透過選擇性基因分型與BSR-Seq探勘控制水稻幼苗根系發育之遺傳結構

Use selective genotyping and BSR-Seq to investigate genetic architecture controlling rice seedling root development

杜芸真(Yun-Chen Du)

指導教授 ： 董致韡

國立臺灣大學/生物資源暨農學院/農藝學系/碩士(2022年)

https://doi.org/10.6342/NTU202204044

摘要

水稻 (Oryza sativa L.) 為世界重要糧食作物之一，而乾旱是氣候變遷下影響水稻產量的逆境之一，因此加速提升水稻乾旱耐受性至關重要。由於根系是植物汲取水分與養分的來源，透過改善根系結構可能有助於提升水稻乾旱耐受性。本研究目標在於，透過選擇性基因分型 (selective genotyping)、群集分離分析法 (bulk segregant analysis) 分析兩極端群體的 DNA 與 RNA 資訊，在減少時間與金錢成本的前提下，加速找到可能控制最大根長 (maximum root length, MRL) 之候選基因。一共有24個極端根長 (long and short) 的重組自交系 (recombinant inbred lines, RILs) 從 Nipponbare 與 IR64 雜交後的 F11 重組自交系族群中被篩選出來，分別稱作 Long (L) bulk (n=12) 與 Short (S) bulk (n=12)。首先，選擇性基因分型透過 general linear model (GLM) 偵測到 5、8、9 與 10 號染色體上皆有顯著的 SNP peak。其中，位於染色體 5 號的顯著單核甘酸多型性 (single nucleotide polymorphism, SNP) 位點 (S5_15750938) 可解釋70.20% 的外表型變異。接著群集分離分析之RNA 定序資料 (bulked segregant RNA-Seq, BSR-Seq) 則用於差異表現分析以及 RNA QTL-seq 分析。結果顯示，長根與短根兩個群集間共有 160 個差異表現基因 (differentially expressed genes, DEGs) 其中108個在 L bulk 是上調基因，52 個在 S bulk 為上調基因。這些基因共參與了13個生物途徑，其上游 2 kb 啟動子區域內共找到 14 種結合位點。另一方面，以 RNA 進行之 QTL-seq 分析，共偵測到 7 個候選區域分別位在染色體 5、8、9、10 與 12 號上。其中 8 號染色體尾端與 10 號染色體中段同時被 selective genotyping 與 QTL-seq 偵測到，因此我們認為重疊區域對最大根長的發育有重要的影響。另外，偵測到 155 顯著的 SNP/Indel 變異則透過 SnpEff 分析說明有 28 個基因帶有中度影響的變異，38 個基因帶有輕度影響的變異。本研究探勘之重要候選區域與候選基因可能控制根系長度的發育，將有助於未來透過調節根系結構以提升水稻耐旱性之目標。

關鍵字

水稻 ； 最大根長 ； 選擇性基因分型 ； RNA 定序集群分離分析法 ； 差異表現基因 ； 數量性狀基因座定位 ； QTL-seq

並列摘要

Rice (Oryza sativa L.) is one of the most important edible food crops in the world. Drought stress under climate change is one of the major dilemmas for the final production of rice. Hence, it is necessary to accelerate the improvement of ricedrought tolerance to drought stressin rice. Due to the function of roots to absorb water and nutrients from the soil, root system architecture (RSA) was regarded as a promising factor to advance drought tolerance in plants. In this study, to accelerate the identification of candidate regions and genes controlling maximum root length (MRL), one of the RSA, selective genotyping, bulked segregant RNA sequencing (BSR-Seq), and RNA-based QTL-seq were adopted to analyze the DNA and RNA of two bulks with extreme phenotypes. phenotypes. A total of 24 recombinant inbred lines (RILs) with extremely long and short roots, called Long (L) bulk (n=12) and Short (S) bulk (n=12) were screened from an F11 RILs population derived from a cross between Nipponbare and IR64. Selective genotyping analysis was conducted with a general linear model (GLM) and revealed 4 four significant SNP peaks located in chromosomes 5, 8, 9, and 10. And the most significant peak (S5_15750938) is located on chromosome 5 explaining 70.20 % phenotypic variation. Second, BSR-Seq was used to identify the differentially expressed genes (DEGs) and conduct RNA-based QTL-seq. A total of 160 DEGs were found between 2 two bulks, containing 108 up-regulated genes in L bulk and 52 up-regulated genes in S bulk. Then, MapMan analysis reveals that 160 DEGs are involved in 13 pathways, and PlantCARE clarifies that a total of 14 types of the binding site are enriched in the 2k upstream sequence of 160 DEGs. RNA-based QTL-seq revealed 7 seven candidate regions on chromosomes 5, 8, 9, 10, and 12. Two candidate regions including the end of chr8 and the middle of chr10 overlapped between the results of selective genotyping and RNA-based QTL-seq, which elucidates the importance of these two regions on MRL development. QTL-seq revealed 155 significant SNPs with an ∆SNP-index greater than 0.8 at a significant level of 0.01. SnpEff revealed that 28 genes with moderate impact variants and 38 genes with low impact variants. In this study, we found important genomic regions and genes that might control the seedling MRL development. It is valuable for improving drought resistance by regulating RSA in rice.

並列關鍵字

Rice ； Maximum root length ； Selective genotyping ； Bulk segregant analysis RNA-Seq ； Differentially expressed gene ； QTL mapping ； QTL-seq

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