藉DNA序列探討稻作基因體之變異

Abstract

基因體學基於分子生物學與遺傳學的研究成果而發展，生物資訊學因為基因體學的資訊處理需求而成長，而且因為定序技術的改良而更顯重要，但工具的選擇往往因為目的或階段性任務而有不同。稻作為人類重要糧食作物之一，也因為基因體大小，與其他作物具有高度共線性 (co-linear) 的演化關係等因素，成為被用來進行全基因體定序的第一個作物。在2005 年第一套高完成率且高品質的全基因組假染色體 (pseudomolecule) 發表之後，進入所謂的後基因體時代 (post genomic era)。研究人員有了序列、位置、結構性的註解與功能性的註解等資訊，超越了過去所用的遺傳圖譜 (genetic map)，進入核苷酸為單位的實體圖譜 (physical map) 時代。生物的資訊從外表型態與周邊資訊開始累積，記錄與操作生物的基因體序列資訊，因為冗長、繁瑣、易於出錯，生物資訊的工具開始被積極地開發，然而工具的開發是基於工作流程中，某一個步驟的需要，當應用到某項工程，則需要適當的安排使用流程。本論文的目的在於，集合利用稻作基因體的生物資訊，採用不同視野及角度，以得到的不同研究成果，闡述基因體學的基礎研究成果，回歸到育種材料的可能性。全論文的生物資訊處理，主要對象為稻作基因體，而操作的資料為參試系的全基因體次世代定序資料。 在第二章，為了尋求臺灣稉稻品種臺中六十五號對光不感等位基因的突變起源 (貢獻親)，本實驗室先前採用分子生物技術，將文件記載的六十個山地陸稻，縮減到剩下三個，已達該工具分析能力的極限，且關鍵的Hd1基因與插入的跳躍子都已經被定序。故藉由次世代定序的結果與生物資訊工具，從取用高可信度的單一核苷酸多型性為分子標幟，畫分屬於各參試系的單倍體基因型區域，到全基因體的整體視野為判斷依據，判定Muteka與Nakabo為最有可能的貢獻親，也就是臺中六十五號在近百年前的育種田中，受到非計畫性的花粉汙染，Hd1及Ehd1被自然地漸滲進入現在的品種之中。 在第三章，主要目的在探討膿桿菌媒介的轉殖過程中，對於稻作基因體的影響是否顯著的大於僅進行組織培養所造成的突變。透過8組不同處理的臺農67樣本，外加臺粳九號與IR64兩不同品種的五個樣本，一共31個各組不等的全基因體定序資料，結果指出膿桿菌為媒介不會造成顯著的突變，但凸顯出Taiwan Rice Insertional Mutants (TRIM)及Tos17的突變系族群，可能是因為大量產生突變系的工作流程，造成有大約10倍的突變率。 在第四章，意在藉由稻作基因體的資訊，提醒稻田內花粉汙染的可能性，即使現代的稻作品種為自交作物也會發生。在本研究舉出本實驗室遇到的兩個例子，一個是在TRIM的族群中，我們在同一個品系同時獲得秈稻漸滲而來的大粒種子等位基因GS3，也同時因為轉殖過程引發的體細胞複製變異，造成D17基因的突變，獲得矮株多分櫱的性狀；另一個是在前一個研究的採樣田間，有一個種子被納入胚培養的流程，兩個都在經過與秈稻基因體比對之後，提出有力證據證明其意外漸滲的未知親本為秈稻。 在第五章，意在結合單一基因的等位基因探勘與擴增遺傳歧異度兩樣工作，以便因應全球氣候環境變化、病蟲害與作物的共同演化，以及40年後人口的倍增。在此研究，將焦點放在近半世紀被廣為採用的單一來源等位基因，也就是成就綠色革命的sd1，在3000個稻作基因體資料中，探勘SD1基因所有已知的與高度可能的同功等位基因。透過IRRI提供的外表型紀錄，驗證了探勘結果的準確性，即使對株高的變異程度，只有不到30%的解釋度。

Rice (Oryza sativa) is one of the most important crops in the world. Since the 1st well assembled rice pseudomolecules were published in 2005, rice study has entered the post genomic era. Taking the advantage of it, we may use the physical map, rather than genetic map, to ping point the position and function of the query sequence. Many bioinformatics tools have being invented for various jobs. In this thesis, I would like to demonstrate how to organize these tools to address the questions. In the second chapter, the purpose is to resolute the mystery of how Taichung 65 loss its photoperiod sensitivity. We used accession-specific single nucleotide olymerphism analysis to reveal the gene flow that occurred between different rice accessions decades ago and demonstrate that two landraces introgressed during the breeding process, which led to the loss of photoperiod sensitivity. Both Hd1 and Ehd1 may be important during artificial selection for flowering time, especially in a subtropical region such as Taiwan. Transgenic plant assays have been used frequently for complementation, overexpression or antisense analysis, but sequence changes caused by callus growth, Agrobacterium incubation medium, virulence genes, transformation and selection conditions are unknown. In the third chapter, we used high-throughput sequencing of DNA from rice lines derived from Tainung 67 to analyze non-transformed and transgenic rice plants for mutations caused by these parameters. From the result, we could not conclusively detect sequence changes resulting from Agrobacterium-mediated transformation in addition to those caused by tissue culture-induced somaclonal variation. However, the mutation frequencies within the two publically available tagged mutant populations, including TRIM transformants or Tos17 lines, were about 10-fold higher than the frequency of standard transformants, probably because mass production of embryogenic calli and longer callus growth periods were required to generate these large libraries. Several rice insertional mutant libraries are publicly available for systematic analysis of gene functions. However, the tagging efficiency of these mutant resources–the relationship between genotype and phenotype is very low. In the fourth chapter, we used whole-genome sequencing to analyze a T-DNA-tagged transformant, M28590, from TRIM and one regenerant. Both showed the introgression occurred from some unknown indica rice. For M0028590, the large-grain trait came from the GS3 gene of the introgressed region and the tillering dwarf phenotype came from a single nucleotide change in the D17 gene that occurred during the callus induction to regeneration of the transformant. In addition to the known sequence changes such as T-DNA integration, single nucleotide polymorphism, insertion, deletion, chromosome rearrangement and doubling, spontaneous outcrossing occurred in the rice field may also explain some mutated traits in a tagged mutant population. Thus, the co-segregation of an integration event and the phenotype should be checked when using these mutant populations. The rice Green Revolution (GR) at the 60’S had increased agricultural production by more than two fold worldwide. In the fifth chapter, we propose the opportunities to maintain the diversity of rice germplasm and mining the information of genes. The key gene for the first GR is a lost-of-function of Semi-dwarf1 (SD1) gene, came from an old Taiwan landrace, Dee-Geo-Woo-Gen (DGWG). We mined multiple Sd1 alleles using the 3K-RGP data and validate them from IRRI’s phenomics records. The result showed about five hundred accessions consisted of DGWG sd1 alleles, including some varieties from Taiwan, IRRI and countries worldwide. The Kasalath type, i.e. full-function SD1 allele, however, were present in one-third of 3K project. The Nipponbare type, i.e. partial-function SD1 allele had present in one-fourth of 3K project. That is, sd1 allele had a narrow diversity in the germplasm. Thus, we suggest rice breeders to use the widely collected genomic information for choosing parental lines to include demanded traits and keep the diversity.