一个新的玉米细胞核雄性不育突变体ms6的鉴定与基因定位

doi:10.3724/SP.J.1006.2023.23062

作物学报 ›› 2023, Vol. 49 ›› Issue (8): 2077-2087.doi: 10.3724/SP.J.1006.2023.23062

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

一个新的玉米细胞核雄性不育突变体ms6的鉴定与基因定位

王兴荣¹(), 张彦军¹, 涂奇奇², 龚佃明²^,^*(), 邱法展²^,^*()

¹ 甘肃省农业科学院作物研究所, 甘肃兰州 730070
² 湖北洪山实验室, 湖北武汉 430070

收稿日期:2022-09-23 接受日期:2023-02-21 出版日期:2023-08-12 网络出版日期:2023-03-10
通讯作者: 龚佃明,邱法展
作者简介:E-mail: wxr_0618@163.com
基金资助:
武汉市生物育种关键技术及新品种培育重大专项(2022021302024852);甘肃省农业科学院科研条件建设及成果转化项目(2021GAAS04);甘肃省省级重点人才项目(农作物种质资源保护利用与人才培养)和国家自然科学基金项目(31960405)

Identification and gene localization of a novel maize nuclear male sterility mutant ms6

WANG Xing-Rong¹(), ZHANG Yan-Jun¹, TU Qi-Qi², GONG Dian-Ming²^,^*(), QIU Fa-Zhan²^,^*()

¹ Crops Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China
² Hubei Hongshan Laboratory, Wuhan 430070, Hubei, China

Received:2022-09-23 Accepted:2023-02-21 Published:2023-08-12 Published online:2023-03-10
Contact: GONG Dian-Ming,QIU Fa-Zhan
Supported by:
Wuhan Major Project of Key Technologies in Biological Breeding and New Variety Cultivation(2022021302024852);Research Condition Construction and Achievement Transformation Project of Gansu Academy of Agricultural Sciences(2021GAAS04);Key Talent Project of Gansu Province(Crop Germplasm Resources Protection and Utilization Talent Training). and National Natural Science Foundation of China(31960405)

摘要/Abstract

摘要：

玉米雄性不育基因的定位、克隆和功能机理研究, 不仅能够加深我们对玉米雄花生长发育的分子调控机理的认识, 而且能够有效推动雄性不育技术体系的发展及在玉米育种和种子生产中的运用。本研究以玉米自交系Mo17为野生型背景材料, 经EMS诱变获得了一个玉米雄性不育突变体, 命名为ms6 (male sterile 6)。表型鉴定结果表明, ms6突变体植株能够正常抽雄, 但雄花颖壳不能正常开裂和散粉, 花粉粒干瘪, 表现为无花粉型不育。同时, ms6与Mo17野生型(wild type, WT)在株型、穗部性状以及籽粒粒形等相关性状上无显著差异, 说明该基因突变后, 仅影响植株的育性, 而不影响其他农艺性状。细胞学观察显示, ms6不育突变体的小孢子发育晚期出现异常, 表现为绒毡层细胞提前降解, 小孢子不能进行有丝分裂并逐渐裂解。扫描电镜观察表明, ms6花药外壁皱缩, 内壁无完整的花粉粒, 无乌氏小体的存在。遗传学分析表明, ms6突变性状受1对隐性核基因控制。以ms6 × B73 F₂遗传定位群体, 利用全基因组约200对多态性SSR分子标记, 结合表型与基因型连锁分析, 将ms6初定位于玉米6号染色体C6-19与C6-30两个标记之间, 进一步利用区间内10对新开发的多态性标记, 最终将ms6定位在分子标记M13~M14之间约480 kb的区间范围内。转录组测序结合qRT-PCR试验验证结果, 初步将Zm00001d035201确定为ms6的关键候选基因。Zm00001d035201基因编码一个酸性核糖体蛋白。本研究结果为ms6后续基因功能的研究打下了坚实的基础。同时ms6作为一个新的核不育突变体也为将来玉米新型核不育基因的生产应用提供了重要材料支持。

关键词: 玉米, 雄性不育, ms6, 基因定位, 候选基因鉴定

Abstract:

The localization, cloning, and functional mechanism research of male sterility genes can not only deepen our understanding of the molecular regulation mechanism of male flower growth and development, but also effectively promote the development of male sterility technology system and its application in maize breeding and seed production in maize. In this study, maize inbred line Mo17 was used as the wild-type background material, and a maize male sterile mutant, named male sterile 6 (ms6), was obtained by EMS mutagenesis. Phenotypic identification of ms6 mutant plants showed that they could perform normal tasseling, but the male flower glume could not dehiscence and powder could not disperse normally, and the pollen grains were dry, indicating pollen free sterility. Meanwhile, there were no significant differences between ms6 and Mo17 wild type (WT) in plant architecture, ear-related traits, kernel size, and other related traits, demonstrating that the ms6 gene only affected plant fertility, but did not affect other agronomic traits. Cytological observation revealed that the microspore of ms6 mutant was abnormal at the late stage of microspore development, which showed that tapetal cells were degenerated in advance, and the microspore could not undergo mitosis and gradually split. Scanning electron microscopy demonstrated that the anther epidermis of mutant ms6 were shrunk, and there was no intact pollen or ubisch on the inner surface of anther locule. Genetic analysis revealed that the ms6 phenotype was controlled by a pair of recessive nuclear genes. Combined with phenotypic and genotypic linkage analysis, ms6 was initially located between C6-19 and C6-30 on maize chromosome 6 by using the ms6 × B73 F₂ genetic mapping population and about 200 pairs of polymorphic SSR markers in the whole genome, and 10 pairs of newly developed polymorphic markers in the interval were further used. Finally, ms6 was located within the interval of about 480 kb between M13-M14. Gene mapping located the mutation site into a 480 kb interval between M13 and M14. Zm00001d035201 was preliminarily identified as the key candidate gene of ms6 by RNA-Seq combined with qRT-PCR. Zm00001d035201 encoded an acid ribosomal protein. This study provides a solid foundation for further studies on the function of ms6 gene. Meanwhile, as a novel male sterile mutant, ms6 also provides the important material support for the production and application of new maize genetic sterile genes in the future.

Key words: maize, male sterile, ms6, gene mapping, identification of candidate genes

王兴荣, 张彦军, 涂奇奇, 龚佃明, 邱法展. 一个新的玉米细胞核雄性不育突变体ms6的鉴定与基因定位[J]. 作物学报, 2023, 49(8): 2077-2087.

WANG Xing-Rong, ZHANG Yan-Jun, TU Qi-Qi, GONG Dian-Ming, QIU Fa-Zhan. Identification and gene localization of a novel maize nuclear male sterility mutant ms6[J]. Acta Agronomica Sinica, 2023, 49(8): 2077-2087.

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群体 Population	总株数 Total number	可育株 Fertile plants	不育株 Sterile plants	可育/不育(3:1) Fertility/sterile (3:1)	χ (3:1)	χ²_0.05
ms6×B73	396	300	96	3.13	0.12	3.84
ms6×Mo17	159	116	43	2.70	0.35

引物 Primer name	序列 Primer sequence (5°-3°)	引物 Primer name	序列 Primer sequence (5°-3°)
IN34-F	AGCACGGAGAAATGGAGTTG	M13-F	GAGGCCAAGCACAATGTATG
IN34-R	AACAGAAAGCTTGGCGTCAT	M13-R	TTTCAGGTGGAACCAGCAAT
MY8860-F	AATGGCAGGAGCACTACACC	C6-11A-F	CAACCAGGAGAAGCTGGCTA
MY8860-R	GGTCTGCCTGATGCTCTTGT	C6-11A-R	GAAGAGCGCGTAGAAGATGC
M10-F	GTGGAGCCCAGACACATTTT	M14-F	ACTGCAGCGTGTGAAAACAA
M10-R	TTTGTTTATGCTTGGCCGTA	M14-R	GCGGGAGACTCATACCTGAA
M11-F	ATGACCACTCCTGGGTTTTG	M17-F	GAGTTCTAAATAAGGGGGATGGA
M11-R	CTGTAGCCAAGAAGCCTTGC	M17-R	TTGGTTTCTGTGATGCATTGA
M12-F	AATTGGAGGAACCAGCTCAA	C6-20-F	ATTCGATCTAGGGTTTGGGTTCAG
M12-R	ATTTGGAGGGCGGTTTTATC	C6-20-R	GATGCAGTAGCATGCTGGATGTAG

基因ID Gene ID	注释 Gene annotation
Zm00001d035199	WPP domain-associated protein
Zm00001d035200	Putative AMP-dependent synthetize and ligase superfamily protein
Zm00001d035201	60S acidic ribosomal protein P0
AC187088.3_FG002	Guanylate kinase/Guanosine monophosphate kinase
Zm00001d035203	Unknown
Zm00001d035204	Unknown
Zm00001d035206	ATP binding protein
Zm00001d035207	Putative uncharacterized protein
Zm00001d035208	BTB/POZ domain-containing protein
Zm00001d035211	Ca(2)-dependent phospholipid-binding protein (Copine) family

基因ID Gene ID	表达差异Expression difference
基因ID Gene ID	花药发育时期 Anther development stage	ms6	野生型 WT	log₂FC	P-value	显著性 Significant
Zm00001d035199	四分体时期The tetrad stage	1024	1157	0.18	0.43	NS
	双核期The binucleate stage	1069	911	-0.31	0.28	NS
Zm00001d035200	四分体时期The tetrad stage	103	140	0.45	0.38	NS
	双核期The binucleate stage	135	75	-0.84	0.26	NS
Zm00001d035201	四分体时期The tetrad stage	9652	8941	-0.11	0.69	NS
	双核期The binucleate stage	6690	17205	1.36	0.00018	DOWN
AC187088.3_FG002	四分体时期The tetrad stage	NS	NS	NS	NS	NS
	双核期The binucleate stage	NS	NS	NS	NS	NS
Zm00001d035203	四分体时期The tetrad stage	NS	NS	NS	NS	NS
	双核期The binucleate stage	NS	NS	NS	NS	NS
Zm00001d035204	四分体时期The tetrad stage	NS	NS	NS	NS	NS
	双核期The binucleate stage	NS	NS	NS	NS	NS
Zm00001d035206	四分体时期The tetrad stage	600	943	0.65	0.0035	NS
	双核期The binucleate stage	1196	1924	0.69	0.088	NS
Zm00001d035207	四分体时期The tetrad stage	736	819	0.15	0.56	NS
	双核期The binucleate stage	763	729	-0.06	0.82	NS
Zm00001d035208	四分体时期The tetrad stage	827	1022	0.31	0.45	NS
	双核期The binucleate stage	1184	877	-4.41	0.41	NS
Zm00001d035211	四分体时期The tetrad stage	167	134	-0.31	0.59	NS
	双核期The binucleate stage	95	40	-1.06	0.16	DOWN

一个新的玉米细胞核雄性不育突变体ms6的鉴定与基因定位

Identification and gene localization of a novel maize nuclear male sterility mutant ms6

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