西藏野生拟南芥开花时间变异的遗传基础

doi:10.11983/CBB23140

植物学报 ›› 2024, Vol. 59 ›› Issue (3): 373-382.DOI: 10.11983/CBB23140

西藏野生拟南芥开花时间变异的遗传基础

杨继轩, 王雪霏, 顾红雅^*()

北京大学生命科学学院, 蛋白质与植物基因研究国家重点实验室, 北京 100871

收稿日期:2023-10-15 接受日期:2024-01-02 出版日期:2024-05-01 发布日期:2024-01-12
通讯作者: *顾红雅, 北京大学生命科学学院教授, 博士生导师。长期从事植物遗传多样性和演化研究, 基因家族功能和演化研究。以通讯作者身份在Cell Research、Plant Cell、New Phytologist和PLoS Genetics等植物遗传及演化相关的国际期刊上发表研究论文80余篇; 参与编著教材3部, 科普专著2部, 翻译教材3部。目前其研究团队以拟南芥为模式生物, 收集了全国约10个省份几十份野生拟南芥居群, 利用遗传学、基因组学、群体遗传学和生物信息学等手段对其遗传多样性、演化历史及适应性机制进行研究。E-mail: guhy@pku.edu.cn
基金资助:
国家自然科学基金(31970242)

Genetic Basis of Flowering Time Variations in Tibetan Arabidopsis thaliana

Jixuan Yang, Xuefei Wang, Hongya Gu^*()

State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China

Received:2023-10-15 Accepted:2024-01-02 Online:2024-05-01 Published:2024-01-12
Contact: *E-mail: guhy@pku.edu.cn

摘要/Abstract

摘要： 开花时间是被子植物生活史中的关键节点。十字花科植物拟南芥(Arabidopsis thaliana)广布于世界各地, 在海拔4 000 m以上的青藏高原也发现了该物种的自然居群, 高原独特的环境塑造了其生活史的独特表型, 在开花时间上表现为中等程度早花。该研究构建了西藏拟南芥Lhasa居群的F₂代作图群体, 基于全基因组测序的QTL-seq定位分析, 在该居群中定位到主效基因FLC, 并且鉴定到其第1个内含子中存在2 307 bp的缺失, 这种单倍型只存在于西藏拟南芥居群。利用CRISPR-Cas9技术构建了Lhasa背景的flc^-/-突变体, 表现为开花时间显著提前。研究结果表明, 西藏拟南芥开花时间改变的主要原因是FLC第1个内含子缺失, 该变异并未使其丧失全部功能, 这可能有利于西藏拟南芥适应青藏高原独特的气候环境。

关键词: 西藏拟南芥, 开花时间, Flowering Locus C, 适应性演化

Abstract: Flowering time is a critical point in the life cycle of angiosperm plants. Arabidopsis thaliana of the Brassicaceae is widely distributed around the world, and the natural populations of this species have been found at altitude 4 000 m in the Qinghai-Tibet Plateau. The cold/short summer plateau climate has shaped their flowering time to be moderately early compared with those living in low altitude areas. In this study, we constructed an F₂ mapping population and utilized whole-genome sequencing-based QTL-seq analysis to locate the major effect QTLs in Lhasa population of A. thaliana, and identified a haplotype-specific deletion of 2 307 bp within the first intron of FLC, which is unique to Tibetan A. thaliana. Lhasa population flc^-/- mutant was constructed by CRISPR-Cas9 gene editing technique. The mutant exhibited significantly earlier flowering time than Lhasa. The above findings suggested that the deletion in the first intron of FLC in Tibetan A. thaliana was most likely the major cause for the early flowering phenotype, although it did not cause complete function loss of the FLC. This variation may have facilitated the adaptation of Tibetan A. thaliana to the unique climatic environment of the Qinghai-Tibet Plateau.

Key words: Tibetan Arabidopsis thaliana, flowering time, Flowering Locus C, adaptive evolution

杨继轩, 王雪霏, 顾红雅. 西藏野生拟南芥开花时间变异的遗传基础. 植物学报, 2024, 59(3): 373-382.

Jixuan Yang, Xuefei Wang, Hongya Gu. Genetic Basis of Flowering Time Variations in Tibetan Arabidopsis thaliana. Chinese Bulletin of Botany, 2024, 59(3): 373-382.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB23140

https://www.chinbullbotany.com/CN/Y2024/V59/I3/373

图/表 7

表1 植物材料

Table 1 Plant materials used in this study

Ecotype and transgenic plant	Source
Col-0	Arabidopsis Biological Resource Center
Lhasa	Collected from Lhasa, Tibet
Dagze	Collected from Dagze, Tibet
JXnfx	Collected from Nanfeng, Jiangxi
SXcgx	Collected from Chenggu, Shaanxi
CQtlx	Collected from Tongliang, Chongqing
flc^-/-(Lhasa)	Constructed by this experiment

表2 引物序列

Table 2 Primers used in this study

Primer name	Primer sequence (5′-3′)
DT1-FLC-BsF	ATATATGGTCTCGATTGCCTTCTCCAAACGTCGCAAGTT
DT1-FLC-BsR	ATTATTGGTCTCGAAACCCGGCGATAAGTACGCCTTC
DT1-FLC-F0	TGCCTTCTCCAAACGTCGCAAGTTTTAGAGCTAGAAATAGC
DT1-FLC-R0	AACCCGGCGATAAGTACGCCTTCAATCTCTTAGTCGACTCTAC
FLC-RT-F	AACGTCGCAACGGTCTCA
FLC-RT-R	TCCCACAAGCTTGCTATCCA
TUB2-RT-F	GTTCTCGATGTTGTTCGTAAG
TUB2-RT-R	TGTAAGGCTCAACCACAGTAT

图1 西藏拟南芥和其它居群开花时间及莲座叶数目统计分析 (A) Col-0和部分中国野生居群开花时间统计(n≥40); (B) 开花时莲座叶数目统计(n≥40); (C) 开花时间与莲座叶数目的相关性分析。*** P<0.001 (Student’s t-test)。

Figure 1 Statistic analyses on the flowering time and number of rosette leaves of Tibetan and other Arabidopsis populations (A) Flowering time of Col-0 and some Chinese wild populations (n≥40); (B) Number of rosette leaves at flowering (n≥40); (C) Correlation analysis between flowering time and number of rosette leaves. *** P<0.001 (Student’s t-test).

图2 F2代群体开花时间频率分布、QTL-seq定位结果和FLC基因序列变异 (A) F2代群体开花时间频率分布; (B) F2代群体QTL定位结果; (C) 西藏拟南芥FLC的变异, 黑色方块表示外显子, 黑色线段表示内含子。B图中的红色线段表示全基因组水平0.01的错误发现率。C图中的VRE表示春化响应元件, 虚线表示西藏居群中缺失的序列。

Figure 2 Frequency distribution of flowering time, QTL-seq mapping result in the F2 population and FLC gene sequence variation (A) Frequency distribution of flowering time in the F2 population; (B) QTL mapping result in the F2 population; (C) Variation in FLC of Tibetan Arabidopsis thaliana, black square indicates exon, and black line indicates intron. The genome-wide false discovery rate of 0.01 is indicated by the red line in Figure B. In Figure C, VRE indicates vernalization response element, and the dashed line indicates the deletion sequence in the Tibetan population.

表3 F2代群体开花时间分布的卡方检验

Table 3 Chi-square test for the distribution of flowering time in F2 populations

Population	Flowering time	Observed	Expected	χ²	P-value
Lhasa-F₂	Early (<26 days)	428	432	0.15	0.70
	Late (≥27 days)	148	144
	Total	576	576

图3 部分拟南芥居群FLC第1个内含子区域的变异(改自Michaels et al., 2003; Strange et al., 2011; Méndez-Vigo et al., 2016) 以Col-0的FLC基因为参考, 黑色方形表示外显子, 黑色线段表示内含子, 红色线段表示序列缺失, 灰色方形表示序列插入。

Figure 3 Variation in the first intron of FLC in some Arabidopsis populations (modified from Michaels et al., 2003; Strange et al., 2011; Méndez-Vigo et al., 2016) FLC gene of Col-0 as a reference, black rectangle indicates exon, and black line indicates intron, red line indicates sequence deletion, and gray rectangle indicates sequence insertion.

图4 Lhasa居群和flc-/- (Lhasa)突变体的开花表型、FLC的表达模式及flc-/- (Lhasa)的序列变异 (A) Col-0和Lhasa居群不同发育时期FLC的相对表达水平(veg1和veg2分别表示长出第5和第10片莲座叶时期, flr表示开花时期); (B) Lhasa居群和flc-/- (Lhasa)突变体的DNA和氨基酸序列变异形式(74表示自ATG开始的第74位碱基, 红色序列表示flc-/- (Lhasa)突变体的氨基酸序列, 红色箭头表示氨基酸开始变异的位点, 黑色方框表示氨基酸的终止位点); (C) Lhasa居群和flc-/- (Lhasa)突变体的开花表型(bar=2 cm), 拍摄于幼苗移栽后25天; (D) Lhasa居群和flc-/- (Lhasa)突变体的开花时间和开花时莲座叶数目(n≥40)。*** P<0.001 (Student's t-test)。

Figure 4 Flowering phenotypes of Lhasa population and flc-/- (Lhasa) mutants, FLC expression pattern, and sequence variation of flc-/- (Lhasa) (A) Relative expression levels of FLC at different development stages in Col-0 and Lhasa population (veg1 and veg2 indicate the time of growing the 5th and 10th rosette leaves, respectively, and flr indicating the time of flowering); (B) Variant forms of DNA and amino acid sequences of Lhasa population and flc-/- (Lhasa) mutants (74 indicates the 74th base from ATG, red sequence indicates the amino acid sequence of flc-/- (Lhasa) mutants, red arrow indicates site where amino acid begin to change in the flc-/- (Lhasa) mutants and black box indicates the termination site in flc-/- (Lhasa) mutants); (C) Flowering phenotypes of Lhasa population and flc-/- (Lhasa) mutants (bar=2 cm), photographed 25 d after seedling transplantation; (D) Flowering time and number of rosette leaves at flowering in Lhasa population and flc-/- (Lhasa) mutants (n≥40). *** P<0.001 (Student’s t-test).

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西藏野生拟南芥开花时间变异的遗传基础

Genetic Basis of Flowering Time Variations in Tibetan Arabidopsis thaliana

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