A Novel F3â²5â²H Allele with 14 bp Deletion Is Associated with High

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Bioactive Constituents, Metabolites, and Functions

A Novel F3'5'H Allele with 14 bp Deletion Is Associated with High Catechin Index Trait of Wild Tea Plants and Has Potential Use in Enhancing Tea Quality Ji-Qiang Jin, Yu-Fei Liu, Chun-Lei Ma, Jian-Qiang Ma, WanJun Hao, Yan-Xia Xu, Ming-Zhe Yao, and Liang Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04504 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 27, 2018

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Journal of Agricultural and Food Chemistry

A Novel F3'5'H Allele with 14 bp Deletion Is Associated with High Catechin Index Trait of Wild Tea Plants and Has Potential Use in Enhancing Tea Quality Ji-Qiang Jin, Yu-Fei Liu, Chun-Lei Ma, Jian-Qiang Ma, Wan-Jun Hao, Yan-Xia Xu, Ming-Zhe Yao, Liang Chen* Tea Research Institute of the Chinese Academy of Agricultural Sciences, Key Laboratory of Tea Plant Biology and Resources Utilization, Ministry of Agriculture and Rural Affairs, 9 South Meiling Road, Hangzhou, Zhejiang 310008, China *Corresponding Author tel: +86 571 86652835, fax: +86 571 86650056. E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: Catechins are important chemical components determining the quality

2

of tea. The catechin index (CI, ratio of dihydroxylated catechin (DIC)/trihydroxylated

3

catechin (TRIC)) in the green leaf has a major influence on the amounts of theaflavins

4

in black tea. In this work, the major catechin profiles of wild tea plants originating

5

from Guizhou Province with high CI trait were investigated. We identified a novel

6

flavonoid 3',5' hydroxylase gene (F3′5′H) allele with a 14 bp deletion in the upstream

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regulation region and developed a insertion/deletion (InDel) marker accordingly. The

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14 bp deletion in the novel F3'5'H allele was associated with low F3′5′H mRNA

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expression, thereby resulting in low TRIC content and high CI value. The allelic

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variant in the novel F3'5'H allele associated with high CI values and DIC contents

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was confirmed by the introgression lines derived from a distant cross population. The

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novel F3'5'H allele in wild tea plants is a valuable gene resource, which could be

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applied to breeding improvement on tea quality.

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KEYWORDS: allelic variants, catechin, flavonoid 3',5' hydroxylase gene, wild tea

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plants, tea quality

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INTRODUCTION

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After water, tea is the most widely consumed drink in the world due to its

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pleasant taste and health benefits.1 Tea is made from fresh leaves of the tea plant. Tea

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plants belong to genus Camellia L., section Thea (L.) Dyer of the family Theaceace,

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and are classified into five species based on morphology, i.e., C. sinensis (L.) O.

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Kuntze, C. tachangensis F. C. Zhang, C. crassicolumna Chang, C. taliensis (W. W.

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Smith) Melchior, C. gymnogyna Chang, and three varieties of C. sinensis, namely, C.

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sinensis var. sinensis, C. sinensis var. assamica (Masters) Kitamura, and C. sinensis

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var. pubilimba Chang.2 Three varieties of C. sinensis are commonly cultivated.3 Tea

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leaves have large amounts of characteristic secondary metabolites, such as

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polyphenols, caffeine, theanine, and volatiles. The major polyphenol compounds in

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tea leaves are catechins, which are a subgroup of flavan-3-ols. According to the

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hydroxylation

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3′4′-dihydroxylated catechins (DIC) [(+)-catechin (C), (−)-epicatechin (EC) and

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(−)-epicatechin

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[(+)-gallocatechin (GC), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate

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(EGCG)]. Among the 403 accessions of representative tea germplasms collected from

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various locations in China, EGCG is the most abundant (94.1 mg/g), followed by

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ECG (28.9 mg/g), EGC (16.1 mg/g), and EC (8.2 mg/g), all of which account for over

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95% of the total catechins (TC) of tea.4 The concentration of TRIC is markedly higher

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than DIC in most Chinese tea germplasms.

pattern

gallate

of

their

(ECG)]

B-ring,

and

catechins

can

3′4′5′-trihydroxylated

be

divided

catechins

into

(TRIC)

38

Based on differences in processing, tea is generally classified as green, oolong,

39

black, dark, white, or yellow tea in China; these teas mainly differ in the oxidization

40

degree of their catechins. Depending on the type of manufacturing process applied,

41

each tea sample has a unique aroma, taste, and chemical profile.5 Different tea



42

manufacturing methods require different tea cultivars to achieve distinct chemical

43

compositions and characteristic metabolites. Catechins are important chemical

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components determining the quality of tea. For example, a higher ratio of (EGCG +

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ECG) × 100/EGC has been suggested to be an index of higher quality green tea.6

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Among the different catechin components, ECG presents the strongest bitterness and

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most astringent taste;7,8 as such, cultivars with low ECG contents are considered

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suitable for processing green tea. In black tea, catechins are polymerized to

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theaflavins and thearubigins via a “fermentation” procedure that leads to catechin

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oxidation;1 among these products, theaflavins significantly influence the quality of

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black tea.9−12 Formation of a theaflavin molecule requires a DIC molecule and a TRIC

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molecule. Maximum theaflavin formation occurs when the concentrations of DIC and

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TRIC quinones are equal.12 Based on in vitro oxidation experiments, Robertson

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concluded that a higher catechin index (CI) [(EC + ECG)/(EGC + EGCG)] increases

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the theaflavin/thearubigin ratio in black tea.9 The CI value of green tea leaves can

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determine the ultimate theaflavin amounts and quality of black tea.11−14 Our previous

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study 4 revealed that the CI values of 93 Chinese tea clones varied from 0.22 to 0.67

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and averaged 0.39 ± 0.09. Based on the availability of most Chinese tea clones, DIC

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content appears to be the limiting factor influencing the amount of theaflavin formed

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during the processing of black tea.

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In the last four years, several wild tea plants originating from the southwestern

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(Xingyi City and Pu’an County) and western (Nayong County and Jinsha County)

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regions of Guizhou Province, China, belonging to C. tachangensis and C. gymnogyna,

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respectively, were found to present lower TRIC contents (3) than the cultivated tea plants by our research group. These transnormal tea

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germplasms contained much more DIC than TRIC, which is contrary to the profile of


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major catechins in regular tea plants. The molecular mechanism of low TRIC

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accumulation within these wild tea plants remains unclear. Catechins are synthesized

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through flavonoid pathways via successive enzymatic reactions, as illustrated in

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Figure 1. Flavonoid 3' hydroxylase (F3'H) and flavonoid 3',5' hydroxylase (F3'5'H),

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which belong to the superfamily of cytochrome P450-dependent monooxygenases, are

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two important enzymes controlling hydroxylation at the 3'- and the 3'- and/or

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5'-positions of the B ring of flavonoids, respectively.15,16 By combining bulked

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segregant RNA-seq with candidate gene association mapping, validating with

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biparental segregation population and differential expression analysis, we revealed

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that allelic variants within F3′5′H governing the ratio of CI values and catechin

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contents in tea plant.17 Interestingly, we elucidated that, once the functional allelic

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variant in F3'5'H induces decreased 5′-hydroxylation activity, the resulting tea leaves

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may exhibit high DIC and low TRIC contents, which enables an increased conversion

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of 4′-hydroxylated flavanones into 3′4′-hydroxylated, rather than 3′4′5′-hydroxylated

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products.17 Thus, wild tea plants with high CI trait may signify a valuable and

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potential resource for increasing DIC contents and CI values in cultivated tea plants

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and enhancing black tea quality. To understand these transnormal tea germplasms

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better and use them effectively for breeding and production, the present study: (1)

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clarifies the major catechin profiles and molecular characteristics of wild tea plants

86

with low TRIC and high CI trait, (2) develops a functional marker to identify the

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favorable F3′5′H allele within these wild tea plants and, (3) uses these rare tea

88

resources to innovate higher DIC and suitable CI cultivars and enhance black tea

89

quality.

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MATERIALS AND METHODS

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Plant Materials. A total of 14 tea accessions with diverse genetic backgrounds



92

were used in this study (Table S1). Two accessions are from C. sinensis var. sinensis,

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one is from C. sinensis var. assamica, one is from C. sinensis var. pubilimba, one is

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from C. taliensis, four are from C. tachangensis, and five are from C. gymnogyna.

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These tea resources were collected from their original regions and grown at the Tea

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Research Institute of the Chinese Academy of Agricultural Sciences (TRICAAS)

97

located at Hangzhou, Zhejiang, China. In April 2016, ‘‘one and a bud’’ of the first

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flush shoots of C. tachangensis ‘Xinyi 6a’ (XY6a) and C. sinensis var. sinensis

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‘Fuding Dabaicha’ (FDDB) were plucked and dried. XY6a is a wild tea plant

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originating from Xinyi City, Guizhou Province. FDDB is a widely planted and

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high-quality tea cultivar in China originating from Fuding County, Fujian Province. It

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is the reference cultivar for green tea testing in the country. To develop and validate a

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functional marker with which to identify wild tea plants with high CI trait, 14 tea

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accessions were sampled in August 2016. 3−4 tea plants of each tea accession were

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independently sampled as different biological duplications. All samples were

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immediately fixed by exposure to hot air at 120 °C for 5 min to deactivate polyphenol

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oxidase and then dried at 75 °C. The dried samples were stored in polyethylene bags

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and kept frozen (−20 °C) until analysis. Meanwhile, all fresh tea samples were

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transported in liquid nitrogen and stored at −80 °C for DNA and RNA extraction.

110

Sample Preparation and HPLC Conditions. Sample preparation was

111

performed and HPLC conditions were maintained as described in our previous study.4

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Chromatographic peaks were identified by UV spectroscopy using a diode array

113

detector, and retention times were compared with those of reference compounds. TC

114

content was calculated as the sum of EC, ECG, EGC, and EGCG.

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Gene Expression Analysis by Real-time PCR. Two accessions sampled in

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April 2016 and 14 tea accessions sampled in August 2016 were used to analyze gene


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expression levels. Total RNA was extracted using the RNeasy Plant Mini Kit (Tiangen

118

Bio, Beijing, China) and treated with RNase-free DNase according to the

119

manufacturer’s instructions. A FastQuant RT Kit (Tiangen Bio, Beijing, China) was

120

used to synthesize cDNA from about 200 ng of total RNA. Real-time quantitative

121

reverse-transcription PCR (qRT-PCR) was conducted to quantify the transcript levels

122

of seven representative genes involved in catechin biosynthesis, including chalcone

123

isomerase (CHI), flavanone 3-hydroxylase (F3H), F3'H, F3'5'H, dihydroflavonol

124

reductase (DFR), anthocyanidin synthase (ANS), and anthocyanidin reductase (ANR).

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The housekeeping gene Cs18S (AY563528.1) was employed as the reference gene,

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and the primer sequences for each gene are listed in Table S2. Quantitative RT-PCR

127

was performed using an ABi7500 real-time PCR machine (Applied Biosystems, USA)

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with SYBR Green reagent (Takara Bio., Dalian, China) following the product

129

manual’s instructions.

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Gene Cloning. RT-PCR analysis was used to identify F3′H and F3′5′H cDNA

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sequence variants in XY6a using FDDB as the control. To determine open reading

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frame (ORF) sequences corresponding to F3′H and F3′5′H, cDNAs of XY6a and

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FDDB were amplified using primer sets (a) F3′5′HcDNA-F and F3′5′HcDNA-R and

134

(b) F3′HcDNA-F and F3′HcDNA-R (Table S2), respectively. PCR was performed in a

135

final reaction volume of 50 µl containing 50 ng of cDNA, 1 U of KOD-Plus-Neo

136

DNA polymerase (TOYOBO, Japan), 0.6 µM of each the forward and reverse primers,

137

5 µl of 10 × PCR buffer, and 0.2 mM of each dNTP. The PCR conditions were as

138

follows: 94 °C for 2 min; 35 cycles of 94 °C denaturation for 15 s, 55 °C annealing

139

for 30 s, and 68 °C extension for 1 min; and a final extension of 68 °C for 5 min. The

140

amplified DNA fragments were purified in 1.0% agarose gels and directly sequenced

141

with an ABI 3730XI DNA analyzer. Manual editing was used to identify heterozygous



142

single nucleotide polymorphisms (SNPs) and validate sequence quality.

143

To determine upstream regulatory region (URR) sequences corresponding to the

144

F3′H and F3′5′H variants in XY6a, genomic PCR analysis was also performed.

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Genomic DNA (gDNA) was isolated from young tea shoots via CTAB extraction.

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Based on the sequence of contig 218535 (unpublished genome sequence of tea plant

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containing the full-length gDNA sequence of F3′H, Dr. Yu-Xiao Chang, Shenzhen

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Agricultural Genome Research Institute, Chinese Academy of Agricultural Sciences,

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personal communication) and F3′5′H (GenBank Accession No. KX792116), two

150

specific primer sets of (c) F3′HURR-F and F3′HURR-R and (d) F3′5′HURR-F and

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F3′5′HURR-R (Table S2) were designed and used to amplify the URR sequences of

152

F3′H and F3′5′H, respectively. PCR reactions were performed as described above,

153

except that the cDNA templates were substituted with gDNA.

154

Marker Development. A gene-tagged marker was developed based on the

155

sequence variation of different F3′5′H alleles between FDDB and XY6a. PCR using

156

primer sets of F3′5′HInDel-F and F3′5′HInDel-F (Table S2) was carried out using the

157

gDNA of all materials. The PCR conditions were as follows: 94 °C for 2 min; 35

158

cycles of 94 °C denaturation for 15 s, 62 °C annealing for 25 s, and 68 °C extension

159

for 5 s; and a final extension of 68 °C for 5 min. PCR products were separated on 3%

160

agarose gels stained with ethidium bromide and then photographed.

161

Germplasm Innovation Using XY6a. C. sinensis var. assamica ‘Xiuhong’

162

(hereinafter referred to as XH) is a cultivar with good black tea quality bred by

163

Guangdong Tea Research Institute, which is a widely planted in the south China

164

region. XH has high TC content (184.9 and 255.5 mg/g in spring and fall,

165

respectively), while CI value is only 0.44 and 0.51 in spring and fall, respectively.

166

Although XY6a is a wild tea plant with rare trait of high CI, it has some obvious


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shortcomings, such as slow growth and late germination. To innovate germplasm with

168

high TC content and suitable CI value for black tea breeding, crossing was conducted

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using XH as the maternal parent and XY6a as the paternal parent from mid-October to

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late November of 2016. Over 200 fruits were harvested in early October of 2017, and

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seeds were sown in perlite two weeks later. Twenty progenies from the “XH × XY6a”

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F1 population were selected in early May of 2018 (at the 3−4 leaf stage, the leaves

173

were young) using 20 progenies of the same stage from the “XH open pollinated”

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population as controls. Two young leaves of each individual were sampled for gDNA

175

extraction and catechin content determination as described above.

176

Statistical Analyses. Data are presented as the mean ± standard error (SD). The

177

statistical significance of differences between groups was determined with Student’s

178

t-test using SPSS software (SPSS, Chicago, IL, USA).

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RESULTS

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Catechin Contents in FDDB and XY6a. HPLC chromatograms of the catechins

181

in the young shoots of FDDB and XY6a are presented in Figure 2A, and the contents

182

of catechins estimated from the peak areas by HPLC analysis are presented in Figure

183

2B. In the young shoots of FDDB, EGCG was the most abundant catechin (93.7 ± 2.6

184

mg/g), followed by ECG (31.4 ± 1.4 mg/g), EGC (19.2 ± 0.8 mg/g), and EC (8.8 ± 0.4

185

mg/g). The TRIC content (112.9 ± 3.0 mg/g) of the FDDB sample was obviously

186

higher than its DIC content (40.2 ± 1.8 mg/g), and its CI value was 0.38 ± 0.01. By

187

contrast, XY6a contained much more DIC (102.1 ± 1.8 mg/g) and less TRIC (18.1 ±

188

0.2 mg/g) than FDDB (P < 0.001, Figure 2B), and the corresponding CI value of the

189

former (5.58 ± 0.12) was remarkably higher than that of the latter (P < 0.001, Figure

190

2C). In XY6a, ECG was the main catechin (95.5 ± 1.7 mg/g); EGCG (15.0 ± 0.2

191

mg/g), EC (6.6 ± 0.0 mg/g), and EGC (3.1 ± 0.1 mg/g) were significantly lower in



192

XY6a than those in FDDB (P < 0.05).

193

Discovery of a Novel F3′5′H Allele in XY6a. To survey the molecular

194

characteristic of the low TRIC and high CI trait of XY6a, relative amounts of the

195

transcripts of seven genes encoding enzymes involved in catechin biosynthesis were

196

investigated using semi-quantitative RT-PCR with FDDB as the control and Cs18S as

197

the reference gene. In the spring sample, the expression levels of CHI, F3H, DFR,

198

ANS, and ANR were similar in young shoots between FDDB and XY6a, but the

199

expression levels of F3′H significantly increased and F3′5′H obviously decreased (P

200

< 0.05) in XY6a compared with that in FDDB, as shown in Figure 3.

201

The full-length cDNA sequences of F3′H were isolated from FDDB and XY6a,

202

respectively. The cDNA sequences of both accessions consisted of 1557 nucleotides

203

encoding 518 amino acids. Eleven nucleotides in XY6a were different from FDDB,

204

but only substitution at nucleotide position 1450 had effect on amino acid sequence

205

(Figure S1). For ORFs of F3′5′H, FDDB and XY6a all were 1533 bp in length and

206

encoded 510 amino acids; four amino acids (at positions 12, 332, 337, and 510)

207

differed between FDDB and XY6a (Figure S2). To survey allelic variations in the

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URR of F3′H within XY6a, a 1385 bp fragment with nine SNPs, one 2 bp insertion,

209

and one 3 bp insertion mutation compared with FDDB (1380 bp, -1380–-1) was

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amplified from XY6a using the primer sets F3′HURR-F and F3′HURR-R (Figure S3).

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Four SNPs, one 2 bp insertion, and one 3 bp insertion mutation between XY6a and

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FDDB were also found in C. sinensis var. sinensis ‘Longjing 43’ (1385 bp, Figure S3).

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For URR of F3′5′H, XY6a had a 1334 bp fragment with 37 SNPs, three 1 bp

214

insertions, one 1 bp deletion, and one 14 bp deletion mutation compared with that of

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FDDB (1350 bp, -1350–-1) (Figure 4). In our previous study,17 the 722 bp (-1349–

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-628) URR of F3′5′H was sequenced and the 14 bp deletion mutation in XY6a was


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not found among 202 tea accessions originating from 14 tea-growing provinces in

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China. Thus, the F3′5′H allele in XY6a was a novel one.

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Functional Marker Development and Validation. Based on the 14 bp deletion

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allelic variant of F3′5′H between XY6a and FDDB, an insertion/deletion (InDel)

221

marker was developed, to genotype 14 tea accessions with diverse genetic

222

backgrounds (Figure 5A). The catechin contents of these tea plants were also

223

determined in August 2016. Among the 14 tea accessions, eight accessions with 136

224

bp bands had remarkably lower TRIC contents (P < 0.001, Figure 5B) than six

225

accessions with 150 bp bands, but DIC contents wasn’t substantially different

226

between the two groups (Figure 5C). Three accessions belonging to C. tachangensis

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and five accessions belonging to C. gymnogyna with shorter bands (136 bp) showed

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high CI values (>2.7); the six other tea accessions (including one from C.

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tachangensis) with longer bands (150 bp) showed low CI values ( 0.05, SNP4030, SNP403 and SNP4562 in the corresponding gDNA sequence)

275

and not functional SNPs associated with catechins content or CI value in the

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association population with 202 tea accession. Another non-synonymous SNP (at

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nucleotide position 34) within F3′5′H and the sole non-synonymous SNP (at

278

nucleotide position 1450) within F3′H did not lead to key amino acid mutation

279

(Figures S1 and S2). The corresponding encoding proteins of transnormal germplasms

280

and regular tea plants may feature similar F3'H and F3'5'H activities. In the URR

281

sequence of F3′5′H, the novel allele of the transnormal germplasm showed numerous

282

allelic variants compared with that of regular tea plants (Figure 4). Thus, a novel

283

functional marker was developed to identify the plants containing this novel F3′5′H

284

allele. The predicted motifs were used in homology search of the Plant Cis-acting

285

Regulatory

286

http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)

287

previously described cis-regulatory elements. There were several different

288

cis-regulatory elements between XY6a and FDDB, which may influence the

289

transcriptional regulation of F3′5′H. For example, one cis-regulatory element (CAAT,

290

CAAT-box) was disappeared in the novel allele among the 14 bp deletion. qRT-PCR

291

analysis revealed that allelic variants in these wild tea plants with high CI trait

Elements


(PlantCARE, for

similarity

to


292

affected the F3′5′H transcript levels (Figure 6). The F3′5′H mRNA levels of these

293

wild tea plants were significantly correlated with both TRIC contents and CI values.

294

This novel F3′5′H allele was also identified in wild tea plants from Pu’an County with

295

low F3′5′H expression levels and high CI trait (data not shown). Furthermore, the

296

allelic variant in the novel F3'5'H allele resulting in both increase of CI value and

297

DIC content was confirmed in introgression lines from the “XH × XY6a” F1

298

population compared with the progenies from the “XH open pollinated” population

299

(Figure 7). These correlations linked to the URR polymorphism observed with F3′5′H

300

expression, TRIC contents, and CI values occurring in genetic materials with diverse

301

backgrounds suggested that this URR polymorphism caused low TRIC contents and

302

high CI trait. The allelic variant in the novel F3'5'H allele could be associated with

303

low TRIC and high CI in wild tea plants.

304

Catechins, the most important chemical components of tea leaves, greatly affect

305

black and green tea quality. Theaflavin is one of the key chemical quality parameters

306

of black tea.19 The amount of individual theaflavins formed are largely influenced by

307

the amounts of the precursor catechins in green leaves, their redox potentials and/or

308

polyphenol oxidase preference of the individual catechins, and activity.11 Theaflavin

309

formation requires a reaction between a TRIC molecule and a DIC molecule. The CI

310

of green leaves, therefore, exert a major influence on the final amounts of the

311

theaflavins in black tea.11−14 Equal concentrations of TRIC and the DIC quinones are

312

necessary to maximize theaflavin amounts.12 However, TRICs are oxidized faster than

313

DICs during the fermentation phase of black tea processing.9 Based on in vitro

314

oxidation experiments,

315

theaflavin/thearubigin ratio in black tea.9 Cultivars of Central and Southern Africa,12

316

India,13 and Sri Lanka14 that produce high-quality black tea are characterized by high

Robertson showed that a


high CI increases the

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CI values, except the Kenya tea clones with more equitable distribution of the

318

individual catechins.11 Owuor and Obanda concluded that the distribution of

319

individual catechins in green leaves may be more critical to theaflavin formation than

320

TC content after comparing the catechin compositions and contents of tea cultivars

321

between Central and Southern Africa and Kenya.11

322

The average DIC and TRIC contents in the spring green leaves of 93 Chinese

323

clones were 37.3 ± 8.1 and 105 ± 12.7 mg/g, respectively.4 Based on availability, DIC

324

content should limit the amount of theaflavins formed during the processing of black

325

tea. Moreover, EGCG was the most abundant catechin (about 63.0% of TC) in these

326

93 Chinese tea clones.4 High levels of EGCG in green leaves are speculated to cause a

327

flooding effect of EGCG quinones during fermentation, leading to formation of other

328

products, such as thearubigins.11,19 Thus, breeders may find it necessary to develop

329

clones with more equitable distributions of individual catechins to enable the

330

formation of more diverse theaflavins and production of high-quality black tea.

331

Fortunately, we found an interesting phenomenon in tea plant: once the functional

332

allelic variant in F3′5′H induces less 5′-hydroxylation activity, the resulting leaves

333

may have higher DIC and lower TRIC contents.17 Wild tea plants with high CI trait

334

may be useful germplasm for breeding. In this study, we introduced the novel F3'5'H

335

allele with low transcript levels from a wild tea plant (XY6a, paternal parent) into a

336

cultivar with high black tea quality (XH, maternal parent) by distant cross. Although

337

over 200 “XH × XY6a” fruits were harvested in early October of 2017, phenological

338

period of progenies from the same population was different. In early May of 2018,

339

only 20-25 progenies were at the same stage (3−4 leaf stage) and could be selected

340

from the “XH × XY6a” F1 population for further research. Even though the number

341

of progenies from either open pollinated or hybridized seeds was small, progenies



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from the “XH × XY6a” F1 population showed significantly higher DIC contents and

343

CI values compared with offspring from the “XH open pollinated” population; and

344

TC contents were very similar between plants (Figure 7). Some progenies showed

345

higher CI values and more DIC contents than the highest one among 93 clones

346

previously identified, and these plants can be used as genetic materials for breeding

347

higher black tea quality. Thus, the novel F3'5'H allele from wild tea plants provides a

348

valuable genetic resource for high-DIC tea cultivar breeding and has potential use in

349

enhancing black tea quality.

350

ABBREVIATIONS USED:

351

CI, catechin index; DIC, dihydroxylated catechin; EC, (−)-epicatechin; ECG,

352

(−)-epicatechin-3-gallate,

EGC,

(−)-epigallocatechin;

EGCG,

353

(−)-epigallocatechin-3-gallate, gDNA, F3'H, flavonoid 3' hydroxylase; F3'5'H,

354

flavonoid 3',5' hydroxylase; FDDB, C. sinensis var. sinensis ‘Fuding Dabaicha’;

355

gDNA, genomic DNA; HPLC, high performance liquid chromatography; InDel,

356

insertion/deletion; ORF, open reading frame; SNP, single nucleotide polymorphism;

357

TC, total catechins; TRIC, trihydroxylated catechin; URR, upstream regulatory region;

358

XH, C. sinensis var. assamica ‘Xiuhong’; XY6a, C. tachangensis ‘Xinyi 6a’.

359

Funding

360

This work was supported by the National Natural Science Foundation of China

361

(No. 31670685), Earmarked Fund for China Agriculture Research System (CARS-19),

362

the Chinese Academy of Agricultural Sciences through the Agricultural Science and

363

Technology

364

CAAS-XTCX2016016-5), Science and Technology Major Project for New Crop

365

Varieties Breeding of Zhejiang Province (2016C02053), and the Fundamental

366

Research Funds for the Central Scientific Research Institute (1610212018008).

Innovation

Programs

(CAASASTIP-2017-TRICAAS,


Page 17 of 29


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Figure captions

433

Figure 1. Schematic presentation of the biosynthesis of main catechins in tea plant.

434

CHS, chalcone synthase; CHI, chalcone isomerase; F3′H, flavonoid 3′-hydroxylase;

435

F3′5′H,

436

dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin

437

reductase; ECGT, epicatechins: 1-O-galloyl-β-D-glucose O-galloyltransferase.

438

Figure 2. Comparison of catechin contents between Camellia sinensis var. sinensis

439

‘Fuding Dabaicha’ (FDDB) and C. tachangensis ‘Xinyi 6a’ (XY6a). (A) HPLC

440

chromatogram; (B) catechin contents; (C) catechin index. CI, catechin index; DIC,

441

dihydroxylated catechin; EC, (−)-epicatechin; ECG, (−)-epicatechin gallate; EGC,

442

(−)-epigallocatechin; EGCG, (−)-epigallocatechin gallate; TC, total catechins; TRIC,

443

trihydroxylated catechin. The data represent the mean ± SD of three independent

444

measurements and *, **, and *** indicate statistically significant differences at P

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