G C A T

T A C G
G C A T
genes
Article
De Novo Transcriptome Assembly and EST-SSR Marker
Development and Application in Chrysosplenium macrophyllum
Niyan Xiang 1,2 , Bojie Lu 2 , Tao Yuan 1 , Tiange Yang 2 , Jiani Guo 1 , Zhihua Wu 3 , Hong Liu 2 , Xing Liu 1,4, *
and Rui Qin 2, *

1 Laboratory of Extreme Environmental Biological Resources and Adaptive Evolution, Research Center for
Ecology, School of Sciences, Tibet University, Lhasa 850000, China
2 Hubei Provincial Key Laboratory for Protection and Application of Special Plant Germplasm in Wuling Area
of China, College of Life Sciences, South-Central Minzu University, Wuhan 430074, China
3 College of Life Sciences, Zhejiang Normal University, Jinhua 321004, China
4 State Key Laboratory of Hybrid Rice, Laboratory of Plant Systematics and Evolutionary Biology,
College of Life Sciences, Wuhan University, Wuhan 430072, China
* Correspondence: [email protected] (X.L.); [email protected] (R.Q.)

Abstract: Chrysosplenium macrophyllum Oliv., belonging to the family Saxifragaceae, is a traditional

and unique Chinese herbal medicine. However, the lack of adequate molecular markers has ham-
pered the progress regarding population genetics and evolution within this species. In this research,
we used the DNBSEQ-T7 Sequencer (MGI) sequencing assay to analyze the transcriptome profiles
of C. macrophyllum. SSR markers were developed on the basis of transcriptomic sequences and
further validated on C. macrophyllum and other Chrysosplenium species. The genetic diversity and
structure of the 12 populations were analyzed by using polymorphic expressed sequence tag simple
sequence repeat (EST-SSR) markers. A potential pool of 3127 non-redundant EST-SSR markers were
identified for C. macrophyllum in this study. The developed EST-SSR markers had high amplification
rates and cross-species transferability in Chrysosplenium. Our results also showed that the natural
populations of C. macrophyllum had a high level of genetic diversity. Genetic distance, principal com-
ponent analysis, and popular structure analysis revealed that all 60 samples clustered into two major
Citation: Xiang, N.; Lu, B.; Yuan, T.;
groups that were consistent with their geographical origins. This study provided a batch of highly
Yang, T.; Guo, J.; Wu, Z.; Liu, H.; Liu,
polymorphic EST-SSR molecular markers that were developed via transcriptome sequencing. These
X.; Qin, R. De Novo Transcriptome
Assembly and EST-SSR Marker
markers will be of great significance for the study of the genetic diversity and evolutionary history of
Development and Application in C. macrophyllum and other Chrysosplenium species.
Chrysosplenium macrophyllum. Genes
2023, 14, 279. https://doi.org/ Keywords: Chrysosplenium macrophyllum; transcriptome; EST-SSR; transferability; genetic diversity;
10.3390/genes14020279 population structure

Academic Editor: Gabriella De

Lorenzis

Received: 12 December 2022 1. Introduction

Revised: 16 January 2023
Chrysosplenium L. is a very small perennial herbaceous genus in the family Saxifra-
Accepted: 17 January 2023
gaceae, with tetramerous flowers and petaloid sepals [1]. This genus consists of around
Published: 21 January 2023
80 species distributed in Asia, Europe, Africa, and America; however, only two species in
Chile have been found in the southern hemisphere, and the rest are concentrated in the
northern hemisphere [2–5]. In the northern hemisphere, Chrysospelnium species, including
Copyright: © 2023 by the authors.
ca. 53 species, are mainly distributed in East Asia, with China being one of the diversity
Licensee MDPI, Basel, Switzerland. centers of this genus, with 39 species, of which 24 are endemic [1,5–7]. In accordance with
This article is an open access article the Flora of China, the literature, and field investigations, Chrysospelnium macrophyllum
distributed under the terms and is endemic to China, mainly distributed in 14 Chinese provinces [8,9]. It is a common
conditions of the Creative Commons folk herbal medicine that can treat infantile convulsions, ecthyma, scalds, and lung and
Attribution (CC BY) license (https:// ear disorders [10]. Only a few studies have been performed on C. macrophyllum, and its
creativecommons.org/licenses/by/ chloroplast genomic data have been obtained [11]. Given the lack of rich molecular markers
4.0/).

Genes 2023, 14, 279. https://doi.org/10.3390/genes14020279 https://www.mdpi.com/journal/genes

Genes 2023, 14, 279 2 of 17

for C. macrophyllum, the population structure and genetic diversity of C. macrophyllum are
still unknown, thus minimizing the exploitation and utilization of this species.
Molecular markers are an extremely popular tool in the analysis of genetic diversity
because of their stability, cost-effectiveness, and facile application [12]. The most used
molecular markers mainly include restriction fragment length polymorphisms (RFLP),
random amplified polymorphic DNA markers (RAPD), amplified fragment length poly-
morphisms (AFLP), inter simple sequence repeats (ISSR), sequence-related amplified poly-
morphisms (SRAP), simple sequence repeats (SSR), and single-nucleotide polymorphism
(SNP) markers [13,14]. SSRs are the most widely used molecular markers, associated with
their codominance, abundance, high polymorphism, good reproducibility, and simple
operation [15–17]. SSRs can be separated into genomic SSR (gSSR) and expressed sequence
tag SSR (EST-SSR) markers, in accordance with their type of sequence source [18]. EST-SSRs
have a lower developmental cost than gSSRs and exhibit cross-species transferability and
direct correlations with gene functions [18,19]. They have been widely used in plant re-
search, such as studies on Carex breviculmis [20], Pinus koraiensis [21], Actinidia eriantha [22],
Zingiber officinale [23], Rosa roxburghii [24], and Dendrobium officinale [25].
Next-generation sequencing technology, especially transcriptome sequencing with
Illumina and MGI, is an effective and reliable tool that provides a low-cost means to
develop SSR markers [26–29]. Transcriptome sequencing and de novo assembly are essential
for studying functional genomics as mining markers, especially markers in non-model
organisms that lack sequenced genomes [30,31]. To date, only several nucleotide sequences
of Cymbidium aureobracteatum have been reported (September 2022), and no C. macrophyllum
ESTs are available in GenBank [32]. In previous studies, only the chloroplast gene matK
was used to examine the genetic variations of the genus Chrysosplenium [33]. However, only
a few researchers have investigated C. macrophyllum.
In this study, (i) we used the DNBSEQ-T7 Sequencer to obtain the global transcriptome
of C. macrophyllum and annotated and functionally classified the transcripts. (ii) Then,
a number of EST-SSRs were built for C. macrophyllum on the basis of these transcripts and
we verified their transferability among different Chrysosplenium species. (iii) Finally, we
evaluated the genetic diversity and structure of 12 populations of C. macrophyllum. This
study will lay a solid resource foundation for studies on functional genomics, metabolomics,
proteomics, and the development and utilization of molecular markers, and also provide
important references and new ideas for related studies on the species of Chrysosplenium.

2. Materials and Methods

2.1. Plant Materials, RNA Isolation, and DNA Extraction
The fresh roots, stems, and leaves of C. macrophyllum were gathered on 10 August
2021, from Xuanen County, Hubei Province, China, and instantly frozen in liquid nitrogen.
Samples were then stored at up to −80 ◦ C until used for RNA isolation. The young
leaves of 60 individuals from 12 wild populations of C. macrophyllum were collected and
placed in sealed bags containing dried silica gel for subsequent DNA isolation. They were
collected from seven provinces that included most of the distribution of this species in
China (Table 1). The distance between each individual in the population was more than
1 m. Sixteen additional Chrysosplenium species were gathered to detected the cross-genome
transferability of EST-SSRs (Table 1).
Genes 2023, 14, 279 3 of 17

Table 1. Characteristics of analyzed Chrysosplenium species in this study.

Species Location Latitude (N)/Longitude (E) Elevation (m) Sample Size Voucher
C. macrophyllum Zhijin, Guizhou N: 26◦ 390 0300 /E: 105◦ 340 2900 1950 5 –
Badong, Hubei N: 31◦ 150 3000 /E: 110◦ 230 0100 1425 5 HSN6460
Hongya, Sichuan N: 29◦ 300 2600 /E: 103◦ 150 2400 1770 5 –
Nanjiang, Sichuan N: 32◦ 410 1800 /E: 106◦ 470 4700 1440 5 –
Guidong, Hunan N: 25◦ 590 3800 /E: 113◦ 430 1900 1220 5 –
Xuanen, Hubei N: 30◦ 010 3500 /E: 109◦ 430 1300 1164 5 HSN5500
Wugang, Hunan N: 26◦ 380 5800 /E: 110◦ 360 4600 1030 5 –
Linan, Zhejiang N: 30◦ 200 1400 /E: 119◦ 260 0300 770 5 –
Yinshan, Hubei N: 30◦ 580 0500 /E: 116◦ 010 3700 740 5 –
Jianning, Fujian N: 26◦ 470 0400 /E: 116◦ 560 0400 690 5 –
Tongshan, Hubei N: 29◦ 210 5300 /E: 114◦ 340 0600 590 5 HSN13118
Panan, Zhejiang N: 28◦ 570 4300 /E: 120◦ 330 4200 510 5 –
Chrysosplenium ramosum Fusong, Jilin N: 42◦ 100 2700 /E: 127◦ 300 3000 400 1 SJH2017052107372
Chrysosplenium serreanum Fusong, Jilin N: 42◦ 100 3200 /E: 127◦ 290 0300 412 1 SJH2017052107371
Chrysosplenium japonicum Hangzhou, Zhejiang N: 30◦ 150 0200 /E: 120◦ 60 5900 19 1 HSN7909
Chrysosplenium griffithii var.
Kangding, Sichuan N: 30◦ 060 3000 /E: 101◦ 480 0600 3640 1 HSN09825
intermedium
Doujiangyan,
Chrysosplenium glossophyllum N: 30◦ 550 4900 /E: 103◦ 280 5400 1049 1 QCS2017102608035
Sichuan
Chrysosplenium alternifolium Fusong, Jilin N: 42◦ 100 2700 /E: 127◦ 300 3000 400 1 SJH2017052107369
Nanchuan,
Chrysosplenium microspermum N: 29◦ 010 0200 /E: 107◦ 110 3200 1987 1 –
Chongqing
Chrysosplenium giraldianum PingWu, Sichuan N: 32◦ 530 1900 /E: 104◦ 090 5000 2430 1 JZ2018042507981
Chrysosplenium qinlingense PingWu, Sichuan N: 32◦ 530 1900 /E: 104◦ 090 5000 2430 1 HSN7980
Chrysosplenium lectus-cochleae Fusong, Jilin N: 42◦ 100 2700 /E: 127◦ 300 3000 400 1 HSN7379
Chrysosplenium axillare Tianzhu, Gansu N: 37◦ 030 3800 /E: 102◦ 460 0600 3275 1 –
Chrysosplenium forrestii Gongshan, Yunnan N: 28◦ 040 2500 /E: 98◦ 450 0900 3900 1 HSN7797
Chrysosplenium lanuginosum Badong, Hubei N: 31◦ 210 4900 /E: 110◦ 230 1700 1777 1 BD2017030507343
Chrysosplenium delavayi Quanzhou, Guangxi N: 25◦ 400 1200 /E: 111◦ 30 1600 250 1 –
Chrysosplenium hydrocotylifolium Quanzhou, Guangxi N: 25◦ 400 1000 /E: 111◦ 30 1600 280 1 –
Chrysosplenium nudicaule Chayu, Xizang N: 28◦ 360 5700 /E: 98◦ 030 3700 4426 1 –

Total RNA was extracted by using the R6827 Plant RNA Kit (Omega Bio-Tek, Inc.,
Norcross, GA, USA) in accordance with the manufacturer’s instructions. RNA contami-
nation and degradation were supervised with 1% agarose gels. RNA integrity and purity
was assayed by using a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA)
and NanoDrop One spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA),
respectively. Qualified RNA from roots, stems, and leaves of C. macrophyllum was mixed in
equal amounts for RNA sequencing.
Genomic DNA was extracted by using a modified cetyltrimethylammonium bromide
(CTAB) method [34]. DNA integrity and concentration were determined by using 1%
agarose gel electrophoresis and NanoPhotometer® NP80 (Implen, München, Germany),
respectively. Then, the extracted DNA was diluted with ddH2 O to the desired working
concentration (50 ng/µL) and stored at −20 ◦ C until PCR amplification.

2.2. Transcriptome Sequencing and De Novo Assembly

The transcriptome sequencing of C. macrophyllum was performed using the DNBSEQ-
T7 platform from Wuhan Benagen Technology Co., Ltd. (Wuhan, China). FASTPv0.23.1 [35]
was used to remove reads with adaptors, those with more than 5% unknown nucleotides
(N), or those with more than 50% low-quality (Q-value 5) bases. Then, the de novo
assembly of the high-quality clean reads was conducted by utilizing Trinity v2.8.3 [36]
with the parameters of min_contig_length = 500, min_kmer_cov = 3, and min_glue = 15.
After assembly, CD-HIT [37] was used for clustering to remove redundant transcripts and
unigenes were obtained.

2.3. Annotation and Functional Classification

Coding regions within unigenes were detected by using TransDecoder (https://
github.com/TransDecoder/TransDecoder/releases, accessed on 10 October 2022), im-
Genes 2023, 14, 279 4 of 17

plemented in Trinity software). For the characterization of all the putative functions of
the unigenes, the unigenes were compared against public databases, including NCBI
nonredundant protein sequences (NR) [38], Kyoto Encyclopedia of Genes and Genomes
(KEGG) [39], Gene Ontology (GO) [40], and Clusters of Eukaryotic Orthologous Groups
(KOG) (E-value < 1.0 × 10−5 ) [41].
Eggnog-mapper v2 [42] and InterProScan v5.0 (https://github.com/ebi-pf-team/interproscan,
accessed on 20 October 2022) were used to obtain GO and KOG annotations. After the
prediction of protein sequences, the unigenes were aligned with the NR, Swiss-Prot, and
KEGG databases by using Diamond (E-value < 1.0 × 10−5 ) [43].

2.4. SSR Identification and Primer Design

The detection and localization of potential SSRs were performed by using the mi-
crosatellite tool [44]. The search standards for SSRs were set to the minimum number of 10,
6, 5, 5, 5, and 5 repeat units for mono-, di-, tri-, tetra-, penta-, and hexanucleotide motifs,
respectively. Primers for the flanking sequences of the identified microsatellite motifs were
designed by using Primer 3 software. The parameters considered for primer designing
were as follows: (a) primer length of 18–23 bp with 20 bp as the optimal length; (b) PCR
product sizes ranging from 100 bp to 250 bp; (c) GC content ranging from 40% to 60% with
the optimum of 50%; (d) annealing temperature between 50 ◦ C and 60 ◦ C with 58 ◦ C as the
optimal temperature; and (e) default values for the other parameters.

2.5. EST-SSR Validation and Cross-Species Amplification

In total, 58 pairs of primers were randomly chosen and synthesized by Beijing TS-
INGKE Biological Technology Co., Ltd. (Beijing, China), to develop polymorphic EST-SSR
markers. Twelve DNA samples from different populations, including ZJ, BD, HY, NJ, GD,
XE, WG, LA, YS, JN, TS, and PA, were used to analyze the primary polymorphisms of the
primers. PCR amplification was performed by using BIO-RAD T100 Thermal CyclerTM,
and the PCR reaction system was prepared with a 10 µL total reaction volume comprising
5 µL of 2×T5 Super PCR Mix (PAGE) (Beijing TsingKe Biotech Co., Ltd., Beijing, China),
0.4 µL (10 µM) each of the forward and reverse primers, 1 µL of genomic DNA (50 ng/µL),
and 3.2 µL of ddH2 O. The PCR procedure was conducted as follows: an initial denaturation
for 2 min at 98 ◦ C; 30 cycles of denaturation at 10 s at 98 ◦ C, annealing at 58 ◦ C for 10 s,
and extension at 72 ◦ C for 10 s; and a final extension cycle of 2 min at 72 ◦ C and holding
at 4 ◦ C. The amplified PCR products were mixed with 10× loading buffer at the ratio of
1:5 or 1:10 and immediately placed into a mixture of ice water after being denatured at
95 ◦ C for 5 min in a BIO-RAD T100 Thermal CyclerTM. The same denaturation process was
performed with PAGE Gel 20 bp ladder marker (Beijing Bio-ulab Biotech Co., Ltd., Beijing,
China) as the molecular size standard. Then, the mixture of PCR products and 10× load-
ing buffer was subjected to 6% denatured polyacrylamide gel electrophoresis at 90 W for
1–1.5 h and visualized by using silver nitrate staining.
After the screening of polymorphic primers, 39 pairs of primers with the expected
band sizes were selected for cross-species amplification validation on other Chrysosplenium
species. The PCR reaction system and conditions were the same as above. After PCR
amplification was completed, gel electrophoresis was performed utilizing 3% agarose.
Moreover, 50 bp DNA Ladder was used as a marker to determine the size of PCR products.
Agarose gel photographs were taken using an automated gel imaging system. Then,
10 pairs of polymorphic primers were further selected for the analysis of genetic diversity
in 60 individuals from 12 C. macrophyllum populations. The PCR amplification conditions
and genotyping methods were the same as those above. The PCR bands of gel images
observed under a light lamp were marked as present (1) or absent (0).

2.6. SSR Data Analysis

GENODIVE version 3.06 [45], which can handle genetic data from polyploids or
mixed-ploidy datasets, was used to calculate the following population genetic parameters:
Genes 2023, 14, 279 5 of 17

the number of alleles (Na), effective number of alleles (Ne), observed (Ho) and expected (He)
heterozygosity, and inbreeding coefficient (Fis). The Ho and He, polymorphic information
content (PIC), and Shannon diversity index (I) of each population and locus were estimated
by using POLYGENE v1.2 [46]. Differentiation between C. macrophyllum populations was
assessed on the basis of GST . Analysis of molecular variance (AMOVA) was performed by
using POLYGENE v1.2 to obtain the genetic variation among populations.
A neighbor-joining tree based on DA genetic distance was established for C. macrophyllum
individuals by using POPTREE v.2 software [47]. Principal coordinate analysis (PCoA)
was performed with Cavalli–Sforza’s chord distances, which have been shown to be the
least biased distance measure in the absence of dosage information [48]. STRUCTURE
version 2.3.4 [49] was used to infer the population structure using an admixture model with
correlated allele frequencies. The potential number of genetic clusters (K) ranged from 1 to
10, and 10 independent replicates were run for each K value with a 100,000 burn-in period
and 1,000,000 Markov chain Monte Carlo iterations. The online program STRUCTURE
HARVESTER [50] was used to infer the optimal K in accordance with the method of Evanno
et al. [51]. The program CLUMPP version 1.1.2 [52] was applied to estimate the averaged
admixture coefficients for each K value. The clustering results were visualized by using
Distruct version 1.1 [53].

3. Results
3.1. De Novo Assembly of the Transcriptome
After adapter removal and low-quality sequence filtering, 40,507,062 high-quality
clean reads were obtained. The Q30 base percentage reached 93.00%, and the GC content
was 42.00%. Then, 63,961 assembled transcripts with the mean length of 1551.85 bp, GC
content of 40.21%, and N50 length of 1901 bp were generated by using Trinity v2.8.3. Subse-
quently, the longest copy of assembled transcripts isomer was extracted. After redundancy
removal, the longest remaining transcripts were regarded as unigenes. Finally, a total of
29,477 unigenes with the mean length of 1341.32 bp, the maximum length of 23,968 bp,
and N50 of 1646 bp (Table 2) were obtained. A total of 14,397 unigenes (48.84%) had
lengths less than 1000 bp; 9878 unigenes (33.51%) had lengths between 1001 and 2000 bp;
and 5202 unigenes (17.65%) had lengths > 2000 bp (Figure 1).

Table 2. Summary of the de novo assembly of C. macrophyllum.

Category Items Number

Raw Reads Total raw reads 40,782,638
Total clean reads 40,507,062
Total clean nucleotides (nt) 6,052,073,283
Clean Reads Q30 (%) 93.00%
N (%) 0%
GC (%) 42.00%
Total trinity transcripts 63,961
Total trinity genes 29,508
GC (%) 40.21
Trancripts N50 (bp) 1901
Maximum length (bp) 23,968
Mean length (bp) 1551.85
Total assembled bases 99,257,989
Total unigenes 29,477
GC (%) 40.06
N50 (bp) 1646
Unigenes
Maximum length (bp) 23,968
Mean length (bp) 1341.32
Total assembled bases 39,538,014
R REVIEW

Genes 2023, 14, 279 6 of 17

Figure 1. Distribution of unigene lengths of C. macrophyllum.

Figure 1. Distribution of unigene lengths of C. macrophyllum.
3.2. Gene Annotation Based on Different Databases
A total of 15,647 protein-coding unigenes were predicted by using TransDecoder and
submitted to the NR, KOG, Swiss-Prot, KEGG, and GO databases for functional annotation.
3.2. Gene Annotation Based on Different Databases
As shown in Table 3, 11,115 unigenes were successfully annotated, including 10,946 (37.13%)
in NR, 6670 (22.63%) in KOG, 8422 (28.57%) in Swiss-Prot, 2021 (6.85%) in KEGG, and 7836
A total of 15,647 protein‐coding unigenes were predicted by usi
(26.58%) in GO.

submitted to Table
the3.NR, KOG,
Functional annotationSwiss‐Prot, KEGG,
of C. macrophyllum in different and GO databases f
databases.

tion. As shown in Table Category 3, 11,115 unigenes Number were successfully

Percentage (%) annota
Nr annotation 10,946 37.13
(37.13%) in NR, 6670 (22.63%) in KOG,66708422 (28.57%) in22.63Swiss‐Prot, 2
KOG annotation
Swiss-prot annotation 8422 28.57
and 7836 (26.58%)KEGG in annotation
GO. 2020 6.85
GO annotation 7836 26.58
All annotated unigenes 11,115 37.71

Table 3. Functional annotation of C. macrophyllum in different databases.

On the basis of functional annotation, the unigenes were divided into three main GO
categories (biological process, molecular function, and cellular component) and 57 sub-
Category Number
categories (Figure 2). In the biological process category, “cellular process” was the largest P
subgroup, followed by “metabolic process”, “single-organism process”, and “biological
Nr annotation 10,946
regulation”. Among the 18 different cellular component categories for C. macrophyllum
unigenes, the categories “cell” and “cell part” were the most abundant. The molecular
KOG annotation 6670
function category contained 16 GO terms, among which “binding”, “catalytic activity”,
and “nucleic acid binding transcription factor activity” were highly represented.
Swiss‐prot annotation 8422
KEGG annotation 2020
GO annotation 7836
, x FOR PEER REVIEW 7 of 18

Genes 2023, 14,and

279
“spliceosome” (50) were the main pathways among the top 50 pathways (Figure 4). 7 of 17

In addition, 23 unigenes were found in the “terpenoid backbone biosynthesis” pathway.

Genes 2023, 14, x FOR PEER REVIEW 7 of 18

and “spliceosome” (50) were the main pathways among the top 50 pathways (Figure 4).
In addition, 23 unigenes were found in the “terpenoid backbone biosynthesis” pathway.

ofGO
Figure 2.
Figure 2. GO classification classification of C. macrophyllum.
C. macrophyllum.

The unigenes were annotated and functionally classified into 25 KOG categories, and a
large number of the unigenes were assigned to more than one category (Figure 3). Among
these categories, “general function prediction only” (1541, 23.10%) was the most dominant.
“Post-translational modification, protein turnover, chaperones” (759, 11.38%) constituted
the second-largest cluster, which was followed by “signal transduction mechanisms” (734,
Figure 2. GO
11.00%). classification
However, of C. unigenes
only two macrophyllum.
were annotated to “cell motility” (Figure 3).

Figure 3. KOG classification of C. macrophyllum.

Figure 3. KOG classification of C. macrophyllum.

 Genes 2023, 14, 279 8 of 17

A total of 2020 unigenes were found in the KEGG database and assigned to 127 KEGG
functional pathways belonging to five large groups (“metabolism”, “genetic information
processing”, “environmental information processing”, “cellular processes”, and “organ-
Genes 2023, 14, x FOR PEER REVIEW ismal systems”). “Ribosome” (95), “protein processing in endoplasmic reticulum” (59), 8 of 18
and “spliceosome” (50) were the main pathways among the top 50 pathways (Figure 4).
In addition, 23 unigenes were found in the “terpenoid backbone biosynthesis” pathway.

Figure 4. KEGG
Figure classification
4. KEGG classification of C.
C. macrophyllum.
macrophyllum.

The E-value distribution revealed that 31.26% of the unigenes yielded significant
The E‐value distribution revealed that 31.26% of the unigenes yielded significant hits
hits in the NCBI NR nucleotide database (Figure 5a), and approximately 21.65% of these
in the NCBIexhibited
unigenes NR nucleotide database
greater than (Figure
80% identity 5a), 5b).
(Figure andTheapproximately 21.65%
sequence alignment of these
results of uni‐
genes exhibited greater than 80% identity (Figure 5b). The sequence alignment
the NR protein revealed that 887 unigenes could be aligned with Vitis vinifera, 637 unigenes results of
the could
NR protein revealed
be aligned that
with Nyssa 887 unigenes
sinensis, could
and 541 could be be aligned
aligned withwith Vitis vinifera,
Vitis riparia 637 uni‐
(Figure 5c).
genes could be aligned with Nyssa sinensis, and 541 could be aligned with Vitis riparia
(Figure 5c).
Genes 2023, 14, x FOR PEER REVIEW 9 of 18
Genes 2023, 14, 279 9 of 17

Figure
Figure 5.
5. Homology
Homology searches
searches of of C.
C. macrophyllum
macrophyllumunigene
unigeneand andcharacteristics
characteristics of of non‐redundant pro‐
non-redundant
tein databases
protein databases(Nr).
(Nr).(a)(a)
The TheE‐value
E-valuedistribution of unigene
distribution of unigeneBLASTx
BLASTxhits hits
forfor every
every assembly. (b)
assembly.
BLASTx
(b) BLASTxhit hit
profiles for
profiles forevery
everyassembly ofunigenes.
assembly of unigenes.(c)(c)Distribution
Distribution of accessions
of accessions hit byhit by BLASTx at
BLASTx
the
at the of of
toptop each
eachassembly
assemblyof of unigenes.
unigenes.

3.3. Frequency
3.3. Frequency and
andDistribution
Distributionof SSRs
of SSRs
A total of 5573 unigenes containing 6985 SSRs were identified among 29,477 uni-
A total of 5573 unigenes containing 6985 SSRs were identified among 29,477 unigenes
genes by using MISA software. Among these unigenes, 1091 contained more than one
by using
SSR, and MISA
500 SSRs software.
presented Among these unigenes,
a compound formation.1091In C.contained more
macrophyllum, thethan
SSRone
motifSSR, and
500
were found to be distributed every 5.67 kb on average (Table 4). In the identified SSRs, found
SSRs presented a compound formation. In C. macrophyllum, the SSR motif were
to
thebe distributed every
mono-nucleotide motifs5.67
were kbtheonmost
average (Table
enriched, with4). In the identified
a proportion of 46.09%,SSRs, the mono‐
followed
nucleotide motifs
by di- (33.34%), tri- were the tetra-
(19.18%), most (0.82%),
enriched, with(0.17%),
penta- a proportion of (0.40%)
and hexa- 46.09%,nucleotide
followed by di‐
motifs (Table
(33.34%), tri‐ 4). A total of
(19.18%), 67 different
tetra‐ (0.82%), repeat motifs
penta‐ were found
(0.17%), in all SSR
and hexa‐ loci. nucleotide
(0.40%) A/T (3170) motifs
(Table 4). A total of 67 different repeat motifs were found in all SSR loci.all
was the dominant motif in mononucleotide repeats, accounting for 45.38% of motifs.
A/T (3170) was
The next most dominant motif was C/G (49), which accounted for 0.70% of all motifs.
the dominant motif in mononucleotide repeats, accounting for 45.38% of all motifs. The
Among the dinucleotide repeats, the most frequent motifs were AG/CT (1433, 20.52%),
next most dominant motif was C/G (49), which accounted for 0.70% of all motifs. Among
followed by AT/AT (788, 11.28%), AC/GT (106, 1.52%), and CG/CG (2, 0.03%). Ten dif-
the dinucleotide
ferent trinucleotide repeats, the most
repeat motifs werefrequent motifs
identified, amongwere AG/CT
which (1433, 20.52%),
ATC/ATG, AAG/CTT, followed
by AT/AT (788, 11.28%),
AAC/GTT, and AGG/CTT accounted for 4.31%, 4.27%, 2.25%, and 2.22%, respectively, trinu‐
AC/GT (106, 1.52%), and CG/CG (2, 0.03%). Ten different
cleotide repeat
on the basis motifs were
of frequency. identified,ofamong
The frequencies the otherwhich ATC/ATG,
six motifs were less AAG/CTT,
than 2%. TheAAC/GTT,
fre-
and AGG/CTT
quencies accountedrepeats
of tetranucleotide for 4.31%,
were 4.27%, 2.25%,
0.82%, and and frequencies
the total 2.22%, respectively, on the basis of
of pentanucleotide
and hexanucleotide
frequency. repeats were
The frequencies 0.57%
of the (Table
other six5).
motifs were less than 2%. The frequencies of
tetranucleotide repeats were 0.82%, and the total frequencies of pentanucleotide and hex‐
anucleotide repeats were 0.57% (Table 5).
Genes 2023, 14, 279 10 of 17

Table 4. Sequence searching for the SSR markers of C. macrophyllum.

Searching Item Number

Total number of identified SSRs 6985
Number of SSR-containing sequences 5573
Number of sequences containing more than 1 SSR 1091
Number of SSRs present in compound formation 500
Frequency of SSR 1/5.67 kb
Mononucleotide 3219
Dinucleotide 2329
Trinucleotide 1340
Tetranucleotide 57
Pentanucleotide 12
Hexanucleotide 28

Table 5. Frequencies of different repeat motifs in SSRs of C. macrophyllum.

Repeats 5 6 7 8 9 10 11 12 13 14 15 16+ Total Percentage (%)

A/T 1780 499 248 132 92 72 347 3170 45.38
C/G 3 7 3 8 5 2 21 49 0.70
AC/GT 64 27 8 3 1 1 1 1 106 1.52
AG/CT 788 338 148 64 35 22 11 3 8 4 12 1433 20.52
AT/AT 261 166 107 82 49 44 79 788 11.28
CG/CG 2 2 0.03
AAC/GTT 107 31 9 6 1 1 1 1 157 2.25
AAG/CTT 195 67 17 10 1 3 2 3 298 4.27
AAT/ATT 76 22 14 4 2 1 1 1 2 123 1.76
ACC/GGT 114 22 14 5 155 2.22
ACG/CGT 30 1 31 0.44
ACT/AGT 41 11 8 2 1 1 64 0.92
AGC/CTG 84 30 4 3 1 1 123 1.76
AGG/CCT 46 13 4 3 1 67 0.96
ATC/ATG 209 50 20 18 1 1 1 1 301 4.31
CCG/CGG 17 4 21 0.30
AAAC/GTTT 1 1 1 1 4 0.06
AAAG/CTTT 1 1 0.01
AAAT/ATTT 18 1 19 0.27
AACC/GGTT 1 1 0.01
AACG/CGTT 1 1 2 0.03
AAGG/CCTT 1 1 0.01
AATC/ATTG 4 1 5 0.07
AATG/ATTC 1 1 2 0.03
AATT/AATT 2 2 0.03
ACAT/ATGT 4 4 0.06
ACCC/GGGT 2 2 0.03
ACCT/AGGT 3 3 0.04
ACTC/AGTG 2 2 0.03
AGAT/ATCT 2 1 1 4 0.06
AGCT/AGCT 1 1 0.01
AGGG/CCCT 1 1 0.01
ATCG/ATCG 3 3 0.04
Other 11 12 1 6 2 0 2 0 0 1 1 4 40 0.57
Total 974 1384 623 323 155 1875 578 347 145 109 80 392 6985 100

3.4. Development and Transferability of EST-SSR Markers

A total of 3127 pairs of primers were successfully designed on the basis of the
6985 SSRs. Of these, 58 pairs, mainly comprising dinucleotide and trinucleotide repeat
units, were selected for amplification and polymorphism assessment. The results showed
that 39 (67.24%) primers generated the expected size bands, including six pairs of monomor-
phic primers and 33 pairs of polymorphic primers. Finally, 10 highly polymorphic primers
were selected to analyze the genetic diversity of 60 C. macrophyllum samples from 12 populations.
Whether the primer pairs designed from the EST sequences of C. macrophyllum could
also effectively amplify the same SSR motifs in 16 Chrysosplenium species was verified.
Genes 2023, 14, 279 11 of 17

Of the 39 EST-SST primers with the expected band size, only three (7.69%) successfully
amplified SSR motifs in all Chrysosplenium species, whereas 33 resulted in amplification
in some but not all species, and three failed to result in amplification in all 16 additional
species (Table S1). The top three species with the highest success rates in cross-amplification
trials were C. hydrocotylifolium (79.49%), C. lanuginosum (64.10%), and C. nudicaule (61.54%).

3.5. Genetic Diversity and Structure

By using the set of 10 SSRs, 94 alleles were detected across the 60 C. macrophyllum
samples for an average of 9.4 alleles per locus. The minimum number of alleles detected
at each locus was five (CsSSR30) and the maximum number was 15 (CsSSR5). The PIC
values ranged from 0.565 (CsSSR44) to 0.855 (CsSSR5), with the average of 0.678 (Table S2).
The values of genetic diversity at the population level are shown in Table 6. Ne ranged
from 1.899 in YS to 3.513 in BD, averaging 2.699 alleles per population. Ho as estimated
by GenoDive ranged from 0.480 (LA) to 0.717 (JN), whereas the Ho estimated by using
Polygene ranged from 0.265 (LA) to 0.710 (JN). He calculated by GenoDive and Poly-
gene ranged from 0.459 (YS) to 0.393 (BD and TS) and 0.392 (LA) to 0.636 (BD), respec-
tively. The observed gene heterozygosity was lower than the expected gene heterozygosity.
The Fis of BD, NJ, LA, and TS was greater than 0, whereas that of the other populations
was less than 0. The overall GST among all populations was 0.218. Pairwise comparisons of
genetic differentiation between populations indicated that GST ranged from 0.043 (between
populations ZJ and HY) to 0.249 (between populations PA and HY) (Table S3). AMOVA
revealed that the genetic variation within populations (65.22%) was higher than that
among populations (34.78%) of C. macrophyllum, suggesting a high level of differentiation
(Table S4).

Table 6. Genetic diversity within C. macrophyllum populations at 10 SSR markers.

GenoDive Polygene
Population
Na Ne Ho He Fis Ho He PIC I
ZJ 4.200 3.034 0.716 0.675 −0.060 0.613 0.591 0.526 1.098
BD 5.400 3.513 0.655 0.693 0.055 0.648 0.636 0.600 1.314
WG 3.800 3.003 0.650 0.631 −0.029 0.644 0.591 0.536 1.089
HY 3.300 2.544 0.640 0.620 −0.032 0.592 0.551 0.483 0.954
NJ 4.200 3.042 0.567 0.659 0.141 0.558 0.590 0.547 1.138
GD 3.900 2.852 0.695 0.677 −0.026 0.684 0.624 0.554 1.113
JN 2.600 2.600 0.717 0.628 −0.141 0.710 0.583 0.484 0.913
PA 2.000 2.000 0.508 0.482 −0.056 0.500 0.425 0.333 0.624
LA 2.200 1.909 0.480 0.530 0.094 0.265 0.392 0.321 0.605
YS 2.000 1.899 0.513 0.459 −0.118 0.435 0.408 0.322 0.599
TS 3.800 2.853 0.688 0.693 0.006 0.636 0.626 0.560 1.117
XE 3.900 3.141 0.691 0.682 −0.013 0.662 0.624 0.575 1.157

The population structure of C. macrophyllum was analyzed by using STRUCTURE

2.3.4, and the optimal K value was observed at K = 2, with the maximum ∆K value
(Figure 6a,b). All collected individuals were divided into two genetic groups (Figure 4c).
Group I contained eight populations (JN, WG, GD, TS, NJ, BD, XE, ZJ, and HY), whereas
Group II included three populations (YS, LA, and PA) (Figure 6c). PCoA based on the
10 EST-SSR markers was used to evaluate the population genetic structure. Consistent
with the results of structure analysis, the PCoA results also revealed two groups based on
genetic distance (Figure 7a). The first and second axes explained 14.51% and 12.02% of the
total variation, respectively. In addition, a neighbor-joining tree was constructed by using
DA distances. In the tree, individuals were divided into two groups, in agreement with the
two genetic groups identified by PCoA and STRUCTURE (Figure 7b).
 markers was used to evaluate the population genetic structure. Consistent with the results
markers was used to evaluate the population genetic structure. Consistent with the results
of structure analysis, the PCoA results also revealed two groups based on genetic distance
of structure analysis, the PCoA results also revealed two groups based on genetic distance
(Figure 7a). The first and second axes explained 14.51% and 12.02% of the total variation,
(Figure 7a). The first and second axes explained 14.51% and 12.02% of the total variation,
respectively. In addition, a neighbor‐joining tree was constructed by using DA distances.
respectively. In addition, a neighbor‐joining tree was constructed by using DA distances.
In the tree, individuals were divided into two groups, in agreement with the two genetic
Genes 2023, 14, 279 In the tree, individuals were divided into two groups, in agreement with the two genetic
12 of 17
groups identified by PCoA and STRUCTURE (Figure 7b).
groups identified by PCoA and STRUCTURE (Figure 7b).

Figure 6.
6. Structure analysis
Structureanalysis ofof6060C.C.macrophyllum
analysisof macrophyllum from
from 12 populations based on on
10 EST‐SSRs. (a)
Figure
Figure 6. Structure 60 C. macrophyllum from 12 12 populations
populations based
based on 10 10 EST-SSRs.
EST‐SSRs. (a)
Distribution
(a) Distributionof ΔK in
of ∆K STRUCTURE
in STRUCTURE analysis. (b)
analysis. The likelihood L(K) values presented for K = 1−10.
Distribution of ΔK in STRUCTURE analysis. (b)(b)
TheThe likelihoodL(K)
likelihood L(K)values
valuespresented
presentedfor
forKK ==1−10.
1–10.
(c) Histogram of the STRUCTURE analysis for the model with K = 2 (showing the highest ΔK). Dif‐
Histogramof
(c) Histogram ofthe
theSTRUCTURE
STRUCTUREanalysisanalysis
forfor
thethe model
model with
with K =K2 = 2 (showing
(showing ΔK). ∆K).
the highest
the highest Dif‐
ferent colors represent genetic stock.
ferent colors
Different represent
colors genetic
represent stock.
genetic stock.

Figure 7. Graphical representation of differentiation between populations. (a) Principal coordinate

analysis (PCoA). (b) Neighbor-joining phylogenetic tree of 60 C. macrophyllum individuals.

4. Discussion
Progress in studies on C. macrophyllum has been very slow compared with that
in studies on other model plants with a reference genome. Access to genomic data
is crucial for comprehending and expanding the study of a species. Transcriptome se-
quencing is more affordable and suitable for studying the genomes of non-model plant
species than whole-genome sequencing [54]. In this study, the transcriptome sequenc-
ing of C. macrophyllum generated 40,507,062 high-quality clean reads (93.00% Q30), which
were assembled into 29,477 non-redundant unigenes with an N50 of 1646 bp and an
average length of 1341.32 bp. The current results were comparatively better than those
previously reported for Actinidia eriantha (average length = 594 bp, N50 = 973 bp) [22]
and Panax vietnamensis (average length = 598.32 bp, N50 = 942 bp) [55] and similar to
Genes 2023, 14, 279 13 of 17

those reported for Pistacia chinensis (average length = 1325 bp, N50 = 2027 bp) [56] and
P. vietnamensis var. fuscidicus (average length = 1304 bp, N50 = 2108 bp) [57]. Compared
with C. aureobracteatum (70,753,963 bp total assembled bases), we obtained more assembled
bases in C. macrophyllum (99,257,989 bp total assembled bases) [32]. These findings indi-
cated that the quality of sequencing and assembly was high and can meet the requirements
of subsequent transcriptomic data analysis.
Among the 29,477 unigenes, 11,478 (38.94%) were successfully annotated in the pub-
lic protein databases of NR, KOG, Swiss-Prot, KEGG, and GO. The annotated unigenes
could provide valuable information for future studies on C. macrophyllum. The remaining
unmatched unigenes in the protein databases may be incomplete sequences lacking key
information for annotation and/or the genes specific to C. macrophyllum without previous
characterization. The BLASTX search against the NR database revealed that although only
7.83% of the identified unigenes of C. macrophyllum were similar to those of V. vinifera,
it was the species with the largest number of hits for C. macrophyllum unigenes. In fact,
C. macrophyllum and V. vinifera are members of Saxifragaceae and Vitaceae, respectively,
and are therefore genetically and evolutionarily distant from each other. This result may
be attributed to the lack of whole-genome sequences for any species of Saxifragaceae in
public databases. The division of the identified unigenes into 25 subterms and 57 subcate-
gories in the GO and KOG databases suggested that the annotated unigenes have a wide
range of important functions in C. macrophyllum. A total of 2020 unigenes were mapped to
127 biological pathways, among which the metabolism category was the largest, followed
by the genetic information processing category. These data revealed the active metabolic
processes and the synthesis of various metabolites. In C. nudicaule, C. carnosum, and other
Chrysosplenium species, flavonoids and triterpenoids are the main active components; these
components help in resistance against biological and environmental stresses, such as cold,
drought, and pests [10,58,59]. In this study, we recorded the unigenes for the terpenoid
backbone biosynthesis pathway.
In this study, 5573 unigene genes contained 6985 SSR loci with the distribution fre-
quency and density of 23.46% and 5.67 kb, respectively. The rate of distribution frequency
found in this work was higher than that reported for Epimedium sagittatum (3.67%) [60]
and Phyllostachys violascens (13.83%) [17] but lower than that reported for Phoebe bournei
(55.57%) [61]. The abundance and distribution of SSRs are influenced by numerous fac-
tors, including species differences, SSR search criteria, dataset size, SSR development tools,
and sequence redundancy [56,62,63]. The SSR types in the transcriptome of C. macrophyllum
were relatively abundant, ranging from mononucleotide repeats to hexanucleotide repeats.
Consistent with the EST-SSR distribution reported in C. aureobracteatum [32], the dinu-
cleotide (33.34%) and trinucleotide (19.18%) repeats became dominant when mononu-
cleotides were excluded. Of the mononucleotide motifs, A/T (45.38%) motifs were far
more abundant than the G/C (0.70%) motif, as in most plants [64]. Among dinucleotide
repeats, AG/CT (13.97%) was the most abundant; this result was identical to previous
findings on monocots and eudicots [65,66]. AT/TA (6.09%) and AC/GT (2.21%) were
the next most abundant motifs. In C. macrophyllum, the most predominant trinucleotide
repeat motif was ATC/ATG (4.31%), followed by AAG/CTT (4.27%). In contrast to those
in C. macrophyllum, the most frequent trinucleotide repeat motifs were AGG/CCT in
Z. officinale [23], AAG/CTT in E. sagittatum [60], and CCG/GGC in Elymus sibiricus [67].
Previous studies on other species indicated that the trinucleotide motif AAG/CTT is a
major motif and that CCG/CGG is a rare motif in dicotyledonous plants, but is a common
motif in monocots [68]. In this study, the trinucleotide CCG/CGG motif (0.30%) was the
least abundant trinucleotide repeat, likely due to the high GC content and consequent
codon usage bias in monocots [69,70].
We successfully designed 3127 (44.77%) primer pairs out of 8658 EST-SSR candidate
loci. The failure of primer design for the remaining SSR loci may be due to the short flanking
sequences of the SSR loci or the inappropriate motif of the required SSR markers. Among the
58 primer pairs selected, 39 (67.24%) resulted in successful amplification in C. macrophyllum,
Genes 2023, 14, 279 14 of 17

among which 33 (56.90%) were polymorphic. The rate of polymorphism in this species
was lower than in Vigna mungo (58.2%; n = 18) [71] but higher than in R. roxburghii (29.4%;
n = 16) [24]. Therefore, in this study, the rate of EST-SSR polymorphisms was relatively high.
The transferability of markers corresponds to the similarity of genomes, which can reflect
the genomic relationships and even the evolutionary relationships between species [72].
In general, close genetic relationships among different species are expected with the high
transferability of EST-SSR markers. In this study, the transferability of the 39 EST-SSRs from
C. macrophyllum to C. hydrocotylifolium was the highest, suggesting that C. macrophyllum had
a closer relationship with C. hydrocotylifolium than with other Chrysosplenium species. This
result was consistent with the close phylogenetic relationship between the two species [5].
Significantly, only 3 (7.69%) out of 39 EST-SSR markers failed to amplify successfully in
all 16 Chrysosplenium species. The high transferability of the markers indicated that the
flanking sequences of EST-SSRs were highly conserved among related species. These results
suggest that the markers developed in our study may provide a powerful molecular tool
for the evolutionary adaptation and phylogenetic analyses of C. macrophyllum and other
species of Chrysosplenium.
In this study, the samples were subdivided into two main groups on the basis of
STRUCTURE analysis, and the phylogenetic analysis of the NJ tree and PCoA analysis
supported the two genetic clusters. The species from the YS, LA, and PA populations
were allocated into one cluster, and geographically originated from the Ta-pieh Mountains,
Tianmu Mountains, and Dapan Mountains, respectively. The classification of species
from the same area into one group is correlated with the geographical distribution and
environmental conditions. Geographic isolation may have contributed to the genetic
differences. In addition, the population structure, NJ tree, and PCoA based on the genotypic
data clearly showed obvious genetic differentiation among C. macrophyllum species. The set
of EST-SSRs obtained in this work would facilitate the diversity analysis of C. macrophyllum.

5. Conclusions
The de novo transcriptome sequencing of C. macrophyllum was performed by using
the DNBSEQ-T7 sequencing platform. We obtained a large number of ESTs and identified
6985 EST-SSRs. Our results provided a potential pool of 3127 non-redundant EST-SSR
markers for C. macrophyllum. The developed EST-SSR markers had high amplification rates
and cross-genome transferability of various Chrysosplenium species. Furthermore, 10 EST-
SSR markers were used to analyze the genetic diversity of 60 C. macrophyllum individuals.
Our results showed that the populations of C. macrophyllum had a high level of genetic
diversity. Cluster analysis demonstrated that all 60 individuals clustered into two groups,
mainly in accordance with their origins. These transcriptome data will provide genetic
resources for the functional study of C. macrophyllum. The numerous EST-SSR markers
developed in this study represent a valuable tool for the genetic diversity and evolutionary
analyses of C. macrophyllum and other Chrysosplenium species.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/genes14020279/s1, Table S1: Cross-species amplification of the
39 microsatellite loci in Chrysosplenium; Table S2: Characteristics of polymorphic SSR loci tested
in 60 individuals of C. macrophyllum; Table S3: Nei’s genetic distance among 12 populations of
C. macrophyllum; Table S4: Analysis of molecular variance for C. macrophyllum populations.
Author Contributions: Conceptualization, N.X., R.Q. and X.L.; methodology, N.X.; software, B.L.,
T.Y. (Tao Yuan) and T.Y. (Tiange Yang); validation, N.X., T.Y. (Tao Yuan) and J.G.; formal analysis, N.X.,
T.Y. (Tao Yuan) and T.Y. (Tiange Yang); resources, R.Q.; data curation, N.X.; writing—original draft
preparation, N.X.; writing—review and editing, Z.W.; visualization, N.X., B.L. and H.L.; supervision,
Z.W. and H.L.; project administration, R.Q.; funding acquisition, H.L. and R.Q. All authors have read
and agreed to the published version of the manuscript.
Genes 2023, 14, 279 15 of 17

Funding: This research was funded by the National Natural Science Foundation of China (Grant
No. 32170207), the Construction Plan of Hubei Province Science and Technology Basic Conditions
Platform (Grant No. 2021DFE021), and the “High-Level Talents Training Program” for postgraduates
of Tibet University (Grant No. 2020-GSP-B008). All funders mentioned provided financial support
for our study.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Transcriptome data created from this research submitted to NCBI
under the SRA ID (SRR23148709), BioProject ID (PRJNA925660) and BioSample ID (SAMN32803129).
Acknowledgments: We are grateful to the National Nature Reserve of Mount Qizimei, National
Nature Reserve of Badong Golden Snub-Nosed Monkey, National Nature Reserve of Guanshan,
National Nature Reserve of Mount Babian, National Nature Reserve of Minjiang Origin, National
Nature Reserve of Mount Dapan, National Nature Reserve of Mount Tianmu, National Nature
Reserve of Mount Jiugong, National Nature Reserve of Mount Xingdou, and National Nature Reserve
of Mount Badagong for their support of our field work and for issuing relevant permits.
Conflicts of Interest: The authors declare no conflict of interest.

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