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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 胡凱康 | |
dc.contributor.author | Chi Kao | en |
dc.contributor.author | 高驥 | zh_TW |
dc.date.accessioned | 2021-06-15T13:31:52Z | - |
dc.date.available | 2019-03-08 | |
dc.date.copyright | 2016-03-08 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-02-02 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51367 | - |
dc.description.abstract | 番椒 (Capsicum annuum L.) 屬茄科 (Solanaceae) 番椒屬,為重要經濟蔬菜作物。目前商業上主要以細胞質與細胞核雄不稔系統 (Cytoplasmic-Genetic Male Sterility, CMS) 來降低雜交種子之成本,而稔性恢復基因 (Restorer-of-fertility, Rf) 為此系統之核心基因。承2012年龔以SSR分子標誌分析204株番椒F2找尋Rf基因連鎖SSR之研究成果,本研究挑選其中80株F2材料與兩親本使用雙限制酶切位點標定法 (double digest Restriction Associated DNA sequencing, ddRAD),簡稱雙重酶切系統,建立具有條碼標示的定序文庫 (library)。次世代定序 (Next generation sequencing, NGS) 採用Illumina HiSeq平台,定序結果以Stacks作為核心程式進行分析,獲得2096組具有多型性且缺值小於10%之SNP分子標誌。結合先前研究之SSR分子標誌,建立同番椒染色體數目12個連鎖群,1277個分子標誌,總長為1623.25 cM之連鎖圖譜。SNP分子標誌在連鎖圖譜上的分布較物理圖譜平均,染色體中段每圖譜單位的序列長度較兩端長,與番椒染色體上異染色質的分布一致;並且在這些區域中所獲得的SNP分子標誌相對較少,顯示本研究中所採用對於甲基化敏感的PstI限制酶可有效避開異染色質區域,使開發獲得的分子標誌能均勻分布於連鎖圖譜。本次研究找尋Rf基因兩端連鎖距離範圍內分子標誌組合,從定序資料設計ASP (Allele-specific PCR)、TaqMan等SNP分析平台引子,驗證204株F2基因型,將Rf基因定位於分子標誌32081與27394之間,遺傳距離分別為5.8 cM與9.5 cM。將此SNP組合用於Rf基因分子輔助回交選種,於回交4代時,仍可確保98%之植株保留Rf基因,與傳統育種使用後裔檢定相比可大幅提升育種之效率。本研究結果顯示雙重酶切系統對於建立複雜基因組物種的高密度連鎖圖譜與其遺傳相關研究是一個有效的工具。 | zh_TW |
dc.description.abstract | Pepper (Capsicum annuum L.) is an important vegetable crop worldwide as well as in Taiwan. Production of commercial hybrid seeds relies on cytoplasmic-genetic male sterility (CMS) system governed jointly by a nuclear Restorer-of-fertility (Rf) gene and male-sterile cytoplasm. Linked molecular markers are useful tool to monitor the transferring of Rf gene during backcross breeding. In this study, a barcode tagged sequencing library was constructed following the double digest Restriction Associated DNA sequencing (ddRAD) scheme with 80 plants randomly selected from a previously mapped F2 population as well as both parents. Sequencing was conducted on an Illumina HiSeq platform and the obtained reads were analyzed by the Stack program, resulting 2096 polymorphic SNP markers with call rate larger than 90%. Combining with the previously mapped SSR markers, a linkage map of 1623.25 cM, consisted 1277 markers on 12 linkage groups was constructed. SNP markers on the linkage map were more uniformly distributed than their locations on the physical map. Drastic increase of base pairs per map unit toward the center regions of chromosomes consistent with the distribution of heterochromatin on pepper chromosomes. The relatively low frequencies of SNP markers discovered in these regions suggested that the use of methylation-sensitive restriction enzyme PstI as the rare cutter in ddRAD may be an effective way for methylation filtration. Positions of SNP markers surrounding Rf gene were validated using 204 F2 plants of the previously SSR mapped population. The Rf gene was located between markers 32081 and 27394, with genetic distances of 5.8 cM and 9.5 cM, respectively. With the aid of these SNP markers, the probability of retaining Rf gene after 4 generations of backcross is increased to 98%. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T13:31:52Z (GMT). No. of bitstreams: 1 ntu-105-R02621125-1.pdf: 4577127 bytes, checksum: 55aebb2faaf0ecbf32d861dfa68f3305 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii Abstract iii 目錄 iv 表目錄 v 圖目錄 vi 第一章 前言1 第二章 前人研究 4 第一節 番椒分子標誌連鎖圖譜建立與稔性恢復基因 (Rf) 之研究 4 第二節 番椒基因體結構相關研究 7 第三節 GBS之相關研究與雙重酶切系統之使用 9 第四節 雙重酶切系統之次世代定序資料處理方式相關研究 12 第三章 材料與方法 14 第一節 試驗材料 14 第二節 葉片DNA萃取、定量與品質檢定 16 第三節 雙重酶切系統建庫準備與流程 16 第四節 分析ddRAD定序資料流程與處理程式 24 第五節 建立連鎖群圖譜 30 第六節 物理連鎖兩圖譜比較與切位片段模擬 32 第七節 驗證找尋之SNP與Rf基因之連鎖關係 35 第四章 結果與討論 39 第一節 番椒F2與F2:3稔性外表型與定序樣品挑選 39 第二節 定序資料以設計條碼區分之情形 40 第三節 讀序比對參考序列結果與多位置比對之讀序特性 44 第四節 SNP分子標誌找尋與篩選 52 第五節 番椒連鎖圖譜建構與Rf基因位置 54 第六節 物理連鎖兩圖譜比較與切位片段模擬對照結果 59 第七節 驗證與Rf基因連鎖之SNP 67 第五章 結論與未來展望 74 參考文獻 75 附錄 81 表目錄 表 1 農友種苗公司提供之番椒品系特性與分類 15 表 2 自行設計之16組條碼序列資訊 18 表 3 以semi-global 比對下G03樣本條碼錯誤型式與比率 42 表 4 定序中大量重複且多重比對之部分讀序 49 表 5 6組讀序針對番椒參考序列與CDS之BLAST比對結果 50 表 6 讀序位置對應參考序列之319 bp片段序列資訊BLAST結果 51 表 7 番椒連鎖圖譜各連鎖群之長度與間隔等資訊 58 表 8 番椒基因組與葉綠體粒腺體之序列各限制酶切位數量 64 表 9 以PstI-MspI模擬番椒參考序列切除片段數量與比率 65 表 10 挑選範圍中16個SNP分子標誌資訊與篩選結果 71 表 11 近年研究與番椒Rf基因遺傳距離最近之分子標誌資訊 72 表 12 CRF-SCAR之引子序列資訊與比對位置 72 表 13 Rf基因緊密連鎖之32801與27394其他14組親本之基因型 72 附表 1 番椒ddRAD建庫使用合成轉接子與PCR引子序列資訊 84 圖目錄 圖 1 觀察花藥花粉與否判斷稔性外表型 15 圖 2 16組條碼(左)與8組(右)核苷酸比率變化圖 18 圖 3 雙重酶切系統建庫流程圖 21 圖 4 建庫過程加入條碼並將多樣品合併之流程 22 圖 5 本研究建立ddRAD文庫架構 23 圖 6 HiSeq定序資料分析處理流程 29 圖 7 Allele specific PCR示意圖 37 圖 8 96個番椒定序樣品配置圖與樣品編號 40 圖 9 TagGD與Stacks兩組程式對於測試檔案之條碼區分差異 41 圖 10 以process_radtags區分88個樣品之讀序數量結果整理 43 圖 11 親本樣品以PE比對至番椒參考序列之插入片段和MAPQ整理 45 圖 12 8個不同親本樣品各自插入片段大小分布 46 圖 13 Stacks共通基因座之深度資訊與篩選分子標誌分佈與數量 53 圖 14 番椒12個連鎖群兩兩分子標誌兩兩重組率與LOD值 56 圖 15 1227個SNP+SSR+CAPS分子標誌建立之番椒連鎖圖譜 57 圖 16 第6、8條分子標誌物理圖譜連鎖圖譜比較圖 62 圖 17 番椒各染色體物理圖譜、連鎖圖譜與SSR連鎖圖譜分子標誌關係圖 63 圖 18 PstI-MspI組合產生不同大小之片段數量 65 圖 19 第6條染色體之模擬片段與實際分析片段數量及對應比率 66 圖 20 第6條染色體前端物理圖譜與兩連鎖圖譜比較 70 圖 21 qASP分析模式 73 圖 22 SNP分子標誌32081第2個SNP TaqMan分析結果 73 附圖 1 Stacks資料處理流程圖 81 附圖 2 兩種不同距離算法之差異 85 附圖 3 物理圖譜與連鎖圖譜和SSR連鎖圖譜比較圖細節 87 附圖 4 Stacks 視覺化介面 93 附圖 5 分子標誌32081設計引子Sanger定序兩親本比對結果 94 | |
dc.language.iso | zh-TW | |
dc.title | 應用雙限制酶切位點標定法定位番椒稔性恢復基因 | zh_TW |
dc.title | Construction of a High Density Linkage Map with Double Digest RADseq and Mapping of Restorer-of-Fertility Gene in Pepper (Capsicum annuum L.) | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳凱儀,董致韡 | |
dc.subject.keyword | 番椒,雙限制?切位點標定法,連鎖圖譜,稔性回復基因, | zh_TW |
dc.subject.keyword | pepper,ddRAD,linkage map,Restorer-of-fertility, | en |
dc.relation.page | 94 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-02-03 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農藝學研究所 | zh_TW |
顯示於系所單位: | 農藝學系 |
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ntu-105-1.pdf 目前未授權公開取用 | 4.47 MB | Adobe PDF |
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