谷子抽穗时间基因SiTOC1的表达与单倍型变异分析

doi:10.3864/j.issn.0578-1752.2021.11.003

摘要/Abstract

摘要：

【目的】谷子抽穗时间的适应性表现是广适性新品种选育的基础,分析抽穗时间关键基因的遗传变异和单倍型效应,为品种适应性改良提供基础信息。【方法】通过全基因组关联分析（genome-wide association study,GWAS）,定位谷子抽穗时间关键基因SiTOC1,利用多组学数据库（multi-omics database for Setaria italica,MDSi）提供的SiTOC1数字表达量,分析SiTOC1的组织时空表达特性,并利用原生质体对SiTOC1蛋白进行亚细胞定位。采用qRT-PCR在短日（10 h光照/14 h黑暗）条件下进行SiTOC1 24 h节律表达模式分析。利用有代表性的99份谷子品种,分析SiTOC1编码区和启动子区的遗传多态性、单倍型以及转录水平,并对单倍型与抽穗时间的关系进行鉴定。【结果】在第1染色体物理位置31 456 761 bp处鉴定到了一个显著的关联信号,与抽穗时间紧密相关,该位点附近存在一个拟南芥抽穗期TOC1的同源基因SiTOC1。SiTOC1在光周期响应组织（根、茎、叶等）中高表达,亚细胞定位于细胞核,在傍晚表达量上调,呈现出24 h节律性表达模式。SiTOC1在不同谷子品种中存在丰富的多态性,但REC和CCT结构域高度保守。SiTOC1编码区2种主要单倍型H-2和H-6分别与启动子单倍型Hp-591C和Hp-591A共分离,其中,启动子单倍型Hp-591C较Hp-591A的相对表达量显著上调了约2.5倍（P=0.014）,并且该单倍型在三亚市、长治市和乌鲁木齐市3个环境下的抽穗时间分别平均延迟9、11和12 d。【结论】SiTOC1启动子区第591 bp处的SNP是引起抽穗时间差异的主效位点,单倍型Hp-591A较Hp-591C早熟,可作为主效单倍型用于分子育种选择。

关键词: 谷子, 抽穗时间, 全基因组关联分析, 单倍型, 遗传变异, 节律性表达

Abstract:

【Objective】 Identification of allelic variations of heading date adaptation related genes and laying foundation for breeding of wide-adapted varieties in foxtail millet. 【Method】In this trial, a vital regulator of heading time in foxtail millet, SiTOC1, was identified using genome-wide association analysis. Spatio-temporal transcription (multi-omics database for Setaria italica,MDSi), sub-cellular localization and 24 hours rhythm expression pattern of SiTOC1 was analyzed. Sequence variations of both promoter and encoding regions in SiTOC1 and relationships between haplotypic variations and heading date were characterized in 99 foxtail millet accessions. 【Result】A significant GWAS signal (Position: 31 456 761 bp) was detected on Chromosome 1 and only one TOC1 homologue was identified (SiTOC1). SiTOC1 highly expressed in root, stem and leaf, and located into cell nucleus. An elevated expression of SiTOC1 was identified at dusk across whole day transcription survey under short-day environment. Many haplotypic variations of SiTOC1 were identified but REC and CCT domains of SiTOC1 were conserved in foxtail millet accessions, and two main haplotypes including H-2 and H-6 in protein encoding regions combined with two co-segregated haplotypes including Hp-591C and Hp-591A were identified. Nearly 2.5 times higher expression of Hp-591C haplotype combined with 9,11 and 12 days delay of heading time through Hainan, Changzhi and Urumuqi were observed. 【Conclusion】The major haplotype Hp-591A identified at 591 bp in the promoter region of SiTOC1 matures earlier than Hp-591C and could be selected as a main effective locus for molecular breeding of foxtail millet.

Key words: Setaria italica, heading time, genome-wide association study, haplotype, genetic variation, rhythmic expression

张林林,智慧,汤沙,张仁梁,张伟,贾冠清,刁现民. 谷子抽穗时间基因SiTOC1的表达与单倍型变异分析[J]. 中国农业科学, 2021, 54(11): 2273-2286.

ZHANG LinLin,ZHI Hui,TANG Sha,ZHANG RenLiang,ZHANG Wei,JIA GuanQing,DIAO XianMin. Characterizations of Transcriptional and Haplotypic Variations of SiTOC1 in Foxtail Millet[J]. Scientia Agricultura Sinica, 2021, 54(11): 2273-2286.

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载体 Vector	引物名称 Primer name	引物序列 Primer sequence (5′-3′)	产物长度 Product length (bp)
GFP-N 端载体 GFP-N terminal vector	GFPG5-F1	GCTGTACAAGACTAGTATGGTGGGCGGCGGCG	1517
	GFPG5-R1	GGGGAAATTCGAGCTCCTACTCTGGAGAAGAAATAATC	1517
	PUCGFPN-TestF	TACAACTACAACAGCCACAA	2163
	PUCGFPN-TestR	CCTCTTCGCTATTACGC	2163
	GFPG5-F1.1	CCATCATCATGATGTCC
	GFPG5-R1.1	TTCAGAGCGACTGCATG
GFP-C端载体 GFP-C terminal vector	G5GFP-F1	TAGTGGATCCATCGATATGGTGGGCGGCGGCG	1514
	G5GFP-R1	TCCCGGGAGCGGTACCCTCTGGAGAAGAAATAATCTC	1514
	PUCGFPC-TestF	ACCTCCTCGGATTCCAT	2354
	PUCGFPC-TestR	TGCCGTTCTTCTGCTTG	2354
	G5GFP-F1.1	CAAGATGCTCAAGTACA
	G5GFP-R1.1	CGATTTACATACCTCACT
RFP-N端载体 RFP-N terminal vector	MYBRFP-F	GCTGTACAAGACTAGTATGGACATGGCGCACGAGAG	1022
	MYBRFP-R	GGGGAAATTCGAGCTCTCACACGGCGGCCTGGGT	1022
	PUCRFPN-TestF	CTCAAGCTCAAGGACGG	1584
	PUCRFPN-TestR	CCTCTTCGCTATTACGC	1584

产物Product	引物名称 Primer name	引物序列 Primer sequence (5′-3′)	产物长度 Product length (bp)
编码区 Coding region	G5-F1	GATCCAGCGACAGTCCA	1450
	G5-R1	AGACAGGTCGGACTGAAATA	1450
	G5-F2	GATGAGTTGCCACAAAGATG	1272
	G5-R2	TGTATTGCCTTTCCCAGTAG	1272
启动子区 Promoter region	G5P-F1	GGCCAAACGAAACCATG	2069
	G5P-R1	CCTAATCCGGGAAACCAG	2069
	G5P-F1.1	ATTGCGTGCACGAATCT
	G5P-R1.1	CAGCAGCAGCCTGCCTCG
SiTOC1互补DNA SiTOC1 cDNA	G5Q-F1	GCCGATCAAGCATCATATGTTAAGT	250
SiTOC1互补DNA SiTOC1 cDNA	G5Q-R1	TTTGGCCTTCATTGCTTCGC	250
Cullin互补DNA Cullin cDNA	Cullin-F	TATGGGTCATCAACAGCTTGTC	112
Cullin互补DNA Cullin cDNA	Cullin-R	GTAGTCCCTCGTGATGAGATCC	112

位点名称 LocusName	拟南芥对应基因 Arabi-symbol	拟南芥基因功能注释 Arabi-defline
Seita.1G235800	N/A	无功能注释N/A
Seita.1G235900	MKM21.12	线粒体核糖体蛋白L27 Mitochondrial ribosomal protein L27
Seita.1G236000	N/A	谷氧还蛋白家族 Glutaredoxin family protein
Seita.1G236100	TOC1	含CCT基序的应答调节蛋白 CCT motif-containing response regulator protein
Seita.1G236200	PAP10	紫色酸性磷酸酶10 Purple acid phosphatase 10
Seita.1G236300	NOP10	核仁RNA结合Nop10p家族蛋白 Nucleolar RNA-binding Nop10p family protein
Seita.1G236400	AtMYB79	含MYB结构域蛋白79 MYB domain protein 79