利用限制酶切位連結核酸定序技術探討野生稻導入系統之基因體多型性

Genomic characterization of wild rice introgression lines using restriction site-associated DNA sequencing

翁子涵(Tzu-Han Weng)

指導教授 ： 黃永芬

國立臺灣大學/生物資源暨農學院/農藝學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201800044

摘要

稻米 (Oryza sativa L.) 為世界上重要的糧食作物，現今育種策略造成稻米之遺傳背景窄化及遺傳脆弱性等問題，使用野生近緣種 (crop wild relatives) 作為雜交親本被視為解決方法之一。本研究利用兩組野生稻導入系統，分別為導入野生稻O. nivara基因體的AA族群及導入野生稻O. officinalis基因體的AC族群，以限制酶切位連結核酸定序技術 (restriction site-associated DNA sequencing, RAD-seq) 建立定序庫，採用Illumina-Hiseq 2500次世代核酸定序平台完成定序後，以Stacks及TASSEL兩種程式進行單一核苷酸多型性 (SNP, single nucleotide polymorphism) 辨識。AA族群共獲得3,428個缺值率小於10%且具有多型性之分子標誌，加上前人使用之157個簡單重覆序列 (Simple sequence repeat, SSR) 分子標誌，經篩選後共以2,489個分子標誌建立總長1,743.3 cM之連鎖圖譜，並以多重區間定位法 (multiple interval mapping, MIM)，針對基本農藝性狀、產量相關性狀、穗與穀粒性狀及抗病表現等性狀進行數量性狀基因座 (quantitative trait loci, QTL) 定位，共定位到178個QTL，平均外表型解釋度為12.1% (介於2.21%至35.7%之間)。AC族群獲得4,601個缺值率小於10%且具有多型性之SNP分子標誌，以單一分子標誌迴歸法 (single marker regression) 定位54個QTL，平均外表型解釋度為10.3% (介於0.2%至52.4%之間)。在千粒重、每穗粒數、穗重、稻熱病抗性、褐飛蝨抗性、紋枯病抗性等許多性狀中皆有發現來自野生稻之有利對偶基因存在，配合搜尋前人文獻後佐證QTL鄰近位置有相關基因存在，且發現未出現於文獻中的QTL，可供進一步研究。

關鍵字

稻米 (Oryza sativa L.) ； 野生近緣種 ； 限制酶切位連結核酸定序技術 ； 數量性狀基因座

並列摘要

Rice (Oryza sativa L.) is one of the most important staple food crop in the world. Breeding strategies for rice nowadays often lead to problems such as narrow genetic background and genetic vulnerability. Using crop wild relatives (CWR) as crossing parents is viewed as one solution to these problems. In this study, two populations of introgression lines, AA and AC, which were generated using O. nivara and O. officinalis as donor parents, respectively, were used. Restriction site-associated DNA sequencing (RAD-seq) libraries were constructed and sequenced using Illumina-Hiseq 2500. Stacks and TASSEL were used for single nucleotide polymorphism (SNP) calling. In AA population, 3,428 SNPs with missing rate less than 10% were discovered. Along with 157 simple sequence repeat (SSR) markers from previous study, after marker filtering, we used a total of 2,489 markers to construct a linkage map spanning 1,743.3 cM. Multiple interval mapping (MIM) was used for mapping quantitative trait loci (QTL) for agronomic traits, yield-related traits and resistance to different pests and diseases. One hundred and seventy-eight QTL, each explained on average 12.1% phenotypic variance (ranging from 2.21 to 35.7%), were identified in AA population. In AC population, 4,601 SNPs with missing rate less than r 10% were discovered and analyzed using single marker regression. Fifty-four QTL, each explained on average 10.3% phenotypic variance (ranging from 0.2% to 52.4%), were identified. Favorable alleles from wild rice were found for several traits such as thousand grain weight, panicle weight, blast resistance and brown plant hopper resistance, etc. Some QTL were confirmed through previous studies, while others have not been reported yet, which may serve as targets for further studies.

並列關鍵字

rice (Oryza sativa L.) ； crop wild relatives ； restriction site-associated DNA sequencing (RAD-seq) ； quantitative trait loci (QTL)

參考文獻

李長沛 (2010) 野生稻Oryza nivara、Oryza officinalis、Oryza australiensis基因導入與育種上之利用。博士論文，國立中興大學，臺中市

Andrews S (2010) FastQC : a quality control tool for high throughput sequence data.

http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Accessed 11 April 2016

Athwal D, Pathak M, Bacalangco E, Pura C (1971) Genetics of resistance to brown planthoppers and green leafhoppers in Oryza sativa L. Crop Sci 11:747-750

Baird NA, Etter PD, Atwood TS, Currey MC, Shiver AL, Lewis ZA, Selker EU, Cresko WA, Johnson EA (2008) Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS One 3:e3376