臺灣博碩士論文加值系統

番茄具多種植物化學因子，其中維生素C與茄紅素最為人們所重視。茄紅素具抗氧化作用，可消除自由基，並減少慢性疾病發生率、降低紫外線對皮膚之傷害及減低發生癌症之機率。許多研究指出番茄與其加工製品對健康有益，故近年來出現許多番茄的加工製品。番茄適合生長於溫暖乾燥且日夜溫差大的氣候。最適之栽培溫度是平均日溫為20∼26℃，平均夜溫為15∼20℃。台灣平地夏季之平均日溫在30℃以上，夜溫在25℃以上，且常有豪雨，在高溫多溼之環境下，由於大部分栽培品種不具耐熱性，因此農民栽培番茄之季節主要集中於乾燥冷涼之秋冬季，故台灣栽培番茄受到溫度之限制甚深。
番茄之耐熱性狀由數量遺傳基因控制，較易受環境影響。2002年夏季種植於文化大學園藝系網室由耐熱之栽培品系CL5915與不耐熱之L4422 (L. Pimpinellifolium)產生之43個F7重組自交系(RIL)。本研究中探討5個耐熱相關性狀：開花數、結果數、結果率、單果重、單株產量，利用混合分離族群分析法(Bulked Segregant Analysis, BSA)及核酸逢機增殖多型性(Random Amplified Polymorphic DNA, RAPD)技術尋找與番茄耐熱數量性狀基因(開花數、結果數、結果率、單果重、單株產量)連鎖之分子標記。以200個OPERON隨機引子進行試驗，發現其中14個引子與DNA模板(耐熱之栽培品系CL5915與不耐熱之品系L4422及43個F7重組自交系)作用後產生明顯之RAPD-PCR多型性。此14個primers共產生88個DNA條帶，其中23個條帶具多型性並以χ2檢定測試(α=0.05)；平均每一引子產生6.3個條帶，其中1.6個條帶具多型性。結合性狀調查數據(phenotype)與RAPD-PCR電泳資料(genotype)，以Mapmaker/Exp 3.0b及Mapmaker/QTL 1.1b軟體進行基因連鎖圖譜分析和數量性狀基因座之定位，結果發現其中引子C09與S13有連鎖，且K06、K14與P08亦連鎖在同一個連鎖群中，但此5個markers並未與任何一個耐熱產量數量性狀基因有連鎖。

Tomato contains many kinds of chemical constituents, such as Vitamin C and lycopene, which are considered as free radicals scavenger and thus protects people from some chronic disorders or reduces the rate of some cancers. Most tomato varieties are adaptive to warm and dry weather, and don’t show heat tolerance. The optimum temperature for growing tomato is 15-20/20-26 °C night/ day. During summer time in Taiwan, the production of tomato is decreased due to high temperature and high rainfall.
Heat tolerance of tomato is a quantitatively inherited trait and easily affected by environmental conditions. A total of 43 F7 recombinant inbred lines derived from a heat tolerant CL 5915 crossed with a heat sensitive L4422 (L. Pimpinellifolium) were grown in the screenhouse at PCCU in the summer of 2002. Five heat tolerance-related traits were scored: flower number, fruit number, fruit set, fruit weight and fruit yield. RAPD analysis was used to identify any primer associated with the flower number, fruit number and fruit weight by bulking DNAs of skewed lines (bulked segregant analysis). A total of 200 random primers were screened. Only 14 RAPD-PCR products generated by OPERON primer with DNA template from L4422, CL5915 and F7 RILs (Recombinant inbred line) were found with polymorphism. Among them, 88 bands were amplified, in which 23 bands showed informative and were used for χ2 test. An average of 1.6 bands out of 6.3 bands was generated by a primer and thought to be informative. Combination of the mentioned 5 traits and the informative markers, we were able to identify and locate the RAPD markers linked to the QTL of traits using Mapmaker/EXP 3.0b and Mapmaker/QTL 1.1b softwares. Two linkage groups were constructed from Mapmaker: C09 and S13; K06, K14, and P08. Nevertheless, all these 5 primers didn’t show any linkage to the five heat tolerance-related traits.

目 錄
摘要 1
ABSTRACT 3
前言 5
前人研究 10
一、 番茄簡介 10
二、 RAPD (Random Amplified Polymorphic DNA) 13
三、 混合分離族群分析法 17
四、 Mapping populations 19
材料與方法 22
一、 試驗材料及生長情況 22
二、 試驗方法 22
(一) 性狀調查 22
(二) Bulked Segregant Analysis 23
(三) RAPD分子標誌之篩選 23
(四) 統計分析 26
結果 29
一、 番茄之性狀調查 29
二、 番茄之外表型性狀分析 29
三、 RAPD之分子性狀分析 30
四、 基因連鎖群之建立與基因之定位 33
討論 51
一、 植株之生長情形 51
二、 耐熱性狀之間的相關係數與決定係數 51
三、 番茄之耐熱性狀表現 52
四、 BSA之實際應用 53
五、 RAPD分子標誌所貢獻之基因效應 54
六、 RAPD分子標誌 55
參考文獻 62

陳正次 (1995) 番茄. 台灣農業要覽(2) p437-636.
陳正次 (1998) 番茄育種、蔬菜育種技術研習會專刊。台灣農試所特刊第73號 p231-284.
曾夢蛟，李本湘 (1988) 高溫逆境與作物品種改良。蔬菜品種改良研究會專輯。p185-194.
蕭吉雄，黃維東，周明燕 (2002) 番茄品種特性與栽培技術全輯。p1-56.
Agrama HAS and ME Moussa (1996) Mapping QTLs in breeding for drought tolerance in maize (Zea mays L.). Euphytica 91:89-97.
Agrama H, G Widle, J Reese, L Campbell, and M Tuinstra (2002) Genetic mapping of QTLs associated with greenbug resistance and tolerance in Sorghum bicolor. Theor. Appl. Genet. 104:1373 — 1378.
Asian Vegetable Research and Deveopment Center (1994) AVRDC Report. AVRDC, Taiwan.
Asian Vegetable Research and Deveopment Center (1997) AVRDC Report. AVRDC, Taiwan.
Berg DE, NS Akopyants, and D Kersulyte (1994) Fingerprinting microbial genomes using the RAPD or AP-PCR method. Methods in Mol. Cell. Biology 5:13—24.
Bett K (2004) Molecular Markers in Plant Breeding. http://www.usask.ca/agriculture/plantsci/classes/plsc416/molecular_markers.pps
Blum E, M Mazourek, M O''Connell, J Curry, T Thorup, K Liu, M Jahn, and I Paran. (2003) Molecular mapping of capsaicinoid biosynthesis genes and QTL analysis for capsaicinoid content in Capsicum. Theor. Appl. Genet. 108:79—86.
Brouwer DJ and DA St Clair (2004) Fine mapping of three quantitative trait loci for late blight resistance in tomato using near isogenic lines (NILs) and sub-NILs. Theor. Appl. Genet. 108(4):628-38.
Chague V, JC Mercier, M Guenard, A Courcel, and F Vedel (1997) Identification of RAPD markers linked to a locus involved in quantitative resistance to TYLCV in tomato by bulked segregant analysis. Theor. Appl. Genet. 95:671—677.
Chaim BA, I Paran, RC Grube, M Jahn, R van Wijk, and J Peleman (2001) QTL mapping of fruit-related traits in pepper (Capsicum annuum). Theor. Appl. Genet. 102:1016-1028.
Chen XH, YC Niu, and BZ Hu (2004) Identification of RAPD markers linked to the resistance gene Yr5 against wheat stripe rust with denaturing PAGE-silver staining. Yi Chuan Xue Bao 31(3):270-4.
Churchill GA , JJ Giovannoni, and SD Tanksley (1993) Pooled-sampling makes high-resolution mapping practical with DNA markers. Proc. Natl. Acad. Sci. 90(1): 16—20.
Doganlar S, A Frary, HM Ku, and SD Tanksley (2002) Mapping quantitative trait loci in inbred backcross lines of Lycopersicon pimpinellifolium (LA1589). Genome 45(6):1189-202.
Doyle, JJ and LL Doyle (1990) Isolation of plant DNA from fresh tissue. Focus 12:13-15.
Fu P, RW Wilen, AJ Robertson, RT Taylor, and LV Gusta (1998) Heat tolerance of cold acclimated Puma winter rye seedling and the effect of a heat shock on freezing tolerance. Plant Cell Physiol. 39:942-949.
Foolad MR, FQ Chen, and GY Lin (1998) RFLP mapping of QTLs conferring salt tolerance during germination in tomato by selective genotyping. Mol. Breeding 3:269-277.
Foolad MR, LP Zhang, and P Subbiah (2003) Genetics of drought tolerance during seed germination in tomato: inheritance and QTL mapping. Genome 46(4):536-45.
Georgiady MS, RW Whitkus, and EM Lord (2002) Genetic analysis of traits distinguishing outcrossing and self-pollinating forms of currant tomato, Lycopersicon pimpinellifolium (Jusl.) Mill. Genetics 161(1):333-44.
Giovanni C, P Dell’Orco, A Bruno, F Ciccarese, C Lotti, and L Ricciardi (2004) Identification of PCR-based markers (RAPD, AFLP) linked to a novel powdery mildew resistance gene (ol-2) in tomato. Plant Sci. 166: 41-48.
Hadrys H, M Balick, and B Schierwater (1992) Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Mol. Ecol. 1:55-63.
Hashizume T, I Shimamoto, and M Hirai (2003) Construction of a linkage map and QTL analysis of horticultural traits for watermelon. Theor. Appl. Genet. 106:779-785.
Hayes PM, TK Blake, THH Chen, S Tragoonrung, F Chen, A Pan, and B Liu (1993) Quantitative trait loci on barley (Hordeum vulgare L.) chromosome 7 associated with components of winter hardiness. Genome 36:66-71.
Kearsey MJ and AGL Farquhar (1998) QTL analysis in plants; what are we now? Heredity 80:137-142.
Kelly JD, P Gepts, PN Miklas, and DP Coyne (2003) Tagging and mapping of genes and QTL and molecular marker-assisted selection for traits of economic importance in bean and cowpea. Field Crops Res. 82:135—154.
Korzun V (2002) Use of molecular markers in cereal breeding. Cell. Mol. Biol. Let. 7:811—820.
Ku HM, S Grandillo, and SD Tanksley (2000) fs8.1, a major QTL, sets the pattern of tomato carpel shape well before anthesis. Theor. Appl. Genet. 101:873—878.
Kuo CG, HM Chen, and HC Chen (1986) Plant hormones in tomato fruit-set and develop at high temperatures. p.53-70. In: Plant Growth Regulators in Agriculture. AVRDC, Taiwan.
Lander ES, P Green, J Abrahamson, A Barlow, MJ Daly, SE Lincoln, and L Newburg (1987) MAPMAKER: an interactive computer package of constructing primary genetic linkage maps of experimental and natural populations. Genomics l:174-181.
LEE, M (1995) DNA markers and plant breeding programs. Adv. Agron. 55:265-343.
Levi A, CE Thomas, TC Wehner, and X Zhang (2001) Low genetic diversity indicates the need to broaden the genetic base of cultivated watermelon. HortScience 36:1096-1101.
Lilley JM, MM Ludlow, SR McCouch, and JC O’Toole (1996) Locating QTL for osmotic adjustment and dehydration tolerance in rice. J. Exp. Bot. 47:1427-1436.
Lin KR, B Martin, and CG Tauer (2002) Identification of quantitative trait loci associated with water-use efficiency and related traits in Lycopersicon. Hwa Kang J. Agr. 9:1-20.
Lippmana Z and SD Tanksley (2001) Dissecting the genetic pathway to extreme fruit size in tomato using a cross between the small-fruited wild species Lycopersicon pimpinellifolium and L. esculentum var. Giant Heirloom. Genetics (158):413-422.
Lohar DP and WE Peat (1998) Floral characteristics of heat-tolerance and heat-sensitive tomato cultivars at high temparture. Sci. Hort. 73:53-60.
Low D, K Brandle, L Nover, and C Forreiter (2000) Cytosolic heat-stress proteins Hsp17.7 class I and Hsp17.3 class II of tomato act as molecular chaperones in vivo. Planta 211:575-582.
Mackay I J and PDS Caligari (2000) Efficiencies of F2 and backcross generations for bulked segregant analysis using dominant markers. Crop Sci. 40:626—630.
Mao CZ, L Yang, BS Zheng, YR Wu, FY Liu, KK Yi, and P Wu (2004) Comparative mapping of QTLs for Al tolerance in rice and identification of positional Al-induced genes. J. Zhejiang Univ. Sci. 5(6):634-43.
Martin B, J Nienhuis, G King, A Schaefer (1989) Restriction fragment length polymophisms associated with water use efficiency in tomato. Science 243:1725-1728.
Martin B, C Tauer, and KR Lin (1999) Carbon isotope discrimination as a tool to improve water-use efficiency in tomato November/December issue. Crop Sci. 39:1775-1783.
Meksem K, VN Njiti, WJ Banz, MJ Iqbal, MM Kassem, DL Hyten, J Yuang, TA Winters, and DA Lightfoot (2001) Genomic regions that underlie Soybean seed isoflavone Content. J. Biomed. Biotechnol. 1(1):38-44.
Mian MAR, MA Bailey, DA Ashley, R Wells, TE Jr. Carter, WA Parrott, and HR Boerma (1996) Molecular markers associated with water use efficiency and leaf ash in soybean. Crop Sci. 36:1252-1257.
Mian MAR, DA Ashley, and HR Boerma (1998) An additional QTL for water use efficiency in soybean. Crop Sci. 38(2): 390-393.
Michelmore RW, I Paran, and RV Kesseli (1991) Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA 88(21):9828-32.
Mohan M, S Nair, A Bhagwat, TG Krishna, M Yano, CR Bhatia, and T Sasaki (1997) Genome mapping, molecular markers and marker-assisted selection in crop plants. Mol. Breed. 3:87-103.
Monforte AJ, MJ Asins, and EA Carbonell (1997) Salt tolerance in Lycopersicon species. VI. Genotype-by-salinity interaction in quantitative trait loci detection: constitutive and response QTLs. Theol. Appl Genet. 95:706—713.
Paran I, R Kesseli, and R Michelmore (1991) Identification of restriction fragment length polymorphism and random amplified polymorphic DNA markers linked to downy mildew resistance genes in lettuce, using near-isogenic lines. Genome 34(6):1021-7.
Paterson AH, S Damon, JD Hewitt, D Zamir, HD Rabinowitch, SE Lincolin, ES Lander, and SD Tanksley (1991) Mendelian factors underlaying quantitative traits in tomato: comparison across species, generations and environments. Genetics 127:181-197.
Paterson AH and RA Wing (1993) Genomic mapping in plants. Cur. Opin. Biotech. 4:142-147.
Rick CM (1976) Natural variability in wild species ofLycopersicon and its bearing on tomato breeding. Genet. Agrar. 30:249-259.
Rick CM (1988) Tomato-like nightshades: Affinities, autoecolory and breeder''s opportunities. Econ. Bot. 42:145-154.
Rao AV and S Agarwal (2000) Role of antioxidant lycopene in cancer and heart diease. J. Am. Coll. Nutr. 19:563-569.
Virlin E (1990) Heat shock protein function and expression in plants. In R Alscher, J Gumming, eds. Stress response in plants: adaptation and acclimation mechanisms. Wiley-Liss, New York, pp 357-375.
Welsh J and M McClelland (1990) Fingerprinting genomes using PCR with arbitrary primers. Nuc. Acids Res. 18:7213-7218.
Wen CS and JY Hsiao (2001) Altitudinal genetic differentiation and diversity of Taiwan lily (Lilium longiflorum var. formosanum; Liliaceae) using RAPD markers and morphological characters. Int. J. Plant Sci. 40:65-71.
Williams JGK, AR Kubelik, KJ Livak, JA Rafalski, and SV Tingey (1990) DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531-6535.
Wu JY, HK Wu, and MC Chung (2002) Co-dominant RAPD markers closely linked with two morphological genes in rice (Oryza sativa L). Bot. Bull. Acad. Sin. 43:171-180.
Saliba CV, M Causse, L Gervais, and J Philouze (2000) Efficiency of RFLP, RAPD, and AFLP markers for the construction of an intraspecific map of the tomato genome. Genome 43(1):29-40.
Spooner D, G Anderson, and R Jansen (1993) Chloroplast DNA evidence for the interrelationships of tomatoes, potatoes, and pepino (Solanaceae). Am. J. Bot. 80:676—698.
Steiner B, M Lemmens, M Griesser, U Scholz, J Schondelmaier, and H Buerstmayr (2004) Molecular mapping of resistance to Fusarium head blight in the spring wheat cultivar Frontana. Theor. Appl. Genet. 109(1):215-24.
Stuber Cw, MD Edwards, and JF Wendel (1987) Molecular marker-facilitated investigations of quantitive trait loci in maize. II. Factors influencing yield and its components traits. Crop Sci. 27:639-648.
Tabor GM, TL Kubisiak, NB Klopfenstein, RB Hall, and HS McNabb (2000) Bulked Segregant Analysis Identifies Molecular Markers Linked to Melampsora medusae Resistance in Populus deltoides. Phytopathology 90:1039-1042.
Tanksley SD (1994) Tomato molecular map. In RL Phillips, IK Yasil, eds, DNA-based Markers in Plants. Kluwer Acad Publ, New York, NY, pp310-326.
Tanksley SD, ND Young, , AH Paterson, and MB Bonierbale, (1989) RFLP mapping in plants: new tools for an old science. Biol. Tech. 7: 257-264.
Tanksley, SD, MW Ganal, JP Prince, MC de Vicente, MW Bonierbale, P Broun, TM Fulton, JJ Giovannoni, S Grandillo, GB Martin, R Messeguer, JC Miller, L Miller, AH Paterson, O Pineda, MS Roeder, RA Wing, W Wu, and ND Young (1992) High density molecular linkage maps of the tomato and potato genomes. Genetics 132:1141-1160.
Teulat B, P Monneveux, J Wery, C Borries, I Souyris, A Charrier, and D This (1997) Relation between relative water content and growth parameters under water stress in barley : a QTL study. New Phytologist 137: 99-107.
Zhang HB, MA Budiman and RA Wing (2000) Genetic mapping of jointless-2 to tomato chromosome 12 using RFLP and RAPD markers. Theor. Appl. Genet. 100:1183-1189.
Zhang Y and JR Stommel (2000) RAPD and AFLP tagging and mapping of Beta (B) and Beta modifier (MoB), two genes which influence ß-carotene accumulation in fruit of tomato (Lycopersicon esculentum Mill.). Theor. Appl. Genet. 100:368—375.
Zhang Y and JR Stommel (2001) Development of SCAR and CAPS markers linked to the Beta gene in tomato. Crop Sci. 41:1602-1608.