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https://doi.org/10.1007/s11295-022-01556-9
ORIGINAL ARTICLE
Chloroplast genomes andnuclear sequences reveal theinterspecific
relationships ofCrataegus bretschneideri C. K. Schneid. andrelated
species inChina
XiaoZhang1· XinyuSun2· TongLi1· JianWang1· MiliaoXue1· ChaoSun1· WenxuanDong1
Received: 27 January 2022 / Revised: 3 April 2022 / Accepted: 13 May 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Crataegus bretschneideri C. K. Schneid. is one of the species cultivated in China. Due to its unclear taxonomic classification
status, the conservation and utilization of this germplasm resource have been limited. In this study, we analyzed the chlo-
roplast genomes and nuclear sequences to reveal the taxonomic relationships among C. bretschneideri and related species.
We assembled the chloroplast genomes of C. bretschneider and related species and varieties, including C. maximowiczii C.
K. Schneid., C. maximowiczii var. ninganensis S. Q. Nie & B. J. Jen., C. pinnatifida Bunge, and C. pinnatifida var. major
N. E. Br. The lengths of the chloroplast genomes ranged from 159,644 bp (C. bretschneideri) to 159,947 bp (C. pinnatifida
var. major). The five Crataegus chloroplast genomes had similar features and possessed 86 to 88 protein-coding genes, 37
tRNA genes, and eight rRNA genes which were arranged in the same order. Eight mutation hotspot regions, including matk,
psaB, accD, petA, clpP, trnD-GUC , psbH-petB, and trnN-GUU-trnR-ACG could be used as potential molecular markers
for further studies of Crataegus genetic diversity. Phylogenetic analyses based on 17 chloroplast genomes of Crataegus and
Amelanchier indicated that C. bretschneideri was related to C. maximowiczii and C. maximowiczii var. ninganensis. However,
the phylogenetic trees constructed by nuclear sequences of 36 Crataegus accessions reflected a closer relationship between
C. bretschneideri and C. pinnatifida. Furthermore, divergence time estimation suggested that C. bretschneideri and C.
maximowiczii diverged in the late Miocene and that speciation of C. pinnatifida occurred during the middle to late Miocene.
These findings revealed that C. bretschneideri is an independent species and may be of hybrid origin.
Keywords Crataegus· C. bretschneideri· Chloroplast genome· Comparative genomics· Interspecific relationships
Introduction
The genus Crataegus belongs to the Rosaceae family. It is
widely distributed in northern temperate zones in eastern
North America, Europe, and East Asia (Phipps 1990; Chang
etal. 2002; Xu etal. 2016). Over 12 Crataegus species are
used as herbal drugs or drug materials worldwide (Chang
etal. 1986). More than 150 compounds, including steroids,
triterpenoids, flavonoids, and organic acids have been identi-
fied and isolated from the Crataegus plant, which benefits
the endocrine, digestive, and cardiovascular systems of the
Communicated by V. Decroocq
* Wenxuan Dong
dongwx63@syau.edu.cn
Xiao Zhang
zhangxiao8866@syau.edu.cn
Xinyu Sun
sunxinyu612@163.com
Tong Li
litong0327@126.com
Jian Wang
botelongma@163.com
Miliao Xue
1250164964@qq.com
Chao Sun
sun13940536629@163.com
1 College ofHorticulture, Shenyang Agricultural University,
Shenyang110866, LiaoningProvince, China
2 Tonghua Horticulture Institute, Tonghua134001,
JilinProvince, China
/ Published online: 25 May 2022
Tree Genetics & Genomes (2022) 18: 24
1 3
human body (Wu etal. 2014; Nazhand etal. 2020; Zhang
etal. 2020a).
China, a major origin of Crataegus, has a long history of
hawthorn cultivation (Guo and Jiao 1995). A total of 20 spe-
cies and seven varieties of Crataegus are widely distributed
across China (Dong and Li 2015). Among these Crataegus,
including C. bretschneideri C. K. Schneid., C. maximowiczii
C. K. Schneid., C. maximowiczii var. ninganensis S. Q. Nie
& B. J. Jen., C. sanguinea Pall., and some populations of
C. pinnatifida Bunge are naturally distributed in Northeast
China (Dong and Li 2015; Du etal. 2019). As a cultivated
species, C. bretschneideri has many excellent characteristics,
such as high yield and cold tolerance. The mature fruit of C.
bretschneideri is sweet and flavorsome with bright colors,
and it is popular among local consumers. Since most of the
C. bretschneideri are polyploidy, their original parents are
still not clear. C. bretschneideri has been recognized as a
taxonomically challenging species (Dong and Li 2015).
Morphological traits are important indices for identifying
Crataegus species (Dickinson etal. 1996). However, the tra-
ditional classification of Crataegus based on morphological
traits has been contested and influenced by the environment
(Christensen 1984; Gosler etal. 1994). The morphological
traits of fruits and leaves between C. bretschneideri and C.
pinnatifida are very similar (Supplementary Fig.1). Accord-
ing to the “Repertorium specierum novarum regni vegeta-
bilis” (Friderico 1903), C. bretschneideri is the synonym
of C. pinnatifida var. major N. E. Br. However, Sokolov
(1954) regarded C. bretschneideri as the synonym of C. pin-
natifida. Moreover, some plant taxonomy websites, such as
“The Plant List” (http:// www. thepl antli st. org/ tpl1.1/ record/
rjp- 18388) and the “Chinese Field Herbarium” (CFH, http://
www. cfh. ac. cn/ 13728 79. sp? AspxA utoDe tectC ookie Suppo
rt=1) defined C. bretschneideri as the synonym of C. pin-
natifida rather than a true species.
Several studies have examined the interspecific relation-
ships of partial Chinese Crataegus accessions. Ten polymer-
ase chain reaction-restriction fragment length polymorphism
(PCR-RFLP) markers amplified the same bands from chloro-
plast DNA (cpDNA) of C. bretschneideri, C. maximowiczii,
C. sanguinea, C. kansuensis E. H. Wilson, and C. dahurica
Koehne ex C. K. Schneid., indicating that these species had
the closer genetic relationships (Wu etal. 2008). C. bretsch-
neideri, C. pinnatifida, and C. pinnatifida var. major N. E.
Br. were clustered into the same branch of the phylogenetic
tree which were constructed by ten apple simple sequence
repeat (SSR) markers (Zhang etal. 2008). Recently, SSRs
and specific locus-amplified fragment sequencing (SLAF-
seq) were used to clarify the origin of Chinese Crataegus
and identify germplasm resources (Du etal. 2019; Zhang
etal. 2021). The results generated from SSRs and SLAF-seq
revealed that C. bretschneideri had a close relationship with
C. pinnatifida. In some early investigations, the complex
origin of C. bretschneideri has not been fully revealed due
to the limited molecular markers.
The chloroplast genome has been the primary objective
for plant phylogeny and evolution studies (Daniell etal.
2021). cpDNA barcode genes, such as psbA-trnH, trnS-
trnG, trnH-rpl2, rpl16, atpF-atpH, trnL-trnF, rpl20-rps12,
matK, and rbcL have been used to estimate the phylogeny
of Crataegus (Verbylaitė etal. 2006; Lo etal. 2009; Zarrei
etal. 2015; Brown etal. 2016; Emami et al. 2018). The
next-generation sequencing has been applied to sequence
the complete plastid genome, and large phylogenetic trees
across green plants have been constructed (Gitzendanner
etal. 2018). The complete or partial chloroplast genomes of
several Crataegus species have been published, including
C. pinnatifida var. major, C. chungtienensis W. W. Smith,
C. marshallii Eggleston, Crataegus sp., C. pinnatifida, C.
kansuensis, and C. hupehensis Sarg. (Zhang etal. 2017; Liu
etal. 2019; He etal. 2020; Zhang etal. 2020b; Hu etal.
2021). With such abundant chloroplast genome data, the
comparative genomic analysis will be an effective tool to
reveal the interspecific relationships among C. bretschnei-
deri and related species.
In this study, five Crataegus chloroplast genomes (C.
bretschneideri, C. maximowiczii, C. maximowiczii va r.
ninganensis, C. pinnatifida, and C. pinnatifida var. major)
were newly sequenced and compared with published Cra-
taegus and Amelanchier chloroplast genomes. We analyzed
potential molecular markers, phylogenetic relationships, and
divergence times based on the above comparison results.
In addition, the phylogenetic relationships of eight species
and two varieties of Crataegus native to China were evalu-
ated by nuclear genes sequencing, including nrDNA internal
transcribed spacer region (ITS) and floral meristem identity
control protein LEAFY intron 1. These results will provide
a reliable basis for revealing the classification status and
possible origin of C. bretschneideri.
Materials andmethods
Plant materials
C. bretschneideri, C. maximowiczii, C. maximowiczii var.
ninganensis, C. pinnatifida, and C. pinnatifida var. major
were used for chloroplast genomes sequencing. In total,
thirty-six accessions of Crataegus were sampled, which
consisted of C. bretschneideri (9 accessions), C. maximo-
wiczii (3 accessions), C. maximowiczii var. ninganensis (1
accession), C. sanguinea (3 accessions), C. altaica (Loudon)
Lange (2 accessions), C. pinnatifida (7 accessions), C. pin-
natifida var. major (4 accessions), C. hupehensis (3 acces-
sions), C. scabrifolia (Franch.) Rehder (3 accessions), and C.
songarica K. Koch (1 accession). The thirty-six Crataegus
24 Page 2 of 16 Tree Genetics & Genomes (2022) 18: 24
1 3
accessions were subjected to ITS and LEAFY sequencing.
Voucher specimen information for these Crataegus acces-
sions was provided in Table1. Young, healthy, and fresh
Crataegus leaves were collected from the National Fruit-tree
Germplasm Resources, Shenyang Hawthorn Repository in
2019. All the samples were immediately frozen in liquid
nitrogen and stored at −80 °C.
Chloroplast genome sequencing andassembly
For genomic DNA extraction, 1 g of frozen leaf material was
ground using the cetyl-trimethylammonium bromide method
(Du etal. 2019). The harvested DNA was detected via aga-
rose gel electrophoresis and quantified using an Agilent
2100 bioanalyzer. The DNA concentration (> 30 ng·μL−1)
Table 1 Biogeographic region and botanical characteristics of Crataegus accessions
*These accessions were used for chloroplast genomes sequencing
# Botanical characteristics of Crataegus taxa were taken “Flora of China 9: 111–117. 2003” as a reference
Taxon ID Biogeographic region Botanical characteristics#
Crataegus bretschneideri C. K. Schneid. JF1H Northeast, China It is mainly distributed in Northeast China and is similar to
C. pinnatifida. Leaves lobed, fruit globose, red, 26 mm in
diameter, pyrenes 3–5.
JF2H Northeast, China
JF3H Northeast, China
JF4H Northeast, China
ZF1H* Northeast, China
ZF2H Northeast, China
ZF3H Northeast, China
SF1H Northeast, China
CH Northeast, China
Crataegus maximowiczii C. K. Schneid. MSZ1H Northeast, China Leaves pubescent on both surfaces. Leaf basally cuneate or
broadly cuneate, occasionally truncate; fruit globose, red or
purplish-brown.
MSZ2H Northeast, China
MSZ3H* Northeast, China
Crataegus maximowiczii var. ninganensis
S. Q. Nie & B. J. Jen.
NASZ* Northeast, China It is a variety of C. maximowiczii. Stipules serrate; pedicel
glabrous; fruit persistent sepals pilose.
Crataegus sanguinea Pall. LNSZ1H Northeast, China It is native to the extreme north of China. Pedicel and peduncle
glabrous. Leaf basally cuneate; fruit red, 1 mm in diameter.
LNSZ2H Northeast, China
LNSZ3H Northeast, China
Crataegus altaica (Loudon) Lange AETSZ2H Northwest, China Leaves deeply pinnatifid to more than 1/2 width of the blade.
Fruit golden-yellow, 8–10 mm in diameter, pyrenes 4 or 5;
leaves glabrous or slightly pubescent.
AETSZ3H Northwest, China
Crataegus pinnatifida Bunge NMGSLH North, China It is known as the Chinese hawthorn which originated in North
China. Leaves deeply pinnatifid to more than 1/2 width of the
blade. Leaves truncate or broadly cuneate, with 3–5 pairs of
lobes, pubescent along midvein and lateral veins; fruit red, 15
mm in diameter, pyrenes 3–5.
WTSSLH North, China
YR5H Northeast, China
YP6H Northeast, China
YP8H Northeast, China
1541SLH Northeast, China
MDFSLH* Northeast, China
Crataegus pinnatifida var. major N. E. Br. QJX* Northeast, China It is a variety of C. pinnatifida. Leaves lobed. Leaves truncate
or broadly cuneate, with 3–5 pairs of lobes, pubescent along
midvein and lateral veins; fruit red, 20–30 mm in diameter,
pyrenes 3–5.
MPSZ North, China
XLZR North, China
MYDJX North, China
Crataegus hupehensis Sarg. HBSZ1H Central, China Leaves lobed or not divided, lateral veins extending to apices
of lobes or teeth only. Fruit red, rarely yellow; inflorescence
pubescent or glabrous.
HBSZ2H Central, China
HBSZ3H Central, China
Crataegus scabrifolia (Franch.) Rehder YNSZ1H Southwest, China The tree is apparently not cultivated outside China. Leaves
lobed or not divided, lateral veins extending to apices of
lobes or teeth only. Fruit red, rarely yellow.
YNSZ2H Southwest, China
YNSZ3H Southwest, China
Crataegus songarica K. Koch ZGESZ Northwest, China Leaves lobed or not divided, lateral veins extending to apices
of lobes or teeth only. Pulp yellow; pyrenes 2 or 3, smooth on
2 inner sides.
Page 3 of 16 24Tree Genetics & Genomes (2022) 18: 24
1 3
was quantified using a NanoDrop 2000 spectrophotometer,
and fragmentation was achieved using a Covaris instrument.
The fragmented DNA was purified and end-repaired, and
the fragment sizes were determined via gel electrophoresis.
After purification, an A-tailing was done at the 3′ end of
the DNA fragments and then adaptors were ligated to the
end of the DNA fragments using the T4 DNA ligase. PCR
was done to amplify the adaptor-ligated DNA for the con-
struction of the sequencing library. The PCR products were
used to produce short-insert (400 bp) libraries, and libraries
sizes were detected using an Agilent 2100 bioanalyzer. The
control library was used to test the quality of sequencing.
We sequenced the complete chloroplast genomes of C.
bretschneider, related species, and varieties using the Illu-
mina NovaSeq platform (Illumina, USA) in paired-end (2 ×
150 bp) sequencing mode (LC-Bio Biotechnologies Co., Ltd,
Hangzhou, Zhejiang, China). Raw reads were filtered using
SOAPec v2.01 (Luo etal. 2012) to obtain high-quality reads.
The chloroplast genomes were assembled with A5-MiSeq
v20150522 (Coil etal. 2015) and SPAdes v3.9.0 (Bankevich
etal. 2012) using clean data. Then the filtered reads were
assembled using BLAST to C. kansuensis (MF784433), with
an >80% match cutoff and gaps filled by filtered reads with
90% similarity over 50% of the gap length.
Chloroplast genome annotation
The whole chloroplast genomes were annotated using PGA-
Plastid Genome Annotator (Qu etal. 2019) and CPGAVAS2
(Shi etal. 2019) with the default parameters. Subsequently,
all tRNAs were verified by tRNAscan-SE v2.0 (Chan and
Lowe 2019). The structure diagram of Crataegus chloroplast
genomes with annotations was obtained using CHLOROP-
LOT (Greiner etal. 2019). The GC content was calculated
using EditSeq (Burland 1999).
Repeat structure andmicrosatellite analyses
The repeat structures, including forward, reverse, comple-
mentary, and palindromic repeats, were identified using
REPuter online program (Kurtz etal. 2001). The REPuter
parameters were set to a minimal repeat size of ≥ 30 bp and
a Hamming distance of 3 (90% or greater sequence iden-
tity). Tandem repeats were identified using Tandem Repeats
Finder v4.07b (Benson 1999), and the alignment parameters
match, mismatch, and indels were set to 2, 7, and 7, respec-
tively. The minimum alignment scores to report repeats and
maximum period size were 70 bp and 500 bp, respectively.
The SSRs within these chloroplast genomes were detected
using MISA-web (Beier etal. 2017). When the SSR motif
length was 1, 2, 3, 4, 5, and 6, the minimum numbers of
repeats in the SSR search parameters were 10, 5, 4, 3, 3,
and 3, respectively. The maximum sequence length between
two SSRs for registration as a compound SSR was 100 bp.
Sequence divergence analysis
Seventeen chloroplast genomes were aligned using MAFFT
v7 (Katoh and Standley 2013) on the FFT-NS-2 module.
DNA polymorphism analyses (Sliding-window analyses)
were calculated using DnaSP v5 (Librado and Rozas 2009)
based on the alignment results to generate the nucleotide
diversity (Pi) of these chloroplast genomes. The window
length was set to 600 bp, with a step size of 200 bp.
Divergence time estimation
The chloroplast genomes of Crataegus and Amelanchier
genera used in this study were downloaded from GenBank
as follows: C. chungtienensis (KY419947), C. hupehensis
(MW201730), C. kansuensis (MF784433), C. marshallii
(MK920293), C. pinnatifida var. major (KY419945), C. pin-
natifida (MN102356), Crataegus sp. (MK920294), Mespi-
lus germanica L. (MK920295), A. alnifolia (Nutt.) Nutt. ex
M.Roem. (MN068255), A. ovalis Medik. (MK920297), A.
sanguinea (Pursh) DC. (MN068262), and A. spicata (Lam.)
K. Koch. (MK920292).
The divergence times of 17 species and varieties
were estimated by BEAST2 (Bouckaert etal. 2014). The
Amelanchier species were selected as the out-group, and the
divergence node of Crataegus and Amelanchier was con-
strained using a lognormal distribution with an offset of 45
Mya (Mya = million years ago) and a mean and standard
deviation of 0.5 (Lo etal. 2009; Lo and Donoghue 2012).
We referred to detailed parameter settings of BEAST2 from
Kim etal. (2020).
ITS andLEAFY intron 1 sequencing
We sequenced the ITS and LEAFY intron 1 of 36 accessions
representing eight species and two varieties from Crataegus.
The primers used in this study were listed in Table2. PCR
amplification was performed in a reaction mixture with a
final volume of 20 μL consisting of 1 μL of template DNA
(40–50 ng), 10 μL of Takara ExTaq® (RR001A), and 2 μL
of primers. The PCR conditions were as follows: initial
denaturation at 94 °C for 3 min; followed by 35 cycles of
30 s at 94 °C, 30 s at 50–70 °C, 1 min at 72 °C; and a final
extension of 10 min at 72 °C. PCR amplification was carried
out in a thermal cycler (Applied Biosystems, USA). PCR
products were separated on a 1.5% agarose gel in 5 × TBE
(Tris-borate-EDTA) buffer and submitted to Sangon Biotech
(Co., Ltd., Shanghai, China) for sequencing. All sequences
were aligned using MAFFT v7 with the FFT-NS-2 module.
24 Page 4 of 16 Tree Genetics & Genomes (2022) 18: 24
1 3
Phylogenetic analyses
Seventeen chloroplast genomes of Crataegus and Amelanch-
ier genera were visualized using mVISTA (Frazer etal.
2004). IQ-TREE 2 (Minh etal. 2020) was used to build a
maximum likelihood (ML) tree with the TVM+F+R3 mod-
ule and 1000 bootstrap replicates. Mrbayes v3.2.7a (Ron-
quist etal. 2012) was used to build a Bayesian inference (BI)
tree. The parameter settings were regarded Raman’s method
(2020) as a reference. The ML and BI trees were visualized
using FigTree v1.4.4 (http:// tree. bio. ed. ac. uk/ softw are/ figtr
ee/). Multiple sequence alignment files of the 36 Crataegus
accessions generated from MAFFT v7 were conversed to
the rdf files using DnaSP 5.0. Network 10.2 software (Ban-
delt etal. 1999) was used to draw the haplotype network of
the ITS and LEAFY intron 1 sequences using the median-
joining module.
Results
Chloroplast genomes features ofCrataegus species
andvarieties
Five Crataegus complete chloroplast genomes sequenced
ranged from 159,644 (C. bretschneideri, ZF1H) to 159,947
bp (C. pinnatifida var. major, QJX) in length, with differ-
ences ranging from 101 to 303 bp (Fig.1, Table3, Supple-
mentary Fig.6–9). The structures of Crataegus chloroplast
genomes were similar to that of most terrestrial plants. The
genomes contained the typical quadripartite structure that
included the inverted repeats a (IRa) and inverted repeats
b (IRb) regions (26,170–26,387 bp) separated by large sin-
gle copy region (LSC, 87,694–88,303 bp) and small single
copy region (SSC, 19,140–19,273 bp). The GC contents of
the complete genomes ranged from 36.60 to 36.68%, 34.31
to 34.37% in the LSC regions, 42.64 to 42.76% in the IR
regions, and 32.73 to 32.91% in the SSC region, revealing
the high level of similarity among different Crataegus spe-
cies and varieties.
Generally, five Crataegus chloroplast genomes encoded
an identical set of 109–113 genes, including 76–79
protein-coding genes, 29 tRNA genes, and four rRNA genes
(Table3). The representative annotated chloroplast genomes
including gene number, order, and names were illustrated by
circular maps in Fig.1 and Supplementary Fig.6–9. Seven
protein-coding genes (rps19, rpl23, rpl2, ndhB, ycf2, rps12,
and rps7), seven tRNA genes (trnR-ACG , trnL-CAA , trnI-
CAU , trnI-GAU , trnA-UGC , trnV-GAC , and trnN-GUU ),
and four rRNA genes (rrn16, rrn23, rrn4.5, and rrn5) were
duplicated in the IR regions. CPGAVAS2 annotations iden-
tified four tRNA (trnG-GCC
, trnG-UCC
, trnfM-CAU
, and
trnS-GCU
) that may be trnG-UCC
, trnG-GCC
, trnM-CAU
,
and trnS-GGA
, respectively.
Among 113 unique genes identified (excluding 22 dupli-
cated genes), eight protein-coding genes (atpF, ndhA, ndhB,
petB, petD, rpoC1, rpl16, and rpl2) and six tRNA genes
(trnA-UGC
, trnG-GCC
, trnI-GAU
, trnK-UUU
, trnL-UAA
,
and trnV-UAC
) had one intron; and two protein-coding genes
(clpP and ycf3) contained two introns (Table4). In addition,
12 genes (trnK-UUU
, rps16, trnG-GCC
, atpF, rpoC1, ycf3,
trnL-UAA
, trnV-UAC
, clpP, petB, petD, and rpl16) that con-
tained one or two introns were distributed in the LSC region,
eight genes (four duplicated genes: rpl2, ndhB, trnI-GAU
, and trnA-UGC
) with one intron were located in the IR
region, and one gene (ndhA) was in the SSC region.
Variations intheborder regions
The adjacent genes and border regions of five Crataegus
chloroplast genomes were analyzed, and C. kansuensis
(MF784433) was used as a reference (Fig.2). Although
the general genomes structures, including the order and
number of genes, were relatively conserved, six Cratae-
gus chloroplast genomes exhibited visible differences at
the LSC/IRb and IRa/LSC borders. The LSC/IRb borders
differed significantly among the six Crataegus chloroplast
genomes. The IRb region expanded into the rpl19 gene
with 120 bp in the IRb region for C. maximowiczii and
C. kansuensis, while it generated distances of 25 bp and
29 bp to the junction in C. bretschneideri and C. pinnati-
fida, respectively. The IRb region expanded into the rpl2
gene by 26 bp in the LSC regions for C. maximowiczii var.
ninganensis. The IRa/LSC borders also presented several
Table 2 Information of nuclear
sequences primers used in this
study
ITS, internal transcribed spacer region; LEAFY, floral meristem identity control protein
Gene Forward and reverse primers Tm Reference sequence
ITS1-5.8S-ITS2 F: 5′-TCC TCC GCT TAT TGA TAT GC-3′
R: 5′-GGA AGG AGA AGT CGT AAC AAGG-3′
58 °C Crataegus laevi-
gata (Poir.) DC.
(EU500466)
LEAFY intron 1 F: 5′-GGA TCC RGA TGC CTT CTC TGC GAA CTT
GTT CAA GTG G-3′
R: 5′-GTT CTT TTT GCC ACG CGC CAC CTC CCC
CGG -3′
70 °C Crataegus sp.
(EU500483)
Page 5 of 16 24Tree Genetics & Genomes (2022) 18: 24
1 3
differences among the six Crataegus chloroplast genomes.
The rpl2 gene was located close to the IRa/LSC junction
with distances of 2 bp, 43 bp, and 47 bp in C. maximowic-
zii var. ninganensis, C. pinnatifida, and C. bretschneideri,
respectively. The trnH gene was located close to the junc-
tion with a distance of 57 bp in C. maximowiczii. The IRa
region expanded into the rpl19 gene by 1 bp in the LSC
region for C. kansuensis. Unlike the above junction, the
SSC/IRa and IRb/SSC junctions were relatively conserved.
The yfc1 gene crossed the SSC/IRa junction and expanded
to the same length in the SSC region (4557 bp) and the IRa
region (1074 bp) in all six Crataegus chloroplast genomes.
The ycf1 gene was located in the IRb region next to the
junction, with no gaps in C. kansuensis. The ndhF gene
was located near the junction, with no gaps. It crossed the
IRb/SSC junction and expanded by 12 bp in the IRb region
in C. maximowiczii, C. maximowiczii var. ninganensis., C.
pinnatifida, C. pinnatifida var. major, C. bretschneideri,
and C. kansuensis. The variation in these boundary regions
was responsible for the differences in length of the six
Crataegus chloroplast genomes and their LSC, IR, and
SSC regions.
Fig. 1 Gene map of Crataegus bretschneideri C. K. Schneid. chloro-
plast genome. Genes shown outside of the outer circle are transcribed
clockwise and those inside are transcribed counterclockwise. Genes
belonging to different functional groups are color-coded. The dashed
area in the inner circle indicates the GC content of the chloroplast
genome
24 Page 6 of 16 Tree Genetics & Genomes (2022) 18: 24
1 3
Table 3 Statistics on the basic features of five Crataegus chloroplast genomes
The numbers in parenthesis indicate the duplicated genes in chloroplast genomes. LSC, large single-copy region; SSC, small single-copy region;
IR, inverted repeat regions; tRNA, transfer RNA; rRNA, ribosomal RNA
Crataegus maxi-
mowiczii C. K.
Schneid.
Crataegus maximowiczii var.
ninganensis S. Q. Nie & B. J.
Jen.
Crataegus pin-
natifida Bunge
Crataegus pinnatifida
var. major N. E. Br.
Crataegus bretsch-
neideri C. K.
Schneid.
Genome size (bp) 159,945 159,916 159,749 159,947 159,644
LSC size (bp) 87,904 88,303 87,841 87,946 87,694
SSC size (bp) 19,273 19,273 19,140 19,234 19,256
IR size (bp) 52,768 52,340 52,768 52,768 52,694
Number of total genes 133 (22) 135 (22) 131 (22) 131 (22) 131 (22)
Protein coding genes 88 (10) 89 (10) 86 (10) 86 (10) 86 (10)
tRNA genes 37 (8) 37 (8) 37 (8) 37 (8) 37 (8)
rRNA genes 8 (4) 8 (4) 8 (4) 8 (4) 8 (4)
Duplicated genes in IR 17 17 17 17 17
GC content (%) 36.60 36.60 36.68 36.63 36.61
GC content in LSC (%) 34.34 34.31 34.41 34.37 34.34
GC content in SSC (%) 32.81 32.73 32.91 32.88 32.76
GC content in IR (%) 42.65 42.76 42.72 42.64 42.73
Table 4 Genes identified in five Crataegus chloroplast genomes
a Genes containing a single intron; bGenes containing two introns; cTwo gene copies in the IR regions
Category of genes Group of genes Name of genes
Genes for photosynthesis Photosystem I psaA, psaB, psaC, psaI, psaJ
Photosystem II psbA, psbB, psbC, psbD, psbE, psbF, psbI, psbJ, psbk, psbL, psbM, psbN, psbT,
psbZ, bycf3
Cytochrome b/f complex petA, apetB, apetD, petG, petL, petN
NADH-dehydrogenase andhA, andhB, ndhB, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
ATP synthase atpA, atpB, atpE, aatpF, atpH, atpI
Rubisco rbcL
Transcription and transla-
tion related genes
DNA dependent RNA polymerase rpoA, rpoB, arpoC1, rpoC2
Ribosome (large submit) rpl14, arpl16, arpl2, rpl20, rpl22, rpl23, rpl23, rpl32, rpl33, rpl36
Ribosome (small subunit) rps11, rps12, rps12, rps14, rps15, rps16, rps18, rps19, rps2, rps3, rps4, rps7,
rps8
RNA genes Ribosomal RNA rrn4.5S, rrn5S, rrn16S, rrn23S
Transfer RNA trnH-GUG , trnK-UUU , trnQ-UUG , trnG-GCC , trnR-UCU , trnC-GCA , trnD-
GUC , trnY-GUA , trnE-UUC , trnT-GGU , trnS-UGA , trnG-UCC , trnfM-CAU ,
trnT-UGU
, trnL-UAA
, trnF-GAA
, trnV-UAC
, trnM-CAU
, trnW-CCA
, trnP-
UGG
, trnL-UAG
, ctrnS-GCU , ctrnL-CAA , ctrnV-GAC , ctrnI-GAU , ctrnI-CAU ,
ctrnA-UGC , ctrnR-ACG , ctrnN-GUU
Other genes Acetyl-CoA-carboxylase accD
c-type cytochrom synthesis gene ccsA
Envelop membrane protein cemA
Protease bclpP
Translational initiation factor infA
Maturase matK
Function-unknown genes Conserved open reading frames ycf1, ycf2, ycf4
Page 7 of 16 24Tree Genetics & Genomes (2022) 18: 24
1 3
Fig. 2 Comparison of the LSC, IRs, and SSC border regions of five
Crataegus chloroplast genomes. The chloroplast genome of Cratae-
gus kansuensis E. H. Wilson is considered as a reference. LSC, large
single copy region; SSC, small single copy region; IRa, inverted
repeats a region; IRb, inverted repeats b region
Fig. 3 Type and distribution of repeated sequences and SSRs in five
Crataegus chloroplast genomes. a Repeat types number. b Number of
repeat sequences by length. c SSR type number. d Number of identi-
fied SSR motifs. Mono., Di., Tri., Tetra., and Penta. represent mono-
nucleotide, dinucleotide, trinucleotide, tetranucleotide, and pentanu-
cleotide short sequence repeats
24 Page 8 of 16 Tree Genetics & Genomes (2022) 18: 24
1 3
Repeat sequences andmicrosatellites assays
The repeat structures within each Crataegus genome, including
forward, reverse, complementary, and palindromic repeats, were
identified. Generally, 39–49 repeat sequences were observed,
including 18–28 forward repeat sequences, 17–22 palindromic
repeat sequences, and 2–4 reverse repeat sequences (Fig.3,
Supplementary Table1). One complementary repeat sequence
was identified in C. pinnatifida var. major. The lengths of the
repeat sequences in these chloroplast genomes ranged from 14
to 95 bp, with sequences between 31 and 39 bp representing
the majority (38.78–65.31%). There were relatively few repeat
sequences shorter than 30 bp (10.20–16.33%), from 40 to 49 bp
(6.12–18.37%), from 50 to 59 bp (4.08–12.24%), and 60 bp or
longer (4.08–12.24%). The most frequent length of repeats in
C. pinnatifida var. major was 32 bp. Meanwhile, 28–39 tandem
repeats were also detected in the genomes, with repeat lengths
of 30 to 150 bp (Fig.3a, b, Supplementary Table2). The repeat
sequences were mainly distributed in the LSC region. Several
protein-coding genes and tRNA genes, such as ycf1, ycf2, ndhA,
clpP, trnS-GCU
, and trnT-UGU
contained repeat sequences
(Supplementary Table1, Supplementary Table2).
We also analyzed the mononucleotide, dinucleotide,
trinucleotide, tetranucleotide, and pentanucleotide short
sequence repeats (SSRs, or microsatellites) (Fig.3c, d, Sup-
plementary Table3). In general, 68–73 microsatellites were
predicted across five Crataegus chloroplast genomes. The
mononucleotides were the most common microsatellites in
each genome, and most of these were T repeats, ranging in
quantity from 27 in C. maximowiczii and C. maximowiczii
var. ninganensis to 30 in C. pinnatifida var. major. The dinu-
cleotides were the second most numerous microsatellites,
and the main dinucleotide type was AT, with seven in C.
maximowiczii, C. maximowiczii var. ninganensis, and C. pin-
natifida and six in C. pinnatifida var. major and C. bretsch-
neideri. The trinucleotides (TAAs) were only detected in
C. pinnatifida and C. pinnatifida var. major. Each chloro-
plast genome contained three to six tetranucleotide SSRs,
of which at least two were TTTA-type SSRs. In addition, a
pentanucleotide SSR of the type ATTTA was predicted in C.
bretschneideri. Similar to the case for the repeat sequences,
73% or more SSRs were distributed in the LSC region, fol-
lowed by the SSC and IR regions. Several protein-coding
and tRNA genes contained SSRs, including matk, atpF,
rpoC1, ndhK, cemA, clpP, ycf1, ycf3, trnG-GCC
, and trnL-
UAA
(Supplementary Table3).
Sequence divergence andmutational hotspots
analyses
DNA polymorphism analyses were conducted to deter-
mine the nucleotide diversity (Pi) from the LSC, SSC,
and IR regions of five Crataegus chloroplast genomes
(Supplementary Fig.2). The SSC region showed the high-
est nucleotide diversity (0.003023), followed by the LSC
region (0.002695) and the IR region (0.000586). The most
diverse region was in the LSC region between 50,000 and
60,000 bp. In total, 16 diverse coding genes and non-cod-
ing sequences had high variability (Pi > 0.007). Twelve
mutational hotspots were located in the LSC region, includ-
ing matk, trnQ-UUG-psbK, atpI-rps2, ndhJ, atpB, atpA,
trnD-GUC
, psaB, accD, petA, clpP, and psbH-petB; three
hotspots were located in the LSC region (ycf1, ndhD, and
trnL-UAG
); trnN-GUU-TrnR-ACG
was the hotspot in the
SSC/IRb boundary region. In addition, six genes (matk,
psaB, accD, petA, clpP, and trnD-GUC
) and two spaces
(psbH-petB and trnN-GUU-trnR-ACG
) exhibited higher
variability, and these hotspots could be regarded as poten-
tial molecular markers for phylogenetic analyses due to Pi
> 0.01.
Phylogenetic analysis anddivergence time
estimation ofCrataegus andAmelanchier
Five newly sequenced chloroplast genomes of Cratae-
gus and 12 chloroplast genomes from the NCBI data-
base were used to evaluate the phylogenetic relationships
among the genera Crataegus and Amelanchier (Supple-
mentary Fig.3). The ML tree and BI tree were highly
congruent (Supplementary Fig.4). Overall, 17 species
and varieties were classified into three major clades.
Crataegus and Amelanchier were separated into two
clades. In the Crataegus clade, 11 species and varieties
were divided into two distinct subclades. C. pinnatifida,
C. pinnatifida var. major, and C. hupehensis clustered
together and showed close relationships; M. germanica
was monophyletic. Furthermore, C. maximowiczii, C.
maximowiczii var. ninganensis, and C. bretschneideri
exhibited a close relationship and clustered into a com-
mon subclade, forming a sister group to the subclade
formed by C. marshallii, Crataegus sp., C. chungtienen-
sis, and C. kansuensis.
To estimate the divergence times for Crataegus species
and varieties, the chloroplast genomes of the Amelanchier
group (A. alnifolia, A. ovalis, A. sanguinea, and A. spicata)
were selected as the out-group. The divergence clades of
these genera were the same as those for the ML and BI trees
(Fig.4, Supplementary Fig.4). It is estimated that the diver-
gence time of the two main clades was approximately 44.989
Mya (middle Eocene). Mespilus germanica and Crataegus
were differentiated around 31.171 Mya (early Oligocene).
The divergence time of C. pinnatifida, C. hupehensis, and
C. pinnatifida var. major was 15.542 Mya (middle Miocene).
C. bretschneideri and C. maximowiczii were differentiated
around 7.867 Mya (late Miocene).
Page 9 of 16 24Tree Genetics & Genomes (2022) 18: 24
1 3
Phylogenetic analyses ofCrataegus accessions
based onnuclear sequences
The ITS and LEAFY intron 1 sequences were used to reveal
the relationships among 36 Crataegus accessions, which
belong to eight species and two varieties. ML and BI trees
had the same structure, constructed using ITS and LEAFY
intron 1 sequences (Fig.5). The ITS sequences resolved
three major clades within Crataegus, labeled A–C (Fig.5a).
Clade A was made up of C. maximowiczii, C. maximowic-
zii var. ninganensis, C. sanguinea, and C. altaica; clade B
contained C. scabrifolia, three C. bretschneideri materials,
two C. pinnatifida var. major materials, one C. hupehensis
material, and one C. pinnatifida material; clade C included
six C. pinnatifida materials, six C. bretschneideri materials,
two C. pinnatifida var. major materials, two C. hupehensis
materials, one C. maximowiczii materials, and C. songarica.
The LEAFY intron 1 sequences resolved three major clades
within Crataegus, labeled A–C (Fig.5b). Clade A included
C. hupehensis, C. pinnatifida var. major, C. scabrifolia, and
four C. pinnatifida materials; clade B included C. maximo-
wiczii, C. maximowiczii var. ninganensis, C. sanguinea, and
C. altaica; clade C was a sister group of clade B, and it
contained C. bretschneideri, C. songarica, and three C. pin-
natifida materials.
The median-joining haplotype networks constructed using
Network 10.0 produced 21 and 30 active haplotypes gener-
ated from the ITS and LEAFY intron 1 sequences, respec-
tively (Supplementary Fig.5, Supplementary Table4). Hap_1
belonged to the network constructed by ITS sequence was the
principal haplotype consisting of C. bretschneideri, C. pinnati-
fida, C. pinnatifida var. major, C. songarica, and C. hupehen-
sis accessions (Supplementary Fig.5a). Hap_2–6 were derived
from Hap_1. The haplotypes generated from the LEAFY
intron 1 sequence were mainly divided into three groups
(Supplementary Fig.5b): group 1 contained Hap_10, Hap_11,
Hap_12, Hap_14, and Hap_15, which mainly consisted of C.
maximowiczii, C. sanguinea, and C. altaica; group 2 con-
sisted mainly of C. bretschneideri (Hap_1, Hap_2, Hap_4,
and Hap_13) and partial C. pinnatifida (Hap_3 and Hap_5);
group 3 contained Hap_6, Hap_7, Hap_8, and Hap_16, which
consisted mainly of C. pinnatifida, C. pinnatifida var. major,
C. songarica, and C. hupehensis accessions.
Discussion
The complete chloroplast genome is remarkably conserved
in size and structure, with a relatively slow evolution rate
involving few gains and losses (Grewe etal. 2013). Five
Fig. 4 Divergence time estimation for Crataegus and Amelanchier
based on the chloroplast genomes. The number at each node repre-
sents the median divergence time, and the node bars represent 95%
HPD (highest posterior density). The accession numbers in Gen-
bank (C. chungtienensis (KY419947), C. hupehensis (MW201730),
C. kansuensis (MF784433), C. marshallii (MK920293), C. pinnati-
fida var. major (KY419945), C. pinnatifida (MN102356), Crataegus
sp. (MK920294), Mespilus germanica (MK920295), A. alnifolia
(MN068255), A. ovalis (MK920297), A. sanguinea (MN068262),
and A. spicata (LMK920292)) are listed here. The ruler on the lower
left represents the geologic timescale. Paleogene (23.03–66 Mya);
Eocene (33.90–55.80 Mya); OLI (Oligocene, 23.03–33.90 Mya);
Neogene (0–23.03 Mya); Miocene (5.33–23.03 Mya); Pliocene
(1.81–5.33 Mya); PLE (Pleistocene, 0.01–1.81 Mya)
24 Page 10 of 16 Tree Genetics & Genomes (2022) 18: 24
1 3
newly sequenced Crataegus chloroplast genomes had typi-
cal quadripartite structures with LSC, SSC, and IR regions.
The structures of these genomes were similar to those of
the previously reported chloroplast genomes of C. pinnati-
fida (He etal. 2020) and C. hupehensis (Hu etal. 2021).
The chloroplast genomes of vegetable and fruit species
are 120–160 kb in length, while those of cereal species are
110–140 kb in length (Daniell etal. 2021). In this study, the
chloroplast genomes were conserved and similar in length,
ranging from 159,644 bp in C. bretschneideri to 159,947
bp in C. pinnatifida var. major. Five Crataegus chloroplast
genomes encoded 131/135 genes. The LSC regions were
87,694–88,303 bp, and the SSC regions were 19,140–19,273
bp. The pair of inverted IRa/IRb regions was 26,170–26,384
bp (Table3). The GC contents and composition of C.
bretschneideri and related species were very similar, indicat-
ing that these Crataegus chloroplast genomes were relatively
conserved.
Fig. 5 Phylogenetic trees of Crataegus accessions using maximum
likelihood (ML) and Bayesian inference (BI) based on ITS (a) and
LEAFY (b) sequences. The midpoints in ML and BI analyses are
listed above the branches (ML/BI), and the root is positioned at the
midpoint between the two longest branches. The color of each acces-
sion represents the different accession of Crataegus.
Page 11 of 16 24Tree Genetics & Genomes (2022) 18: 24
1 3
The fluctuating lengths of IRs are the main contributors
to increases or decreases in the cpDNA sizes of many angio-
sperms (Wolf etal. 2010). Dynamic expansion of IRs in
groups such as Geraniaceae (Weng etal. 2014), Mimosoid
legumes (Dugas etal. 2015), and the contraction of IRs in
the Lauraceae family (Song etal. 2015) have previously been
reported. Our results showed that the length of IRs regions
among five Crataegus chloroplast genomes did not exhibit a
significant change, ranging only from 26,168 to 26,384 bp.
Two junctions across the genomes showed high similarity,
particularly for the ndhF gene located at the border between
IRb and SSC regions, and ycf1 at the border between IRa
and SSC regions (Fig.2). mVISTA results (Supplementary
Fig.3) indicated that the LSC and SSC regions were rela-
tively more divergent than the IRs regions.
Previous researchers have found that larger and more
complex repeat sequences contribute more to sequence
arrangements and the evolution of the chloroplast genome
(Huang etal. 2014; Weng etal. 2014). We have identified
four types of repeat sequences in five Crataegus chloroplast
genomes (Supplementary Tables1, Supplementary Table2).
There were significant differences in the number and posi-
tion of dispersed repeats within the genomes. The repeat
sequences were mainly distributed in the LSC region, and
C. bretschneideri had the lowest number of dispersed repeats
(37), and tandem repeats (37). The copy number variability
of SSRs is highly polymorphic among chloroplast genomes,
and these could be employed as molecular markers in popu-
lation genetics, phylogeography, and species identification
(Xue etal. 2012; Wang etal. 2013). In this study, we identi-
fied numerous SSRs within the five genomes, and 68–73
microsatellites were predicted (Supplementary Tables3).
Among these SSRs, 73% or more SSRs were distributed
in the LSC region. Furthermore, the SSRs were composed
mainly of thymine (T) or adenine (A) repeats. These results
were consistent with those of related studies and indicate
that SSRs contribute to the AT richness of plastid genomes
(Nie etal. 2012). Furthermore, our findings were identical to
those produced by previous studies that the protein-coding
genes, clpP and ycf1, contained both repeat sequences and
SSRs (Curci etal. 2015; Zhao etal. 2015; Li etal. 2020a).
Overall, these polymorphic sites may be considered as
potential molecular markers in further studies of species
delimitation and phylogeny in Crataegus.
Multigenome comparisons can facilitate explorations of
mutational hotspots which were used for interspecies dis-
crimination and phylogenetic studies at the species level
(Yang etal. 2018; Abdullah etal. 2019). Several plas-
tid DNA markers generated from coding and non-coding
regions with higher levels of variation could be applied to
resolve the phylogenetic problems of different plant species.
Prior successes have been found with the coding genes ycf1
in Debregeasia (Wang etal. 2020); accD in Artemisia (Kim
etal. 2020); the first and second exons of clpP, the first
intron of clpP, the first exon of atpF, the second intron of
ycf3, matK, and ndhF in Abelmoschus (Li etal. 2020b). In
this study, we proposed a set of eight divergent coding genes
and non-coding sequences (Pi > 0.01) from Crataegus,
including matk, psaB, accD, petA, clpP, trnD-GUC , psbH-
petB, and trnN-GUU-trnR-ACG . These mutational hotspots
could resolve taxonomic discrepancies and provide genetic
barcodes for the Crataegus genus.
Phylogenetic relationships in Rosaceae have been prob-
lematic because of frequent hybridization, apomixis, pre-
sumed rapid radiation, and complex historical diversifica-
tion (Xue etal. 2019). The chloroplast genome has typical
maternal inheritance characteristics. A growing number of
studies have used the complete chloroplast genome to evalu-
ate phylogenetic relationships among plants (Daniell etal.
2021). In this study, 17 chloroplast genomes were used to
construct the ML tree and BI tree (Supplementary Fig.4).
The clade B included the East Asian species C. maximow-
iczii, C. bretschneideri, C. chungtienensis, and C. kansuen-
sis and eastern North American species C. marshallii and
M. germanica. These results supported Phipps’s hypothesis
(1990) that Crataegus has migrated eastward from East Asia
to North America. In addition, Mespilus and Crataegus are
sister genera in the Rosaceae tribe Pyrea based on the phy-
logeny analyses of nuclear sequences and intergenic cpDNA
regions (Lo etal. 2007; Talent etal. 2008). The phyloge-
netic trees in this study also reflect the close relationship
between Mespilus and Crataegus (Supplementary Fig.4).
However, Phipps (2016) clarified the morphological distinc-
tion between Mespilus and Crataegus and argued for the
retention of a monotypic Mespilus.
The divergence times of 17 Crataegus and Amelanchier
accessions were estimated. One constraint was based on the
oldest fossil record of Amelanchier leaves from the Mid-
dle Eocene, or approximately 40 Mya, around One Mile
Creek, Princeton, British Columbia (Wolfe and Wehr 1988).
Another constraint was that Amelanchier and Crataegus
were expected to have differentiated from each other around
45 Mya (Lo and Donoghue 2012). Our results found that
Amelanchier and Crataegus differentiated around 44.989
Mya; Mespilus germanica and Crataegus were differentiated
around 31.171 Mya. These two divergence times were quite
similar to those reported by Lo and Donoghue (2012). Mon-
soon weather patterns during the Miocene affected Asian
vegetation due to the slightly warmer and wetter climate
(Su etal. 2013). In the late Miocene (5.333–11.63 Mya),
intraspecific migration and interspecific divergence events
occurred among Crataegus species (Wen etal. 2016; Du
etal. 2019). The divergence time of C. pinnatifida origi-
nated in Southwest China was around 15.542 Mya while
the divergence time of C. pinnatifida originated in Northeast
China was around 7.148 Mya. This result indicated that C.
24 Page 12 of 16 Tree Genetics & Genomes (2022) 18: 24
1 3
pinnatifida may have migrated from Southwest to North-
east China. Moreover, C. bretschneideri and C. maximow-
iczii were differentiated around 7.867 Mya. This divergence
time was overlapped with that of C. pinnatifida originated
in Northeast China.
Hybrids are often found in areas where different spe-
cies overlap and crossbreed (Bugaj-Nawrocka etal. 2020).
The intraspecific hybridization of Crataegus species often
occurs where the distribution areas of different species over-
lap (Talent and Dickinson 2007). The conflicts detected in
the sequencing results of chloroplasts, ITS, and LEAFY
intron 1 suggested that three species (C. marshalli, Cratae-
gus spathulata Michx., and Crataegus phaenopyrum (L. f.)
Medik.) from the southeastern USA were hybrids derived
from European and North American ancestors (Lo etal.
2009). Thus, Phipps (2005) posited that hybridization is a
potential explanatory factor for speciation in Crataegus. C.
bretschneideri, C. maximowiczii, and partial C. pinnatifida
originated in Northeast China. Several studies consider that
C. bretschneideri was the variety of C. pinnatifida (Dai etal.
2007; Guo and Jiao 1995). Du etal. (2019) found that C.
bretschneideri was closely related to C. pinnatifida based
on SSRs and SLAF-seq data and that gene flow occurred
from C. maximowiczii to C. bretschneideri. In this study, the
structure of the chloroplast phylogenetic tree was similar to
those of Wu etal. (2008). C. bretschneideri and C. maximo-
wiczii were closely related from the perspective of maternal
inheritance (Fig.4). However, the phylogenetic trees con-
structed by LEAFY intron 1 and ITS sequences showed that
C. bretschneideri and C. pinnatifida had a closer relation-
ship (Fig.5). C. bretschneideri and C. pinnatifida accessions
shared the same haplotype in the network constructed by
ITS sequences (Supplementary Fig.5). On the contrary, the
LEAFY intron 1 haplotype of C. bretschneideri was inde-
pendent in the middle of the network, which was linked with
group 1 haplotypes (C. maximowiczii and C. sanguinea) and
group 3 haplotypes (MDFSLH, C. pinnatifida). In general,
our studies revealed that C. bretschneideri was an independ-
ent species. C. maximowiczii may be the maternal origin
of C. bretschneideri. MDFSLH (C. pinnatifida) may be the
paternal origin of C. bretschneideri.
Conclusion
The comparative analyses of five Crataegus chloroplast
genomes have provided rich genome data for further stud-
ies of Crataegus genetic diversity. In total, 403 repeats
sequences, 352 SSRs, and 8 mutational hotspots could be
applied for the development of molecular markers related
to Crataegus phylogeny. Furthermore, the chloroplast
genomes and nuclear sequences supported the proposal that
C. bretschneideri is an independent species and may be of
hybrid origin. The present work will promote the identifica-
tion and conservation of Crataegus in the future.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s11295- 022- 01556-9.
Funding This work was supported by “The Conservation and Utiliza-
tion of Crop Germplasm Resource–Hawthorn (Project Nos. 19190178;
19200357).”
Declarations
Conflict of interest The authors declare no competing interests.
Data archiving statement The raw sequences of chloroplast genomes
reported in this paper have been deposited in the Genome Sequence
Archive (Genomics, Proteomics & Bioinformatics 2017) in National
Genomics Data Center (Nucleic Acids Res 2021), China National
Center for Bioinformation/Beijing Institute of Genomics, Chinese
Academy of Sciences, under accession number CRA004494 that are
publicly accessible at https:// ngdc. cncb. ac. cn/ gsa. All the nuclear
sequences have been uploaded to the GenBank (https:// www. ncbi.
nlm. nih. gov/ genba nk/) with the accession numbers: MZ688339–
MZ688374; MZ686456–MZ686491.
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