臺灣博碩士論文加值系統

番木瓜原產於中美洲，因高產且營養豐富，現已推廣於全球熱帶及亞熱帶地區種植，番木瓜具有3種性別，包括雄株、雌株以及兩性株，其中以兩性株果實最具利用價值，據信由X及Y性染色體上一對性別決定Sex基因座的3種對偶基因型調控，雌株、雄株以及兩性株分別為XX、以及XYh染色體組成，但目前還未能確認實際控制性別基因內容，僅知Y(h)染色體上Short Vegetative Phase (SVP) -like基因座與雄株及兩性株性別調控密切有關。番木瓜種苗7號是從一般兩性株中發現的自然突變品系SR*所選拔得來的，其自交後裔可產生100%兩性株後裔與一般兩性自交產生2兩性:1雌性後裔明顯不同，根據前人雜交後裔性別組成結果，推論受X染色體上ml隱性致死對偶基因型所調控，但對其成因尚缺乏深入研究，因此，本篇內容以解析番木瓜種苗7號全兩性性狀成因為主要目的，首先基於X、Y以及Yh染色體上SVP-like對偶基因序列差異，新設計番木瓜性別鑑定分子標誌，藉以分析種苗7號自交或與一般兩性株雜交後裔的種子及小苗，並且基於前人提出的種子敗育假說及新提出的種子發芽抑制展開兩種後裔性別分離模式，結果顯示種苗7號自交後裔種子測得雌株及兩性株基因型，惟在小苗僅測得兩性株基因型，符合種苗7號全兩性係因雌株種子發芽抑制所致，新提出全兩性由發芽抑制Germinating suppressor (Gs)基因座調控。接續導入全基因體分析方法，比較種苗7號X染色體序列與公開資料中X染色體BAC殖系參考序列間的小片段序列差異，結果顯示在種苗7號monodehydroascorbate reductase 4 (MDAR4)基因上發現一處3 bp Indel可能對產物功能有影響，且此一缺失也未見於資料庫中48個廣泛蒐集系X染色體序列，符合罕見缺失事實。已知MDAR4蛋白參與種子發芽過程中過氧化物質清除，扮演種子順利發芽重要角色，基因表現分析顯示番木瓜MDAR4基因表現在浸種後明顯提升，符合阿拉伯芥模式植物的表現特徵，是首次提出MDAR4基因參與番木瓜種子發芽過程。最後為了將此一發現應用於番木瓜品種改良，也針對MDAR4基因3 bp Indel設計對偶基因專一性分子標誌，可於苗期鑑別具有全兩性特性之後裔小苗，有助於提升選拔效率，此外，也發現對偶基因帶有3 bp同質結合缺陷的後裔雌株種子比重較輕，可能與MDAR4蛋白參與種子儲藏性油脂氧化有關。綜上，基於基因序列差異與功能性影響，在此提出MDAR4基因就是番木瓜種苗7號全兩性調控Gs候選基因。

Papaya, Carica papaya L. originated from Central America, is an economically important fruit crop due to its high yield and nutritional value. Nowadays, papaya is cultivated in tropical and subtropical regions worldwide. Sex forms of papaya have female, male, and hermaphrodite and are determined by a pair of sex chromosomes including XX for female, XY for male and XYh for hermaphrodite plants. The fruits from the hermaphrodite flowers tend to be more elongated and characterized with smaller fruit cavity than female flower. Therefore, farmers tend to grow only hermaphrodite papaya plants in the field. To date, sex determinating gene in papaya has yet been vilided and previous studies revealed Short Vegetative Phase (SVP) –like locus on Y(h) chromosome is highly related to male-hermaphrodite transformation. The H*-TSS No.7, an inbred line derived from a rare X chromosome mutant SR*, produced all-hermaphrodite progeny. Sex segregation in populations generated from cross-pollinating H*-TSS No.7 with a normal hermaphrodite showed a 2 hermaphrodite (H): 1 female (F) segregation ratio, whereas self-pollinated produced 1 H: 0 F ratios. This unique phenomenon is manifested by a lethal allele, ml, on the X chromosome was proposed. The result was based on morphology characters while no genotyping at the DNA level was conducted. Hence, the major objective was to dissect the control of this unique all-hermaphrodite phenomen at genetics level. Firstly, SCAR markers derived from the SVP-like gene which can discriminate the three sex forms: female, male and hermaphrodite papaya were conducted to progeny analysis of H*-TSS No.7 derived seeds and seedlings. Two mechanisms might result in the all-hermaphrodite phenomenon in H*-TSS No. 7: seed abortion during zygote development as previous study or, alternatively, lost germination ability in female plants were proposed. The results revealed female progeny was only detected in self-pollinated seedlings but nor in seeds suggested that seed abortion should not the one responsible for all-hermaphrodite phenomenon. Hence, a Germinating suppressor (Gs) locus responsed for all-hermaphrodite phenomenon in H*-TSS No. 7 was proposed. Subsequently, whole genome comparsion between H*-TSS No. 7 and reference genomes revealed a 3 bp Indel in monodehydroascorbate reductase 4 (MDAR4) gene might inference the protein function. Among 48 widly collected papaya lines no one harbor the same rare 3 bp deletion as H*-TSS No.7 did. It is known MDAR4 is one of the enzymes involving in the seed germinating process which play an important role in H2O2 scavenging. Furthermore, gene expression analysis revealed MDAR4 gene showed higher expression levels in imbibed seeds than those in dry seeds as observed in Arabidopsis germinating process. This is the very first report providing the evidences that MDAR4 involed in papaya germinating process. Finally, allele specifc SCAR markers were developed for marker-assisted selection which could identified all-hermaprodite progeny at seedling stage. Moreover, female progeny harbor momologus 3 bp deletion tened to have less specific weight might interfere composition of seed storage oil, since MDAR4 enzyme is also involving in fatty acid metabolism in seed. All mention above, this study has identified papaya MDAR4 gene being involved in all-hermaphrodite characteristic in H*-TSS No.7 as the most candidate of Gs locus.

中文摘要 i
Abstract iii
目次/Contents v
表目次/Contents of table vi
圖目次/Contents of figure viii
縮寫表/ Abbreviation x
第一章、前言及前人研究:番木瓜性別決定之研究現況 1
Chapter 1. Introduction and literature review：Progress of sex determination in papaya
第二章、解析X染色體缺陷全兩性番木瓜種苗7號之成因 22
Chapter 2. Dissecting the all-hermaphrodite phenomenon of a rare X chromosome mutant in papaya (Carica papaya L.)
第三章、全基因組探勘顯示monodehydroascorbate reductase 4基因為番木瓜種苗7號之全兩性調控候選基因 50
Chapter 3. Genomic characterization of a rare Carica papaya X chromosome mutant reveals a candidate monodehydroascorbate reductase 4 gene involved in all-hermaphrodite phenomenon
第四章、結論 88
Chapter 4. Conclusions

第一章、前言及前人研究:番木瓜性別決定之研究現況
Chapter 1. Introduction and literature review: Progress of sex determination in papaya
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第二章、解析X染色體缺陷全兩性番木瓜種苗7號之成因
Chapter 2. Dissecting the all-hermaphrodite phenomenon of a rare X chromosome mutant in papaya (Carica papaya L.)
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第三章、全基因組探勘顯示monodehydroascorbate reductase 4基因為番木瓜種苗7號之全兩性調控候選基因
Chapter 3. Genomic characterization of a rare Carica papaya X chromosome mutant reveals a candidate monodehydroascorbate reductase 4 gene involved in all-hermaphrodite phenomenon
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