葡萄醇酰基转移酶编码基因遗传变异研究

doi:10.3864/j.issn.0578-1752.2022.14.010

Abstract

Abstract:

【Objective】The purpose of this study was to develop molecular markers suitable for the identification of grape aroma phenotypes, so as to provide a theoretical basis for grape molecular assisted breeding. 【Method】 The aroma components and content of 45 grape varieties were determined by solid-phase microextraction combined with gas-phase mass spectrometry, and the structural variation and SNP variation of VvAAT gene were analyzed by Sanger sequencing and amplicon sequencing, respectively. 【Result】A total of 65 aroma components were found in 45 grape varieties, which could be divided into four types: esters, alcohols, terpenes and aldehydes. Among them, the content of esters showed significant variety differences, and 20 grape varieties such as Kyoho were rich in volatile esters, but little volatile esters was detected in other 25 grape varieties such as 87-1. A total of 5 structural variants have been found in the VvAAT gene. Types I, II, IV and V could not be translated correctly due to premature termination codons or fragment insertion mutations. Only type III could be translated aright. According to the results of the phylogenetic tree analysis of their amino acid sequences, Type III could be divided into two types: III.1 and III.2. Combining the aroma data, it could be inferred that III.1 was functional, while the rest were non-functional. Amplicon sequencing and bioinformatics analysis revealed 8 SNP sites (T32C, A69T, G436C, A1247G, A1818T, G1929T, A1959G, and C1975G) located in the exon region of the VvAAT gene, all of which caused mutations in the coding amino acids. It could accurately distinguish between ester-rich varieties and ester-poor varieties, with an accuracy rate of 97.8%. 【Conclusion】VvAAT gene locus had abundant genetic variation, including gene structure variation and SNP variation; 8 SNP loci located in the coding region of VvAAT gene could accurately determine the phenotype of volatile esters, which could be applied to grape molecular assisted breeding.

Key words: grape, aroma, alcohol acyltransferase, VvAAT, amplicon sequencing, marker-assisted selection

JI XiaoHao,LIU FengZhi,WANG BaoLiang,LIU PeiPei,WANG HaiBo. Genetic Variation of Alcohol Acyltransferase Encoding Gene in Grape[J].Scientia Agricultura Sinica, 2022, 55(14): 2797-2811.

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Figures/Tables 13

Table 1

Fig. 1

Table 2

Table 3

VvAAT genotype statistics of 27 grape varieties"

品种 Variety	基因名称 Gene name	NCBI注册号 GenBank accession number	基因结构类型 Gene structure type	表型 Phenotype
巨峰 Kyoho	VvAAT-Kyoho-1	OK559426	III.2	酯香型 Esters-rich
巨峰 Kyoho	VvAAT-Kyoho-2	OK559427	III.1	酯香型 Esters-rich
阳光玫瑰 Shine-Muscat	VvAAT-Shine_Musca-1	OK559428	IV	非酯香型 Esters-poor
阳光玫瑰 Shine-Muscat	VvAAT-Shine_Musca-2	OK559429	V	非酯香型 Esters-poor
着色香 Zhuosexiang	VvAAT-Zhuosexiang-1	OK559430	III.1	酯香型 Esters-rich
着色香 Zhuosexiang	VvAAT-Zhuosexiang-2	OK559431	V	酯香型 Esters-rich
87-1	VvAAT-87_1	OK559432	III.2	非酯香型 Esters-poor
晚17 Wan17	VvAAT-Wan17	OK559433	II	非酯香型 Esters-poor
8611	VvAAT-8611-1	OK559434	III.2	非酯香型 Esters-poor
	VvAAT-8611-2	OK559435	III.2	非酯香型 Esters-poor
香妃 Xiangfei	VvAAT-Xiangfei	OK559436	III.2	非酯香型 Esters-poor
京早晶 Jingzaojing	VvAAT-Jingzaojing	OK559437	III.2	非酯香型 Esters-poor
郑州早红Zhengzhouzaohong	VvAAT-Zhengzhouzaohong	OK559438	V	非酯香型 Esters-poor
索罗门 Madeleine_Salomon	VvAAT-Madeleine_Salomon-1	OK559439	III.2	非酯香型 Esters-poor
索罗门 Madeleine_Salomon	VvAAT-Madeleine_Salomon-2	OK559440	III.2	非酯香型 Esters-poor
京秀 Jingxiu	VvAAT-Jingxiu-1	OK559441	III.2	非酯香型 Esters-poor
京秀 Jingxiu	VvAAT-Jingxiu-2	OK559442	II	非酯香型 Esters-poor
早巨选 Zaojuxuan	VvAAT-Zaojuxuan-1	OK559443	III.2	酯香型 Esters-rich
	VvAAT-Zaojuxuan-2	OK559444	III.1
	VvAAT-Zaojuxuan-3	OK559445	III.2
	VvAAT-Zaojuxuan-4	OK559446	III.2
奥利文 Irsay_Oliver	VvAAT-Irsay_Oliver	OK559447	III.1	酯香型 Esters-rich
香槟 Champion	VvAAT-Champion	OK559448	V	非酯香型 Esters-poor
紫早 Zizao	VvAAT-Zizao	OK559449	III.1	酯香型 Esters-rich
里扎马特 Rizamat	VvAAT-Rizamat-1	OK559450	III.2	非酯香型 Esters-poor
里扎马特 Rizamat	VvAAT-Rizamat-2	OK559451	III.2	非酯香型 Esters-poor
228	VvAAT-228	OK559452	III.2	非酯香型 Esters-poor
安尼斯基 Ayaneskeal	VvAAT-Ayaneskeal	OK559453	III.1	酯香型 Esters-rich
阿登纳玫瑰 Muscat_Adda	VvAAT-Muscat_Adda	OK559454	II	非酯香型 Esters-poor
红富士 BenniFuji	VvAAT-BenniFuji	OK559455	III.1	酯香型 Esters-rich
吉香 Jixiang	VvAAT-Jixiang	OK559456	III.1	酯香型 Esters-rich
藤稔 Fujiminori	VvAAT-Fujiminori	OK559457	III.1	酯香型 Esters-rich
尼加拉 Nigara	VvAAT-Nigara-1	OK559458	III.2	酯香型 Esters-rich
尼加拉 Nigara	VvAAT-Nigara-2	OK559459	III.1	酯香型 Esters-rich
乌兹别克玫瑰 Ojin_Kokn	VvAAT-Ojin_Kokn	OK559460	III.2	非酯香型 Esters-poor
井川1014 Jingchun1014	VvAAT-Jingchun1014-1	OK559461	III.2	酯香型 Esters-rich
井川1014 Jingchun1014	VvAAT-Jingchun1014-2	OK559462	III.1	酯香型 Esters-rich
秦龙大穗 Qinlongdasui	VvAAT-Qinlongdasui-1	OK559463	III.1	酯香型 Esters-rich
秦龙大穗 Qinlongdasui	VvAAT-Qinlongdasui-2	OK559464	III.2	酯香型 Esters-rich
累克基特 Leikejite	VvAAT-Leikejite-1	OK559465	I	非酯香型 Esters-poor
累克基特 Leikejite	VvAAT-Leikejite-2	OK559466	II	非酯香型 Esters-poor

Table 3

Fig. 2

Fig. 3

Table 4

Table 5

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Table 6

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化合物类型 Type of compound	种类 Number	变异范围 Variation range (ng∙g^-1)	平均值 Mean (ng∙g^-1)	方差 Variance
酯类Esters	22	0—3238.77	465.67	777.60
醇类Alcohols	2	0—164.84	12.58	31.91
醛类Aldehydes	12	642.80—5485.97	1890.40	1171.33
萜类Terpenoids	29	0—8548.37	471.41	1601.95
总计 Total	65	800.43—11387.75	2835.44	2381.58

酯香型品种 Varieties rich in volatile esters	非酯香型品种 Varieties poor in volatile esters
红香蕉 Hongxiangjiao (6, 244.27)	8611 (0, 0)
高砂 Gaosha (9, 278.59)	累克基特 Leikejite (0, 0)
秦龙大穗 Qinlongdasui (8, 220.90)	蓓蕾玫瑰 Muscat Bailey (0, 0)
黑蜜 Heimi (9, 199.58)	克瑞森无核 Crimson_Seedless (0, 0)
秋蜜 Qiumi (13, 402.83)	8612 (0, 0)
琥珀 Hupo (9, 330.06)	齐查卡普列 Qichakapulie (0, 0)
红丹 Hongdan (16, 920.17)	牧丹红 Mudanhong (0, 0)
火星无核 Mars_Seedless (9, 408.72)	红意大利Red_Italian (0, 0)
着色香 Zhuosexiang (6, 543.78)	玫瑰花 Meiguihua (0, 0)
黑虎香 Heihuxiang (14, 591.16)	泽玉 Zeyu (0, 0)
藤稔 Fujiminori (13, 701.42)	巧保2号 Qiaobao2 (0, 0)
红富士 BenniFuji (10, 723.95)	红巴拉多蒂 Hongbaladuodi (0, 0)
井川1014 Jingchun1014 (9, 800.74)	乌兹别克玫瑰 Ojin_Kokn (0, 0)
巨峰 Kyoho (15, 1453.39)	阿登纳玫瑰 Muscat_Adda (0, 0)
尼加拉 Nigara (13, 1697.20)	228 (0, 0)
奥利文 Irsay_Oliver (7, 1708.50)	里扎马特 Rizamat (0, 0)
安尼斯基 Ayaneskeal (9, 1920.01)	香槟 Champion (0, 0)
吉香 Jixiang (9, 2200.84)	京秀 Jingxiu (0, 0)
紫早 Zizao (9, 2370.34)	索罗门 Madeleine_Salomon (0, 0)
早巨选 Zaojuxuan (19, 3238.77)	郑州早红 Zhengzhouzaohong (0, 0)
	87-1 (0, 0)
	晚17 Wan17 (0, 0)
	香妃 Xiangfei (0, 0)
	京早晶 Jingzaojing (0, 0)
	阳光玫瑰 Shine_Musca (0, 0)

品种 Variety	Clean reads (K)	Clean bases (M)	Q20 (%)	Q30 (%)	覆盖度 Coverage (K)
奥利文 Irsay_Oliver	235.22	30.18	90.66	87.57	15.09
早巨选 Zaojuxuan	407.65	52.72	90.77	86.72	26.36
火星无核 Mars_Seedless	359.00	46.58	90.83	87.73	23.29
红香蕉 Hongxiangjiao	445.26	56.83	90.85	87.80	28.41
琥珀 Hupo	553.36	71.05	91.05	88.03	35.52
红富士 BenniFuji	373.19	47.93	90.47	87.29	23.96
秋蜜 Qiumi	295.73	37.96	90.76	87.69	18.98
巨峰 Kyoho	379.10	48.84	90.93	87.88	24.42
井川1014 Jingchun1014	518.11	66.35	90.89	87.84	33.17
黑虎香 Heihuxiang	729.92	95.28	90.87	87.79	47.64
黑蜜 Heimi	601.47	78.07	91.05	88.01	39.03
秦龙大穗 Qinlongdasui	693.37	90.00	90.50	87.34	45.00
红丹 Hongdan	338.81	42.87	90.80	87.78	21.43
高砂 Gaosha	343.88	43.87	91.00	88.01	21.93
红巴拉多蒂 Hongbaladuodi	521.69	67.30	91.20	88.17	33.65
巧保2号 Qiaobao2	355.88	45.40	91.04	88.00	22.70
泽玉 Zeyu	1187.71	155.61	90.83	87.68	77.80
玫瑰花 Meiguihua	1771.53	234.87	90.68	87.46	117.43
红意大利 Red Italian	627.85	79.59	89.91	86.61	39.79
牧丹红 Mudanhong	1348.14	173.77	89.67	86.65	86.88
8612	1342.69	173.45	89.86	86.51	86.72
克瑞森无核 Crimson Seedless	569.85	70.73	90.29	87.14	35.36

Genetic Variation of Alcohol Acyltransferase Encoding Gene in Grape

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基因名称 Gene name	SNPs								基因结构类型 Gene structure type
基因名称 Gene name	32	69	438	1247	1818	1929	1959	1975	基因结构类型 Gene structure type
VvAAT-Kyoho-1	T	A	G	A	A	G	A	C	III.2
VvAAT-Kyoho-2	C	T	C	G	T	T	G	G	III.1
VvAAT-Shine_Musca-1	T	A	G	A	A	G	A	C	IV
VvAAT-Shine_Musca-2	T	A	G	A	A	G	A	C	V
VvAAT-Zhuosexiang-1	C	T	C	G	T	T	G	G	III.1
VvAAT-Zhuosexiang-2	T	A	G	A	A	G	A	C	V
VvAAT-87_1	T	A	G	A	A	G	A	C	III.2
VvAAT-Wan17	T	A	G	A	A	G	A	C	II
VvAAT-8611-1	T	A	G	A	A	G	A	C	III.2
VvAAT-8611-2	T	A	G	A	A	G	A	C	III.2
VvAAT-Xiangfei	T	A	G	A	A	G	A	C	III.2
VvAAT-Jingzaojing	T	A	G	A	A	G	A	C	III.2
VvAAT-Zhengzhouzaohong	T	A	G	A	A	G	A	C	V
VvAAT-Madeleine_Salomon-1	T	A	G	A	A	G	A	C	III.2
VvAAT-Madeleine_Salomon-2	T	A	G	A	A	G	A	C	III.2
VvAAT-Jingxiu-1	T	A	G	A	A	G	A	C	III.2
VvAAT-Jingxiu-2	T	A	G	A	A	G	A	C	II
VvAAT-Zaojuxuan-1	T	A	G	A	A	G	A	C	III.2
VvAAT-Zaojuxuan-2	C	T	C	G	T	T	G	G	III.1
VvAAT-Zaojuxuan-3	T	A	G	A	A	G	A	C	III.2
VvAAT-Zaojuxuan-4	T	A	G	G	T	T	G	G	III.2
VvAAT-Irsay_Oliver	C	T	C	G	T	T	G	G	III.1
VvAAT-Champion	T	A	G	A	A	G	A	C	V
VvAAT-Zizao	C	T	C	G	T	T	G	G	III.1
VvAAT-Rizamat-1	T	A	G	A	A	G	A	C	III.2
VvAAT-Rizamat-2	T	A	G	A	A	G	A	C	III.2
VvAAT-228	T	A	G	A	A	G	A	C	III.2
VvAAT-Ayaneskeal	C	T	C	G	T	T	G	G	III.1
VvAAT-Muscat_Adda	T	A	G	A	A	G	A	C	II
VvAAT-BenniFuji	C	T	C	G	T	T	G	G	III.1
VvAAT-Jixiang	C	T	C	G	T	T	G	G	III.1
VvAAT-Fujiminori	C	T	C	G	T	T	G	G	III.1
VvAAT-Nigara-1	T	A	G	A	A	G	A	C	III.2
VvAAT-Nigara-2	C	T	C	G	T	T	G	G	III.1
VvAAT-Ojin_Kokn	T	A	G	A	A	G	A	C	III.2
VvAAT-Jingchun1014-1	T	A	G	A	A	G	A	C	III.2
VvAAT-Jingchun1014-2	C	T	C	G	T	T	G	G	III.1
VvAAT-Qinlongdasui-1	C	T	C	G	T	T	G	G	III.1
VvAAT-Qinlongdasui-2	T	A	G	A	A	G	A	C	III.2
VvAAT-Leikejite-1	T	A	G	G	T	T	G	G	I
VvAAT-Leikejite-2	T	A	G	A	A	G	A	C	II

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	I	II	III.1	III.2	IV	V
I	0	-	-	-	-	-
II		0	-	0	-	-
III.1			1604.18	1482.20	-	543.78
III.2				0	-	-
IV					-	0
V						0