调控玉米株高和穗位高候选基因Zmdle1的定位

doi:10.3864/j.issn.0578-1752.2023.05.002

Abstract

Abstract:

【Objective】 Plant height is one of the important target traits in maize plant type breeding, which is closely related not only to mechanized grain harvesting and lodging resistance, but also to maize yield. Therefore, it is of great theoretical and breeding value to isolate QTL/gene of maize plant height and analyze its function. This study aims to locate a novel maize dwarf gene ZmDLE1, clarify its biological function, and provide important theoretical basis and gene resources for accelerating the improvement of maize plant type. 【Method】 A single recessive mutant was derived in maize inbred line LY8405, from Crop Research Institute of Gansu Academy of Agricultural Sciences, by chemical mutagenic agent Ethyl Methyl Sulfonate (EMS). A maize dwarf and low ear mutant was isolated from the M₂ progeny, and the M₃ and M₄ progeny could stably inherit, which was named dwarf and low ear mutant1 (Zmdle1). The F₂ population was constructed by hybridization with Mo17 and was identified by bulked segregate analysis-sequencing (BSA-seq) and target segment recombination exchange. Based on the Mo17 reference genome, the genes in the target region were obtained and functionally annotated to locate candidate genes.【Result】Phenotypic identification of Zmdle1 was carried out, and the phenotype of Zmdle1 at seedling stage was not significantly different from that of the control LY8405. The plant height and ear height of Zmdle1 at mature stage were reduced by 87.2 cm and 55.4 cm, respectively, accounting for about 35.0% and 62.9%, difference is extremely significant. Morphological observation showed that the decrease of internode number and the shortening of internode cell length were the main reasons for the significant decrease of plant height and ear height of Zmdle1. The genetic analysis of the mutant gene was conducted by using the F_2:3 genetic populations. The Zmdle1 mutant is inherited in a 3﹕1 (χ²=2.854) ratio and is a single recessive gene. Therefore, according to the results of BSA-seq, the candidate gene ZmDLE1 was initially located in the 15 Mb region of Bin1.09-1.10 on chromosome 1 of maize. The polymorphism molecular markers were further developed using the re-sequencing results of Mo17 and Zmdle1, and the target gene was accurately cloned by map-based cloning. Finally, the candidate genes were mapped to the size range of 600 kb, and there were 16 candidate genes in this range. By comparing the re-sequencing data, it was found that Zm00001d033231 gene changed into A at the 2062 position G, which resulted in amino acid changing from glycine to serine, and the transcription level expression was significantly reduced compared with LY8405. Zm00001d033234 changed from T to C at the 223rd position leading to the 75th amino acid changed from serine to proline, and there was no significant difference in transcription level. Through association analysis of natural populations and the predicted genes for functional annotation, it was found that Zm00001d033231 and Zm00001d033234 were related to the growth and development of maize. 【Conclusion】 The candidate gene ZmDLE1 in the Bin1.09 region at the end of chromosome 1, was identified to effectively regulate maize plant height and ear height, and the target region was reduced to 600 kb by fine localization. Association analysis showed that Zm00001d033231 was genetic locus significantly associated with plant height within the target region.

Key words: maize, plant height, ear height, BSA-seq, fine mapping, association analysis

ZHOU WenQi, ZHANG HeTong, HE HaiJun, GONG DianMing, YANG YanZhong, LIU ZhongXiang, LI YongSheng, WANG XiaoJuan, LIAN XiaoRong, ZHOU YuQian, QIU FaZhan. Candidate Gene Localization of ZmDLE1 Gene Regulating Plant Height and Ear Height in Maize[J].Scientia Agricultura Sinica, 2023, 56(5): 821-837.

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引物名称Primer name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
Umc2047	GACAGACATTCCTCGCTACCTGAT	CTGCTAGCTACCAAACATTCCGAT
Umc1914	CAACATGAGCGTGCTAAATACTCG	ACAGGAACACATGAGGTCATCAAA
TIDP6556	TCAAGGAGGTCATCCTCACC	ATCACATATCTTGACGCGAGG
Umc1968	CTTCCCCTCCGCTACTGCTC	GTACTTGGTGTTGTCGCTCTTCTTC
Umc2612	AACTCAGGTACTTCAATCCCACCC	TGTTGCCATCAACAGGTTAAAGAA
Umc2510	TCTGCGAATAGAACTTAAACAGGTTG	AGAGGTCCGGGAAGAACAAGTAGT
BKN2-8	GTGTCTGCGCTCGATCTGT	TGTGTGATGGTGGCCTGTTA
1.10-14	AGACGTAAGACGATGCGACA	TCATGGGTTGCAATGGTAAA
MJD-52	AGAACCACCACTGCCAAAAA	GCTCTCATCTTGCCAAAACC
MJD-64	GCCGCTTGTTTCTTTTCCTT	TTCCTTCATGGCTCACCTGT
InDel-1	GTTGAGTTGAGTGGAAGGTG	GACCAGAGGAGAAAGCACCC
InDel-2	CGCTTCTCGTCGGCTGTCTG	CAGCCCTCGACCACGGTAA
InDel-3	CGAATGGAGATGGAGGCGACAG	GGAAAACCGCCCGGATCGCTT
InDel-5	TCCCAATCTGTTCCGAGCCAG	CACCTCCGCCGAAAGCCCCT
InDel-9	GGACCAATTACACGTAGCCG	CTTCTGGACGGTTCTGTTGC
InDel-13	TTAGTCCTATTTAGAACCTC	CAAACTCACCCAATCCACAT
InDel-22	CCCGACATCCTCTGCTCACG	TAGAGCACCTCCGATAGTTC
InDel-25	ACTTCTTGGGTTGTTGTGC	ATCTGGCACGAGTCTGAGAGG
InDel-26	CCTCTGATCCTATCTCGTGCTT	CATTGCGTTTGTTCCTTTG

类型Type	性状Trait	LY8405	Zmdle1	P值P-value	n
株型 Plant type	株高Plant height (cm)	248.9±21.8	161.7±23.8**	1.40931E-08	100
株型 Plant type	穗位高Ear height (cm)	88.1±16.5	32.7±11.4**	1.82449E-09	100
雄穗 Tassel	雄穗分枝数Number of tassel branches	11.1±3.1	8.1±1.8**	0.001072267	30
雄穗 Tassel	雄穗长Tassel length (cm)	50.4±2.6	50.3±2.3	0.545429534	30
雌穗 Ear	穗长Ear length (cm)	13.2±1.8	12.3±2.3	0.164088327	30
	穗粗Ear diameter (cm)	4.2±0.2	4.4±0.4	0.156854806	30
	轴粗Corncob thickness (cm)	2.6±0.2	3.2±0.2*	0.116425574	30
	穗行数Number of rows per ear	16.7±1.7	16.0±2.0	0.543525975	30
	行粒数Row number	18.8±2.3	16.9±3.4	0.173632692	30
叶型 Blade profile	倒3叶长The third top leaf length (cm)	38.1±6.0	45.0±3.8*	0.032741731	30
	倒3叶宽The third top leaf width (cm)	8.2±0.9	6.7±0.4**	0.002881444	30
	剑叶长Flag leaf length (cm)	25.2±3.7	30.0±5.6	0.119644254	30
	剑叶宽Flag leaf width (cm)	4.4±0.4	4.5±0.9	0.938315361	30
粒型 Grain dimension	粒长Grain length (cm)	1.13±0.30	1.04±0.20**	4.07025E-05	100
	粒宽Grain width (cm)	0.78±0.03	0.84±0.04*	0.01070802	100
	千粒重Thousand seed weight (g)	289.5±15.6	305.1±22.7	0.55039481	1000

群体 Population	总株数 Total plant number	正常表型 Normal phenotype	矮化表型 Dwarfing phenotype	分离比 Segregation ratio	P值 P value	χ²
(LY8405×Zmdle1) F₂	538	400	138	2.9﹕1	0.73	1.975
(Mo17×Zmdle1) F₂	2381	1821	560	3.2﹕1	0.102	2.854

候选基因 Candidate gene	蛋白类型 Protein type and putative function	基因位置 Position (bp)	表达差异（P值） Differential expression (P value)
Zm00001d033229	假设蛋白Hypothetical protein	255233719—255234639	0.620542
Zm00001d033230	类受体蛋白29 Receptor like protein 29	255243582—255245348	0.505878
Zm00001d033231	扩张蛋白B4 Expansin B4	255266246—255275500	0.001218**
Zm00001d033232	花粉Ole e1过敏原和扩展蛋白家族蛋白 Pollen Ole e1 allergen and extensin family protein	255276609—255280338	0.974205
Zm00001d033233	类水解酶蛋白质Hydrolase-like protein	255287266—255290302	0.008914**
Zm00001d033234	蛋白激酶PINOID Protein kinase PINOID	255293638—255295053	0.1486
Zm00001d033235	未知蛋白Unknown	255315224—255316506	0.48863
Zm00001d033236	未知蛋白Unknown	255319161—255321117	0.373901
Zm00001d033237	L型植物凝集素类受体蛋白激酶 L-type lectin-domain containing receptor kinase	255361226—255391452	0.466955
Zm00001d033238	未知功能Unknown	255391827—255391899	#DIV/0!
Zm00001d033239	未知功能Unknown	255392565—255392637	#DIV/0!
Zm00001d033240	感光性NPH3家族蛋白Phototropic-responsive NPH3 family protein	255510009—255515376	0.125879
Zm00001d033241	阳离子氨基酸转运体4 Cationic amino acid transporter 4	255565519—255569235	0.139972
Zm00001d033244	转导蛋白/WD40重复样超家族蛋白 Transducin/WD40 repeat-like superfamily protein	255748790—255757682	0.698459
Zm00001d033246	HD-Zip蛋白Class Ⅲ HD-Zip protein Ⅲ	255864303—255869980	0.770706
Zm00001d033247	转导蛋白/WD40类重复超家族蛋白 Transducin/WD40 repeat-like superfamily protein	255988480—256003180	0.137846

Candidate Gene Localization of ZmDLE1 Gene Regulating Plant Height and Ear Height in Maize

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