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Validation of two novel primers for the promising amplification of the mitogenomic Cytochrome c Oxidase subunit I (COI) barcoding region in Artemia aff. sinica (Branchiopoda, Anostraca)

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Due to the lack of a taxonomic key for the identification of Artemia species, molecular markers have been increasingly used for phylogenetic studies. The mtCOI marker is a regularly considered marker for the molecular systematics of Artemia populations. The proposed universal and specific primers have mostly failed to amplify the Artemia aff. sinica mtCOI marker, and on the whole, the successfully amplified products of the PCR were inefficient, primarily through the representation of poly-peak or incomplete sequences. We presumed that if a forward primer could be developed regarding the joint regions of the last part of the previous gene (tRNA Tyr) and the beginning of the target gene mtCOI, the sequence could be relevant to the target-sequence of mtCOI. Thus, here, we describe a new set of primers, which could be used to amplify the mtCOI barcoding region of Artemia aff. sinica Cai, 1989, with a high performance of sequencing. The new primer set worked well also for other Artemia bisexual species, as well as for parthenogenetic populations. It is recommended that joint regions between the previous/next gene(s) and the target marker, could be aimed at when designing specific primers for other markers and taxa. RÉSUMÉ En raison de l'absence d'une clé taxonomique pour l'identification des espèces d'Artemia, les marqueurs moléculaires ont été de plus en plus utilisés pour les études phylogénétiques. Le marqueur mtCO est régulièrement considéré pour la systématique moléculaire des populations d'Artemia. Les amorces proposées, universelles ou spécifiques, ont la plupart échoué à amplifier le marqueur Artemia aff. sinica mtCOI, et dans l'ensemble, les produits amplifiés avec succès par PCR ont été inefficients, principalement par la représentation de poly-pic ou de séquences incomplètes. Nous supposons que si une amorce anti-sens pouvait être développée pour les régions jointives de la dernière partie du gène précédent (tRNA Tyr) et le début du gène cible mtCOI, la séquence pourrait être pertinente de la séquence cible de mtCOI. Ainsi, nous décrivons ici un nouvel ensemble d'amorces qui pourraient être utilisées pour amplifier la région du code-barre mtCOI d' Artemia aff. sinica Cai, 1989, avec une performance de séquençage élevée. Ce nouvel ensemble d'amorces a bien fonctionné aussi pour les autres espèces bisexuelles d'Artemia, ainsi que pour les populations parthénogénétiques. Il est recommandé que la région de jonction entre les gènes précédents et suivants et le marqueur cible, pourrait être visée dans le design d'amorces spécifiques pour d'autres marqueurs ou taxa.
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Crustaceana 95 (5-6) 585-592
VALIDATION OF TWO NOVEL PRIMERS FOR THE PROMISING
AMPLIFICATION OF THE MITOGENOMIC CYTOCHROME COXIDASE
SUBUNIT I (COI) BARCODING REGION IN ARTEMIA AFF. SINICA
(BRANCHIOPODA, ANOSTRACA)
BY
ALIREZA ASEM1,2), CHUN-ZHENG FU3), NING YANG2), AMIN EIMANIFAR4),
YING CAO2), PEI-ZHENG WANG5)and CHUN-YANG SHEN1,6)
1)Department of Biology, Chengde Medical University, Chengde 067000, P.R. China
2)Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources,
Hainan Tropical Ocean University, Sanya 572000, P.R. China
3)Institute of Sericulture, Chengde Medical University, Chengde 067000, P.R. China
4)Independent Senior Scientist, Industrial District, Gaithersburg, MD 20878, U.S.A.
5)Key Laboratory for Coastal Marine Eco-environment Process and Carbon Sink of Hainan
Province, Hainan Tropical Ocean University, Yazhou Bay Innovation Institute, Sanya 572000,
P.R. China
ORCID iD: Asem: 0000-0002-8991-4903
ABSTRACT
Due to the lack of a taxonomic key for the identification of Artemia species, molecular markers
have been increasingly used for phylogenetic studies. The mtCOI marker is a regularly considered
marker for the molecular systematics of Artemia populations. The proposed universal and specific
primers have mostly failed to amplify the Artemia aff. sinica mtCOI marker, and on the whole, the
successfully amplified products of the PCR were inefficient, primarily through the representation
of poly-peak or incomplete sequences. We presumed that if a forward primer could be developed
regarding the joint regions of the last part of the previous gene (tRNATy r ) and the beginning of the
target gene mtCOI, the sequence could be relevant to the target-sequence of mtCOI. Thus, here, we
describe a new set of primers, which could be used to amplify the mtCOI barcoding region of Artemia
aff. sinica Cai, 1989, with a high performance of sequencing. The new primer set worked well also
for other Artemia bisexual species, as well as for parthenogenetic populations. It is recommended
that joint regions between the previous/next gene(s) and the target marker, could be aimed at when
designing specific primers for other markers and taxa.
Key words. Artemia aff. sinica, PCR production, sequencing, mtCOI, barcoding region,
specific-primers
6)Corresponding author; e-mail: scyshenchunyang@163.com
©KONINKLIJKE BRILL NV, LEIDEN, 2022 DOI 10.1163/15685403-bja10207
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586 ALIREZA ASEM ET AL.
RÉSUMÉ
En raison de l’absence d’une clé taxonomique pour l’identification des espèces d’Artemia,les
marqueurs moléculaires ont été de plus en plus utilisés pour les études phylogénétiques. Le marqueur
mtCO est régulièrement considéré pour la systématique moléculaire des populations d’Artemia.
Les amorces proposées, universelles ou spécifiques, ont la plupart échoué à amplifier le marqueur
Artemia aff. sinica mtCOI, et dans l’ensemble, les produits amplifiés avec succès par PCR ont
été inefficients, principalement par la représentation de poly-pic ou de séquences incomplètes.
Nous supposons que si une amorce anti-sens pouvait être développée pour les régions jointives
de la dernière partie du gène précédent (tRNATyr ) et le début du gène cible mtCOI, la séquence
pourrait être pertinente de la séquence cible de mtCOI. Ainsi, nous décrivons ici un nouvel ensemble
d’amorces qui pourraient être utilisées pour amplifier la région du code-barre mtCOI d’ Artemia
aff. sinica Cai, 1989, avec une performance de séquençage élevée. Ce nouvel ensemble d’amorces a
bien fonctionné aussi pour les autres espèces bisexuelles d’Artemia, ainsi que pour les populations
parthénogénétiques. Il est recommandé que la région de jonction entre les gènes précédents et
suivants et le marqueur cible, pourrait être visée dans le design d’amorces spécifiques pour d’autres
marqueurs ou taxa.
Mots clés. Artemia aff. sinica, production par PCR, séquençage, mtCOI, région à code-barre,
amorces spécifiques
INTRODUCTION AND METHODS
The genus Artemia Leach, 1819 (Branchiopoda, Anostraca), that comprises a
unique taxon of zooplankton organisms, is the most conspicuous inhabitant of
hypersaline environments, with a world-wide geographical distribution except on
Antarctica (Zheng & Sun, 2013). The genus Artemia contains both bisexual species
and a large number of parthenogenetic populations with different ploidy levels
(Asem et al., 2010, 2016a, b; Eimanifar et al., 2014).
In an ongoing comprehensive project on the phylogeography and population
genetics of Artemia in the provinces Hebei and Qinghai (China), we have surveyed
the biological communities of several inland salt lakes, especially those hosting
Artemia aff. sinica Cai, 1989. The mitochondrial COI (mtCOI) marker has been
utilized to identify the taxonomic status and population genetic structure, using
a universal pair of primers for metazoan invertebrates (Folmer et al., 1994)
and two specific primers designed especially for Artemia (Munoz et al., 2008;
Xu, 2018). Although, these three primer pairs have successfully amplified the
barcoding region of mtCOI in our previous studies on different species/populations
of Artemia, in the current project, the PCR failed for 50% to 60% of the samples
examined. On the other hand, almost all successful PCR products represented poly-
peak or incomplete sequences that could not be referred to a reliable sequence
of mtCOI for any phylogenetic study (figs. 1-2). Previously, we had made this
observationinmtCOI sequences of the pentaploid parthenogenetic population
from the Yinggehai Saltern (China). The same challenges have also been noted
for the Mongolian bisexual Artemia aff. sinica (S.C. Sun, pers. comm.) as well as
for the majority of parthenogenetic populations from inland salt lakes in Rumania
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TWO NOVEL PRIMERS FOR COI MARKER OF ARTEMIA AFF. SINICA 587
Fig. 1. Examples of sequencing results of the COI barcoding region of Artemia aff. sinica Cai, 1989
using different primers: A, cf. Folmer et al. (1994); B, cf. Munoz et al. (2008); C, cf. Xu (2018).
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588 ALIREZA ASEM ET AL.
Fig. 2. An example of an incomplete sequence of the COI barcoding region of Artemia aff. sinica Cai,
1989 for amplification of the COI marker, with only <700 bp length successful, on electrophorese
gel using previously described primers (Folmer et al., 1994; Munoz et al., 2008; Xu, 2018).
(A. Berindean, pers. comm.), in which the PCR products were not completely
sequenced.
Recently, a bisexual population from Mongolia has been named Artemia frame-
shifta Naganawa & Mura, 2017, using a single sequence of the mtCOI gene
(LC195588) without any confirmation from either population genetic, or biologi-
cal analyses. A main problem determined in the submitted COI sequence, was the
identification of several stop codons in the corresponding protein translation. Over-
all, due to those reasons, the taxonomic status of the form named A. frameshifta
could not be confirmed (Asem et al., 2020; Eimanifar et al., 2020; D.C. Rogers,
pers. comm.). Additionally, Wang et al. (2008) have reported poly peaks sequences
for the mtCOI marker of an Artemia population from Co Qen in Tibet (China). This
matter might be attributed to nuclear copy/ies of mtDNA gene(s) (Srinivasainagen-
dra et al., 2017; Eimanifar et al., 2020) and/or the existence of pseudogene(s).
In order to solve the problem, we aimed at amplifying the assured mtCOI
barcoding region of Artemia aff. sinica. We hypothesized that, if a forward
primer could be designed to start amplification from the joint regions of the last
part of the previous gene (tRNATyr ) and the beginning of the target mtCOI,the
obtained sequence can most probably be referred to an errorless sequence of the
mitogenomic COI (fig. 3).
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TWO NOVEL PRIMERS FOR COI MARKER OF ARTEMIA AFF. SINICA 589
Fig. 3. Schematic arrangement of the tRNATyr /COI positions and the relative regions of the newly
designed F and R primers. The numbers show the positions on the Artemia sinica Cai, 1989
mitogenome. The purple line represents the COI barcoding region in metazoan invertebrates (to
represent clear positions in this schematic figure, actual scales were not considered).
RESULTS AND DISCUSSION
The complete mitochondrial genome of Artemia sinica (MK069595) was cho-
sen as a reference sequence (Asem et al., 2019). The online primer designing tool
of NCBI was employed to design the desired primers (NCBI, 2021-2022). Accord-
ing to the gene order of the Artemia mitogenome, tRNATyr is placed at the rear of
COI. Because tRNATyr is located on the L-strand (Asem et al., 2019, 2021a), its
reverse sequence on the H-strand was followed to design the F-primer. In practice
this meant, that the last 13 bp of the reverse sequence of tRNATy r on the H-strand
(bp 1347–1359) and 7 bp of the beginning of COI (bp 1361–1367) were chosen
to design the F-primer. Then, based on the barcoding region of the COI marker
(Folmer et al., 1994), a 21-bp-long fragment was chosen on the mitogenome of A.
sinica (MK069595), from bp 2139 to 2160, to design the R-primer (fig. 3). A novel
primer set:
ArtCOI-F (5-CGGCCACTTTACTATGCAACG-3)and
ArtCOI-R (5-CCGAATGCTTCCTTTTTCCCTC-3)
was designed for the barcoding region of COI. A fragment of 750 bp was amplified
using the following PCR programme: 4 min. at 95°C, followed by 35 cycles of 60 s
at 95°C, 60 s at 57-60°C and 90 s at 72°C, with a final extension step of 7 min.
at 72°C. The performance of the newly designed primer set was examined for two
populations of Artemia aff. sinica from China, as well as for all bisexual species
and parthenogenetic populations with different ploidy levels. For parthenogenetic
populations, offspring of the second generation were used to extract DNA and
check ploidy levels.
The designed primers were tested and it appeared that they could successfully
amplify the COI target region in Artemia aff. sinica, with a high quality per-
formance in sequencing especially R reaction (fig. 4). This primer set has also
exhibited its efficiency in amplification of the COI marker in other bisexual species
and in parthenogenetic populations of Artemia. The list of studied species and
populations has been summarized in table I. We thus designed the forward and
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590 ALIREZA ASEM ET AL.
Fig. 4. An example of sequencing results of the COI barcoding region of Artemia aff. sinica Cai,
1989 using the new primer set (ArtCOI-F/ArtCOI-R) described herein.
reverse primers, ArtCOI-F and ArtCOI-R, that completely cover the COI barcod-
ing region for metazoan invertebrates (Folmer et al., 1994) (see fig. 3).
Due to the lack of unequivocal morphological characters, there is no classic
identification key for Artemia species (Asem et al., 2010, 2020). Therefore,
molecular phylogenetic methods have been employed to delimit the taxonomic
status of Artemia populations (Munoz et al., 2008; Maccari et al., 2013; Eimanifar
et al., 2014, 2020; Asem et al., 2016b, 2019, 2021b; Saji et al., 2019; Shen
TABLE I
Artemia species/populations tested using ArtCOI Artemia-specific primers
Species/population Locality GenBank accession number
Artemia sinica Cai, 1989 Yuncheng Lake, P.R. China OM737895-99
Artemia aff. sinica Hebei, P.R. China OM737900-04
Artemia aff. sinica Qinghai, P.R. China OM737905-09
Artemia tibetiana Abatzopoulos,
Zhang & Sorgeloos, 1998
Lagkor Co, P.R. China OM737910-14
Artemia urmiana Günther, 1889 Urmia lake, Iran OM737915-19
Artemia salina (Linnaeus, 1758) Wadi Natron, Egypt OM737920-24
Artemia persimilis Piccinelli &
Prosdocimi, 1968
ARC1321, Argentina OM737925-29
Artemia franciscana Kellogg, 1906 Great Salt Lake, U.S.A. OM737930-34
Parthenogenetic pop. (2n) Ga Hai Lake, P.R. China OM737935-39
Parthenogenetic pop. (3n) Ankiembe, Madagascar OM737940-44
Parthenogenetic pop. (4n) Hoh Lake, P.R. China OM737945-49
Parthenogenetic pop. (5n) Yinggehai Saltern, P.R. China OM737950-54
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TWO NOVEL PRIMERS FOR COI MARKER OF ARTEMIA AFF. SINICA 591
et al., 2021). Hence, it is necessary to provide a dataset of a large number
of sequences from each population, to reliably analyse population genetic and
phylogenetic relationships among them (Maccari et al., 2013). While all primers
hitherto suggested for the COI marker have failed, the new primer set we developed
has presented consistent and most promising results for Artemia aff. sinica.
In conclusion, we may state that, as all bisexual species and parthenogenetic
populations tested in this study could be successfully sequenced in regard of their
COI mitochondrion marker, the primer set ArtCOI-F/ArtCOI-R definitely consti-
tutes a useful tool in phylogenetic studies of Artemia. It is therefore suggested in
general, that joint regions between the previous/next gene(s) and the target marker,
be explicitly considered when designing specific primers for other markers and
taxa.
ACKNOWLEDGEMENTS
The authors thank Prof. Shi-Chun Sun (Ocean University of China, China) and
Prof. Gilbert Van Stappen (Artemia Research Center, Ghent University, Belgium)
for preparing Artemia samples. This study has been supported by 2021 Hebei
Province introduced foreign intelligence project and Natural Science Foundation
of Hebei Province [Grant No. C2020406016].
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ResearchGate has not been able to resolve any citations for this publication.
Review of the biogeography of Artemia Leach, 1819 (Crustacea: Anostraca) in China
  • B S C Zheng
  • Sun
ZHENG, B. & S. C. SUN, 2013. Review of the biogeography of Artemia Leach, 1819 (Crustacea: Anostraca) in China. International Journal of Artemia Biology, 3: 20-50. First received 21 March 2022. Final version accepted 7 May 2022. Published online 29 July 2022.