臺灣博碩士論文加值系統

高粱(Sorghum bicolor)穀粒富含澱粉，其澱粉品質為評斷高粱穀粒優劣的主要項目，澱粉品質進而影響營養價值與加工的應用性。本研究採用7個高粱品種，分析品種間澱粉相關性狀(澱粉與直鏈澱粉含量、千粒重、榖粒長、榖粒寬與澱粉糊化特性)差異性，並選定澱粉生合成路徑Soluble starch synthases (SSIIa)、Granule-bound starch synthase (Wx)與Starch branching enzyme (Ae1)三個候選基因，進行序列多型性的分析，最後以關聯分析(association analysis)方法尋找與表型具關聯性的等位基因。結果顯示SSIIa序列上具有9個多型性位點，而Wx 共具有7個多型性位點，其中一些位點與澱粉糊化特性有相關。Ae1增幅的兩片段上具有29個多型性位點。Ae1增幅片段的單一核苷酸多型性(single nucleotide polymorphisms, SNP)及插入與缺失(insertion and deletion, InDel)片段與澱粉糊化起始溫度(gelatinization temperature)相關，平均溫度相差9.4℃。在Ae1內插子區域的SNP與直鏈澱粉含量之含量具關聯性(p<0.05)，腺嘌呤鹼基 (adenine base)的缺失導致直鏈澱粉含量甚低。綜合實驗結果，序列多型性的鑑定可說明高粱澱
粉相關特性的遺傳變異。

Sorghum is rich in starch content, the starch quality contributes to its nutritional value and processing attributes tremendously. In this study, seven varieties of soghum were collected for analysis in their starch content, amylose content, pasting properties, thousand grain weight, and grain size. To identify the correlation of gene variation and the phenotype in starch content, genes including soluble starch synthases (SSIIa), granule-bound starch synthase (Wx) and starch branching enzyme (Ae1) were chosen for association analysis using statistical software. A total of 9 polymorphisms in SSIIa and 7 polymorphisms in Wx were identified, which were greatly associated with starch pasting properties. A total of 29 polymorphisms in two fragments of Ae1 were identified. Single nucleotide polymorphisms(SNP) and InDel in Ae1 were found associated with gelatinization temperature, which with a difference of 9.4℃ in gelatinization temperature. One SNP in non-coding region of Ae1 were found associated with amylase content (p<0.05), inferred deletion of a adenine base would cause a decrease in non-branching starch. The polymorphism we identified in this study may provide a genetic explanation to variation in sorghum starch properties.

中文摘要 I
Abstract II
目錄 III
圖目錄 VI
表目錄 VII
壹、前言 1
貳、文獻回顧 2
一、高粱之相關性質 2
(一) 高粱之介紹及現況 2
(二)高粱穀粒組成分 4
(三)高粱之應用 5
二、植物澱粉生合成 5
(一)植物澱粉 5
(二)澱粉生合成路徑 8
(三)澱粉生合成之相關酵素 9
三、關聯分析研究 12
(一)關聯分析的基本方法 13
(二)關聯分析的分析步驟 13
(三)植物關聯分析研究應用 13
(四)關聯分析應用之分子標誌 16
四、 研究目的與實驗架構 16
參、材料與方法 18
一、高粱Wx, SssIIA, Ae1 基因序列分析 18
(一)試驗材料 18
(二)高粱Genomic DNA 萃取 19
(三) Genomic DNA 濃度測定 20
(四)聚合酶連鎖反應(PCR) 21
(五)瓊脂膠體電泳分析 21
(六) PCR產物純化與濃縮 26
(七) TA cloning 27
(八) Ligation 28
(十)定序與序列分析 29
(十一)聚類分析樹圖 29
二、高粱穀粒澱粉相關性狀之測定與評估 31
(一)碘染法糯性鑑定 31
(二)一般成分分析 31
1.水分 31
2.灰分 31
3.粗脂肪 32
(三)單寧含量 32
(四)高粱穀粒澱粉組成 33
1.總澱粉含量測定 33
2.直鏈澱粉含量測定 33
(五)高粱穀粒物理性狀測定 34
1.穀粒千粒重 34
2.穀粒長、寬 34
(六)高粱澱粉糊化性質檢測 34
(七)高粱澱粉物性分析 35
1. 澱粉萃取 35
2. 澱粉膨潤力(swelling power)、溶解度(solubility)與水結合力(water binding capacity) 35
(八)統計分析 36
四、高粱Wx、SssIIA、Ae1 基因序列與穀粒性狀之相關性分析 36
肆、結果與討論 37
一、高粱SSIIa 、Wx、Ae1 基因序列分析 37
(一) SSIIa序列多態性 37
(二) Wx序列多態性 38
(三) Ae1序列多態性 39
二、高粱穀粒澱粉相關性狀之測定與評估 53
(一)碘染法糯性鑒定 53
(二)一般成分分析 55
(三)單寧含量 56
(四)高粱穀粒澱粉組成 57
(五)高粱穀粒物理性狀 58
(六)高粱澱粉糊化性質 59
(七)高粱澱粉物性分析 62
三、高粱SSIIa、Wx、Ae1 基因序列與穀粒澱粉相關性狀之關聯性分析 71
伍、總結 75
陸、參考文獻 76
附錄一、高粱試樣SSIIa專一性引子對SSIIa8F/9R增幅片段之排序比對 85
附錄二、高粱試樣Waxy專一性引子對GBSS1F/1R增幅片段之排序比對 88
附錄三、高粱試樣Waxy專一性引子對GBSS9F/11R增幅片段之排序比對 91
附錄四、高粱試樣Ae1專一性引子對SBE1F/2R增幅片段之排序比對 93
附錄五、高粱試樣Ae1專一性引子對SBE9F/10R增幅片段之排序比對 95

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