如下：CBM複合益生菌可以淨化水質,並且提高溶氧量，CBM有抑致水中常在病原菌的效果，以複合益生菌製作無抗發酵飼料可節約飼料消耗：配製後的飼料消化吸收率能提高3%∼5%(按提高4%計算)；CBM發酵植物蛋白源優點：活性小肽增加，酵素增加，抗營養因子下降，適口性增加，抗氧化物、維生素、礦物質增加，無乳鏈球菌。經田間實驗證明，於吳郭魚上使用，可有效提高存活率與10%的飼料轉換效率。經此疫苗接種後，有85%的魚具保護效期超過30週，將篩選出的分子標記與SRAP-PCR擴增進行不同親魚品系的檢驗，可以快速、準確地挑選出抗海豚鏈球菌病感染的親魚，初步試驗顯示經過此分子標記選育，成魚感染鏈球菌病的敏感性降低了36%，發病後成魚的成活率提高了51%，通過物聯網智能水產養殖雲平台系統項目實施，可減少水產養殖過程中人力、物力投入。前期田間試驗結果顯示通過的水產病害診斷專家系統施行，可有效水產動物疾病提高活存率30%以上，經濟效益顯著。通過的資訊監控系統和水溫冷熱調節機、增氧機、pH滴定設備、抽水泵、取樣電磁閥等終端控制系統，結合預警預報系統，可極大避免了因人為管理不當造成的經濟損失，從而實現了水產養殖的智慧化控制和管理，綜合減少成本20%以上，具有良好的經濟效益，通過的水產養殖動物的成長表現監控與管理系統施行，可提供即時監測水產養殖動物生長過程，以達到節約飼料與促進養殖動物生長。
農業副產物於臺灣鯛複方免疫調節劑之開發與應用
本計畫執行擬定，為利用農業副產物提升臺灣鯛之健康管理策略，降低養殖過程中化學藥品之使用，並且有效擴充臺灣鯛生物技術產業技能，發展臺灣周邊海域常見之藻類海木耳，以及農業副產物赤芝與巴西蘑菇生長榖糧基質直接進行其生物活性之效能分析，進而應用於臺灣鯛之健康管理。試驗過程針對藻類及赤芝與巴西生長榖糧基質對養殖生物免疫細胞活性、基因表現量與對抗病原菌之評估，完整呈現一個活性物質開發的過程，有助於提升臺灣鯛產品的優質化與水產替代性藥物發展，達到友善海洋、環境保育、疾病預防控制、水產食品安全及替代性醫學，發展轉譯農學，藉以提升生產經濟效益，開創前瞻且精緻性的養殖科技產業。
耐鹽之臺灣鯛營養組成比較與其加工副產品之加值利用
臺灣鯛製成冷凍魚片加工每年約會產生4500公噸的魚類廢棄物，目前副產品及廢棄物多作為低度利用的飼料使用，本計畫擬探討以耐鹽臺灣鯛及市售臺灣調(淡水)為原料，收集魚片加工場所生產之副產品(去鱗)，如魚皮、魚骨、魚頭等新鮮(經過清洗)之材料為試驗原料，分析比較其營養成分之差異，並運用簡易食品加工技術將其開發成具有多樣性的產品，如臺灣鯛調味粉、臺灣鯛魚露等，或著是進一步酵素水解加工為生醫美容原料，如膠原蛋白與魚骨之活性胜肽產品，期望藉由此技術更進一步廣泛地運用至其他大宗水產加工品的副產品，如鱸魚或虱目魚等。
本計畫目標與工作事項為收集耐鹽台灣鯛加工副產品之樣品(魚皮、魚頭、魚骨)，進行樣品前處理(清洗、分裝)並詳細記錄其重量(推其算產品之產率及利用率)。首先針對不同部位的副產品進行主成分分析與建立其化學組成份的特徵並與市售臺灣鯛營養成分進行比較，以利後續研發之參考依據。首先針對本計畫實驗流程兩大主軸的副產品樣品(1.魚頭、2.魚骨)，研發並建立其最適化之熱加工、酵素水解及乾燥條件，同時並尋求最佳化兩大類產品(1.臺灣鯛調味粉、2.臺灣鯛魚露)之產率的可行性與商品化。
預期效益一、完成耐鹽臺灣鯛之加工副產品的利用。二、達成減少漁業廢棄物之目的。三、建立利用耐鹽臺灣鯛加工副產品的加工技術。四、藉由生產多樣化之產品，進而增加耐鹽臺灣鯛加工副產品的附加價值。
促進臺灣鯛健康飼料產品之開發
飼料中的蛋白質是最昂貴的原料，為降低飼料成本，飼料中的魚粉添加量逐漸被其他蛋白源取代，但魚粉取代研究多著重在對成長的影響，對魚肉品質和養殖生物的健康則較少著墨。本計畫結合物性特性、營養成分和生理指標分析技術，以胺基酸平衡的方法探討機能性飼料添加物對臺灣鯛品質和健康的影響，在不使用魚粉的前提下提高水產品品質，產業升級更具國際競爭力，並兼顧飼料成本和環境友善。 Application of Genomic Marker-Assisted Selection Developing Technologies for Stress-Resistance Strains Breeding in Taiwan Tilapia
At present, selective breeding for the genetic improvement of quality-based Taiwan tilapia is an urgent task in Taiwan. Genetics and brood stock management strategies of genomic and molecular marker-assisted selection and breeding are important for promoting improved varieties of seeds of Taiwan tilapia species. However, it remains important to overcome the potential problems of quality-based variety selection and development efficiency during seedling cultivation for genetic improvement. To effectively resolve these problems, this research project will use radio frequency identification and molecular marker-assisted breeding techniques to develop the stress-resistance strains with high economic value and great potential for developing quality-based Taiwan tilapia species in the international market. This project, which is expected to span over 2 years, will include the following objectives: (1) establishment of a pedigree trace system and a relationship between the phenotype (identifiable traits) and genotype (heritable information) of Taiwan tilapia brood stocks, and determination of the economic importance of cold and thermal stress tolerance traits related functional gene markers and genotype information and development of microsatellite or single nucleotide polymorphism molecular markers for assisted quality-based Taiwan tilapia selection and breeding; and (2) screening and development of functional molecular markers with functional nucleotide sequence variations to facilitate the family selection of Taiwan tilapia and determination of their effects on important economic traits of stress-resistance in family breeding experiments. Our findings will shorten the breeding platform for integrated molecular marker-assisted selection for the industrial applications of quality-based Taiwan tilapia, which requires large space, long time, and genetic stability. In addition, this study will assess the results of basic research for their value in industrial applications. The results of this study will help the Taiwan tilapia industry to scientifically manage complete traceability and branding certification and to create a new approach for increasing productivity and providing a competitive advantage in the future.
Implementation of analysis and integration platform of tilapia genome selection for application in marker-assisted selection of disease-resistant Taiwan tilapia
The purpose of this project is to establish the genome selection platform for disease resistance of Taiwan tilapia strain by markers assisted selection. We would like to establish the database of microsatellte DNA markers related with functional genes involved in immune regulation and disease resistance by using previously established Nile tilapia genomics and transcriptomics database to select for polymorphic microsatellite DNA markers. Different tilapia strains including NTOU Nile tilapia NT1, NT2, NTH hybrid strains and commercial Taiwan tilapia strains (YH and SD strains) were challenged with virulent <em>Streptococcus </em><em>iniae</em> or <em>S. </em><em>agalactiae</em><em> </em>in the dose of high mortality and analyzed by polymorphic microsatellite DNA markers to obtain disease-resistant DNA markers. Highly disease-resistant Taiwan tilapia strain will be selected and confirmed in their offspring generation assisted by these disease-resistant genes related molecular markers. To pave the way for sustainable development of Taiwan Tilapia industrial, we will integrate genome, transcriptome, other omics, and cloud-based information technology to develop Taiwan tilapia omic database for genome breeding. Via the collections of specific and important phenotypes like a disease, cold/ heat, and salt resistance, expression quantitative trait loci (eQTLs) can be conducted to correlate genetic association with gene expression. Such an approach will facilitate our understanding of the molecular mechanisms of these traits and indicate those genes/ biomarkers which can be used to evaluate their genetic variants for the best strain for specific environmental status. In this study, one of our goals is to unveil the complex relationship among the gut metagenome, health, and texture of tilapia’s meat to decrease the use of drugs and antibiotics after introducing the specific feed additives. Meanwhile, web databases created by this study will become portal to tilapia aquatic research community for acceleration of molecular selection of specific strain with important traits.
Establishment of antibiotics-free tilapia culture technique
The aim of this project is to establish a non-resistant breeding mode for tilapia to achieve this long-term goal. This subprogram aims to establish a breeding system for the environmental safety, biosafety and food safety of wu guo fish with the network of biological technology and intellectualization. In the next two years to build the following the implementation of project: including: 1) to establish tilapia environment safety system - will apply complex microbial preparation and bio-floc technology (BioFloc Technology) and water quality management, improve the aquatic environment. 2) to establish tilapia biosecurity system - apply molecular breeding technology (Marker Assist Selection, MAS) breeding high temperature resistant Taiwan tilapia strains; To develop Streptococcus sp. and tilapia lake virus vaccines and rapid streptococcus and tilapia lake virus detection kit, allow tilapia farmer can quickly test the pathogenic bacteria and immediate symptomatic medicine in the tilapia culture site. 3) establish a tilapia food safety production systems - using the antimicrobial peptide, plant immune stimulants, microbial fermented soybean meal, fish waste-derived small peptides, probiotics to develop antibiotic-free aquafeed, to provide a green antibiotic-free aquaculture method, can also promote tilapia growth and reduce the incidence of a disease. 4) Construct smart tilapia culture system- which employed modern IoT technology and biotechnology strategies to revolutionize aquaculture. Three sub-systems were developed to fulfill the goal; 1）Advanced monitoring of aquaculture environment by ICT technology, i.e. IoT+ aquaculture system means extensive measuring, monitoring and automatic control by driving various regulators; 2）Automatic species recognition, weight determination and precise feeding system, By using a vision-based catch registration system can automatically recognize fish species and measure the body length and then converts to body weight. Collection of biological data on individual length and weight of specimen has always been an important part of aquaculturists. 3）Tele-diagnosis system for fish diseases. A clinical sign-based diagnosis aid system is applied by sending infected animals images, text description through mobile phone to the diagnosis center. To implement the designed diagnosis process, fish disease database and disease cause/prevention/treatment database were established. Candidate disease by comparing the observed and selected clinical signs to the information on the remote database. Information such as cause, diagnosis, treatment and prevention method of candidate disease are provided as a medical advice through internet to user PC or mobile devices. This system will support fish farmers and veterinarians by provide easy and rapid diagnosis of fish diseases. Since ICT and IoT technology are used to collect information from farmers and biotechnology can provide the solution for the problems of commercial fish farming.
Development and application of agricultural by product as immunomodulation compound on Taiwan tilapia
The present project aims to improve the health management strategies, reduce the abuse of chemicals and expand the application of biotechnology in Taiwan Tilapia via the application of algae <em>Sarcodia ceylanica</em> that commonly seen in the sea water around Taiwan and agricultural byproduct such as growing medium of <em>Ganoderma multipileum</em> and <em>Agaricus blazei </em>into daily feed to examine their potential for immunomodulation in Taiwan Tilapia. Through this study the course of development of an algae <em>S. ceylanica</em> and growing medium of <em>G. multipileum</em> and <em>A. blazei</em> as immunomodulators will be shown by examination of their impacts on the immune cell activity, gene expression and anti-pathogenic bacteria activity. Furthermore, this study will have impacts on improving the quality of Taiwan Tilapia production and alternative aquatic drugs development. In this project target, we hope to achieve the issue of environmental conservation, disease prevention and control, aquatic food safety and alternative medicine further to enhance the economic benefits of tilapia production, and create a forward-looking and sophisticated aquaculture technology industry.
Study of comparison of nutrition composition from salt durable Taiwan tilapia, and utilization of its processing by products with added value
Around 4.5 thousand tons of Taiwan tilapia by-products and wastes were produced yearly from frozen tilapia fillet industries. Currently these materials are under-utilized as animal feeds. The object of this project is aimed to collect fillets and by-products of the salt durable Taiwan tilapia and commercial tilapia as experimental materials such as Tilapia skin (de-scaled), bones, and heads, and to use simplified food processing technologies to create diverse products such as Taiwan tilapia seasoning powder, Taiwan tilapia fish sauce, healthy organic calcium, and highly-functional feeds or as collagen and bioactive peptides products. Eventually this approach can be extensively applicable to other marine processing products like sea bass or milkfish in Taiwan.
The objective and the projected work of this project are firstly to collect the salt durable Taiwan tilapia by-products (skin, head and bone), to perform the sample preparation for further proximate analysis, and to compare the nutritional values between salt durable Taiwan tilapia and fresh water Taiwan tilapia. Accordingly the major by-products of head and bone will be developed to figure out the optimal thermal processing, enzymatic hydrolysis and drying parameters in order to produce two major products of Taiwan tilapia seasoning powder and Taiwan tilapia fish sauce.
Predictive outcomes are listed below:
(1)Accomplish the utilization of salt durable Taiwan tilapia by-products.
(2)Reduce the fishery wastes.
(3)Establish the critical processing technology for salt durable Taiwan tilapia by-products.
(4)Produce diverse value added products from salt durable Taiwan tilapia by-products.
The study on healthy additives for tilapia feed
Protein is the most expensive ingredient in fish feed. To reduce the cost of artificial feed, the content of fish meal has been replaced with other protein sources by degrees. However, the research of fish meal replacement mainly focused on fish growth, and rarely investigated the effect on flesh quality or fish health. We design additives formula according to amino acids requirement of tilapias, and combine analysis technologies which include physical characters, nutrient compositions and physiological parameters in order to evaluate the effect of functional additives on tilapias quality and health status. Under the premise of no fish meal using, elevating the quality of aquaculture products and upgrading industry will achieve the balance of sustainable aquaculture and feed cost.
