高粱早抽穗數量性狀基因座檢定

Abstract

高粱(Sorghum bicolor (L.) Moench)為世界栽培面積第五大作物，其具備多種用途，包括芻料、生質能源及主食等，同時也相對其他作物更耐旱節水。高粱抽穗為重要的農藝性狀，利用光週期調整抽穗期，為熱帶作物如高粱適應溫帶氣候的重要關鍵，也因此相關研究較多。然而，其他重要的環境因子如溫度，相關研究卻很少。為了找尋光週期以外的相關抽穗基因，將高粱早熟地方種品系V9 與晚熟光不敏感參考基因體品系BTx623雜交，產生兩個族群進行後續的研究。F2 共238個個體在2019夏季種植於嘉義調查其抽穗期，接著使用雙限制酶簡化基因體定序(ddGBS)獲得其中192個個體序列及基因型，並用4個連鎖圖譜定位模型進行數量性狀基因座（Quantitative trait locus, QTL）定位及計算其信賴區間。結果顯示，該族群中總共定位到四個QTL，分別是qHD1.1、 qHD1.2、qHD3及 qHD8，這些QTL在不同模型分別解釋5.3803-11.071%、8.549%-14.8873%、3.650%-5.9638%、17.960-21.4272%的變異量。取4個連鎖圖譜定位模型的QTL區間交集分別為chr1: 6191963-13427114、chr01:61900965-64055038、chr03: 231634-59781645及chr08: 59410431-60136254。為了進一步調查這些QTL區間中控制抽穗期性狀的基因，包含6個早抽穗及7個晚抽穗的品系的13個F8 品系以及親本V9被全基因體定序以取得基因型。期田間試驗即溫度試驗結果顯示溫度與基因型有交感以及外表型早抽以及晚抽穗兩族群外表型已經固定。接著對其序列變異進行註解及計算子代\DeltaSNP頻率（∆SNP-index）。結果顯示，總共有14個區域被modified QTL-seq方法找到。為了去除選拔過程中出現有意或無意造成的∆SNP-index顯著的情況，我們僅用連鎖圖譜基因定位結果、序列變異註解、Esembl plant 同源基因資料庫及Plant Reactome資料庫用來找尋基因。然而，僅有qHD3 區間中的SORBI_3003G202200 同時符合所有條件，且該變異被預測為高度影響基因功能。這個基因被預測涉及細胞分裂素（cytokinin, CK）生合成相關途徑以及花芽發育（flower development）。根據序列變異註解結果顯示，其編碼序列（coding sequence, CDS）區域出現InDel 導致移碼突變 (frameshift mutation)。變異預測為中等影響者，有與水稻及阿拉伯芥CRY1同源的SORBI_3006G101600，及高粱的PHYB基因SORBI_3001G394400，在同源基因有溫度控制開花的相關，未來還需要更多研究來繼續深入探討高粱溫度調控抽穗相關調控路徑。若能夠找到溫度相關的相關基因，未來可以利用分子標誌將早抽穗基因導入豐產品系中，加速開發節水且可與水稻輪作的品種。

Sorghum (Sorghum bicolor (L.) Moench) is fifth place of plant area in the world. It has miscellaneous utilization, inclusive of forage, bioenergy, and staple, and it also save water compared to other crops. Heading date is a critical phenotype. Heading date controlling by photoperiod is a key feature for a tropical crop like sorghum to adapt to the temperate environment, so it is relatively clearly explored. Nonetheless, other important environmental factor like ambient temperature remains unknown. To investigate the heading date genes controlling by other factor in sorghum, an early heading (EH) landrace V9 is crossed with photoperiod insensitive reference genome variety late heading date (days) (LH) BTx623, and then two populations are developed to allow further investigation. F2 is grown in Chiayi in 2019 summer to record their heading date. ddGBS is applied to obtain genotype from 192 individuals of this population, then 4 models are applied to get QTL peak and later their confidence interval. Results show that 4 QTL are mapped with this population, they are qHD1.1, qHD1.2, qHD3, and qHD8. These QTLs explain 5.3803-11.071%, 8.549%-14.8873%, 3.650%-5.9638%, and 17.960-21.4272% respectively. They are in the intersection interval of four model of these QTLs are chr1: 6191963-13427114, chr01:61900965-64055038, chr03: 231634-59781645 and chr08: 59410431-60136254 respectively. To further investigate underlie genes, whole genome sequenced (WGS) F8 population composed of 6 EH and 7 LH lines and parent V9 are genotyped. We observe fixation of phenotype contrasting group and temperature and genotype interaction in field and temperature experiment. Their variants are then annotated and calculate SNP-index. 14 regions are identified with this modified QTL-seq method. To get rid of intentional and unintentional fixation during selection which may cause the significance of SNP-index, linkage mapped result, variant annotation, Plant Reactome and Ensembl Plants ortholog databse are integrated to hunt the candidate genes. However, only SORBI_3003G202200 (in qHD3 region) on chromosome 3 are qualified all the condition described so far. This gene is predicted to be involved in CK biosynthesis and flower development. According to annotation result, an InDel cause frameshift in its CDS. Variant annotation also reveals that sorghum CRY1 ortholog of Arabidopsis thaliana and Oryza sativa Japonica group, SORBI_3006G101600, and sorghum PHYB gene SORBI_3001G394400 have MODERATE impact variants. Othologs of these two genes have temperature related report in previous study. More investigation about temperature controlling pathway is necessary in the future. If genes involved ambient temperature controlling pathway are identified, molecular markers can be developed and help accelerate the breeding program to develop a variety that can not only save water but also adapt to rotation choice with rice.