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R E S E A R C H A R T I C L E Open Access
Characterization of genome-wide genetic
variations between two varieties of tea
plant (Camellia sinensis) and development
of InDel markers for genetic research
Shengrui Liu
1†
, Yanlin An
1†
, Wei Tong
1
, Xiuju Qin
2
, Lidia Samarina
3
, Rui Guo
1
, Xiaobo Xia
1
and Chaoling Wei
1*
Abstract
Background: Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) are the major genetic
variations and are distributed extensively across the whole plant genome. However, few studies of these variations
have been conducted in the long-lived perennial tea plant.
Results: In this study, we investigated the genome-wide genetic variations between Camellia sinensis var. sinensis
‘Shuchazao’and Camellia sinensis var. assamica ‘Yunkang 10’, identified 7,511,731 SNPs and 255,218 InDels based on
their whole genome sequences, and we subsequently analyzed their distinct types and distribution patterns. A total
of 48 InDel markers that yielded polymorphic and unambiguous fragments were developed when screening six tea
cultivars. These markers were further deployed on 46 tea cultivars for transferability and genetic diversity analysis,
exhibiting information with an average 4.02 of the number of alleles (Na) and 0.457 of polymorphism information
content (PIC). The dendrogram showed that the phylogenetic relationships among these tea cultivars are highly
consistent with their genetic backgrounds or original places. Interestingly, we observed that the catechin/caffeine
contents between ‘Shuchazao’and ‘Yunkang 10’were significantly different, and a large number of SNPs/InDels
were identified within catechin/caffeine biosynthesis-related genes.
Conclusion: The identified genome-wide genetic variations and newly-developed InDel markers will provide a
valuable resource for tea plant genetic and genomic studies, especially the SNPs/InDels within catechin/caffeine
biosynthesis-related genes, which may serve as pivotal candidates for elucidating the molecular mechanism
governing catechin/caffeine biosynthesis.
Keywords: Molecular markers, Genetic diversity, SNP, InDel, Catechin/caffeine biosynthesis, Camellia sinensis
Background
Tea is the most popular non-alcoholic beverage and pos-
sesses numerous crucial properties including attractive
aroma, pleasant taste, and helpful and medicinal benefits
[1–3]. The tea plant (Camellia sinensis (L.) O. Kuntze) is
a perennial evergreen woody plant (2n = 2x = 30) belong-
ing to the section Thea of the genus Camellia in the
family Theaceae [4,5]. Evidence is accumulating that the
tea plant was originated from Yunnan Province in
southwestern China [4–7]. Currently, cultivated tea plant
varieties primarily belong to two groups, Camellia sinen-
sis var. sinensis (CSS) and Camellia sinensis var. assa-
mica (CSA), are extensively cultivated in tropical and
subtropical regions around the world [6,8]. Generally,
CSS is a slower-growing shrub with a relatively higher
cold-resistance capacity, while CSA is quick-growing
with larger leaves and high sensitivity to cold climate [9].
With the successive release of two draft genome se-
quences, CSA ‘Yunkang 10’[10] and CSS ‘Shuchazao’
[9], this plant is rapidly becoming another tractable ex-
perimental model for genetics and functional genomics
research on tea trees. It is known that self-
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
* Correspondence: weichl@ahau.edu.cn
†
Shengrui Liu and Yanlin An contributed equally to this work.
1
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural
University, 130 Changjiang West Road, Hefei, China
Full list of author information is available at the end of the article
Liu et al. BMC Genomics (2019) 20:935
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incompatibility and long-term allogamy contributed con-
siderably to the highly heterogeneous and abundant gen-
etic variation of tea plant [11,12]. Therefore, it is highly
important to characterize genome-wide genetic variation
between the two varieties.
Molecular markers, based on DNA polymorphisms,
are useful and powerful tools for genetic and breeding
research. Numerous molecular markers have been
successfully developed and applied in genetic and
genomic research in tea plant, such as restriction
fragment length polymorphisms (RFLPs), amplified
fragment length polymorphisms (AFLPs), random
amplification of polymorphic DNAs (RAPDs), cleaved
amplified polymorphic sequences (CAPS), inter-simple
sequence repeats (ISSRs), and simple sequence repeats
(SSRs) [12,13]. With the rapid development of the
high-throughput sequencing approaches, the third-
generation single nucleotide polymorphism (SNP) and
insertion/deletion (InDel) markers are gradually be-
coming the most widely used molecular markers,
demonstrating a promising future in plant genetic
and breeding research.
SNPs are the most abundant genetic variations in most
plant species, and the exploitation of SNP markers in
single-copy regions is considerably easier than use of the
other DNA markers [14–16]. InDel markers have prac-
tical value for those laboratories with limited resources,
which also showed reliable transferability between dis-
tinct populations [14,17,18]. Both SNPs and InDels
have been extensively applied for breeding programs and
genetic studies including pedigree analysis, origin and
evolutionary analysis, population structure and diversity
analysis, construction of linkage maps, QTL mapping,
and marker-assisted selection [14,19–22]. Several stud-
ies have also reported the development and application
of SNP/InDel markers in tea plant genetic studies. For
instance, 16 expressed sequence tag (EST)-SNP based
CAPS markers were developed and applied for tea plant
cultivar identification [23]. A set of SNPs from EST da-
tabases was identified and verified [24]. Fang et al.
(2014) validated 60 EST-SNPs, and constructed genetic
relationships among tea cultivars and their specific DNA
fingerprinting [25]. Based on specific locus amplified
fragment sequencing (SLAF-seq), a total of 6042 SNP
markers were validated and a final genetic map contain-
ing 6448 markers was constructed [26]. Through restric-
tion site-associated DNA sequencing (RAD-Seq)
approach, Yang et al. (2016) identified a vast number of
SNPs from 18 cultivated and wild tea accessions, and
found that 13 genes containing non-synonymous SNPs
exhibited strong selective signals suggesting artificial se-
lective footprints during domestication of these tea ac-
cessions [27]. By harnessing the two reference genomes,
it is now suitable for identifying genome-wide SNPs/
InDels between them to guide rapid and efficient devel-
opment of markers for high-resolution genetic analysis.
The whole genome sequences of tea trees can provide
an elegant platform for identifying abundant genetic
variation and developing many genetic markers. The
completion of the two reference genome sequences is a
notable advance for genetic and genomic studies and a
basis for this study. The tea plant whole genome CSA
‘Yunkang 10’was first reported based on the Illumina
next-generation sequencing platform, producing a ~ 3.02
Gb genome assembly containing 37,618 scaffolds with
N50 length of 449 Kb [10]. Subsequently, the genome
assembly of CSS ‘Shuchazao’was released by combined
Illumina and PacBio sequencing platforms, yielding a ~
3.14 Gb genome assembly that consists of 36,676 scaf-
folds with N50 length of 1.39 Mb [9]. In this study, sev-
eral principal objectives were completed. Genome-wide
genetic variation and distribution patterns were investi-
gated. A number of polymorphic and stable InDel
markers were developed, providing informative molecu-
lar markers for genetic and genomic studies. The cat-
echin and caffeine contents of the two tea cultivars were
detected, and SNPs/InDels within catechin/caffeine
biosynthesis-related genes were characterized. The iden-
tified genome-wide genetic variations and newly devel-
oped InDel markers provide valuable resources for tea
plant genetic and genomic studies, and the identification
of SNPs/InDels within catechin/caffeine biosynthesis-
related genes can serve as important candidate loci for
functional analysis.
Results
Mapping of clean reads to the reference genome
‘Shuchazao’
CSS ‘Shuchazao’has been observed to have significant
differences in bud, leaf and budding flower size com-
pared with CSA ‘Yunkang 10’(Fig. 1). The completion
of the two reference genome sequences (‘Shuchazao’and
‘Yunkang 10’) is a notable advance for comparative gen-
omic studies on tea plants in Thea section. Therefore,
genome-wide genetic variations were identified between
the two genome assemblies. After filtering the raw data,
a total of 324,154,064 clean reads from the CSA whole
genome sequencing data were generated; these reads
had a coverage depth of 10.4X the ‘Yunkang 10’genome
with a 100 bp length and 43% GC content. Through
alignment, a total of 317,878,025 clean reads were
mapped to the reference genome, accounting for 98.1%
of total reads. The mapped clean reads contained two
types of sequencing reads: pair-end and single-end reads.
The former was predominantly type (317,063,284,
99.7%), while single-end reads accounted for only 0.3%
(814,741 clean reads).
Liu et al. BMC Genomics (2019) 20:935 Page 2 of 16
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Fig. 1 Comparison of bud and leaf size between ‘Shuchazao’and ‘Yunkang 10’. Young buds and leaves were collected on April 2019, while
mature leaves were collected from branches of last-year autumn
Fig. 2 Classification and distribution of identified SNPs/InDels in ‘Yunkang 10’/‘Shuchazao’comparison. aFrequency of different substitution
types in the identified SNPs; the x-axis and y-axis represent the types and number of SNPs, respectively. bDistribution of the length of InDels
identified between the two tea cultivars; the x-axis shows the number of nucleotides of InDels, and the y-axis represents the number of InDels at
each length
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Identification and distribution of SNP and InDel loci
After a series of filtering, a total of 7,071,433 SNP loci
were generated, with an average SNP density in the tea
genome being estimated to be 2341 SNPs/Mb. Based on
nucleotide substitutions, the detected SNPs were classi-
fied as transitions (Ts: G/A and C/T) and transversions
(Tv: A/C, A/T, C/G, and G/T), which accounted for
77.46% (5,818,773) and 22.54% (1,692,958), respectively
(Fig. 2a), with a Ts/Tv ratio of 3.44. In transitions, the
number of A/G is equivalent to the C/T type, which in-
cluded 2,905,203 and 2,913,570, respectively. For trans-
versions, the number of four types (A/C, A/T, C/G and
G/T) are almost evenly distributed with an insignificant
difference among them, which accounted for 27.23%
(460,988), 24.72% (418,536), 20.84% (352,802) and
27.21% (460,632), respectively (Fig. 2a).
A total of 255,218 InDels were identified, with an
average density of 84.5 InDels/Mb. The length distri-
bution of InDels was analyzed by dividing the lengths
into different groups and calculating the ratios for the
corresponding length groups (Fig. 2b). It is obvious
that mononucleotide InDels is the most abundant
type, accounting for 44.27% (112,976) of the total
number. The length of InDels ranging from 1 to 20
bp was predominant, accounting for more than 95.5%
(243,749) of the total InDels. A clear tendency was
that the number of InDels gradually decreased with
increasing InDel length.
Location and functional annotation of SNPs and InDels
The annotation of the ‘Shuchazao’reference genome
was used to uncover the distribution of SNPs and InDels
within distinct genomic regions. According to the gene
structure of the reference genome, the overwhelming
number of SNPs (94%) was identified in intergenic re-
gions, while only 6% (440,298) of SNPs were located in
genic regions (Fig. 3a). Among the SNPs located in genic
regions, 89,511 SNPs were detected in the CDs region,
which contained 38,670 synonymous and 50,841 non-
synonymous SNPs, respectively. Similarly, a small pro-
portion of InDels were located in the genic regions,
which accounted for only 12% (31,130) of the total num-
ber (Fig. 3b). Remarkably, 3406 InDels were located in
the CDs region, which can be regarded as the preference
for developing InDel markers.
To better understand the potential functions of these
genetic variations within genes, GO term enrichment
analysis of genes containing SNPs/InDels within CDs re-
gion was performed. These genes were classified into
biological process, cellular component and molecular
function categories (Additional file 2: Figure S2). Regard-
ing the genes containing SNPs, the GO terms of cellular
process, metabolic process and single-organism process
were dominantly abundant in the biological process
(Additional file 2: Figure S2A). In the cellular compo-
nent category, the top three enriched GO terms were
membrane, cell and cell part. Based on the molecular
function category, catalytic activity and binding are pre-
dominantly enriched, while others accounted for a small
proportion (Additional file 2: Figure S2A). Interestingly,
a nearly consensus result was obtained for GO terms
analysis of genes containing InDels, nothing but the
number of genes is less compared with the number of
genes containing SNPs (Additional file 2: Figure S2B).
Fig. 3 Annotation of SNPs and InDels identified between ‘Shuchazao’and ‘Yunkang 10’.aAnnotation of SNPs. bAnnotation of InDels. SNPs and
InDels were classified as intergenic and genic on the ‘Shuchazao’reference genome, and locations within the gene models were annotated
Liu et al. BMC Genomics (2019) 20:935 Page 4 of 16
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Validation and polymorphism of newly-developed InDel
markers
Initially, all InDels were used for designing primer pairs
using Primer3.0. To validate the InDels and develop
polymorphic InDel markers, we selected 100 InDel
markers that were distributed on different scaffolds. To
facilitate the screening and development of more prac-
tical markers, the lengths of all selected InDels ranged
from 5 to 20 bp in length. To determine the reliability
and polymorphisms of the primers, six tea cultivars were
selected for testing their amplified fragments using Frag-
ment Analyzer™96. Of the total primer sets tested, 48
primer pairs were successfully amplified with unambigu-
ous bands and length polymorphisms among the six tea
cultivars, 19 primer sets generated non-polymorphic or
empty amplifications, and 33 primer pairs yielded non-
specific amplification or ambiguous bands.
Consequently, the 48 primer sets were regarded as ele-
gant InDel markers and used for further analysis.
To test cross-cultivars/subspecies transferability, the
48 InDel markers were conducted on a panel of 46 tea
cultivars belonging to section Thea of genus Camellia.
The detailed information of the 46 tea cultivars is listed
in Additional file 4: Table S1. The results of 18 InDel
markers testing on various tea cultivars are shown in
Fig. 4, demonstrating that unambiguous and poly-
morphic bands were obtained based on these markers.
The amplified results of the remaining 30 markers were
also demonstrated (Additional file 3: Figure S3). For the
newly developed markers, 20, 25 and 3 InDel markers
generated high polymorphism, moderate polymorphism,
and low polymorphism in the 46 tea cultivars, respect-
ively. The PIC value of each InDel marker was presented
in Table 1. The amplified allele sizes across them were
Fig. 4 Exhibition of transferability and polymorphism detected by 18 out of 48 InDel markers among 46 tea cultivars
Liu et al. BMC Genomics (2019) 20:935 Page 5 of 16
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Table 1 Characteristics of 48 newly developed InDel markers
Marker ID Scaffold location Fragment size (bp) Na MAF Ho He PIC
CsInDel01 Scaffold 5: 236696 139–156 3 0.787 0.383 0.361 0.327
CsInDel02 Scaffold 5: 1208833 186–205 4 0.489 1.000 0.633 0.555
CsInDel03 Scaffold 12: 195263 332–354 3 0.500 0.489 0.577 0.478
CsInDel04 Scaffold 30: 3820588 214–242 5 0.532 0.532 0.636 0.576
CsInDel05 Scaffold 39: 128636 236–264 4 0.479 0.979 0.556 0.448
CsInDel06 Scaffold 41: 2074123 280–295 3 0.808 0.180 0.319 0.273
CsInDel07 Scaffold 46: 249178 176–189 3 0.734 0.362 0.405 0.336
CsInDel08 Scaffold 51: 314982 206–215 6 0.394 0.638 0.691 0.627
CsInDel09 Scaffold 51: 760768 201–248 7 0.532 0.660 0.679 0.645
CsInDel10 Scaffold 52: 469482 288–306 3 0.745 0.255 0.394 0.329
CsInDel11 Scaffold 60: 843530 292–332 6 0.383 0.213 0.748 0.701
CsInDel12 Scaffold 60: 843632 240–275 5 0.426 0.660 0.704 0.645
CsInDel13 Scaffold 64: 151635 270–289 3 0.404 0.617 0.643 0.559
CsInDel14 Scaffold 66: 500052 203–232 4 0.436 0.064 0.621 0.535
CsInDel15 Scaffold 77: 505984 185–207 2 0.500 1.000 0.505 0.375
CsInDel16 Scaffold 89: 1202911 231–248 2 0.819 0.149 0.300 0.252
CsInDel17 Scaffold 98: 664107 306–354 6 0.395 0.256 0.731 0.677
CsInDel18 Scaffold 114: 416691 283–326 6 0.489 0.809 0.703 0.661
CsInDel19 Scaffold 129: 540746 180–214 6 0.422 1.000 0.652 0.579
CsInDel20 Scaffold 154: 767901 285–297 5 0.266 0.979 0.763 0.709
CsInDel21 Scaffold 225: 80286 191–204 2 0.649 0.362 0.461 0.352
CsInDel22 Scaffold 1000: 52494 216–288 3 0.532 0.404 0.612 0.537
CsInDel23 Scaffold 1001: 123324 236–326 6 0.628 0.489 0.568 0.526
CsInDel24 Scaffold 1001: 149678 190–199 2 0.798 0.021 0.326 0.271
CsInDel25 Scaffold 1001: 155681 195–218 2 0.649 0.319 0.461 0.352
CsInDel26 Scaffold 1001: 1251845 341–363 3 0.583 0.833 0.511 0.399
CsInDel27 Scaffold 1001: 1261469 273–290 3 0.777 0.064 0.359 0.306
CsInDel28 Scaffold 1001: 1400899 213–253 6 0.660 0.383 0.537 0.501
CsInDel29 Scaffold 1001: 1491192 182–226 4 0.457 1.000 0.586 0.489
CsInDel30 Scaffold 1001: 1691928 238–258 4 0.745 0.362 0.411 0.363
CsInDel31 Scaffold 1001: 1982826 284–316 4 0.489 0.915 0.619 0.539
CsInDel32 Scaffold 1452: 285463 272–299 3 0.596 0.426 0.511 0.406
CsInDel33 Scaffold 1539: 196438 271–280 2 0.798 0.404 0.326 0.271
CsInDel34 Scaffold 1541: 138532 265–286 3 0.564 0.851 0.523 0.413
CsInDel35 Scaffold 1543: 253456 172–207 2 0.915 0.128 0.157 0.144
CsInDel36 Scaffold 1551: 196819 157–237 3 0.606 0.745 0.499 0.391
CsInDel37 Scaffold 1553: 529121 211–237 4 0.564 0.511 0.547 0.451
CsInDel38 Scaffold 1555: 5209 109–340 14 0.298 0.489 0.869 0.849
CsInDel39 Scaffold 1579: 1466247 261–272 2 0.606 0.787 0.483 0.363
CsInDel40 Scaffold 1592: 672899 276–329 7 0.596 0.979 0.666 0.489
CsInDel41 Scaffold 1593: 1022219 172–187 2 0.957 0.085 0.082 0.078
CsInDel42 Scaffold 1594: 195199 184–206 3 0.691 0.426 0.454 0.380
CsInDel43 Scaffold 1611: 1270988 226–254 5 0.426 0.319 0.684 0.619
CsInDel44 Scaffold 2220: 166816 292–328 3 0.543 0.575 0.521 0.402
Liu et al. BMC Genomics (2019) 20:935 Page 6 of 16
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within the ranges detected in the donor tea cultivar, im-
plying that the amplified fragments were derived from
the same loci and that the primer binding sites of the al-
leles were highly conserved among distinct tea cultivars/
subspecies. Several crucial parameters for evaluating
polymorphism of markers were subsequently conducted,
such as the number of alleles (Na) per locus ranged
from 2 (CsInDel15, CsInDel16, CsInDel21, CsInDel24,
CsInDel25, CsInDel33, CsInDel35, CsInDel39, CsIn-
Del41, CsInDel46, and CsInDel47) to 14 (CsInDel38)
with an average of 4.02 alleles, the major allele frequency
(MAF) ranged from the lowest 0.266 (CsInDel20) to the
highest at 0.957 (CsInDel41 and CsInDel47) with an
average of 0.585, the observed heterozygosity (Ho)
ranged from 0.021 (CsInDel24) to 1.000 (CsInDel15,
CsInDel19, and CsInDel29) with an average of 0.524 and
the expected heterozygosity (He) ranged from 0.082
(CsInDel41 and CsInDel47) to 0.869 with an average of
0.528, the polymorphic information content (PIC) values
were from the lowest value 0.078 (CsInDel41 and CsIn-
Del47) to the highest 0.849 (CsInDel38) with an average
of 0.457 (Table 1). Notably, the value of He has a similar
variation trend as the PIC value, while it has a distinct
variation trend with Ho values. The primer sequences
and genomic locations of these newly developed markers
are listed in Additional file 5: Table S2. These results
showed that these newly developed InDel markers are
informative and possess good transferability among vari-
ous tea subspecies/cultivars.
Population structure and genetic relationship analysis
Population structure analysis was performed on the 46
tea cultivars using Structure 2.3.3 software based on 48
newly-developed InDel markers. The Q-plot output pre-
sented our grouping results, indicating that the two
groups were the optimal classification at K = 2 (Fig. 5a).
Apparently, tea cultivars from southern and southwest-
ern China (Guangxi, Guangdong, Yunnan and Sichuan
Provinces) belonging to Camellia sinensis var. assamica
were clustered tightly together. In comparison, the tea
cultivars possessing smaller leaf sizes and shorter heights
that were cultivated in several other provinces were clas-
sified into another group (Fig. 5b).
To further confirm the applicability of the developed
InDel markers for classification, we constructed a phylo-
genetic tree based on their genetic distances (Fig. 5c).
Two major branches were generated (designated as α
and βgroups), which contained 17 and 29 tea cultivars,
respectively. Group αcan be further divided into two
subgroups, which were designated as α-1 and α-2 sub-
groups and consisted of 13 and 4 members, respectively.
The dendrogram reflects that the phylogenetic relation-
ships among them are highly consistent with their back-
grounds or places of origin, as well as displaying
consistency with the results from population structure
analysis although a small discrepancy was observed (Fig.
5c).
Identification of genetic variation in catechin/caffeine
biosynthesis-related genes
Tea cultivars belonging to Camellia sinensis var. assa-
mica possess significant differences in phenotypes (plant
height, leaf size and flower) and major characteristic sec-
ondary metabolites (such as catechin and caffeine, which
contributed tremendously to tea quality) compared with
Camellia sinensis var. sinensis. Therefore, we detected
the contents of catechin (flavan-3-ols) and caffeine in
both ‘Shuchazao’and ‘Yunkang 10’based on HPLC ana-
lysis. The total content of catechin in both buds and the
second leaf from ‘Yunkang 10’was higher than from
‘Shuchazao’(Fig. 6a). To understand the potential mo-
lecular mechanism of difference, we performed the cat-
echin biosynthesis pathway based on several previous
studies (Fig. 6b). After search, we identified a number of
SNPs and InDels in some crucial genes that are involved
in the catechin biosynthesis pathway, including phenyl-
alanine ammonia-lyase (PAL), cinnamic acid 4-
hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chal-
cone synthase (CHS), chalcone isomerase (CHI), flava-
none 3-hydroxylase (F3H), flavonoid 3′-hydroxylase
(F3’H), flavonoid 3′,5′-hydroxylase (F3’5’H), dihydrofla-
vonol 4-reductase (DFR), leucoanthocyanidin reductase
(LAR), anthocyanidin synthase (ANS), anthocyanidin re-
ductase (ANR), and 1-O-galloyl-β-D-glucose O-
galloyltransferase (ECGT, which belongs to subclade 1A
of serine carboxypeptidase-like (SCPL) acyltransferases)
(Table 2).
Table 1 Characteristics of 48 newly developed InDel markers (Continued)
Marker ID Scaffold location Fragment size (bp) Na MAF Ho He PIC
CsInDel45 Scaffold 15,285: 211487 281–321 5 0.333 0.952 0.752 0.699
CsInDel46 Scaffold 15,433: 302840 190–253 2 0.638 0.468 0.467 0.355
CsInDel47 Scaffold 15,579: 267174 176–186 2 0.957 0.043 0.082 0.078
CsInDel48 Scaffold 15,650: 137667 228–266 6 0.489 0.596 0.671 0.614
Average ––4.02 0.585 0.524 0.528 0.457
Na number of alleles, MAF major allele frequency, Ho observed heterozygosity, He expected heterozygosity, PIC polymorphism information content
Liu et al. BMC Genomics (2019) 20:935 Page 7 of 16
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Detection of caffeine content in the two tea var-
ieties demonstrated that the caffeine in both bud
and the second leaf from ‘Yunkang 10’is lower than
that from ‘Shuchazao’(Fig. 7a). In Fig. 7b, the well-
studied caffeine biosynthesis pathway was also per-
formed based on previous studies [10,28–31]. Simi-
larly, a number of genetic variations within some
critical regulatory genes were also detected, such as
in IMP dehydrogenase (IMPDH), guanosine synthase
(GMPS), 5′-nucleotidase (5′-Nase) and tea caffeine
synthase (TCS) genes (Fig. 7candTable2). Collect-
ively, these results indicate that certain genetic varia-
tions within these genes may explain the significant
difference in catechin/caffeine synthesis between
‘Shuchazao’and ‘Yunkang 10’.
Discussion
Identification of genetic variations in tea plant whole
genome
The recent release of the ‘Shuchazao’and ‘Yunkang 10’
genome sequences will strongly facilitate the efficiency
of comparative genomics and functional research in tea
plants. This advance may enable researchers to study
numerous agronomic traits associated with the perennial
tea trees with a complete set of tools, including identifi-
cation and development of SNP/InDel markers. Never-
theless, genome-wide identification and development of
SNP/InDel markers are still in infancy, especially genetic
variations related to important agronomical traits. By
mapping the clean reads of ‘Yunkang 10’to the reference
genome assembly ‘Shuchazao’, we comprehensively
Fig. 5 Population structure and phylogenetic relationship analysis based on 48 InDel markers. aEstimation of the optimal group number through
ΔK, the number of Kwas set from 2 to 9. bQ-plot of the population structure when K= 2. Each tea cultivar is represented by a horizontal bar. c
The dendrogram was constructed based on genotypes using neighbor-joining algorithm with 1000 bootstrap replicates
Liu et al. BMC Genomics (2019) 20:935 Page 8 of 16
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surveyed DNA polymorphisms at the genome-wide scale
and revealed the high level of genetic diversity between
them. The vast number of SNPs and InDels identified in
this study will provide valuable resources for tea plant
genetics and breeding studies.
After filtering, a total of 7,071,433 SNPs and 255,218
InDels were identified, and their densities distributed in
the tea plant genome were estimated to be 2341 SNPs/
Mb and 84.5 InDels/Mb, respectively. The densities of
SNP and InDel in the tea plant were significant differ-
ences compared with in other plant species, such as in
Arabidopsis [32], Brassica rapa [17], quinoa [19], and
soybean [33]. These significant differences in SNP/InDel
density among different plant species may be due to the
distinct filtering protocols and/or the different genomic
composition. It is known that tea cultivars belonging to
distinct varieties are highly heterogeneous with broad
genetic variation due to their self-incompatibility and
long-term allogamy [11]. In terms of SNPs, our results
showed that A/G and C/T transitions are the most
common pattern of nucleotide substitution, which is
consistent with the results obtained in other plant spe-
cies, such as foxtail millet [34], citrus [35], and soybean
[33]. For InDels, the most prevalent types in the tea
plant genome are short InDels. The number of 1–5bp
InDels is the predominant types, accounting for 76% of
all InDels, and similar results were displayed in several
other plant species [14,33–35].
Knowing the genomic positions of genetic variations
in genetic markers or functional genes is highly import-
ant. It was shown that only minimal SNPs and InDels
were distributed in the CDs region, which can be ex-
plained by the fact that the CDs region only accounted
for a small proportion of the whole genome sequences
and had relatively higher conservation compared with
other regions. Among the 89,511 SNPs located in the
CDs region, a total of 50,841 SNPs were non-
synonymous variations. Non-synonymous variations can
usually have several functional impacts due to an altered
amino acid sequence, such as hampering the interaction
Fig. 6 Detection of catechin content and genetic variations within catechin biosynthesis-related genes. aDetection of catechin content of the
bud and leaf of both ‘Shuchaza’and ‘Yunkang 10’. T-test was employed for significant analysis and two asterisks represent p< 0.01. Each sample
was tested with three independent biological replicates and two technical replicates. bThe flavonoid biosynthesis pathway. PAL, phenylalanine
ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate-CoAligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone
3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′,5′H, flavonoid 3′,5′-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; ANS,
anthocyanidin synthase; ANR, anthocyanidin reductase; LAR, leucocyanidin reductase; SCLP1A, subclade 1A of serine
carboxypeptidase-like acyltransferases
Liu et al. BMC Genomics (2019) 20:935 Page 9 of 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Table 2 Statistics on SNPs and InDels within catechin biosynthesis-related genes
Gene
name
Gene ID SNP InDel Gene
name
Gene ID SNP InDel
DNA CDs DNA CDs DNA CDs DNA CDs
PAL TEA014056.1 2 2 0 0 F3H TEA004906.1 0 0 1 0
TEA034008.1 6 6 0 0 TEA010326.1 1 1 0 0
TEA003137.1 16 16 0 0 TEA032907.1 3 3 1 1
TEA023243.1 3 3 0 0 TEA028622.1 75 1 3 0
TEA024587.1 3 3 0 0 TEA009737.1 4 4 0 0
TEA003374.1 2 2 1 0 TEA000753.1 1 1 0 0
C4H TEA034001.1 16 8 1 1 TEA023937.1 1 1 0 0
TEA016772.1 5 1 1 0 TEA016601.1 4 2 1 0
TEA034002.1 6 6 0 0 TEA023790.1 10 3 1 0
4CL TEA018887.1 1 1 0 0 TEA000474.1 8 1 0 0
TEA034012.1 9 4 1 1 TEA026443.1 1 1 0 0
TEA019275.1 14 10 0 0 TEA004898.1 1 1 0 0
TEA027829.1 12 3 1 0 TEA006643.1 15 15 0 0
TEA025906.1 2 1 0 0 TEA014951.1 29 8 2 0
TEA009431.1 42 10 4 2 DFR TEA032730.1 2 0 1 0
TEA018045.1 22 3 4 0 TEA023829.1 13 1 0 0
TEA006577.1 6 1 0 0 TEA021807.1 2 0 0 0
TEA031627.1 11 8 0 0 TEA021815.1 2 2 0 0
TEA022274.1 2 1 0 0 ANS TEA010322.1 1 1 0 0
TEA010681.1 8 4 0 0 TEA015762.1 1 1 0 0
TEA002100.1 13 0 1 0 TEA015769.1 1 0 0 0
CHS TEA018665.1 1 1 0 0 ANR TEA030023.1 1 1 0 0
TEA034046.1 34 10 0 0 TEA022960.1 6 2 0 0
TEA034011.1 6 4 0 0 TEA007646.1 1 0 1 0
TEA034045.1 1 1 0 0 TEA003247.1 1 1 0 0
TEA023331.1 2 2 0 0 LAR TEA021535.1 1 1 0 0
TEA023340.1 3 3 2 0 TEA027582.1 0 0 2 0
TEA034013.1 2 2 0 0 TEA009266.1 3 3 1 0
TEA034043.1 31 7 0 0 SCPLA1 TEA034031.1 4 2 0 0
TEA034019.1 3 3 0 0 TEA034032.1 11 5 0 0
TEA034014.1 1 1 0 0 TEA010715.1 6 5 0 0
TEA011908.1 6 1 0 0 TEA034056.1 33 1 0 0
TEA019029.1 4 4 0 0 TEA009664.1 4 0 0 0
CHI TEA034003.1 10 2 1 0 TEA016469.1 2 0 0 0
TEA033023.1 127 4 10 0 TEA016463.1 9 1 0 0
TEA033031.1 2 1 0 0 TEA034055.1 59 1 0 0
F3’H TEA016718.1 2 2 0 0 TEA034034.1 4 0 0 0
TEA010133.1 5 2 0 0 TEA034036.1 1 1 0 0
TEA006847.1 14 10 1 1 TEA023444.1 3 0 0 0
F3’5’H TEA013315.1 12 12 0 0 TEA034039.1 31 2 0 0
TEA034021.1 6 1 0 0 TEA023451.1 4 1 0 0
TEA034051.1 32 4 4 0 TEA000223.1 4 0 0 0
Liu et al. BMC Genomics (2019) 20:935 Page 10 of 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
between proteins and affecting gene expression due to
the functional consequences of distinct motif binding at
variation sites [33,36]. It is worth noting that a total of
3406 identified InDels were located in the CDs region.
InDels tend to have more impact on protein structure
and function than single base changes, especially those
in the CDs region [33]. Nevertheless, genetic variations
at UTRs may also play important roles, such as modifi-
cation of regulatory elements affecting the interaction of
the UTRs with proteins and miRNAs [37]. Overall, these
SNPs and InDels can serve as important candidates for
functional research, especially those InDels in the CDs,
which can be considered as a valuable resource for de-
veloping phylogenetic and/or functional markers.
Development and application of InDel markers
Molecular markers are becoming indispensable tools for
evolutionary analysis, germplasm identification and con-
servation, and marker-assisted selection (MAS). SSR is
an extensively used marker type among genetic markers,
and a large number of highly polymorphic SSR markers
have been developed and applied in various genetic stud-
ies in tea plants [8,13]. These SSR markers, however,
could easily result in non-specific amplifications and
cause confusion in genotyping scoring [19], especially
for plant species with large genome and high repetitive
sequences. In fact, InDel markers are also PCR-based
markers and are similarly affected by genomic complex-
ity. However, they gave relatively less stutter bands due
to the variations are more conservative compared with
SSR markers [18,19]. Through a series of screenings, we
developed a final of 48 polymorphic and stable InDel
markers with 5–20 bp in length based on the genomic
assembled sequences (Table 1). The length of fragments
of the alleles amplified across tea cultivars was consist-
ent with the expected sizes of the products, implying
that the primer binding sites of the alleles were highly
conserved. The large proportion of InDel markers dis-
played a moderate PIC value (0.25 < PIC< 0.5), and the
average of PIC was 0.4. It is obvious that the PIC values
of most InDel markers were lower than the PIC of the
majority SSR markers [2,8,38,39], supporting that the
InDel markers are stable and bi-allelic throughout the
genome. Therefore, these newly developed InDel
markers are suitable for germplasm identification and
conservation, genetic diversity analysis, population struc-
ture and phylogenetic relationship analysis. In addition,
InDels can affect gene functions by causing the gain or
loss of a frameshift and/or a stop codon, it is therefore
suitable for developing functional markers that might be
particularly valuable for MAS [19,40].
Population structure analysis and phylogenetic trees
can reflect the genetic diversity, pedigree relationships,
and geographic distances among plant species and/or
varieties [2,16,22]. They can also be used to evaluate
the reliability of molecular markers. To test the reliabil-
ity and practicability of the newly-developed InDel
markers, population structure and phylogenetic
Fig. 7 Detection of caffeine content and genetic variations within caffeine biosynthesis-related genes. a. Detection of catechin content of the
bud and leaf of both ‘Shuchaza’and ‘Yunkang 10’. T-test was employed for significant analysis and one asterisk represents p< 0.05. Each sample
was tested with three independent biological replicates and two technical replicates. b. The caffeine biosynthesis pathway. IMP, Inosine
monophosphate; XMP, Xanthosine monophosphate; GMP, Guanosine monophosphate; IMPDH, IMP dehydrogenase; GMPS, Guanosine synthase;
5′-Nase, 5′-nucleotidase; 7-NMT, 7-methylxanthosine synthase; SAM, S-Adenosyl-L-methionine; N-MeNase, N-methylnucleotidase; TCS, tea caffeine
synthase. c. SNPs and InDels calling in caffeine biosynthesis-related genes
Liu et al. BMC Genomics (2019) 20:935 Page 11 of 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
relationship analysis were employed, and a consistent re-
sult was established (Fig. 5). Apparently, the tea cultivars
from southern and southwestern China were clustered
together, which originated from C. sinensis var. assamica
populations. In comparison, most tea cultivars from cen-
tral China had relatively close relationships with each
other, which have distinct phenotypes, including small
leaf size and short height of tea trees. These results indi-
cate that the population structure analysis and phylogen-
etic tree reflect the relationships of the 46 tea cultivars,
demonstrating the high reliability of these InDel markers
for genetic analysis.
Genetic variations within catechin/caffeine biosynthesis-
related genes
Catechin and caffeine are among the most important
components in tea plant leaves, which enormously affect
the quality of tea products and pharmacy [9,41]. It is
well-known that the contents of catechin and caffeine
are influenced by genotypic factors, and significant dif-
ferences can be observed among distinct tea varieties/
cultivars [31,42,43].
Based on HPLC detection, we found that the total cat-
echin content from ‘Yunkang 10’was significantly higher
than that from ‘Shuchazao’in both bud and the second
leaf (Fig. 6a). Evidence has shown that the total catechin
content of tea varieties tended to decline from the
southern to the northern regions [42,43], and our result
is consistent with this tendency. Because catechins are
important factors for the oxidation degree and dark tea
was produced with severe fermentation during process-
ing [41,43], our results supported the fact that most tea
cultivars belonging to Camellia sinensis var. assamica
are more suitable for producing dark tea. To understand
the potential molecular mechanisms, genetic variations
within key genes associated with the catechin biosyn-
thesis pathway were investigated between the two var-
ieties. Unsurprisingly, a large number of SNPs and
InDels were identified and some of them were located in
the CDs (Table 2). Combining the results of detection of
catechin constitutes, it is likely to successfully select cer-
tain candidate genetic variations associated with the
genotypic factors. For instance, a study reported that a
number of candidate allelic variants relating to catechin
traits at the F3’5’H locus were identified, and the genetic
effects of SNP840/848 were the most robust among
them [41].
The result of HPLC detection showed that the caffeine
content from ‘Yunkang 10’was significantly lower than
from ‘Shuchazao’(Fig. 7a). Remarkably, a number of
SNPs and InDels were found within some genes associ-
ated with the caffeine biosynthesis pathway (Fig. 7c).
Previously, a study reported that a 252 bp InDel muta-
tion in the 5′-UTR of TCS1 plays a crucial role in
caffeine biosynthesis [44]. Thus, our results can provide
valuable candidates for identifying variations within
genes related to caffeine biosynthesis. Overall, these
valuable resources can be used for further validation,
such as functional characterization, association analysis,
or development of functional markers for marker-
assisted selection.
Conclusions
Comparison of the whole genome sequences between
‘Yunkang 10’and ‘Shuchazao’revealed a large amount of
genetic variations, including SNPs and InDels, demon-
strating that the tea plant genome is highly variable. The
types of SNPs and InDels were subsequently investi-
gated, and their distributions and annotations were also
analyzed. Based on these InDel loci, a total of 48 novel
InDel markers with moderate polymorphism and high
stability were developed. Population structure and phylo-
genetic relationship analyses were conducted based on
these markers, revealing that tea cultivars from Camellia
sinensis var. assamica were apparently clustered to-
gether, while the other tea cultivars from Camellia
sinensis var. sinensis were clustered into another group.
Remarkably, significant differences were observed in cat-
echin and caffeine content between ‘Yunkang 10’and
‘Shuchazao’, and a number of SNPs and InDels were
identified within genes related to the catechin/caffeine
biosynthesis pathways.
Methods
Plant materials and DNA extraction
A total of 46 clonal tea cultivars were collected from the
main tea-growing regions in China, and we obtained
permission to collect all the tea samples. The details of
these samples, including cultivar name, subspecies,
germplasm type, registration number in China and culti-
vation region are listed in Additional file 4: Table S1.
Two individuals (‘Keke 1’and ‘Keke 2’) were collected
from the local natural population in Guangdong Prov-
ince with the local government’s permission; three clonal
tea cultivars (‘Liubaoxiye’,‘Lingyun 2’and ‘Zihong’) were
collected from the Tea Germplasm Repository of the
Tea Research Institute of Guangxi Province with permis-
sion; the rest of 41 clonal tea cultivars were commercial
cultivars and cultivated widespread in China, which were
deposited in the Tea Plant Cultivar and Germplasm Re-
source Garden in Guohe Town (N31°49′, E117°13′,
Hefei, China) of our Institute (Anhui Agricultural Uni-
versity). Until now, a total of 107 national tea cultivars
(NTCs) and 139 provincial tea cultivars (PTCs) were
registered in China [45]. In this study, 20 NTCs and 13
PTCs were used (the deposition numbers of NTCs are
included in Additional file 4: Table S1), and the
remaining 13 local tea cultivars (LTCs) were registered
Liu et al. BMC Genomics (2019) 20:935 Page 12 of 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
by the corresponding provincial government, while the
subspecies type of four tea cultivars (‘Keke 1’,‘Keke 2’,
‘Ziyan’and ‘Zixian’) was still undetermined.
Young leaves of these tea cultivars were collected and
immediately frozen in liquid nitrogen, and subsequently
stored at −80 °C until further use. Total genomic DNA
was extracted using the EZgene™CP Plant Miniprep Kit
(Biomiga, USA) following the manufacturer’s protocol.
The quality and quantity of DNA samples were deter-
mined by 1% agarose gel electrophoresis and the Nano-
Drop 2000 UV-Vis spectrophotometer, respectively. The
concentration of each sample was adjusted to approxi-
mately 30 ng/ul for further use in the subsequent PCR
amplifications.
Identification of SNPs and InDels by genome-wide
comparison
Considering the quality of genome assemblies of ‘Shu-
chazao’is better than the assemblies of ‘Yunkang 10’[9,
10], it is reasonable to choose the assemblies of ‘Shucha-
zao’as the reference genome. The clean reads of ‘Yun-
Kang 10’were retrieved from the NCBI Sequence Read
Archive under project number PRJNA381277 (Only the
reads with library insert size equal to 500 bp (~ 10 ×)
were applied for the further variation calling).
Subsequently, several steps were applied to identify gen-
etic variations between the two assemblies: aligning the
clean reads of ‘Yunkang 10’to the reference using BWA-
MEM (version 0.7.17) with parameter ‘-M –R-t40’,remov-
ing PCR duplicates with Picard program, calling SNPs and
InDels using GATK-HaplotypeCaller method with param-
eter ‘-stand_call_conf 30’, and the combination method of
Samtools-mpileup with parameters ‘-ugf -t DP -t SP’and
Bcftools-call with parametes ‘-v –m-O’, respectively. Then
take the intersection of the two results and use the GATK
software to filter according to the following parameters:
‘QD <20.0|| ReadPosRankSum <-8.0|| FS >10.0||QUAL
<$MEANQUAL (the first filter)’and ‘DP < 50.0||GQ <
10.0||QD <20.0||FS >200.0||SOR >10.0||MQRankSum
<-12.5||ReadPosRankSum <-8.0||QUAL <$MEANQUAL’,
finally get a high quality variation locus set (Additional file
1: Figure S1). Annotation for the remaining variations was
conducted using snpEFF, and statistics of variations with
Vcftools. The genes containing SNPs/InDels in CDs were
selected by SnpSift, and their GO term enrichment analysis
were performed using the free online platform OmicShare
tools (http://www.omicshare.com/tools)(Additionalfile1:
Figure S1). These software programs have been accurately
and expediently applied in SNP calling from next-
generation sequencing data [46,47].
Validation and development of InDel markers
To develop suitable InDel markers for genetic research,
the InDel lengths ≥5 and ≤20 bp were used as candidate
loci. Specific primers were designed based on the se-
quences flanking the InDel loci through the Primer 3.0
program with the following parameters: amplicons
length (bp) 150–350; primer length 20–22, with the
optimum length being 20 bp; Tm (°C) 50–60, with 55 °C
being the optimum; GC content (%) 40–60, with 50%
being the optimum.
A total of 100 primer pairs were randomly selected
and preliminarily screened on six tea cultivars (‘Guyux-
iang’,‘Longjing 43’,‘Echa 5’,‘Guilv’, Yungui’, and
‘Fudingdabaicha’) using the Fragment Analyzer™96 (Ad-
vanced Analytical Technologies, Inc., Ames, IA). Primers
that gave polymorphic and unambiguous bands were
further screened for identification against the 46 tea cul-
tivars. Details refer to PCR reagents and amplification
conditions were performed according to our previous
study [2]. If more than two fragments were amplified
against some individuals using certain markers, only two
fragments were collected based on the following criteria:
selecting the higher peak value, the higher concentration
of amplified products, and the more frequency of frag-
ments occurred among other individuals.
Genetic diversity analysis
The PROSizeTM 2.0 included in the Fragment Analyzer™
96 system was applied to visually select strong and clearly
polymorphic DNA fragments for scoring, with the same
strategy as described previously [8]. The values of ex-
pected heterozygosity (He) and observed heterozygosity
(Ho) were determined by Popgene 32 version software.
The number of alleles (Na), major allele frequency (MAF),
and polymorphism information content (PIC) were calcu-
lated using PowerMarker 3.25 [48]. Based on the PIC
value, markers were divided into three types: highly in-
formative (PIC> 0.5), moderately informative (0.25 < PIC<
0.5) and slightly informative (PIC< 0.25) [19].
Population structure analysis
Genetic structure analysis of distinct tea accessions was
performed using the Structure 2.3.4 program [49]. To
minimize Hardy-Weinberg and linkage disequilibrium
within each group, the model-based Bayesian clustering
algorithm was employed to assign individuals to groups
with a predetermined number (K, it represents the num-
ber of inferred populations). Ten independent runs for
each Kranging from 2 to 9 were employed and 10,000
iterations were conducted for estimation after a 10,000
iterations burn-in period [19]. Estimation of the sub-
groups and the best Kvalue was performed according to
a previous study [50].
Phylogenetic analysis
Nei’s genetic distances of the 46 tea cultivars based on
48 InDel markers were calculated using PowerMarker
Liu et al. BMC Genomics (2019) 20:935 Page 13 of 16
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
3.25. The dendrogram was constructed using the
neighbor-joining (NJ) algorithm as implemented in
MEGA 7.0 [51], with bootstrap values at the default set-
ting of 1000 replicates. Pairwise gap deletion mode was
employed to guarantee that the divergent domains could
contribute to the topology of the tree [52].
Detection of catechin content using HPLC
The contents of catechin and caffeine were extracted
and examined according to the previous study [53]. All
samples were detected with three independent biological
replicates and each independent sample was examined
with two technical replicates. The content of (+)-Gallo-
catechin (GC), (+)-Gallocatechin gallate (GCG), (−)-Epi-
catechin (EC), (−)-Epicatechin gallate (ECG),
(−)-Epigallocatechin gallate (EGCG), and caffeine were
detected. The catechin biosynthesis pathways were
established according to previous studies [41,54–57].
The number of SNP/InDel within the catechin/caffeine
biosynthesis-related genes was also identified based on
the result of alignment and functional annotation.
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12864-019-6347-0.
Additional file 1: Figure S1. Flowchart diagram for identifying
genome-wide genetic variations between ‘Shuchazao’and ‘Yunkang 10’
and functional annotation.
Additional file 2: Figure S2. Functional categorization of the genes
containing genetic variations within the CDs region. aFunctional
annotation of genes containing SNPs within in the CDs region. b
Functional annotation of genes containing InDels within in the CDs
region. These genes were categorized based on GO annotation, and the
number of each category is shown based on biological process, cellular
component and molecular function.
Additional file 3: Figure S3. Exhibition of transferability and
polymorphism detected by the remaining 30 InDel markers among 46
tea cultivars.
Additional file 4: Table S1. Detailed information for the 46 tea cultivars
used in this study.
Additional file 5: Table S2. Primer sequences of 48 newly developed
InDel markers.
Abbreviations
AFLPs: Amplified fragment length polymorphisms; CAPS: Cleaved amplified
polymorphic sequence; EST: Expressed sequence tag; He: Expected
heterozygosity; Ho: Observed heterozygosity; InDels: Insertions/Deletions;
ISSRs: Inter-simple sequence repeats; MAF: Major allele frequency;
Na: Number of alleles; PIC: Polymorphism information content; RAD-
seq: Restriction site-associated DNA sequencing; RAPDs: Random
amplification of polymorphic DNAs; RFLPs: Restriction fragment length
polymorphisms; SLAF-seq: Specific locus amplified fragment sequencing;
SNPs: Single nucleotide polymorphisms; SSRs: Simple sequence repeat
Acknowledgments
The authors thank the other members of our groups for technical assistance
and appreciate the anonymous reviewers for constructive comments on this
manuscript.
Authors’contributions
SRL performed data analysis and manuscript drafting. YLA conducted DNA
extraction, primer design, PCR amplification, and InDel marker validation. WT
were involved in the identification and analysis of variation loci. XJQ and LS
were involved in sample collection and data analysis. XBX and RG are
involved in DNA extraction and PCR amplification. CLW conceived and
designed the research. All authors read and approved the final manuscript.
Funding
This work was financially supported by the Key R&D Program of China
(2018YFD1000601), the Anhui Provincial Natural Science Foundation
(1808085QC92), the China Postdoctoral Science Foundat ion (2017 M621991),
the Natural Science Foundation of Anhui Provincial Department of Education
(KJ2018A0131), and the National Natural Science Foundation of China
(31800585). The funding bodies had no role in the design of the study,
collection, analysis, and interpretation of data, and in writing the manuscript.
Availability of data and materials
Most of the important data generated or analyzed during this study are
included in the article and its supplementary information files. The other
data and materials associated with the current study are available from the
corresponding author on reasonable request.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural
University, 130 Changjiang West Road, Hefei, China.
2
Guangxi LuYI Institute
of Tea Tree Species, 17 Jinji Road, Guilin, China.
3
Department of
Biotechnology, Russian Research Institute of Floriculture and Subtropical
Crops, Sochi, Russia.
Received: 25 August 2019 Accepted: 28 November 2019
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