西瓜果实表皮蜡粉的化学成分与基因定位

doi:10.3864/j.issn.0578-1752.2019.09.010

Abstract

Abstract:

【Objective】Wax powder is the first protective barrier for plants to resist external stress. In order to find out the physiological and biochemical mechanism of this trait and to get candidate genes, the structure, chemical compositions and inheritance of wax powder on watermelon fruit epidermis were investigated in this research, and the candidate genes were predicted. 【Method】Six-generation populations (P₁, P₂, F₁, F₂, BC₁P₁, and BC₁P₂) were constructed by crossing the inbred line ‘Meijiaxuanhei’ (in glossy) and ‘FH’ (in waxy). Scanning electron microscopy (SEM) was used to observe the structure of wax powder on mature watermelon fruit epidermis. The composition of wax powder was determined by gas chromatography-mass spectrometry (GC-MS), and the contents of different chemical components were quantitatively calculated based on the peak area. The wax powder trait was preliminary mapped by BSA-seq, and BLAST software was used to annotate the coding genes in the localization interval in many databases. By analysis of detailed gene annotation information and mutation site, candidate genes were quickly screened. 【Result】The color of wax powder on ‘Meijiaxuanhei’ fruit was gray-white, and the structure was compact plate shaped with about 5 μm in length, while ‘FH’ fruit was found smooth epidermis and no similar structure. 24 kinds of aliphatic compounds were detected in the wax powder, which were classified as hydrocarbons, alcohols, esters, acids, phenols and aldehydes. There were 10 kinds of hydrocarbons were found, which accounted for 77.72% of the total effective chemical extracts of epidermal wax powder and chain length ranged from C17 to C36. Among them, most of the saturated n-alkanes were C27, C28, C29, C32, C33, C34 and C36, 5 kinds of alcohols accounted for 12.60%, 5 kinds of esters accounted for 0.43%, 2 kinds of acids accounted for 0.53%, 1 kind of phenol accounted for 0.80%, and 1 kind of aldehydes accounted for 3.99%, respectively. The top five chemical components in watermelon wax powder were n-34, n-29, 1,30-triacontanediol, n-33 and n-octacosane. The fruit of F1 and BC₁P₂ progenies had wax, the segregation of waxy and glossy in F₂ progenies corresponded to the Mendelian ratio of 3:1, and the segregation ratio in BC₁P₁ progenies was 1:1, so it could be concluded that the hereditary mode of waxy was the dominant heredity of single gene, and the waxy type was the dominant character. By SNP and InDel correlation analysis, 3.16-4.84Mb association region of chromosome 1 was obtained, which contained 144 genes, 138 genes in them were functionally annotated, including 10 non-synonymous mutations and 1 frameshift mutation. Combining with the literature reports, there were five non-synonymous mutation genes might be related to the formation of watermelon epidermal wax powder: Cla002367 was an enoyl ACP-reductase (ECR) gene, which was essential for the synthesis of extra-long chain fatty acids. Cla011514, Cla002337 and Cla002342 were cytochrome P450 (CYP) family genes, and some proteins encoded by the CYP family genes could catalyze the formation of aliphatic compounds through alkyl hydroxylation and other reactions; Cla002353 gene annotation information was ABC transporter, and ABC transporters were closely related to the transport of waxy molecules.【Conclusion】Wax powder on watermelon fruit epidermis was a plate structure, which was mainly composed of aliphatic compounds derived from long chain fatty acids. The trait conformed to the single gene inheritance model, and wax powder was dominant. The association region of chromosome 1 in the range of 1.68Mb was obtained by BSA correlation analysis. 5 non-synonymous mutation genes Cla002367, Cla011514, Cla002337, Cla002342 and Cla002353 were predicted as candidate genes for wax powder on watermelon fruit epidermis.

Key words: watermelon, wax powder, epidermis structure, chemical compositions, genetic analysis, gene mapping

GONG ChengSheng, ZHAO ShengJie, LU XuQiang, HE Nan, ZHU HongJu, DOU JunLing, YUAN PingLi, LI BingBing, LIU WenGe. Chemical Compositions and Gene Mapping of Wax Powder on Watermelon Fruit Epidermis[J].Scientia Agricultura Sinica, 2019, 52(9): 1587-1600.

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References 38

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化合物类别 Categories	化合物名称 Compounds	保留时间 Retention time (min)	相对含量 Relative content (%)
	正二十四烷* Tetracosane*	17.20
烃类 Hydrocarbons	2,6,10-三甲基十四烷 Tetradecane,2,6,10-trimethyl-	4.92	0.05
	3-乙基-5-（2-乙基丁基）十八烷 Octadecane, 3-ethyl-5-(2-ethylbutyl)-	19.00	0.01
	正二十八烷 Octacosane	23.45	5.89
	正二十九烷 Nonacosane	27.92	16.24
	17-三十五烯 17-Pentatriacontene	28.58	0.60
	正二十七烷 Heptacosane	29.90	1.14
	正三十四烷 Tetratriacontane	32.31	40.76
	正三十二烷 Dotriacontane	34.02	1.07
	正三十三烷 Tritriacontane	36.12	10.67
	正三十六烷 Hexatriacontane	39.79	0.93
酯类 Esters	(E)-3,7-二甲基-2,6-辛二烯醇3-甲基丁酸酯 Geranyl isovalerate	5.28	0.10
	顺式-9-十八碳烯酸-2-苯基-1,3-二氧杂环己烷-4-甲基酯 9-Octadecenoic acid, (2-phenyl-1,3-dioxolan-4-yl)methyl ester, cis-	7.58	0.09
	俞酸甘油酯 Docosanoic acid, 1,2,3-propanetriyl ester	35.83	0.11
	胆酸乙酯 Ethyl iso-allocholate	12.21	0.05
	十八烯酸-3（十八烷氧基）丙酯 Oleic acid, 3-(octadecyloxy)propyl ester	40.79	0.07
醇类 Alcohols	1-三十七烷醇 1-Heptatriacotanol	9.64	0.15
	E,E,Z-1,3,12-十九烯-4,14-二醇 E,E,Z-1,3,12-Nonadecatriene-4,14-diol	13.04	0.21
	1,30-三十烷二醇 1,30-Triacontanediol	34.99	11.55
	1-三十五醇 1-Pentatriacontanol	36.86	0.48
	乙酰氧基-7,8-环氧羊毛甾烷-11-醇 7,8-Epoxylanostan-11-ol, 3-acetoxy-	37.58	0.21
酸类 Acids	顺式-1-二十碳烯酸 cis-13-Eicosenoic acid	6.83	0.33
酸类 Acids	五氟丙酸八碳五烯酸Octatriacontyl pentafluoropropionate	40.29	0.20
酚类Phenols	2,4-二叔丁基苯酚Phenol,2,4-bis(1,1-dimethylethy)-	5.701	0.80
醛类Aldehyde	十八醛 Octadecanal	26.42	3.99

世代 Generations	植株总数 Number of total plants	有蜡粉株数 Number of plants with wax	无蜡粉株数 Number of wax-free plants	有﹕无 Ratios	P值 P value	卡方值 χ² values
P₁	50	-	50
P₂	50	50	-
F₁	50	50	-
F₂	714	529	185	2.86:1	0.57	0.32
BC₁P₁	279	150	129	1.16:1	0.21	1.58
BC₁P₂	50	50	-

功能注释数据库Annotated_databases	有注释信息基因数 Annotated information gene number	存在非同义突变的基因数Number of genes with nonsynonymous mutations	存在移码突变的基因数 Number of genes with frameshift mutation
GO	42	6	0
KEGG	46	4	0
COG	63	8	1
NR	137	10	1
NT	133	10	1
trEMBL	136	10	1
SwissProt	118	10	1
Total	138	10	1

突变类型 Mutation type	基因 Gene Id	基因注释 Gene annotation
非同义突变 Non synonymous mutation	Cla011514	细胞色素P450 Cytochrome P450
非同义突变 Non synonymous mutation	Cla011577	乳酸/苹果酸脱氢酶 Lactate/malate dehydrogenase,
	Cla011542	铜胺氧化酶,酶结构域 Copper amine oxidase, enzyme domain
	Cla011561	3-Oxoacyl-[酰基载体蛋白(ACP)]合成酶Ⅲ 3-Oxoacyl-[acyl-carrier-protein (ACP)] synthase III
	Cla002342	含IPRO结构域（S）的IPRO1128细胞色素P450 Contains Inerpro domain(s) IPR001128 Cytochrome P450
	Cla002367	烯酰基-（酰基载体蛋白）还原酶 Enoyl-(Acyl carrier protein) reductase
	Cla002326	木聚糖酶抑制剂N端 Xylanase inhibitor N-terminal
	Cla002353	ABC转运体 ABC transporter
	Cla002337	细胞色素P450 Cytochrome P450
	Cla011554	无顶端分生组织（NAM）蛋白 No apical meristem (NAM) protein
移码突变 Frameshift mutation	Cla002327	rRNA/tRNA 2′-O-甲基转移酶类纤维蛋白样蛋白 rRNA/tRNA 2'-O-methyltransferase fibrillarin-like protein

Chemical Compositions and Gene Mapping of Wax Powder on Watermelon Fruit Epidermis

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