基于BSA-seq和RNA-seq挖掘水稻株高相关QTL

doi:10.13560/j.cnki.biotech.bull.1985.2023-0386

生物技术通报 ›› 2023, Vol. 39 ›› Issue (8): 173-184.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0386

基于BSA-seq和RNA-seq挖掘水稻株高相关QTL

吴元明¹(), 林佳怡¹, 柳雨汐¹, 李丹婷², 张宗琼², 郑晓明³^,⁴^,⁵, 逄洪波¹()

1.沈阳师范大学生命科学学院，沈阳 110034
2.广西壮族自治区农业科学院水稻研究所广西水稻遗传育种重点实验室，南宁 530007
3.中国农业科学院作物科学研究所，北京 100081
4.海南三亚中国农业科学院国家南繁研究院，三亚 571700
5.国际水稻研究所，菲律宾马尼拉 DAPO box 7777

收稿日期:2023-04-22 出版日期:2023-08-26 发布日期:2023-09-05
通讯作者: 逄洪波，女，博士，副教授，研究方向：植物逆境分子生物学；E-mail: panghb@synu.edu.cn
作者简介:吴元明，男，硕士研究生，研究方向：生物化学与分子生物学；E-mail: 19861602091@163.com
基金资助:
海南省自然科学基金项目(2021JJLH0075);国际（地区）合作与交流项目(32261143465);海南省重点研发项目(ZDYF2022XDNY260);国家自然科学基金项目(31970237);辽宁省自然科学基金面上项目(2022-MS-309);沈阳师范大学“百人计划”拔尖人才项目(SSDBRJH2002012);沈阳师范大学重大项目孵化工程(ZD202104);省级大学生创新创业训练计划资助项目(S202310166005)

Identification of Rice Plant Height-associated QTL Using BSA-seq and RNA-seq

WU Yuan-ming¹(), LIN Jia-yi¹, LIU Yu-xi¹, LI Dan-ting², ZHANG Zong-qiong², ZHENG Xiao-ming³^,⁴^,⁵, PANG Hong-bo¹()

1. College of Life Science, Shenyang Normal University, Shenyang 110034
2. Rice Research Institute, Guangxi Academy of Agricultural Sciences, Nanning 530007
3. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081
4. Sanya National Research Institute of Breeding in Hainan, Chinese Academy of Agricultural Sciences, Sanya 571700
5. International Rice Research Institute, Metro Manila DAPO box 7777, Philippines

Received:2023-04-22 Published:2023-08-26 Online:2023-09-05

摘要/Abstract

摘要：

株高是影响水稻产量稳定性的重要因素之一，水稻株高相关QTL的定位及候选基因的挖掘，有助于深入了解株高分子调控机制，进而为培育理想株型的水稻品种奠定基础。以油占8号及广西野生稻为亲本构建的285个CSSL群体为试验对象，结合NGS与BSA-seq等方法，利用SNP和InDel 两种分子标记进行关联分析，对可能与株高相关的基因组区段进行定位。结果显示，Δ（SNP-index）分子标记在Chr.7和Chr.10中分别关联到3.205和1.311 Mb大小的候选基因组区域。Δ（InDel-index）标记关联到的基因组区域大小分别为2.848和1.292 Mb，且全部包含于Δ（SNP-index）关联到的区间内。结合GO、KEGG、Uniprot、eggNOG等数据库中的功能注释、高质量多态性位点筛选以及已有的株高相关转录组数据，最终关联到Chr.7中的5个候选基因，包括功能和机理已知的OsTCP21。RT-qPCR结果显示，OsTCP21在高株和矮株中的表达差异与已有研究结果相一致，LOC_Os07g05050和LOC_Os07g02850在高株中表达量较高，LOC_Os07g04220和LOC_Os07g02770在矮株中表达量较高。这5个候选基因在水稻株高性状的调控中起重要的作用，OsTCP21是一个关键调控基因。

关键词: 水稻, 株高, 定位, BSA-seq, RNA-seq

Abstract:

The stability of rice yield is significantly influenced by plant height, making it as a crucial factor. The identification of plant height-associated QTLs and mining of candidate genes are conducive to comprehending the molecular regulatory mechanisms that determines plant height, which may lay a foundation for breeding ideal rice varieties. This study employed 285 CSSL populations derived from the parental strains of Oryza sativa var. Youzhan 8 and wild rice from Guangxi as the experimental cohort. The study utilized SNP and InDel molecular markers, in conjunction with NGS and BSA-seq techniques, to conduct an association analysis aimed at identifying genomic regions that are potentially associated with plant height. As results, the molecular markers Δ（SNP-index）were discovered to have an association with candidate genomic regions of 3.205 and 1.311 Mb in Chr.7 and Chr.10, respectively. The genomic regions linked with Δ（InDel-index）markers were of sizes 2.848 and 1.292 Mb, and were entirely encompassed within the intervals associated with Δ（SNP-index）. Through the integration of functional annotation in GO, KEGG, Uniprot, and eggNOG databases, as well as high-quality polymorphic site screening and existing transcriptome data pertaining to plant height, five candidate genes located in Chr.7 were ultimately linked to this trait, including the previously characterized OsTCP21 function and mechanism. The qRT-PCR findings were consistent with prior research, revealing differential expression of OsTCP21 in tall and dwarf plants. Specifically, LOC_Os07g05050 and LOC_Os07g02850 demonstrated elevated expressions in tall plants, whereas LOC_Os07g04220 and LOC_Os07g02770 had higher expressions in dwarf plants. The regulation of rice plant height is significantly influenced by five candidate genes, with OsTCP21 being identified as a pivotal regulatory gene.

Key words: rice, plant height, mapping, BSA-seq, RNA-seq

吴元明, 林佳怡, 柳雨汐, 李丹婷, 张宗琼, 郑晓明, 逄洪波. 基于BSA-seq和RNA-seq挖掘水稻株高相关QTL[J]. 生物技术通报, 2023, 39(8): 173-184.

WU Yuan-ming, LIN Jia-yi, LIU Yu-xi, LI Dan-ting, ZHANG Zong-qiong, ZHENG Xiao-ming, PANG Hong-bo. Identification of Rice Plant Height-associated QTL Using BSA-seq and RNA-seq[J]. Biotechnology Bulletin, 2023, 39(8): 173-184.

图/表 11

参考文献 52

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样品Sample	原始数据 Raw base/bp	过滤后的有效数据 Clean base/bp	比对率Mapping rate/%	平均测序深度Average depth（×）	至少覆盖1个碱基的位点的比例Coverage at least 1×/%	至少覆盖4个碱基的位点的比例Coverage at least 4×/%	Phred 数值大于20的碱基比例Q20/%	Phred 数值大于30的碱基比例Q30/%	碱基G和C的含量GC/%
YZ8	11 134 196 100	11 134 054 800	99.00	23.81	82.46	79.06	97.57	93.31	44.60
GCWR	14 338 273 200	14 338 088 700	98.65	28.82	85.26	82.67	97.78	93.79	44.23
H-pool	5 067 688 200	5 067 637 200	99.04	11.36	82.27	74.08	97.11	92.15	43.59
L-pool	4 969 848 300	4 969 806 000	99.02	11.43	81.81	74.14	97.14	92.13	43.61

样品Sample	原始数据 Raw base/bp	过滤后的有效数据 Clean base/bp	比对率Mapping rate/%	平均测序深度Average depth（×）	至少覆盖1个碱基的位点的比例Coverage at least 1×/%	至少覆盖4个碱基的位点的比例Coverage at least 4×/%	Phred 数值大于20的碱基比例Q20/%	Phred 数值大于30的碱基比例Q30/%	碱基G和C的含量GC/%
YZ8	11 134 196 100	11 134 054 800	99.00	23.81	82.46	79.06	97.57	93.31	44.60
GCWR	14 338 273 200	14 338 088 700	98.65	28.82	85.26	82.67	97.78	93.79	44.23
H-pool	5 067 688 200	5 067 637 200	99.04	11.36	82.27	74.08	97.11	92.15	43.59
L-pool	4 969 848 300	4 969 806 000	99.02	11.43	81.81	74.14	97.14	92.13	43.61

染色体 Chromosome	起始位置 Start/bp	终止位置 End/bp	大小 Size/Mb	基因数量 Gene number
7	1	2 848 000	2.848	141
10	3 796 001	5 088 000	1.292	135
总计Total	-	-	4.140	276

染色体 Chromosome	起始位置 Start/bp	终止位置 End/bp	大小 Size/Mb	基因数量 Gene number
7	1	2 848 000	2.848	141
10	3 796 001	5 088 000	1.292	135
总计Total	-	-	4.140	276

基因 Gene ID	基因注释 Gene annotation
LOC_Os07g02770	含F-box和亮氨酸富集重复序列的蛋白 Protein containing F-box and leucine-enriched repeats
LOC_Os07g02850	含DUF538结构域的蛋白 Protein containing DUF538 domain
LOC_Os07g04220	光敏色素信号与损伤相关的受体类激酶 Wound and phytochrome signaling involved receptor like kinase
LOC_Os07g05050	含DEAD-box的ATP依赖的RNA解旋酶 ATP-dependent RNA helicase containing DEAD-box
LOC_Os07g05720	TCP转录因子 TCP family transcription factor

基于BSA-seq和RNA-seq挖掘水稻株高相关QTL

Identification of Rice Plant Height-associated QTL Using BSA-seq and RNA-seq

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基因 Gene	引物序列 Primer sequence（5'-3'）	产物大小 Product size/bp
LOC_Os07g02770	F: GAGAGCATGGCAAGGAAGGT	130
LOC_Os07g02770	R: TAGTGCGGTGCATTTGTCCT	130
LOC_Os07g02850	F: GCGTCTGCAGGAAGAAGTCC	125
LOC_Os07g02850	R: CTGCACCAGCCACATGAATC	125
LOC_Os07g04220	F: CCCGCCATTAGTCCACAGAG	128
LOC_Os07g04220	R: TTGCTGGTGGGCCTGATATG	128
LOC_Os07g05050	F: CGAACTCCCAAACACCTCGG	241
LOC_Os07g05050	R: GGTTCTTGGGCTTCCAGCTAA	241
LOC_Os07g05720	F: TCTCCATGTCCTCGGGTCTT	175
LOC_Os07g05720	R: GGGCTGAAGAGGAACGGAAT	175
OsActin	F: AGACCTTCAACACCCCTGCT	110
OsActin	R: GTGGCTGACACCATCACCAG	110

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功能注释数据库 Annotated database	注释基因数目 Number of annotated genes
eggNOG	26
Uniprot	41
GO	48
KEGG	20
总计Total	78

功能注释数据库 Annotated database	注释基因数目 Number of annotated genes
eggNOG	26
Uniprot	41
GO	48
KEGG	20
总计Total	78