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作物学报 ›› 2024, Vol. 50 ›› Issue (4): 871-886.doi: 10.3724/SP.J.1006.2024.34124

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

茶树橙花叔醇和芳樟醇樱草糖苷含量全基因组关联分析及候选基因预测

张力岚1,2(), 杨军1,2, 王让剑1,2,*()   

  1. 1福建省农业科学院茶叶研究所, 福建福州 350013
    2国家茶树改良中心福建分中心, 福建福州 350013
  • 收稿日期:2023-07-18 接受日期:2023-10-23 出版日期:2024-04-12 网络出版日期:2023-11-13
  • 通讯作者: * 王让剑, E-mail: wangrj@faas.cn
  • 作者简介:E-mail: lilanzhang0114@foxmail.com
  • 基金资助:
    福建省科技计划项目(2023R1090);福建省农业科学院科技专项(ZYTS202408)

Genome-wide association study and candidate gene prediction of nerolidol and linalool primeveroside content in tea plants

ZHANG Li-Lan1,2(), YANG Jun1,2, WANG Rang-Jian1,2,*()   

  1. 1Tea Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, Fujian, China
    2Fujian Branch, National Center for Tea Improvement, Fuzhou 350013, Fujian, China
  • Received:2023-07-18 Accepted:2023-10-23 Published:2024-04-12 Published online:2023-11-13
  • Contact: * E-mail: wangrj@faas.cn
  • Supported by:
    Technology Plan Project of Fujian Province(2023R1090);Science and Technology Specific Project of Fujian Academy of Agricultural Science(ZYTS202408)

摘要:

橙花叔醇与芳樟醇是广泛分布于植物中的挥发性萜烯醇类化合物, 在茶树新梢中主要以樱草糖苷形式存在, 提高其含量对茶叶香气品质改良具有重要意义。为揭示茶树橙花叔醇和芳樟醇樱草糖苷的遗传机制, 本研究以169个茶树自然杂交后代单株为关联群体, 利用均匀分布在茶树染色体上的675,245个SNP标记, 对3个年份下茶树新梢中橙花叔醇与芳樟醇樱草糖苷含量进行全基因组关联分析(genome-wide association study, GWAS)。结果表明, 橙花叔醇与芳樟醇樱草糖苷含量的表型变异系数为60.83%~80.08%, 广义遗传力分别为51.29%与61.87%, 基本符合正态分布, 具有典型的数量性状遗传特征。全基因组关联分析共检测到50个显著关联位点, 各位点分别对橙花叔醇和芳樟醇樱草糖苷含量变化的贡献率均超过20%, 其中橙花叔醇樱草糖苷含量(nerolidol primeveroside content, NPC)变化位点最大贡献率为38.73%, 芳樟醇樱草糖苷含量(linalool primeveroside content, LPC)变化位点最大贡献率为39.07%。通过等位变异效应分析, 鉴定出4个主效SNP位点及其优异等位变异, 并发现1个可同时调控NPC和LPC的一因多效位点。结合已有的文献报道和基因的功能注释, 筛选出每个显著位点置信区间内最有可能的候选基因共59个, 主要涉及茶树的糖类代谢、转录调控、萜烯类生物合成等多个生物学过程, 其中26个基因在适制绿茶品种和适制乌龙茶品种单芽中的表达水平存在显著差异。本研究为深入解析茶树橙花叔醇和芳樟醇樱草糖苷含量的遗传机制提供了新的信息, 为加速培育高香型优异茶树新品种提供了重要的基因资源。

关键词: 茶树, 橙花叔醇樱草糖苷, 芳樟醇樱草糖苷, 全基因组关联分析, 候选基因

Abstract:

Nerolidol and linalool are volatile terpene alcohols compounds widely distributed in plants. They are mainly existing in the form of primeveroside in tea plant tender shoots, and increasing their content is of great significance for improving the aroma quality of tea. The objective of this study is to reveal the genetic mechanism of nerolidol and linalool primeveroside in tea plants. 169 natural hybrid progenies were used as associated populations, and the contents of nerolidol and linalool primeveroside in tea plant tender shoots in three years were analyzed by using 675,245 single nucleotide polymorphism (SNP) markers evenly distributed uniformly on the chromosomes of tea genome. The results showed that the phenotypic variation of nerolidol and linalool primeveroside content were 60.83%-80.08%, and the broad-sense heritability were 51.29% and 61.87% respectively. Nerolidol and linalool primeveroside content were in normal distribution, suggesting that the traits have typical genetic characteristics of quantitative traits. A total of 50 significantly associated loci were detected by GWAS, and each locus contributed more than 20% to the variations of nerolidol and linalool primeveroside content, of which the maximum contribution rate of nerolidol primeveroside content (NPC) variation site was 38.73%, and the maximum contribution rate of linalool primeveroside content (LPC) variation site was 39.07%. Furthermore, the elite alles of the four major SNPs was identified by allelic variation effect analysis, among which one locus that could affected NPC and LPC simultaneously. Finally, a total of 59 genes were annotated in the confidence intervals of each significantly associated loci, and the most likely candidate genes were predicted according to the comparison with previous reports and gene functional annotations. These candidate genes were mainly involved in multiple biological processes such as sugar metabolism, transcriptional regulation, terpene biosynthesis. Among them, there were significant differences in the relative expression levels of 26 genes between green tea varieties and oolong tea varieties. This study provides new information for further dissecting the genetic mechanism of nerolidol and linalool primeveroside content in tea plants, and provides important gene resources for accelerating the breeding of new tea varieties with high quality.

Key words: Camellia sinensis, nerolidol primeveroside, linalool primeveroside, genome-wide association study, candidate gene

附表1

169份茶树自然杂交后代单株名称及其信息来源"

序号
No.
名称
Name
母本来源
Maternal parent
序号
No.
名称
Name
母本来源
Maternal parent
1 04-1 FT104(♀) 86 16-15 FT516(♀)
2 04-10 FT104(♀) 87 16-16 FT516(♀)
3 04-11 FT104(♀) 88 16-17 FT516(♀)
4 04-12 FT104(♀) 89 16-18 FT516(♀)
5 04-13 FT104(♀) 90 16-19 FT516(♀)
6 04-14 FT104(♀) 91 16-2 FT516(♀)
7 04-15 FT104(♀) 92 16-20 FT516(♀)
8 04-16 FT104(♀) 93 16-21 FT516(♀)
9 04-17 FT104(♀) 94 16-22 FT516(♀)
10 04-18 FT104(♀) 95 16-23 FT516(♀)
11 04-19 FT104(♀) 96 16-24 FT516(♀)
12 04-2 FT104(♀) 97 16-25 FT516(♀)
13 04-20 FT104(♀) 98 16-26 FT516(♀)
14 04-21 FT104(♀) 99 16-27 FT516(♀)
15 04-22 FT104(♀) 100 16-28 FT516(♀)
16 04-23 FT104(♀) 101 16-29 FT516(♀)
17 04-24 FT104(♀) 102 16-3 FT516(♀)
18 04-25 FT104(♀) 103 16-30 FT516(♀)
19 04-26 FT104(♀) 104 16-31 FT516(♀)
20 04-27 FT104(♀) 105 16-32 FT516(♀)
21 04-28 FT104(♀) 106 16-33 FT516(♀)
22 04-29 FT104(♀) 107 16-34 FT516(♀)
23 04-3 FT104(♀) 108 16-35 FT516(♀)
24 04-30 FT104(♀) 109 16-36 FT516(♀)
25 04-31 FT104(♀) 110 16-37 FT516(♀)
26 04-32 FT104(♀) 111 16-38 FT516(♀)
27 04-33 FT104(♀) 112 16-39 FT516(♀)
28 04-34 FT104(♀) 113 16-4 FT516(♀)
29 04-35 FT104(♀) 114 16-40 FT516(♀)
30 04-36 FT104(♀) 115 16-41 FT516(♀)
31 04-37 FT104(♀) 116 16-42 FT516(♀)
32 04-38 FT104(♀) 117 16-43 FT516(♀)
33 04-39 FT104(♀) 118 16-44 FT516(♀)
34 04-4 FT104(♀) 119 16-45 FT516(♀)
35 04-40 FT104(♀) 120 16-46 FT516(♀)
36 04-41 FT104(♀) 121 16-47 FT516(♀)
37 04-42 FT104(♀) 122 16-48 FT516(♀)
38 04-43 FT104(♀) 123 16-49 FT516(♀)
39 04-44 FT104(♀) 124 16-5 FT516(♀)
40 04-45 FT104(♀) 125 16-50 FT516(♀)
41 04-46 FT104(♀) 126 16-51 FT516(♀)
42 04-47 FT104(♀) 127 16-52 FT516(♀)
43 04-48 FT104(♀) 128 16-53 FT516(♀)
44 04-49 FT104(♀) 129 16-54 FT516(♀)
45 04-5 FT104(♀) 130 16-55 FT516(♀)
46 04-50 FT104(♀) 131 16-56 FT516(♀)
47 04-51 FT104(♀) 132 16-57 FT516(♀)
48 04-52 FT104(♀) 133 16-58 FT516(♀)
49 04-53 FT104(♀) 134 16-59 FT516(♀)
50 04-54 FT104(♀) 135 16-6 FT516(♀)
51 04-55 FT104(♀) 136 16-60 FT516(♀)
52 04-56 FT104(♀) 137 16-61 FT516(♀)
53 04-57 FT104(♀) 138 16-62 FT516(♀)
54 04-58 FT104(♀) 139 16-63 FT516(♀)
55 04-59 FT104(♀) 140 16-64 FT516(♀)
56 04-6 FT104(♀) 141 16-65 FT516(♀)
57 04-60 FT104(♀) 142 16-66 FT516(♀)
58 04-61 FT104(♀) 143 16-67 FT516(♀)
59 04-62 FT104(♀) 144 16-68 FT516(♀)
60 04-63 FT104(♀) 145 16-69 FT516(♀)
61 04-64 FT104(♀) 146 16-7 FT516(♀)
62 04-65 FT104(♀) 147 16-70 FT516(♀)
63 04-66 FT104(♀) 148 16-71 FT516(♀)
64 04-67 FT104(♀) 149 16-72 FT516(♀)
65 04-68 FT104(♀) 150 16-73 FT516(♀)
66 04-69 FT104(♀) 151 16-74 FT516(♀)
67 04-7 FT104(♀) 152 16-75 FT516(♀)
68 04-70 FT104(♀) 153 16-76 FT516(♀)
69 04-71 FT104(♀) 154 16-77 FT516(♀)
70 04-72 FT104(♀) 155 16-78 FT516(♀)
71 04-73 FT104(♀) 156 16-79 FT516(♀)
72 04-74 FT104(♀) 157 16-8 FT516(♀)
73 04-75 FT104(♀) 158 16-80 FT516(♀)
74 04-76 FT104(♀) 159 16-81 FT516(♀)
75 04-77 FT104(♀) 160 16-82 FT516(♀)
76 04-78 FT104(♀) 161 16-83 FT516(♀)
77 04-79 FT104(♀) 162 16-84 FT516(♀)
78 04-8 FT104(♀) 163 16-85 FT516(♀)
79 04-9 FT104(♀) 164 16-86 FT516(♀)
80 16-1 FT516(♀) 165 16-87 FT516(♀)
81 16-10 FT516(♀) 166 16-88 FT516(♀)
82 16-11 FT516(♀) 167 16-89 FT516(♀)
83 16-12 FT516(♀) 168 16-9 FT516(♀)
84 16-13 FT516(♀) 169 16-90 FT516(♀)
85 16-14 FT516(♀)

图1

关联群体在不同年份下表型值的箱线图"

表1

不同年份间橙花叔醇与芳樟醇樱草糖苷含量的描述统计分析"

性状
Trait
年份
Year
变异范围
Range (µg mL-1)
均值±标准差
Mean (µg mL-1) ± SD
变异系数
CV (%)
峰度
Kurtosis
偏度
Skewness
遗传力
H2 (%)
橙花叔醇樱草糖苷含量
Nerolidol primeveroside content
E2018 0.0023-0.0508 0.0144±0.0115 80.08 1.56 1.49 51.29
E2019 0.0018-0.0278 0.0070±0.0052 75.24 2.81 1.69
E2020 0.0013-0.0167 0.0049±0.0036 72.55 1.29 1.45
芳樟醇樱草糖苷含量
Linalool primeveroside content
E2018 0.0024-0.0500 0.0139±0.0092 66.71 1.96 1.39 61.87
E2019 0.0014-0.0280 0.0083±0.0053 64.30 1.43 1.27
E2020 0.0014-0.0229 0.0073±0.0045 60.83 0.77 1.06

图2

不同年份间橙花叔醇与芳樟醇樱草糖苷含量的相关性分析 A: 橙花叔醇樱草糖苷含量(NPC); B: 芳樟醇樱草糖苷含量(LPC)。**表示在0.01概率水平差异显著。"

表2

橙花叔醇与芳樟醇樱草糖苷含量显著关联的SNP位点"

编号
Number
标记
SNP
染色体
Chromosome
位置
Position (bp)
等位
基因
Allele
贡献率1)
R2 (%) 1)
性状
Trait
共同环境a
Common environments a
SNP_1 Sca.1199:748834 Scaffold1199 748834 G/A 24.78 NPC E2018 (7.5)
SNP_2 Sca.2168:1897613 Scaffold2168 1897613 A/C 38.73 NPC E2018 (8), E2019 (11.2), E2020 (9.48)
SNP_3 Sca.2542:459511 Scaffold2542 459511 A/C 25.69 NPC E2018 (6.8), E2019 (8.39), E2020 (7.35)
SNP_4 Sca.5635:459399 Scaffold5635 459399 T/C 25.66 NPC E2018 (7.11)
SNP_5 Sca.1219:1790059 Scaffold1219 1790059 C/T 31.64 NPC E2019 (7.53), E2020 (10.04)
SNP_6 Sca.1520:1733255 Scaffold1520 1733255 G/A 32.53 NPC E2019 (7.12), E2020 (9.71)
SNP_7 Sca.1979:279352 Scaffold1979 279352 G/C 29.88 NPC E2019 (6.96), E2020 (9.57)
SNP_8 Sca.2438:547979 Scaffold2438 547979 C/G 31.22 NPC E2019 (7.81), E2020 (10)
SNP_9 Sca.278:1059711 Scaffold278 1059711 G/A 29.91 NPC E2019 (7.14), E2020 (9.63)
SNP_10 Sca.3798:966881 Scaffold3798 966881 T/C 29.90 NPC E2019 (6.84), E2020 (9.45)
SNP_11 Sca.4003:297821 Scaffold4003 297821 C/T 32.81 NPC E2019 (8.63), E2020 (10.44)
SNP_12 Sca.558:2642171 Scaffold558 2642171 G/A 32.12 NPC E2019 (7.83), E2020 (10.18)
SNP_13 Sca.635:692228 Scaffold635 692228 C/G 30.06 NPC E2019 (7.03), E2020 (9.54)
SNP_14 Sca.682:1668595 Scaffold682 1668595 C/T 21.87 NPC E2019 (7.03)
SNP_15 Sca.70:876326 Scaffold70 876326 G/A 32.07 NPC E2019 (6.9), E2020 (9.36)
SNP_16 Sca.889:283250 Scaffold889 283250 A/G 33.74 NPC E2019 (7.45), E2020 (9.86)
SNP_17 Sca.986:1314724 Scaffold986 1314724 G/A 29.95 NPC E2019 (6.79), E2020 (9.41)
SNP_18 Sca.1618:424426 Scaffold1618 424426 C/T 22.09 NPC E2020 (7.24)
SNP_19 Sca.2184:835086 Scaffold2184 835086 G/A 37.75 NPC E2020 (8.58)
SNP_20 Sca.2616:169719 Scaffold2616 169719 C/T 37.82 NPC E2020 (8.6)
SNP_21 Sca.3180:797058 Scaffold3180 797058 C/G 22.41 NPC E2020 (6.86)
SNP_22 Sca.3342:374264 Scaffold3342 374264 A/C 37.28 NPC E2020 (8.74)
SNP_23 Sca.387:1481497 Scaffold387 1481497 T/C 30.41 NPC E2020 (9.76)
SNP_24 Sca.1271:799628 Scaffold1271 799628 T/C 29.65 NPC E2019 (6.95), E2020 (9.56)
SNP_25 Sca.2348:501688 Scaffold2348 501688 G/A 30.81 NPC E2019 (7.36), E2020 (9.88)
SNP_26 Sca.3286:410383 Scaffold3286 410383 T/G 30.50 NPC E2019 (7.26), E2020 (9.79)
SNP_27 Sca.4005:1000551 Scaffold4005 1000551 T/C 29.90 NPC E2019 (7.1), E2020 (9.63)
SNP_28 Sca.408:698032 Scaffold408 698032 C/T 29.96 NPC E2019 (7.09), E2020 (9.64)
SNP_29 Sca.4208:1208863 Scaffold4208 1208863 C/T 29.60 NPC E2019 (6.93), E2020 (9.54)
SNP_30 Sca.7623:206978 Scaffold7623 206978 C/T 31.21 NPC E2019 (7), E2020 (9.55)
SNP_31 Sca.247:794502 Scaffold247 794502 T/C 37.92 NPC E2020 (8.79)
SNP_32 Sca.336:1433036 Scaffold336 1433036 T/A 38.63 NPC E2020 (8.78)
SNP_33 Sca.348:1050518 Scaffold348 1050518 G/A 23.33 NPC E2020 (6.79)
SNP_34 Sca.411:1462612 Scaffold411 1462612 T/C 22.74 NPC E2020 (6.92)
SNP_35 Sca.502:3196094 Scaffold502 3196094 G/T 38.27 NPC E2020 (8.68)
SNP_36 Sca.5364:1037305 Scaffold5364 1037305 G/T 28.75 NPC E2020 (9.09)
SNP_37 Sca.874:532016 Scaffold874 532016 C/T 28.69 NPC E2020 (8.71)
SNP_38 Sca.878:1091319 Scaffold878 1091319 C/T 22.70 NPC E2020 (6.98)
SNP_39 Sca.1970:1955068 Scaffold1970 1955068 T/A 21.06 NPC E2019 (6.82)
27.59 LPC E2018 (8.45), E2019 (7.03)
SNP_40 Sca.1858:573611 Scaffold1858 573611 C/T 39.07 LPC E2018 (6.97), E2019 (9.73), E2020 (11.57)
SNP_41 Sca.3968:194068 Scaffold3968 194068 T/C 37.99 LPC E2018 (7.12), E2019 (10.56)
SNP_42 Sca.1026:543252 Scaffold1026 543252 A/G 25.68 LPC E2019 (7.07), E2020 (8.04)
SNP_43 Sca.2322:1086470 Scaffold2322 1086470 C/T 26.33 LPC E2019 (6.93), E2020 (8.12)
SNP_44 Scaf.257:1761348 Scaffold257 1761348 C/T 26.34 LPC E2019 (6.78), E2020 (8.14)
SNP_45 Sca.7573:246109 Scaffold7573 246109 G/A 27.34 LPC E2019 (6.78), E2020 (8.17)
SNP_46 Sca.1125:229901 Scaffold1125 229901 C/T 25.32 LPC E2020 (7.97)
SNP_47 Sca.1713:1655993 Scaffold1713 1655993 G/A 26.62 LPC E2020 (7.76)
SNP_48 Sca.1911:518191 Scaffold1911 518191 C/T 27.03 LPC E2020 (8.03)
SNP_49 Sca.2268:2160703 Scaffold2268 2160703 T/A 24.98 LPC E2020 (7.28)
SNP_50 Sca.2982:1181264 Scaffold2982 1181264 C/T 27.12 LPC E2020 (7.67)

图3

主效SNP位点的关联分析及单倍型分析 NPC: 橙花叔醇樱草糖苷含量; LPC: 芳樟醇樱草糖苷含量。"

附图1

GWAS候选基因在茶树各组织中的表达"

表3

候选基因鉴定"

序号
Number
候选基因
Candidate gene
物理位置
Physical position (bp)
性状
Trait
功能注释
Function annotation
1 TEA028249.1 Scaffold1199: 818045-820839 NPC WRKY转录因子 WRKY transcription factor
2 TEA005261.1 Scaffold5635: 513652-518770 NPC 细胞色素P450 Cytochrome P450
3 TEA020225.1 Scaffold2438: 629180-630643 NPC UDP糖基转移酶 UDP-glycosyltransferase
4 TEA010008.1 Scaffold70: 830891-835051 NPC 葡萄糖基转移酶 Glucosyltransferase
5 TEA029804.1 Scaffold986: 1389566-1393613 NPC 细胞色素P450 Cytochrome P450
6 TEA022017.1 Scaffold1271: 787923-796373 NPC α-半乳糖苷酶 Alpha-galactosidase
7 TEA008260.1 Scaffold2348: 490385-492318 NPC 细胞色素P450 Cytochrome P450
8 TEA027002.1 Scaffold3286: 462879-469437 NPC 细胞色素P450 Cytochrome P450
9 TEA013172.1 Scaffold4005: 1056917-1064631 NPC 半乳糖基转移酶 Galactosyltransferase
10 TEA026690.1 Scaffold408: 689261-702026 NPC 鼠李糖基转移酶 Rhamnosyltransferase
11 TEA005230.1 Scaffold4208: 1227172-1232799 NPC β-半乳糖苷酶 Beta-galactosidase
12 TEA018377.1 Scaffold7623: 242686-249624 NPC 半乳糖基转移酶 Galactosyltransferase
13 TEA005213.1 Scaffold247: 765403-771112 NPC WRKY转录因子 WRKY transcription factor
14 TEA031275.1 Scaffold336: 1366825-1368276 NPC 葡萄糖基转移酶 Glucosyltransferase
15 TEA016084.1 Scaffold348: 1150211-152775 NPC UDP糖基转移酶 UDP-glycosyltransferase
16 TEA016178.1 Scaffold411: 1489977-1491356 NPC UDP糖基转移酶 UDP-glycosyltransferase
17 TEA022548.1 Scaffold502: 3111388-3123387 NPC 葡萄糖基转移酶 Glucosyltransferase
18 TEA017006.1 Scaffold5364: 1032472-1040796 NPC 细胞色素P450 Cytochrome P450
19 TEA017007.1 Scaffold5364: 993754-999940 NPC 细胞色素P450 Cytochrome P450
20 TEA026818.1 Scaffold874: 583227-591047 NPC 细胞色素P450 Cytochrome P450
21 TEA026846.1 Scaffold874: 626136-642573 NPC 细胞色素P450 Cytochrome P450
22 TEA022489.1 Scaffold878: 1081413-1084684 NPC UDP糖基转移酶 UDP-glycosyltransferase
23 TEA028423.1 Scaffold1026: 471732-473370 LPC 葡萄糖基转移酶 Glucosyltransferase
24 TEA021834.1 Scaffold1713: 1746139-1754677 LPC NAC转录因子 NAC transcription factor
25 TEA021094.1 Scaffold2268: 2095978-2097423 LPC UDP糖基转移酶 UDP-glycosyltransferase
26 TEA027095.1 Scaffold2982: 1184278-1189401 LPC β-半乳糖苷酶 Beta-galactosidase

图4

转录组与GWAS筛选共有基因的表达水平热图 Fuyun 6_1、Fuyun 6_2、Fuyun 6_3: 候选基因在绿茶品种‘福云6号’单芽中的表达水平; Yuemingxiang_1、Yuemingxiang_2、Yuemingxiang_3: 候选基因在乌龙茶品种‘悦茗香’单芽中的表达水平。"

图5

候选基因调控模型示意图 TFs: 转录因子; TPS: 萜烯合成酶; CYP450: 细胞色素P450; GHs: 糖苷水解酶; GTs: 糖基转移酶。"

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