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The chloroplast genome features and phylogenetic
relationships of Platycarya longipes
(Juglandaceae), an important woody species within
karst forests of eastern Asia
Yingliang Liu
Guizhou Normal University
Lijuan Hu
Guizhou Normal University
Xiaoshuang Wang
Guizhou Normal University
Ya Tan
Guizhou Normal University
Lei Gu ( leigu1216@nwafu.edu.cn )
Guizhou Normal University
Article
Keywords: Chloroplast, Platycarya longipes, Genome comparsion, Illumina reads, Juglandaceae
Posted Date: May 9th, 2022
DOI: https://doi.org/10.21203/rs.3.rs-1602797/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
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Abstract
Platycarya longipes
of the Juglandaceae family is an important woody species in maintaining the
stability of community structure of karst forests. However, its phylogenetic relationship within
Juglandaceae is still unclear. In this study we assembled the complete cp genome of
P. longipes
. The
genome comprises a 158,592 bp quadripartite circular that includes a large single copy (LSC) region of
88,066 bp and a small single copy (SSC) region of 18,524 bp separated by a pair of inverted repeats (IRA
and IRB) with 26,001 bp. The genome contains 113 unique genes, including 80 protein-coding genes, 29
tRNAs and 4 rRNAs. Additionally, we detected 49 long repeat sequences and 66 simple sequence repeats
(SSRs). Analysis of the Ka/Ks substitution rate values in the comparison of
P. longipes
VS.
Platycarya
strobilacea
, supported that
P. longipes
and
P. strobilacea
are two species. Compared with other species of
Juglandaceae, the cp genome of
P. longipes
has a conserved gene order and structure. Phylogenetic
analysis based on ML and BI methods using genomes of the Fagales order showed that
P. longipes
is
most closely related to
Platycarya strobilacea
. Our research provides a critical genetic resource for
P.
longipes
supporting future phylogenetic and population genetics studies.
Introduction
The karst landscape results from the action of rainfall and groundwater on carbonate bedrock1 and is
widespread globally, accounting for 12% of the world land area2. More karst landscape occurs in China
than anywhere else in the world, and it is mainly distributed in mountainous regions in the south-western
part of the country, particularly in the province of Guizhou3,4. Karst regions generally contain fragile
ecosystems due to soils that form extremely slowly, have weak water retention capacity, and have
shallow, patchy coverage. Karst ecosystems are maintained in part by karst forests, which provide
valuable ecosystem services5, and within these forests, woody species comprise vital biodiversity6,7.
Therefore, understanding the genetic diversity and phylogenetic relationships of woody species of karst
forests is critical for modern approaches to management and conservation.
Juglandaceae, the walnut family, comprises nine genera and 71 species of which seven genera and 27
species occur in karst regions of China8. Thus, this family plays an important role in maintaining the
community structure of karst forest ecosystems especially due to adaptations of species to the
challenging edaphic environment4.
Platycarya longipes
, as a member of Juglandaceae family, is widely
distributed in karst forests of southern China and represents a critical element within the karst
ecosystem4. Additionally, this species is valued for its bark and leaves, which is rich in gallic and ascorbic
acid9–12 and consequently, has antioxidant and pro-oxidant properties13. Nevertheless, despite the
ecological and medicinal importance of
P. longipes
, there have been no studies of its plastid genome,
genetic diversity, or phylogenetic relationships with other species of Juglandaceae or the Fagales order to
our knowledge.
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The chloroplast (cp) genome, which is maternally inherited in angiosperms, is highly conserved in gene
content and genome structure14 and is an ideal system for deciphering genome evolution15,16, performing
DNA barcoding, and inferring phylogenetic relationships in angiosperm families that have evolutionary
histories recalcitrant to traditional morphological approaches or molecular phylogenetic approaches
using a few DNA markers17–22. The cp genome of angiosperms generally comprises a quadripartite,
circular molecule including one large single copy (LSC) region and one small single copy (SSC) region,
which were separated by two inverted repeat regions (IRA and IRB)23. Most cp genomes range from 120
to 160 kb in length and harbor 110–130 unique genes that are essential to photosynthesis and the
biosynthesis of starch, amino acids, fatty acids, and pigments24. Recently, owing to the advances of high-
throughput sequencing, thousands of cp genome sequences are now publicly available via the National
Center for Biotechnology Information (NCBI), since the rst complete chloroplast genome was sequenced
in tobacco (
Nicotiana tabacum
L.) in 198625. Among these, the cp genome of
Platycarya strobilacea
(KX868670) has provided valuable information for resource conservation9. However, the cp genome of
P.
longipes
has not been sequenced.
In this study, we assembled the complete cp genome of
P. longipes de novo
from Illumina short reads.
Within the assembled cp genome, we identied a total of 66 simple sequence repeats (SSRs) loci and 49
long duplicates repeats. We used the complete chloroplast genome sequence of
P. longipes
and related
species of Fagales to perform phylogenetic analysis by ML and BI methods. Overall, our results provide
valuable information for the further development of genetic resources to support ecological and
evolutionary studies of
P. longipes
and its close relatives.
Materials And Methods
Ethics statement
During the leaf samples collection, no harms was done to the environment, this study did not involve
endangered or protected species, and no specic permits were required for collection.
Plant materials and sequencing
We collected a total of 5g of young fresh leaves of
P. longipes
on campus at Guizhou Normal University
of China (26°23'.12"N, 106°38'32" E). We extracted total DNA from the leaves using the DNeasy Plant
Mini Kit (Qiagen, USA) according to manufacturer instructions and assessed the quality and quantity of
the DNA by agarose gel electrophoresis. We used the extracted DNA to construct a library from fragments
~ 450 bp in size for the Illumina HiSeq X Ten (Illumina, USA) platform following manufacturer’s protocols.
Genome assembly and gene annotation
We obtained 150 bp paired-end reads through Illumina HiSeq X Ten sequencing. After removing
sequencing adapters and low-quality reads, we selected out sequences representing the cp genome by
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aligning reads to the closely related species,
P. strobilacea
9 using BLASR 26 with default parameters. We
used the selected reads to construct the draft cp genome of
P. longipes
in SOAPdenovo (v2.04)27,
performed sequence extension in SSPACE28, and accomplished gap lling in GapCloser using default
parameters 29.
Then we employed the software of Dual Organellar GenoMe Annotator (DOGMA)30 to annotate the genes
within the cp genome, including protein-coding genes, tRNAs, and rRNAs, and we manually identied
coding sequence boundaries according to the positions of start and stop codons. We used OGDraw
v1.231 to circularize the annotated gene map, and we deposited the annotated cp genome of
P. longipe
s
in GenBank (accession number MT032191).
Identication of long repeat sequences and simple
sequence repeats
We used the REPuter webserver (https://bibiserv.cebitec.uni-bielefeld.de/reputer/)32 to identify long
repeats of at least 30 bp, with sequence identity above 90% or greater including forward, palindrome,
reverse, and complement repeats. We detected simple sequence repeats (SSR) using Misa-web
(https://webblast.ipk-gatersleben.de/misa/)33 with the following settings: ten minimal repeats for mono-
nucleotides, ve for di-, four for tri-, and three for tetra-, penta-, and hexa- nucleotides.
Analysis of codon usage
Analysis of codon usage not only reects the origin, evolution and mutation mode of species or genes,
but also has an important inuence on gene function and protein expression34–36. CodonW1.4.2
(http://downloads.fyxm.net/CodonW-76666.html) was used to calculate the relative synonymous codon
usage (RSCU) of
P. longipes
chloroplast protein-coding genes under the default parameters.
Comparisons of the whole cp genomes of related species
We compared sequence divergence of the complete cp genome of
P. longipes
with
Carya illinoinensis
,
Castanopsis echinocarpa
,
Cyclocarya paliurus
,
Juglans hopeiensis, Quercus acutissima
and
P.
strobilacea
using mVISTA in the Shue-LAGAN mode37. The SNPs and indels between the
P. longipes
and
P. strobilacea
cp genome were detected by Mummer3.23 with the default settings (maxgap = 500,
mincluster = 100). Additionally, we visualized comparisons of the LSC/IRB/SSC/IRA junctions in seven
species of Juglandaceae, including
C. illinoinensis
,
C. paliurus
,
J. hopeiensis
,
J. cinerea
,
J. major
,
P.
strobilacea
, and
P. longipes
, according to their annotations of chloroplast genomes deposited in GenBank
using IRscope (https://irscope.shinyapps.io/irapp/).
Molecular evolution analysis
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To assess the synonymous (Ks) and nonsynonymous (Ka) substitution rates, We calculated pairwise
comparisons of 62 commonly conserved protein-coding genes between
P. longipes
and the six closely
related species mentioned above in mVISTA analysis, and the Ka/Ks rations were computed by TBtools38
using the default parameters of Simple Ka/Ks calculator mode.
Phylogenetic analysis
We obtained a total of 31 cp genomes (nucleotide level) of the Fagales including 15 species of
Juglandaceae, four species of Fagaceae, and 12 species of Betulaceae from GenBank and used these
together with
P. longipes
for phylogenetic reconstruction. The complete chloroplast genome sequence of
these 32 species were aligned using the MAFFT software with default parameters, we performed
phylogenetic reconstruction of the selected species of Fagales in MEGA7.039 using the maximum
likelihood (ML) method based on the Tamura-Nei model. And 1000 bootstrap replicates were set to infer
node support, branches corresponding to partitions reproduced in less than 50% bootstrap replicates are
collapsed. Meanwhile, the Mrbayes 3.2.740 under GTRGAMMA model was used to construct a
phylogenetic tree with the Bayesian inference (BI) method, four chains of the Markov Chain Monte Carlo
were run each for 1,000,000 generations and were sampled every 100 generations.
Results
Assembly and features of the
P. longipes
cp genome
We obtained a total of 8.46 Gb raw reads from Illumina sequencing platform. After trimming, we retained
1.15 Gb of clean reads, from which we performed
de novo
assembly of the complete cp genome of
P.
longipes.
The cp genome showed a typical circular quadripartite structure that was 158,459 Qbp in
length, contains 113 unique genes, including 80 protein-coding genes, 29 tRNAs and 4 rRNAs. It included
a large single copy (LSC) region of 87,898 bp, a small single copy (SSC) region of 18,521 bp, which were
separated by two inverted repeats (IRa and IRb) having a total of 26,020 bp. The overall GC content of the
P. longipes
cp genome was 36.16%. The two IR regions had the highest GC content of 42.54%, followed
by 33.76% in the LSC region, and 29.67% in the SSC region (Table 1; Fig. 1).
Among the 113 unique genes, ten genes, comprising four protein-coding genes and six tRNA genes, had
one intron; and only two genes (ndhB and trnR-UCU) possessed two introns (Table 2).
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Table 1
Summary of the complete chloroplast genomes of
P. longipes
and ve closely related species
Genome
Features
P. longipes P.
strobilacea C.
illinoinensis J.
hopeiensis C.
paliurus Q.
acutissima
Length
(bp) 158,459 160,994 160,819 159,714 160,562 161,129
GC
content
(%)
36.16 36.04 36.14 36.14 36.08 36.78
LSC
length
(bp)
87,898 90,225 90,042 89,316 90,007 90,423
LSC GC
content
(%)
33.76 33.59 33.74 33.71 33.66 34.62
SSC
Length
(bp)
18,521 18,371 18,791 18,352 18,477 19,070
SSC GC
content
(%)
29.67 29.72 29.89 29.79 29.71 31.31
IR
length
(bp)
26,020 26,199 25,993 26,023 26,039 25,817
IR GC
content
(%)
42.54 42.47 42.58 42.56 42.55 42.77
Total
genes 113 112 107 112 116 114
Protein
genes 80 79 77 79 81 79
tRNA
genes 29 29 26 29 31 31
rRNA
genes 4 4 4 4 4 4
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Table 2
Gene composition in the chloroplast genome of
P. longipes
Category of
genes Group of genes Name of genes
photosynthesis Subunits of
NADH-
dehydrogenase
ndhJ, ndhK, ndhC, ndhBa,c
,
ndhH, ndhA, ndhI, ndhG,ndhE, ndhD,
ndhF
Large subunit
of Rubisco
rbcL
Subunits of
photosystem
psbA, psbK, psbI, psbD, psbC, psbZ, psbF, psbE, psbB, psbH
Subunits of
photosystem
psaB, psaA, psaI, psaJ, psaC
Subunits of
ATP synthase
atpA, atpF, atpH, atpI, atpE, atpB
,
Subunits of
cytochrome
b/f complex
petA, petB, petD, petL ,petG
photosystem
assembly
ycf3b
,
ycf4
Self-replication Ribosomal
RNA genes
rrn16a
,
rrn23a
,
rrn4.5a
,
rrn5a
Transfer RNA
genes
trnG-GCC, trnS-GGA, trnL-UAAb
,
trnF-GAA, trnM-CAU, trnI-GAUb
,
trnA-UGCa,b
,
trnR-ACGa
,
trnN-GUUa
,
trnR-UCUa
,
trnC-GCA, trnT-
GGU, trnS-UGA, trnE-UUC, trnY-GUA, trnD-GUC, trnS-GCU, trnQ-
UUG, trnH-GUG, trnV-GACa
,
trnI-GAUa,b
,
trnA-UGCb
,
trnR-ACG, trnL-
UAG, trnR-UCUc
,
trnL-CAAa
,
trnM-CAU, trnP-UGG, trnW-CCA, trnC-
ACAb
,
trnT-UGU
Small subunit
of ribosome
Large subunit
of ribosome
rps16b
,
rps2, rps14, rps4, rps18, rps11, rps8, rps3, rps19, rps7,
rps15, rps7a
,
rps12b
rpl33, rpl20, rpl14, rpl16, rpl22, rpl2a
,
rpl23a
a indicates genes duplicated in the IR regions
bindicates the genes containing a signal intron
cindicates the genes containing two signal introns
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Category of
genes Group of genes Name of genes
DNA-
dependent
RNA
polymerase
rpoC2, rpoC1, rpoB, rpoA
Translation
initiation factor
infA
Other genes Maturase
matK
Subunit of
acetyl-CoA
accD
Protease
ClpPb
Envelope
membrane
protein
cemA
C-type
cytochrome
synthesis
ccsA
Functionally
unknown
genes
Conserved
Open reading
frames
ycf1, ycf2a
a indicates genes duplicated in the IR regions
bindicates the genes containing a signal intron
cindicates the genes containing two signal introns
Detection of long repeat sequences and SSRs
We detected a total of 49 long repeats in the cp genome of
P. longipes
ranging from 37 to 78 bp in length.
These included 32 forward, 13 palindromic, and four reverse repeats, but we detected no complement
repeat was detected. Most repeats (34, 69.39%) were located in intergenic spacer (IGS) regions, 14
repeats (28.57%) occurred within coding sequences (CDS), and 11 repeats (22.45%) were in introns (Table
S1). Among these repeats, 10 were of 30–39 bp in size, 14 were 40–49 bp, 13 were 50–59 bp, nine were
60–69 bp, and three were 70–79 bp (Table S1).
In the complete cp genome of
P. longipes
, we detected 66 SSR loci of 15 different types with lengths of at
least 10 bp, including 47 mononucleotides, 11 dinucleotides, three trinucleotides, four tetranucleotides,
and one pentanucleotide (Table S2). Of the 47 mononucleotides, 46 were A or T types, and only one was
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a G type as is consistent with observations in other cp genomes of angiosperms21,22,41. Among the
dinucleotide repeats, AT (6, 54.5%) was observed more frequently than TA, AG, CT and TC, the
trinucleotides repeats comprised ATT and TAT, the tetranucleotides were TTTA, AATA, CTTT and AAAG,
and the pentanucleotide was AATAT. Out of the 66 SSRs, 51 SSR loci occurred in the LSC region (77.27%),
nine in the SSC region (13.64%), and six among the two IR regions (9.09%) (Table S2). 14 identied SSRs
were within the coding regions, while 51 were located in the intergenic regions and only one was located
in the intron regions.
Codon usage analysis
The codon usage frequency and RSCU were analyzed based on the sequence of 80 protein-coding genes
in the
P. longipes
chloroplast genome (Figure S1), a total of 25529 codons were detected. The statistics
analysis of all protein-coding cpDNA and amino acid sequences showed obvious codon preferences. Of
these codons, 2693 (10.54%) encoded leucine, whereas only 298 (1.16%) encoded cysteine, indicating the
most and the least frequently used amino acids in the
P. longipes
cp genome, as observed in the
plastomes of other angiosperms such as the early diverging species42. The codon usage frequency and
RSCU were used as a relative intuitionistic to measure the extent of codon bias43, based on sequences of
80 distinct protein-coding genes in the
P. longipes
chloroplast genome. The results showed that the AUU
had the highest frequencies and the UGC had the lowest frequencies. 20 amino acids were encoded by 61
codons, the RSCU value of 31 codons were > 1, indicating that these codons exist preference. Moreover,
among the preferred codons, except UUG and UCC, all of the preferential codons ended with A/U,
supporting the idea that such biased usage of certain degenerate codons was likely a result of adaptive
evolution of cp genome.
Analysis of genome divergence
We determined genomic similarity and divergence among
P. longipes
and six related species in mVISTA,
using the cp genome of
P. longipes
as a reference. The result showed that more than 95% of regions were
well conserved among these species, indicating a high degree of sequence similarity. In addition, the non-
coding regions are more variable than coding regions, however, we observed lower levels of sequence
conservation in
rp122
,
rpoC1
, and
petD
(Fig. 2).
A total of 2667 (616 SNPs and 2051 indels) variable sites were observed between the
P. longipes
and
P.
strobilacea
chloroplast genomes, among them, 2.40% variations (1712 SNPs and 401 indels) were within
the LSC region, 2.04% (213 SNPs and 165 indels) were within the SSC region, while 0.34% (126 SNPs and
50 indels) were within the region of IRs (Figure S2). The results suggested that the IR regions were more
conserved than SC regions in the cp genome of
Platycarya
. In spite of this, the chloroplast genome
sequences of
P. longipes
and
P. strobilacea
still showed signicant differences.
Comparison of boundaries regions
We used seven cp genomes of species of Juglandaceae to compare the boundaries of the SSC, LSC, and
IR regions using the IRscope webserver. The result showed that the size of the IR was highly conserved,
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ranging from 25,993 bp to 26,199 bp and that the genes located in the LSC/IRb and SSC/IRa border
regions were also highly conserved. In particular, the LSC/IRb boundaries were located between
rps19
and
rpl2
genes in all seven cp genomes, and the IRa/SSC boundaries were located within the pseudogene
ycf1
. However, genes in IRb/SSC and IRa/LSC junctions were inconstant (Fig. 3). The IRa/LSC border was
located between
rpl2
and
trnH
genes in ve of the cp genomes, including
P. longipes
,
P. strobilacea
,
C.
illinoinensis
,
C. paliurus
, and
J. hopeiensis
, whereas the boundary was between
rpl23
and
trnH
in
J.
cinerea
and
J. major
. In
P. longipes
,
P. strobilacea
, and
C. paliurus
, the border of IRb/SSC was located
between
ycf1
and
ndhF
genes, however, either
ycf1
or
ndhF
gene was absent from IRb in the other four cp
genomes.
Phylogenetic analysis
Chloroplast genomes have been widely used to determine the phylogenetic relationships because they
are highly conserved in terms of gene size and content, genome structure, and linear order of the genes.
We employed 32 selected species of Fagales (Table S3) for phylogenetic reconstruction. The Maximum
Likelihood phylogenetic tree possessed a total of 28 branches with bootstrap values of above 85%.
Among these branches, 26 branches were supported by values above 90% (Fig. 4A). As expected,
P.
longipes
was most closely related to the congeneric species,
P. strobilacea.
The genus
Platycarya
formed
a monophyletic clade with 100% bootstrap support, showed the most closed relationship to
Cyclocarya
genus. Moreover, both the ML and BI phylogenetic (Fig. 4B) tree showed nearly identical topologies in
identifying the taxonomic status of 32 species.
Analysis of selection pressure
The Ka/Ks ratio is widely used to infer rates of genomic evolution and selection pressure on individual
genes44–46. The ratio of Ka/Ks < 1, Ka/Ks = 1, and Ka/Ks > 1 indicate that genes underwent purifying,
neutral, and positive selection, respectively39. In this study, we calculated the pairwise Ka/Ks ratios of 62
common protein-coding genes between the
P. longipes
cp genome and six related species (Table S4),
including
C. illinoinensis, C. echinocarpa, C. paliurus, J. hopeiensis, Q. acutissima
and
P. strobilacea.
Overall, the average Ka/Ks value of these genes in the seven genomes was 0.246. The majority of
common genes (40 of 62 genes) had an average Ka/Ks ratio of 0 and 0.3 when compared to
P. longipes
,
suggesting that these genes were subject to strong purifying selection. The average Ka/Ks ratio of all
comparisons of the
atpF
gene was 1.52, ranging from 0.668 (
P. longipes
vs.
P. strobilacea
) to 1.863 (
P.
longipes
vs.
C. paliurus
and
P. longipes
vs.
J. hopeiensis
), indicating that this gene has undergone strong
positive selection. Moreover,
matK
,
rpoA
,
petD
,
atpF
,
rpl22
, and
ycf2
also exhibited high ratios, with Ka/Ks
> 0.5 among the six pairwise comparisons (Table S4, Fig. 5).
Comparison analysis of SSR and long repeats
Simple sequence repeats (SSRs), also known as microsatellites, are frequently used as molecular
markers in population genetics and evolutionary studies of higher eukaryote genomes15. In the present
study, we detected complete SSRs among the six cp genomes of species of Fagales (Fig. 6), the results
revealed a total of 66, 61, 62, 72, 78 and 83 SSRs in the
P. longipes
,
C. illinoinensis
,
P. strobilacea
,
J.
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cinerea
,
Corylus yunnanensis
and
Q. acutissima
cp genomes, respectively.
Q. acutissima
of Fagaceae
had the largest number of SSRs, followed by
C. yunnanensis
of Betulaceae. Similarly, hexanucleotide
SSRs (AACAGA and TTTTAT) were detected in the cp genome of
C. yunnanensis
and
Q. acutissima
but
not in the family of Juglandaceae (
P. longipes
,
C. illinoinensis
,
P. strobilacea
and
J. cinerea
). Furthermore,
we observed a signicantly larger number of A and T microsatellites than G and C as expected based on
reports from other species of angiosperms47–49. These results suggest that SSRs can be used to conduct
evolutionary analysis and are powerful for identifying the genetic diversity among different species.
Longer repeat sequences facilitate base substitutions, evolution of genome size, and genomic
rearrangements in cp genomes and are useful for phylogenetic studies50,51. We detected a total of 294
long repeat sequences across the six genomes with a length distribution of 30–109 bp, most of them
were 30–60 bp long and accounted for 87.41% of the total, and two duplicates with a length greater than
100 were only detected in
J. cinera
. Each species possessed 49 long repeats, the number of F (Forward,
156) and P (Palindromic, 110) reached 266 among four types of repetition, accounting for 90.48% of the
total, and we detected only one complement repeat, which was in
C. illinoinensis
(Fig. 7). The number and
pattern of repeat sequences were highly similar and conserved within the six cp genomes of Fagales.
Taken together, the long repeats and SSRs may represent valuable lineage-specic markers for
population biology and molecular phylogenetic studies in this plant order41,48.
Discussion
Genome features
In general, the size of cp genomes in photosynthetic land plants ranges from 108 kb to 165 kb47,52−54,
most cp genomes of the angiosperm are considered to be conserved. The size of the cp genome of
P.
longipes
was 158,459 bp and is similar to the sizes of cp genomes previously reported in other species of
Juglandaceae, such as
C. illinoinensis
(160,819 bp),
P. strobilacea
(160,994 bp),
J. hopeiensis
(159,714
bp), and
C. paliurus
(160,562 bp). Among the species we compared,
Quercus acutissima
of Fagaceae had
the largest cp genome (161,129 bp), indicate that the length of cp genomes within Juglandaceae family
is conservative. The LSC regions in the genomes compared were varied from 88,066 bp to 90,423 bp in
lengths, the SSC ranged from 18,352 bp to 19,070 bp, and the IR regions were from 25,817 bp to 26,199
bp (Table1). Notably,
Q. acutissima
has the longest overall length (161,129 bp) but the shortest IR
regions (25,817 bp), which may be attributed to the contraction of the IR regions. The overall GC content
of these cp genomes was approximately 36% and was unevenly distributed among the LSC, SSC, and IR
regions, which had 34%, 30%, and 42% GC content, respectively. Compared with the LSC and SSC regions,
the GC content is greater in IR regions of all Fagales, this unequal distribution of GC content is typical for
angiosperms55,56, in which the presence of ribosomal RNA (rRNA) sequences appears to increase the GC
content of the IR regions57,58.
Page 12/25
The expansion and contraction of the IR regions was the main reasons for variation of cp genomes size,
and evaluating this difference could reveal the evolution of related taxa59,60. The size of IR regions was
relatively conserved, but there were some differences in adjacent genes and junctions. The junctions of
P.
longipes, P. strobilacea
and
C. paliurus
were nearly identical with only slight differences in the distance of
the boundary, whereas there were signicant differences in the boundaries of genes in
P. longipes
compared to
C. illinoinensis, J. hopeiensis, J. cinerea
, and
J. major
. Although there were some changes in
the cp IR boundary regions, the size of the overall genome, base composition of the LSC, SSC and IR
regions of
P. longipes
was similar to those closely related species. Based on comparisons of the complete
cp genome of studied species, the number of genes, genome size, gene order and genome structure were
similar, this further indicates that cp genomes are generally conserved.
Codon usage bias and selection pressure
Codon usage bias was considered to be the consequence of the balance between gene mutation and
natural selection. Generally, the GC content at the rst, second and third base positions per codon is
largely different, and it is consider that the rst base position has the highest GC content, following by
second and third position61. Additionally, the dicot plants mostly ending with A or T, while the monocot
plants mostly ending with G or C62. The analysis of codon usage revealed that codons encoding proteins
in
P. longipes
chloroplast genomes tend to end with A/T, this result is consistent with previous
studies63,64. The GC content varies differently in three positions, indicating the chloroplast genome in
P.
longipes
mostly affected by natural selection, while little affected by gene mutations or other factors.
The synonymous and nonsynonymous substitution incidents were widely occured in the process of gene
evolution, which can be used, to evaluate the rates of genomic evolution and determine whether the
protein-coding gene has a selective effect. It is believed that the
Platycarya
genus comprises of two
closely related species,
P. longipes
and
P. strobilacea
, for a long time65. Chen et al. implemented a
phylogeographical study on
P. strobilacea
using
psbA-trnH
and
atpB-rbcL
intergenic spacer sequences of
cpDNA to demonstrate that
Platycarya
is likely a monotypic genus66. But a later study which employed
both nuclear genetic marker and cpDNA marker showed that the interspecic genetic divergence was
more tting with 'two species' scenario67. In the present study, the cp genome of
P. longipes
has 158,592
bp in length, shorter than the cp genome of
P. strobilacea
(160,994 bp in length)9. Additionally, the Ka/Ks
values of these genes (
ycf3
,
rpoB
,
rpl2
,
matK
,
accD
,
petD
, and
clpP
) in the comparison of
P. longipes
VS.
P.
strobilacea
were even higher than the comparisons between
P. longipes
and other species in
Juglandaceae, likely supported the idea that
P. longipes
and
P. strobilacea
are two species. We noticed
that the
petD
gene, which controls the cytochrome b6/f complex, affecting photosynthetic eciency68,
always showed a signicant positive selection (average Ka/Ks value of 2.995) in
Platycarya
. This gene
can be considered as a glimpse of response of
Platycarya
on the drought habitat of karst. Moreover, most
genes involved in the functional category “Subunits of photosystem”, such as
psbA
,
psaC
,
psbE
,
psaB
,
psbC
and
psbD
genes, have undergone lower purifying selection pressure.
Relationship analysis
Page 13/25
Both ML and BI phylogenetic tree revealed that the 16 species representing Juglandaceae comprised of
multiple clades and that
P. longipes
was most closely related to
P. strobilacea
(Fig.4). The tree topology
was consistent with the traditional tribal-level classication and nuclear RAD-Seq data of
Juglandaceae24, 69. Furthermore, the ML and BI tree showed that Juglandaceae was more closely related
to Betulaceae than to Fagaceae, this is consistent with the ndings in prior studies70.
Conclusion
In this study, we assembled the complete chloroplast genome of
P. longipes
using a
de novo
approach
and found that it was consisted of 158,459 bp in total and exhibited a typical quadripartite, circular
structure comprising an LSC, SSC and two IR regions, including 80 protein-coding genes, 29 tRNAs and
four rRNAs. We detected 49 long repeats and 66 SSRs in the cp genome of
P. longipes
that may be useful
for development of molecular markers as well as phylogenetic and polpulation studies in
P. longipes
. Our
analyses of selection pressure revealed strong positive selection on
atpF
gene in
P. longipes.
The relative
high Ka/Ks values of
ycf3
,
rpoB
,
rpl2
,
matK
,
accD
,
petD
, and
clpP
were observed in the comparison
between
P. longipes
and
P. strobilacea
, likely support the idea that
P. longipes
and
P. strobilacea
were two
different species. The result of our phylogenetic analysis based on ML and BI method showed that
P.
longipes
was most closely related to the congeneric species,
P. strobilacea.
Our results provide insight
into the evolutionary relationships of Juglandaceae and genomic evolution in Fagales, as well as
represent a new genetic resource for future phylogenetic, taxonomic, ecological, population biology, and
conservation studies. However, it is limited to study the taxonomic status and phylogenetic relationship
of Fagales only based on chloroplast genome. With the development of high-throughput sequencing
technology, the nuclear genome information will also be integrated in future studies.
Declarations
Guidelines Statement:The collection of plant material is in comply with relevant institutional, national,
and international guidelines and legislation.
Data Availability Statement: The annotated chloroplast genome data that support the ndings of this
study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov under the accession
number MT032191.
Funding:This research was Supported by National Natural Science Regional Fund Project (31760124),
The Joint Fund of the National Natural Science Foundation of China and the Karst Science Research
Center of Guizhou province (Grant No. U1812401).
Author Contributions
Conceptualization:Lei Gu, Yingliang Liu
Data curation:Lijuan Hu,Xiaoshuang Wang
Page 14/25
Funding acquisition: Yingliang Liu
Resources: Xiaoshuang Wang, Ya Tan
Writing-review & editing:Yingliang Liu, Lijuan Hu, Lei Gu
Conicts of Interest:The authors declare no conict of interest.
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Figures
Page 19/25
Figure 1
Gene map of the complete chloroplast genome of
P. longipes.
Genes on the outside of the circle are
transcribed clockwise, while genes inside are counterclockwise. Genes belonging to different functional
groups are shown in different colors. The thick lines indicate the extent of inverted repeats (IRa and IRb),
which separated the genomes into large signal-copy (LSC) and small signal-copy (SSC) regions. In the
inner circle, the dark gray corresponds the GC content and the light gray corresponds to the AT content.
Page 20/25
Figure 2
Visualization of alignment of the seven Fagales chloroplast genome sequences using
P. longipes
as a
reference. Gray arrows and thick black lines above the alignment indicate gene orientation. Purple bars
represent exons, blue bars represent untranslated regions (UTRs), pink bars represent non-coding
sequences (CNSs), gray bars represent mRNA, and white peaks represent differences in genomics. The y-
axis represents the percentage identity (shown: 50–100%).
Page 21/25
Figure 3
Comparison of the borders of large signal-copy (LSC), inverted repeats (IRa and IRb) and small signal-
copy (SSC) between
P. longipes
and other six related species. Boxes above the main line indicate the
adjacent border genes. The gure is not to scale regarding sequence length, and only shows relative
changes at or near the IR/SC borders.
Page 22/25
Figure 4
The Maximum Likelihood (ML) phylogenetic tree (A) and Bayesian Inference (BI) phylogenetic tree (B)
were constructed based on complete chloroplast genome sequence of 32 species. 1,000 replicates were
tested to conrm the stability of each tree node, numbers at the left of nodes are bootstrap support
values. Four chains of the Markov Chain Monte Carlo were run each for 1,000,000 generations and were
sampled every 100 generations.
Page 23/25
Figure 5
The Ka/Ks ratios of 64 protein-coding genes of the
P. longipes
cp genome versus six closely related
species of Fagales.
Page 24/25
Figure 6
The type and size of simple sequence repeats among six chloroplast genomes. a. Numbers of SSRs
detected in six Fagales chloroplast genomes, b. Frequencies of identied SSRs in LSC, IR and SSC
regions, c. Numbers of SSR types detected in six Fagales chloroplast genomes.
Page 25/25
Figure 7
Analysis of long repeated sequences in the chloroplast genomes between
P. longipes
and other ve
Fagales species. a. frequency of repeat type, b,c. frequency of repeat length.
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