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研究生:黃瑋琪
研究生(外文):Wei-Chi Huang
論文名稱:台灣東部澤蛙(Fejervarya limnocharis)完整粒線體基因組序列及其親緣演化之探討
論文名稱(外文):The studies of complete mitochondrial genomic sequence of eastern Taiwan Fejervarya limnocharis and phylogenetic evolution
指導教授:劉阜果
指導教授(外文):Fu-Guo Robert Liu
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:中文
論文頁數:88
中文關鍵詞:東台灣粒線體DNA分子演化親緣關係
外文關鍵詞:DicroglossidaeFejervarya limnocharis
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澤蛙(Fejervarya limnocharis)被認為為一複合群,過去有研究曾提及台灣東、西部澤蛙為不同族群,且台灣東部澤蛙在分類上之定位尚未十分明瞭,因此本研究採集來自台灣¬台東之澤蛙作為樣本(F. limnocharisETW),完成其完整粒線體基因組之序列,並將該序列資料存放至NCBI之GenBank中,登錄碼為MZ666929。 F. limnocharisETW完整粒線體基因組序列長度為17,663 bp,其中包含13 個蛋白質編碼基因、23個tRNA基因、2個rRNA 基因和2個非編碼控制區(Dloop和L鏈複製起點)。F. limnocharisETW之基因組組成A + T 含量較高,為 57.1%(A 27.3%,T 29.8%,G 15.8%,C 27.1%)。利用台東F. limnocharisETW和叉舌蛙科下21個物種及一外群之完整粒線體基因組序列,以鄰近連接法(Neighbor joining)、最大概率法(Maximum likelihood)及貝葉氏推論分析(Bayesian inference)之運算法建立其親緣關係。親緣關係之結果大多高度支持台東之F. limnocharisETW 與F. kawamurai形成一單系群;來自中國之F. limnocharisCN與F. multistriata為一單系群。16S rRNA之遺傳差異結果顯示台東之F. limnocharisETW 與F. kawamurai之差異為3.977%;與F. limnocharisCN為4.792%;與F. multistriata為4.664%,差異皆大於3%之物種建議門檻。遺傳差異、遺傳距離與親緣演化之結果,皆顯示F. limnocharisETW高度可能為一隱蔽種。此外,發現tRNA基因之演化資訊不足,不適於探討Fejervarya屬內物種分子演化之問題。本研究之台東澤蛙完整粒線體基因組序列將可進一步為Fejervarya 屬及叉舌蛙科之遺傳學、分類學和分子演化學等未來之研究提供更多資訊。
Fejervarya limnocharis, rice frog, is considered a species complex. Previous studies have mentioned that the eastern and western Taiwan rice frogs are different ethnic groups and the classification of the eastern Taiwan rice frogs (F. limnocharisETW) is not very clear. In this study the complete mitochondrial genome sequence of F. limnocharisETW collecting from Taitung, Taiwan was deposited in GenBank with accession number MZ666929. The whole mitochondrial genome of F. limnocharisETW contains 17,663 bps in length, including 13 protein-coding genes, 23 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and 2 non-coding control region (Dloop and origin of L-strand replication). The mitogenome base composition of F. limnocharisETW was biased toward A + T content at 57.1% (A 27.3% , T 29.8% , G 15.8% , C 27.1% ). Phylogenetic relationships were constructed based on complete mitogenome sequences data of F. limnocharis and other 21 members in family Dicroglossidae by Neighbor-joining, Maximum likelihood and Bayesian inferences. The results of phylogeny highly supported that F. limnocharisETW from eastern Taiwan formed a monophyletic group with F. kawamurai ; F. limnocharisCN from China formed a monophyletic group with F. multistriata. The genetic differences of 16S rRNA showed that the differences between F. limnocharisETW to F. kawamurai, F. limnocharisCN from China and F. multistriata were 3.977%, 4.792% and 4.664%, respectively. The results of this study showed F. limnocharisETW highly likely to be a cryptic species. In addition, tRNA genes were not suitable for the studies of genus Fejervarya phylogeny and evolution due to insufficient evolutionary information. The complete mitochondrial genome sequence of F. limnocharisETW in this study provided information for further phylogenetic or genetic evolutionary researches on genus Fejervarya or family Dicroglossidae.
中文摘要 ............................................................................................................ i
ABSTRACT ...................................................................................................... ii
致謝 .................................................................................................................. iii
目錄 .................................................................................................................. iv
圖目錄 .............................................................................................................. vi
表目錄 ............................................................................................................. vii
縮寫表 ............................................................................................................ viii
一、前言 ........................................................................................................... 1
1-1 澤蛙(Fejervarya limnocharis)相關描述 ................................................. 1
1-1-1 物種分類 ............................................................................................ 1
1-1-2形態特徵 ............................................................................................ 2
1-1-3生態分布 ............................................................................................ 4
1-2分子技術及粒線體基因組 ....................................................................... 5
1-3 親緣關係分析方法概述 ........................................................................... 7
1-3-1鄰近連接法(Neighbor joining) ....................................................... 7
1-3-2 最大概率法(Maximum likelihood) ................................................ 7
1-3-3 貝葉氏推論分析(Bayesian inference) ............................................. 8
1-3-4 親緣關係樹之支持度分析 ................................................................. 8
1-4 文獻回顧 ................................................................................................ 11
1-5 研究目的 ............................................................................................... 15
v
二、材料與方法 .............................................................................................. 16
2-1樣本採集 ................................................................................................ 16
2-2 DNA萃取 ............................................................................................... 18
2-3 聚合酶連鎖反應(PCR) ......................................................................... 19
2-4 整合序列資料 ........................................................................................ 21
2-5 分子親緣關係分析 ................................................................................. 21
三、結果 ......................................................................................................... 24
3-1 Fejervarya limnocharisETW完整粒線體基因組序列 .......................... 24
3-2 蛋白質編碼基因(protein coding genes, PCGs) .................................. 30
3-3 非編碼基因(Noncoding genes) ............................................................ 37
3-4 非編碼控制區(Noncoding region) ....................................................... 49
3-5 叉舌蛙科下之親緣關係樹(Phylogenetic tree) .................................... 51
四、討論 ......................................................................................................... 60
4-1 Fejervarya limnocharisETW之親緣關係 ............................................. 60
4-2叉舌蛙科之親緣關係樹與基因排列結構 .............................................. 62
4-3 F. limnocharisCN與F. multistriata之關係 ......................................... 64
4-4 Occidozyga屬與Phrynoglossus屬之關係 ............................................ 64
五、結論 ......................................................................................................... 66
六、參考文獻 .................................................................................................. 67
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