- 學位論文
水稻臺農71號導入秈稻全染色體置換系之族群建構與評估
Construction and Evaluation of Rice Whole Chromosome Substitution Lines of Japonica Cultivar ‘Tainung 71’ Carrying Indica Cultivar ‘Habataki’
摘要
我國水稻栽培品種大多係稉稻遺傳背景，為有效導入秈稻優良農藝性狀基因，提高國內稻作產量潛能與逆境耐性，選育一套稉稻背景之秈稻全染色體置換系族群，以作為台灣稉稻分子輔助選育效益之評估平台，便於完整探討目標性狀之堆疊效應以及與環境交感影響，有助於提高融合秈、稉兩亞種間之優良基因及其評估效益。在規劃全染色體置換系族群的最佳選拔策略前，以所估算完整染色體條件機率進行模擬研究探討各世代最低族群數與試驗效率，結果顯示在BC1F1世代涵蓋全套完整染色體提供親染色體的最小族群數為80株，而BC2F1與BC3F1世代中12個次族群分別需維持30株才能確保目標完整染色體不會在世代推進中遺失，之後各BC3F2次族群的最低族群數需223株挑選目標植株。另用以輔助選拔全染色體置換系的基因型分析平台上，開發了一套506個涵蓋全基因組的InDel標誌，InDel相對於PCR產物大小的比值範圍介於8.8%至45.6%，片段差異大小為21.6%，該結果有助於在2%至3%濃度的洋菜膠體電泳中，在30分鐘內以非浸潤式的電泳模式有效區分PCR產物並進行條帶判別。族群親本係以稉稻臺農71號 (Tainung 71, TNG71) 為輪迴親，日本秈稻品種Habataki為提供親；自2008年第2期作建立雜交組合起，連續回交3個世代並維持導入目標完整染色體，在2011年第一、二期作均進行自交固定各染色體品系 (BC3F2)，品系選拔係仰賴自行開發均勻散布於全基因體151個InDel與SSR標誌輔助偵測遺傳重組。由於秈稻Habataki第四號染色體上攜有稔性基因，因而無法導入完整單一染色體，而其他11條染色體均完整個別轉移至輪迴親臺農71號中，品系分別編為TARI71_1至TARI71_12等13個品系。在2012年第2期作農藝性狀初步評估上，可見TARI71_2品系因攜有hbd2雜種弱勢秈稻對偶基因，該置換系顯出矮株 (70.3 cm)、低稔實率 (48%) 等弱勢現象，使得分蘗數由臺農71號之10.8枝降至3.5枝，未來應避免在稉稻背景下導入該區間的秈稻片段以降低對其他產量基因之干擾。高產量導入效應之評估包括：TARI71_1品系因攜有Habataki的多粒數 (Grain number 1, Gn1) 與半矮性 (Semi-dwarf 1, sd1) 對偶基因，每穗粒數由臺農71號之116.4粒提升為128粒，而株高則由94.7矮化為74.3cm，充實率亦隨之下降至74%；但品系TARI71_6可視為對臺農71號導入Habataki的1次枝梗數並維持充實率之影響，每穗粒數提升為151.2粒且充實率可達90%。生育期長短影響碳水化合物積存能力，在品系TARI71_10可觀察抽穗期Ehd1早熟對偶基因之置換效應，使得抽穗期由76天提早成熟至53天。本試驗最後以粒數基因Gn1為例，說明染色體置換系之應用，分別探討粒數基因置換定位分析與高粒數分子輔助育種模式，期望在有限的試驗資源下配置最高的選拔效率，並評估Habataki的Gn1對偶基因在我國稉型品種上之選拔增進，以提升優良後裔選拔效率；期望上述品系選育試驗能加速與國際育種研究相互接軌，並累積稻作分子輔助選育能力。
並列摘要
To facilitate the introgression of elite indica genes into japonica cultivars and the improvement of grain yield potential and stress tolerance in Taiwanese rice, we developed a novel mapping population consisting of 13 ‘Whole Chromosome Substitution Lines’ (WCSLs). In each line, common genetic background of the japonica cultivar ‘Tainung 71 (TNG71)’ (Taiwanese elite cultivar) was substituted by different single or large segment of chromosome of the indica cultivar ‘Habataki’. In order to determine the selection strategy, simulation studies based on the conditional probabilities of obtaining whole chromosome were conducted to estimate the minimum population size of each generation. The results indicated the minimum BC1F1 population size would be 80 plants to avoid the loss of any chromosome, and each BC2F1 and BC3F1 subpopulation needed at least 30 plats to maintain target whole chromosome. Subsequently, subpopulation size for each BC3F2 would be 223 plants for development of substitution line with targe whole chromosome. In order to enhance the selection efficiency of genotyping platform, a set of 506 PCR-based novel insertion/deletion (InDel) markers were designed. The InDel to amplicon size ratios, based on the japonica template, were in the range of 8.8%-45.6%, with an average of 21.6%. As a result, the PCR product can be efficiently separated and easily scored by running 30 min of non-submerging electrophoresis on 2%-3% agarose gels. For development of WCSLs, recurrent parent TNG71 was crossed with Habataki in the second crop season of 2008, selected progenies carrying target whole chromosomes were then backcrossed for three generations. Subsequently, 13 candidate lines carrying homozygous chromosome segments of Habataki in the target regions were selected from BC3F2 populations in 2011. The genotypes of the 151 InDel and SSR markers for the selected WCSLs were dtermined to confirm the substitution of the target chromosome. Results of preliminary field evaluation of the WSCLs in the second crop season of 2012 showed that the Line ‘TARI71_2’, which carried hybrid breakdown gene hbd2 on chromosome 2, showed dwarf plant height (70.3 cm), low fertility (48%) and panicle number per plant was reduced from 10.8 to 3.5. The chromosome segment containing hbd2 gene should be eliminated during introgression of other high yielding traits to TARI71. In the introgression of other genes with high yield, line ‘TARI71_1, which carried high grain number gene (Gn1) and semi-dwarf gene (sd1) of Habataki on chromosome 1, showed that when comparing with TARI71, the grain number per panicle was increased from 116.4 to 128 and the plant height was reduced from 94.7 to 74.3 cm. Line ‘TARI71_6’, which contained primary branch number gene qPBN6 of Habataki onr chromosome 6, showed the grain number per panicle could arrive at 151.2 and the ripening rate still maintained 90%. Line ‘TARI71_10’, which possess can be observed the substation effect of the early heading date gene Ehd1 of Habataki on chromosome 10, showed that the heading days was reduced from 76 to 53 and the grain number per panicle also was reduced from 116.4 to 78.0. In addition, to demonstrate the potential usage of these WCSLs, we used the ‘Stepped Aligned inbred Recombinant Strains’ (STAIRS) to detect the effect and location of Gn1 locus on the short arm of chromosome 1 and developed a near isogenic line of TARI71 containing Gn1 allele from Habataki which showed significant increases on both number of secondary branches and number of grains. We believed that the construction and evaluation results of WCSL under Taiwanese cultivar background will be beneficial to rice breeding and genetic studies and to accumulate the ability of marker assisted breeding for target traits.
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