Biodiversity Data Journal 11: e100068 CO)
doi: 10.3897/BDJ.11.e100068 open access

Research Article

Genomic microsatellite characteristics analysis of
Dysomma anguillare (Anguilliformes,
Dysommidae), based on high-throughput
sequencing technology

Ziyan Zhu?, Yuping Liu+, Shufei Zhang§, Sige Wang*, Tianyan Yang*

$+ Zhejiang Ocean University, Zhoushan, China
§ Guangdong Provincial Key Laboratory of Fishery Ecology and Environment, South China Sea Fisheries Research Institute,
Guangzhou, China

Corresponding author: Tianyan Yang (hellojellyi130@163.com)

Academic editor: Yahui Zhao

Received: 09 Jan 2023 | Accepted: 31 Mar 2023 | Published: 07 Apr 2023

Citation: Zhu Z, Liu Y, Zhang S, Wang S, Yang T (2023) Genomic microsatellite characteristics analysis of

Dysomma anguillare (Anguilliformes, Dysommidae), based on high-throughput sequencing technology.
Biodiversity Data Journal 11: e100068. https://doi.org/10.3897/BDJ.11.e100068

Abstract

Microsatellite loci were screened from the genomic data of Dysomma anguillare and their
composition and distribution were analysed by bioinformatics for the first time. The results
showed that 4,060,742 scaffolds with a total length of 1,562 Mb were obtained by high-
throughput sequencing and 1,160,104 microsatellite loci were obtained by MISA screening,
which were distributed on 770,294 scaffolds. The occurrence frequency and relative
abundance were 28.57% and 743/Mb, respectively. Amongst the six complete
microsatellite types, dinucleotide repeats accounted for the largest proportion (592,234,
51.05%), the highest occurrence frequency (14.58%) and the largest relative abundance
(379.27/Mb). A total of 1488 microsatellite repeats were detected in the genome of D.
anguillare, amongst which the hexanucleotide repeat motifs were the most abundant (608),
followed by pentanucleotide repeat motifs (574), tetranucleotide repeat motifs (232),
trinucleotide repeat motifs (59), dinucleotide repeat motifs (11) and mononucleotide repeat
motifs (4). The abundance of microsatellites of the same repeat type decreased with the
increase of copy numbers. Amongst the six types of nucleotide repeats, the preponderance

© Zhu Z et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

2 Zhu Z et al

of repeated motifs are A (191,390, 43.77%), CA (150,240, 25.37%), AAT (13,168, 14.05%),
CACG (2,649, 8.14%), TAATG (119, 19.16%) and CCCTAA (190, 19.16%, 7.65%),
respectively. The data of the number, distribution and abundance of different types of
microsatellites in the genome of D. anguillare were obtained in this study, which would lay
a foundation for the development of high-quality microsatellite markers of D. anguillare in
the future.

Keywords

Dysomma anguillare, genome, microstatellite, high-throughput sequencing

Introduction

Shortbelly eel (Dysomma anguillare Barnard, 1923) is a small-sized warm water eel that is
widely distributed in the Indian Ocean and the western Pacific Ocean (Nelson et al. 2016).
In China, it is also one of the preponderant bycatch in the offshore waters of the southern
East China Sea (Zhao et al. 2016). As an intermediate to high trophic-level species in the
coastal food webs, it is of great significance in the offshore marine ecosystem and
biodiversity. However, the limited studies of D. anguillare were mainly focused on the
nutrition and feeding habits (Zhang and Tang 2003), the spatial-temporal pattern of
community structure (Liu and Xian 2009) and the effects of lipid removal on the stable
isotopes (Yang et al. 2020).

The explicit germplasm genetic characteristics of fishery species are considered to be the
indispensable prerequisite for effective fisheries management (Hemmer-Hansen et al.
2018). However, the available genetic data for this species are still scarce and only partial
mitochondrial and nuclear gene sequences have hitherto been reported and analysed
(Chen et al. 2014, Chang et al. 2016, Wang et al. 2019). Microsatellite DNA, also named
simple sequence repeats (SSRs) are short tandem duplications (typically 1-6 nucleotide
repeats and mostly less than 100 bp in length), ubiquitous occurring in eukaryotic
organisms. Besides, the repetitions vary drastically amongst different genotype of the
same species (Tautz and Renz 1984). The co-dominant microsatellite molecular markers,
based on polymerase chain reaction (PCR) techniques, have overriding advantages in high
polymorphism, good repeatability, simple operation and low experimental cost. Therefore, it
has possessed important applied worth in gene mapping and QTL analysis, population
genetics and evolutionary research, as well as molecular marker-assisted breeding
(Messier et al. 1996, Schlotterer 2000). At present, the conventional development
strategies of representative microsatellite loci mainly include anchored-PCR-based
method, selective hybridisation enrichment method, database search and relative species
selection method (Sun et al. 2009). Nevertheless, these above-mentioned technical means
not only are time-consuming and expensive, but also reflect incomplete distribution of
microsatellites and develop limited molecular markers.

Genomic microsatellite characteristics analysis of Dysomma anguillare (Anguilliformes, ... 3

In recent years, along with the rapid progress of high-throughput sequencing (HTS)
technology and the reduction of sequencing cost, developing numerous high-polymorphism
SSR markers from multi-omics data has become more and more convenient. In this study,
the genome-wide sequences of Dysomma anguillare were obtained, based on HiSeq ™
4500 platform for the first time; meanwhile, the SSR loci distribution and characteristics
were also analysed by bioinformatics tools. The findings will help to provide useful
references and basic information for germplasm resources conservation, population
genetic evaluation and phylogenetic relationships analysis amongst related species of
Anguilliformes.

Material and Methods
Sample collection and genomic DNA extraction

Fifty-three samples of Dysomma anguillare were collected by trawling in the coastal waters
of Zhoushan, Zhejiang Province in September 2022. After preliminary morphological
identification, muscle tissues from five male and five female individuals were randomly
selected for the genomic DNA extraction by the traditional Tris-saturated phenol method
(Maniatis et al. 1982). Subsequently, the DNA barcode method, based on the mitochondrial
COI sequence, was further conducted to ensure the species accuracy . The 1% agarose-
gel electrophoresis and NanoDrop 2000 ultraviolet spectrophotometer (USA, Thermo
Fisher Scientific) were performed to detect the integrity and purity of the genomic DNA,
respectively. The obtained DNA samples were stored at -20°C for further analysis.

Library construction and high-throughput sequencing

Equal amounts of DNA (2 yg each) were mixed for library construction and next-generation
sequencing by Onemore Technology (Wuhan) Co., Ltd. The genomic DNA was randomly
fragmented using Covaris Ultrasonic Processor into small 200 to 350 bp fragments. Two
pair-end DNA libraries were constructed through terminal repair, adding Poly-A tails and
sequencing adapters, purification and PCR amplification and then sequenced using the
Illumina HiSeq'™ 4500 sequencing technology.

Sequence cleaning and genome assembly

Raw data output from Illumina platform were firstly transformed into sequence reads by
base calling and recorded in a FASTQ format. Subsequently, clean reads were obtained
after filtering adaptor sequences and low quality read by Cutadapt v.1.16 (Martin 2011).
SOAPdenovo v.2.04 was used to assemble the clean data with the setting parameters “-K
53 -R -M 3 -d 1”, which employed the de Bruijn graph-based assembly strategy (Kajitani et
al. 2014). First, reads sequenced from the small-fragment library were divided into smaller
substrings (K-mers) to construct a preliminary de Bruijn diagram. Then, the simplified de
Bruijn graph was obtained after removing the low-coverage branches and branches that
cannot be connected further due to sequencing errors and the sequences at every

4 Zhu Z et al

bifurcation locus were truncated to obtain the initial contigs. By mapping the paired-end
reads back to the contigs, the connectivity relationships between the reads and the
information of the inserted fragment size were used to further assemble the contigs into
scaffolds and obtain the primary genomic sequence.

Screening and identification of SSRs

MicroSatellite identification tool (MISA) software (http://pgrc.ipk-gatersleben.de/misa/)
written by Perl script was implemented to scan the assembled scaffolds to identify the
genome-wide microsatellite repeat units and to analyse the length, location and quantity of
the SSRs (Thiel et al. 2003). The occurrence frequency of SSR loci, average distribution
distance and density of microsatellites, type and length of repeat motifs were calculated
using Microsoft Excel 2019. The default parameters of MISA were set as follows: the
repeat motif length was from 1 to 6 nucleotides and the minimum thresholds of repeat
counts were 1-10, 2-6, 3-5, 4-5, 5-5 and 6-5, which meant the number of mononucleotide
repeats was less than 10, number of dinucleotide repeats was less than 6 and numbers of
remaining repeats were all less than 5, respectively. Besides, the number of bases
interrupting two SSRs in a compound microsatellite should be less than 100. Considering
the Watson-Crick complementary condition and the difference in the base arrangement,
the repeat sequences and their complementary sequences were grouped together. For
example, the (AC),, (CA),, (TG), and (GT), were treated as the same SSR repeat types.

Results
Genome sequencing and assembly

The information of contigs and scaffolds of the Dysomma anguillare genome was listed in
the Table 1. About 11,805,379 contigs with the total length 1,960 Mb were obtained after
splicing and the average GC content was about 42.2%. The number of scaffolds produced
by the SOAPdenovo v.2.0 assembly was 4,060,742 and the full length was 1,561 Mb, with
the average GC content 39.6%.

Table 1.

The contig and scaffold assembly results statistics.

Assembly — The totallength The sequence Lengthnumberof The maximum N50 N90 GC

level (bp) number sequences length (bp) (bp) (bp) content
2 2Kb (%)

Contig 1,960,673,378 11,805,379 30,667 9,646 272 ~=60 42.2

Scaffold 1,561,530,495 4,060,742 95,727 23,878 709 134 39.6

N50 value is a widely used metric for measuring the quality of sequences by the assembly
algorithms’ output. It refers to the contig or scaffold length value when the accumulated
fragment length (from long to short) exceeds 50% of the total length of all contigs or

Genomic microsatellite characteristics analysis of Dysomma anguillare (Anguilliformes, ... 5

scaffolds for the first time. The greater the N50 value, the smaller the quantity and the
better the assembly quality. In this study, the N50 values of contig and scaffold assembly
were 272 bp and 709 bp, respectively. Compared with the assembled genomes of related
species Anguilla japonica (Henkel et al. 2012), A. anguilla (Jansen et al. 2017) and A.
rostrate (Pavey et al. 2017), the assembly effect of Dysomma anguillare was relatively
good and developing microsatellite markers could reflect the genome-wide characteristics
of SSRs.

SSR repeat types and distribution

A total of 1,160,104 microsatellites with 1-6 bp nucleotide motifs were detected in 770,294
unigenes and 234,959 of them contained more than one SSR locus, with the occurrence
frequency (total number of SSRs detected/total number of unigenes) of 28.57%. The
density of distribution (total length of unigenes/total number of SSRs screened) was on
average 1/1.35 kb and the relative abundance (total number of SSRs screened/total length
of unigenes) was 743/Mb.

These SSR loci can be classified into six repeat types: mononucleotide, dinucleotide,
trinucleotide, tertranucleotide, pentanucleotide and hexanucleotide. The most abundant
type of repeat motif was dinucleotide, accounting for 51.05% in the all SSR loci and then
followed by mononucleotide (37.69%), trinucleotide (8.08%), tertranucleotide (2.71%) and
pentanucleotide (0.25%), while hexanucleotide was the minimum (0.21%) of all (Fig. 1).
The occurrence frequency of dinucleotide repeats was highest, while hexanucleotide was
observed the lowest, representing 14.58% and 0.06% of the total genome, respectively.
The relative abundance of dinucleotide reached 379.27/Mb, with an average of one SSR
locus per 2.64 kb and the next was mononcleotide (280.00/Mb). By comparison, the
relative abundance of hexanucleotide was the lowest (1.59/Mb) (Table 2).

Repeat numbers of different SSRs

The number of repeats of SSR loci mainly ranged from 5 to 24. The predominant repeat
number of the SSR loci was 10 times, comprising 17.52% of the total number of SSR loci.
In general, the number of repeat types decreased with the increase in repeat numbers (Fig.
2). The repeats of mononucleotide, dinucleotide and trinucleotide were mainly distributed in
10-19 times (96.83%), 6-15 times (95.15%) and 5-9 times (85.34%), respectively. However,
the repeat times of the rest of the repeat types were all within 13 times, which were mainly
in the range of 5-8 times and separately accounted for 92.40%, 96.70% and 99.56% (Table
3).

In summary, the repeat numbers of SSR loci were mainly concentrated in 10-15 times and
5-8 times, with a total number of 1,016,359 (87.61%). Few SSR loci with more than 25
repeats were identified and the type of base repeats was monotonous, only composing of
mononucleotide repeat.

6 Zhu Z et al

Copy numbers of repeat units

Amongst the detected 1,488 repeat units, hexanucleotide repeats possessed the most
types and pentanucleotide repeats took second place. Nevertheless, the type of
mononucleotide repeats was the least limited to the base number (Table 4). Amongst all
these repeats, the dominant repeat motifs in mononucleotide, dinucleotide, trinucleotide,
tetranucleotide, pentanucleotide and hexanucleotide were A (191,390, 43.77%), CA
(150,240, 25.37%), AAT (13,168, 14.05%), CACG (2,649, 8.14%), TAATG (119, 19.16%)
and CCCTAA (190, 7.65%), respectively (Fig. 3, Table 4).

Table 2.

Proportions of each SSR repeat types in the genome of D. anguillare.

Repeat type Number Occurrence frequency Relative abundance (per: Average length Total length
(%) Mb ~') (bp) (bp)
Mononucleotide 437,234 10.77% 280.00 0.87 379,455
Dinucleotide 592,234 14.58% 379.27 0.50 298,350
Trinucleotide 93,734 2.31% 60.03 0.72 67,533
Tetranucleotide 31,481 0.78% 20.16 0.72 22,680
Pentanucleotide 2,936 0.07% 1.88 0.82 2,409
Hexanucleotide 2,485 0.06% 1.59 0.71 1,774
Total 1,160,104 28.57% 742.93 4.35 772,201
0.25%

2.71% 0.21%

™@ mononucleotide
@ dinucleotide

= trinucleotide

@ tetranucleotide
@ pentanucleotide

@ hexanucleotide

Figure 1. EES]

Distribution of SSRs repeat types in genomes of D. anguillare.

Genomic microsatellite characteristics analysis of Dysomma anguillare (Anguilliformes, ... 7

SSR length distribution and polymorphism evaluation

The sequence length amongst different types of SSRs varied a lot, from 10 to 54 bp (Fig.
4). The minimum and maximum variations in length were detected in hexanucleotide and
mononucleotide repeats, respectively. The former was in the range of 30-54 bp with the
total length of 1,774 bp, while the latter was in the range of 10-51 bp with total length of
379,455 bp, which constituted approximately 49.14% of the total length of SSRs. Amongst
the six types of nucleotide repeat, dinucleotide and trinucleotide were dominant in the
distribution of microsatellites from the perspective of sequence length, which were 677,805
bp in total and accounting for 87.78% in all SSRs.

Table 3.

Distribution interval of the copy number in different microsatellite motif for D. anguillare.

Repeat Mononu Dinu Trinu Tetranu Pentanu Hexanu Total Proportion
number cleotide cleotide cleotide cleotide cleotide cleotide (%)

0 32,413 17,143 2,071 972 52,599 4.53%
6 0 162,916 18,834 7,300 498 394 189,942 16.37%
7 0 101,359 13,353 3,184 176 270 118,342 10.20%
8 0 72,287 9,325 1,460 94 838 84,004 7.24%

57,111 6,070 895 46 11 64,133 5.53%
10 152,127 46,524 3,962 594 51 0 203,258 17.52%
11 90,414 38,631 2,619 422 0 0 132,086 11.39%
12 58,458 30,798 1,896 430 0 0 91,582 7.89%
13 40,161 23,987 1,488 53 0 0 65,689 5.66%
14 27,543 17,686 1,203 0 0 0 46,432 4.00%
15 18,717 12,237 1,451 0 0 0 32,405 2.79%
16 13,469 8,578 1,069 0 0 0 23,116 1.99%
17 9,925 5,919 51 0 0 0 15,895 1.37%
18 7,271 3,962 0 0 0 0 11,233 0.97%
19 5,276 2,745 0 0 0 0 8,021 0.69%
20 3,800 2,077 0 0 0 0 5,877 0.51%
21 2,605 1,480 0 0 0 0 4,085 0.35%
22 1,889 1,344 0 0 0 0 3,233 0.28%
23 1,297 1,670 0 0 0 0 2,967 0.26%
24 853 878 0 0 0 0 1,731 0.15%
25 697 45 0 0 0 0 742 0.06%
>25 2,712 0 0 0 0 0 2,712 0.23%

The length of the microsatellite was one of the main factors affecting its polymorphism.
Temnykh et al. (2001) divided SSR sequences into two categories: the high-polymorphic

8 Zhu Z et al

type | (length 2 20 bp) and the moderate-polymorphic type Il (12 bp < length < 20 bp). The
microsatellites with length less than 12 bp owned lower polymorphism, but higher mutation
potential. In the present study, there were 21,347 type | SSRs (19%) and 294,373 type II
SSRs (54%), respectively. SSR loci with low mutation potential accounted for 27%.

Discussion

Number and relative abundance of microsatellites in the genome of
Dysomma anguillare

The bioinformatics software was used to search and analyse the various types and
numbers of six perfect microsatellites in the genome of Dysomma_ anguillare.
Approximately 1,160,104 microsatellite loci were revealed across the 1.56 Gb genome
sequence, with a total length of 24,707,980 bp (occupying 58% of the full genome length).
In contrast to other published genomes of bony fishes, it was higher than Takifugu rubripes
(0.77%) (Cui et al. 2006), Scleropages formosus (0.78%) (Duan et al. 2019) and Bagarius
yarrelli (1.23%) (Yang et al. 2021), but lower than Pelteobagrus fulvidraco (1.80%) (Xu et
al. 2020) and Harpadon nehereus (2.01%) (Yang et al. 2021), indicating that genome-wide
microsatellites content was not directly related to the genetic relationship and the reasons
might involve different retrieval tools, parameter settings and databases (He et al. 2015).
Hancock (1996) speculated that the numbers of microsatellites increased with the
chromosome length and the disproportional relationship between the genome size and
microsatellite numbers was also confirmed in our study.

250000
200000
150000

100000

Number of SSRs

50000

Nm rFunwornr~ WO Dd O
SAF a a Aa a ast N

AN mM TO WY
NNNN NN
A

Repeat number

@ mononucleotide @ dinucleotide trinucleotide

@tetranucleotide ™ pentaxucleotide ™ hexanucleotide

Figure 2. EES]

SSR repeats distribution of D. anguillare.

Genomic microsatellite characteristics analysis of Dysomma anguillare (Anguilliformes, ...

Table 4.

Dominant base types and the proportion in genome of D. anguillare.

Repeat type Number of Maximum Minimum
t
NBE® Repeat Number Proportion Repeat motif Number Proportion
motif (%) (%)
Mononucleotide 4 A 191,390 43.77 G 43,065 9.85
Dinucleotide 12 CA 150,240 25.37 GC 604 0.1
Trinucleotide 59 AAT 13,168 14.05 ACG 26 0.03
Tetranucleotide 232 CACG 2,649 8.41 ACCC/ACTT / 1 0.00
AGGT/CCAC
ICCGAICGAT /
TACG/TGGG
Pentanucleotide 574 TAATG 110 19.16 - 1 0.17
Hexanucleotide 608 CCCTAA 190 7.65 - 1 0.04

(1) mononucleotide

(2)dinucleotide

160000

200000 140000
120000

150000
100000
100000 80000
60000

50000
40000
. 20000

A c 6 T

@10 @11 @12 813 #14 B15 B16 B17 wis 819 B20 B21 B22 223 24 m25 m>2S 0

7 A AC AG AT CA CG cT GA GC GT TA Tc TG
(3)trinucleotide

(4)tetranucleotide

14000
3000
12000
10000 2500
8000 2000
6000 1500
4000 1000
2000 500 '
0 0 \ lds hal j |) j |
vu vo Vv er @Y@ zqreuo Vv VM ere uoYv
SSGESESZSESES SESREZ ESSE YUSPESPER EY EY BREESSLESVSHSE
qaqatieqewtg pe”) OoUV OO UFR FE $298 os oSh 2 Oo OES oO Be
SEQLELSSSSSCCsSESEGES SEE
5 86 87 68 wo 10 wil 12 w13 14 15 16 17
@5 86 87 @8 w9 w10 wil w12 813
5)pentaxucleotide ‘
(S)p (6)hexancleotide
120
200
100 180
160
80 140
120
60 100
80
40 60
40
20 20
j 0
0 ai SOSH oe ee SBE ESV Ve SUSE ESEPYVBSEBL
VOZZEFGYVUOEFUFOZTSZOVUSZOFOCOFROUSGEUE
VUEUrFUrFUFRSA udtuu VuUYVYYeagetor ce q@auwu =) 235653 qaeactreatvyverv VoOqaOoOYrEUa 0 =
SEESSEESESSZSRESESSSESSESESES SER ES SSPeSsSSSSsoyessessssereyogs
SSS SEV Ve eSSSSSCYSCSC SESS ZBYSGEEEYPLPEE SS ee Ve ESS SOCCOURSSEGSYEOGSGEFFFE
e566 87 88 #9 #10 5 e687 88 99

Figure 3. EES

The distribution of microsatellite repeats in genome of D. anguillare.

10 Zhu Z et al

Relative abundance was an important feature to measure microsatellite richness. It was
calculated to be 743/Mb of Dysomma anguillare, which was much higher than that of other
marine fishes, such as Scatophagus argus (653/Mb) (Wang et al. 2020), Cociella
crocodilus (428/Mb) (Zhao et al. 2021), Tridentiger bifasciatus (347/Mb) (Zhao et al. 2022)
and four species of pufferfishes (365/Mb in Takifugu rubripes, 369/Mb in Takifugu flavidus,
397/Mb in Takifugu bimaculatus and 525/Mb in Tetraodon nigroviridis) (Xu et al. 2021). The
above result showed that abundant microsatellites existed in the genome of D. anguillare,
which would provide sufficient molecular markers for the further germplasm identification
and genetic diversity studies.

140000
120000

*E 100000
~ 0000
60000

40000 = <12bp

| ™ type II (12~20bp

typeI (>20bp)

20000

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 >25
The length of SSR
@ mononucleotide m dinucleotide trinucleotide

Mtetranucleotide #pentaxucleotide @® hexanucleotide

A B

Figure 4. EES]

Length distribution of genes in D. anguillare.
A SSR length distribution; B Distribution types of SSR (type | and type II).

Distribution characteristics of microsatellites in the genome of Dysomma
anguillare

Varied microsatellite types composing of 1-6 nucleotide repeats were discovered in the
genome of Dysomma anguillare and dinucleotide repeats were the most frequent, followed
by mononucleotide repeats, while the percentages of SSRs containing 3-6 nucleotide
repeats were no more than 10%. Therefore, priority should be given to dinucleotide repeats
when designing SSR primers of D. anguillare. Mononucleotide and dinucleotide repeats
were regarded as the most abundant types of SSRs in most species. It was reported that
mononucleotide repeats tended to dominate in the genomes of higher grade organisms
(Gao and Kong 2005). However, dinucleotide repeats contained higher proportions in fish
genomes, which probably related to the differences in gene expression and regulation.

The CA repeat motif was the most abundant amongst dinucleotide repeats and occupied
25.37% of them, which was consistent with Scophthalmus maximus (Ruan 2009) and
pufferfishes (Cui et al. 2006, Xu et al. 2021), but different from /cta/urus punctatus (Tang et
al. 2022), while the number of GC repeat motifs was the least. The base sliding might

Genomic microsatellite characteristics analysis of Dysomma anguillare (Anguilliformes, ... 11

generate microsatellites more easily at the low melting temperature (T,,). Two hydrogen
bonds between A-T base pairs were more likely to be broken than three hydrogen bonds
between G-C base pairs, resulting in reduction of the GC repeats (Huang et al. 2020).
Some other scholars pointed out that the methylation of CoG might cause the spontaneous
deamination of cytosine to thymine in order to maintain the thermodynamic stability of the
DNA molecule. In this study, the proportion of GC repeats motif was only 0.1% and from
this aspect, the lower GC content in the whole genome also reflected the small amount of
GC repeats (Schorderet and Gartler 1992).

The structural instability and composition of trinucleotide repeats were closely related to
some genetic diseases in humans (Sinden et al. 2002). It was found that AAT repeat motif
was the most numerous of the trinucleotide repeats in the Dysomma anguillare genome,
the same as for humans and primates (Kelkar et al. 2008). Therefore, in-depth analysis of
trinucleotide repeats would contribute to predict some gene loci associated with human
diseases and thereby reduces the occurrence of certain illness by changing gene
expression.

Copy numbers and length variations in the genome of Dysomma
anguillare

The repeat unit length was in inverse proportion to the copy number of microsatellite DNA
(Harr and Schlotterer 2000). Commonly, the higher the copy number of SSRs meant the
more alleles and the richer polymorphism. The number of microsatellite repeats in the
Dysomma anguillare genome was mainly in the range of 5 to 25. Motifs that showed more
than 25 reiterations were very rare (only 2,712 SSRs) and all of them were composed of
mononucleotide repeats. Previous studies proved that the mutation rate of microsatellites
was positively correlated to the copy number of the repeat motif (Wierd! et al. 1997) and
longer microsatellites were expected to have higher mutation rate owing to more chances
of replication slippage (Calabrese and Sainudiin 2005). The results demonstrated that the
number of SSRs decreased as the repeat number increased. In addition, tetranucleotide,
pentanucleotide and hexanucleotide microsatellites might have higher mutation rates than
those of the mononucleotide, dinucleotide and trinucleotide microsatellites.

The length of microsatellites in the Dysomma anguillare genome was generally 10-18 bp
and the number of microsatellites was inversely proportional to the repeat motif length. The
structure and its characteristics analysis of a parthenogenic gastropod Melanoides
tuberculata concluded that the longer the repeat sequence length was, the greater the
selection pressure undergoing and the lower numbers of repeats was (Samadi et al. 1998).
This phenomenon had been verified by various kinds of plants and animals, for instance,
Juglans regia (Liao et al. 2014), Patinopecten yessoensis (Ni et al. 2018) and
Phrynocephalus axillaris (Song et al. 2019). According to Temnykh et al. (2001), SSR
polymorphism could be considered low, medium and high and the SSRs with lengths
longer than 12 bp were potential molecular markers with high polymorphism. In the study,
the type | and type II SSRs in the D. anguillare genome occupied about 73% of the total,
showing great potential for polymorphism microsatellite development.

12 Zhu Z et al

Conclusions

In conclusion, MISA software was used for the first time to search and analyse six types of
perfect microsatellite loci from the whole genome survey data of Dysomma anguillare. The
results showed that both the relative abundance and density of various microsatellite types
were very high. Amongst the 1,160,104 SSR loci, the number of different repeat types
presented a trend as: dinucleotide > mononucleotide > trinucleotide > tetranucleotide >
pentanucleotide > hexanucleotide. The dominant repeat motifs of them were A, CA, AAT,
CACG, TAATG and CCCTAA, respectively. The results supplemented the genetic marker
database of marine fishes and provided valuable information resources for further genetic
analysis of D. anguillare.

Acknowledgements

We are grateful to the National Innovation and Entrepreneurship Training Program for
College Students (202210340023); Science and Technology Innovation Project of College
Students in Zhejiang Province (2023R411006); Science and Technology Planning Project
of Zhoushan (2022C41022); and Fund of Guangdong Provincial Key Laboratory of Fishery
Ecology and Environment (FEEL-2021-7).

Author contributions

Tianyan Yang conceived and designed the study. Shufei Zhang collected and provided
samples. Yuping Liu preformed the DNA extraction and bioinformatics analysis. Ziyan Zhu
wrote and edited the manuscript. All authors contributed to the preparation of the
manuscript.

Conflicts of interest

The authors have declared that no competing interests exist.

References

° Calabrese P, Sainudiin R (2005) Models of microsatellite evolution. In: Loughin TM (Ed.)
Statistical Methods in Molecular Evolution. Springer, New York, 289-305 pp.

[ISBN 978-0-38722-333-9].

° Chang CH, Shao KT, Lin HY, Chiu YC, Lee MY, Liu SH, Lin PL (2016) DNA barcodes of
the native ray-finned fishes in Taiwan. Molecular Ecology Resources 17 (4): 796-805.
https://doi.org/10.1111/1755-0998.12601

° Chen JN, Lopez JA, Lavoue S, Miya M, Chen WJ (2014) Phylogeny of the
Elopomorpha (Teleostei): Evidence from six nuclear and mitochondrial markers.
Molecular Phylogenetics and Evolution 70: 152-161. htips://doi.org/10.1016/j.ympev.20
13.09.002

Genomic microsatellite characteristics analysis of Dysomma anguillare (Anguilliformes, ... 13

Cui JZ, Shen XY, Yang GP, Gong QL, Gu QQ (2006) The analysis of simple sequence
repeats in Takifugu rubripes genome. Periodical of Ocean University of China 36 (2):
249-254. https://doi.org/10.3969/j.issn.1672-5174.2006.02.015

Duan YN, Liu Y, Hu YC, Liu C, Song HM, Wang XJ, Sun JH, Mu XD (2019) Distribution
regularity of microsatellites in Scleropages formosus genome. Chinese Agricultural
Science Bulletin 35 (23): 152-158. https://doi.org/10.11924/j.issn.1000-6850.casb
18030101

Gao H, Kong J (2005) Distribution characteristics and biological function of tandem
repeat sequences in the genomes of different organisms. Zoological Research 26 (5):
555-564. https://doi.org/10.3321/j.issn:0254-5853.2005.05.017

Hancock JM (1996) Simple sequences and the expanding genome. BioEssays 18 (5):
421-425. https://doi.org/10.1002/bies.950180512

Harr B, Schlétterer C (2000) Long microsatellite alleles in drosophila melanogaster have
a downward mutation bias and short persistence times, which cause their genome-wide
under representation. Genetics 155 (3): 1213-1220. https://doi.org/10.1016/1350-4533
(94)90010-8

Hemmer-Hansen J, Hu s, Baktoft H, Huwer B, Eero M (2018) Genetic analyses reveal
complex dynamics within a marine fish management area. Evolutionary Applications 12
(4): 830-844. https://doi.org/10.1111/eva.12760

Henkel CV, Dirks RP, de Wijze DL, Minegishi Y, Aoyama J, Jansen HJ, Turner B,
Knudsen H, Bundgaard M, Hvam KL, Boetzer M, Pirovano W, Weltzien FA, Dufour S,
Tsukamoto K, Spaink HP, van den Thillart GE (2012) First draft genome sequence of
the Japanese eel, Anguilla japonica. Gene 511: 195-201. htips://doi.org/10.1016/j.gene.
2012.09.064

He XD, Zheng JW, He KY, Wang BS (2015) Comparison of different search tools to find
microsatellites sites in unigene sequences of Salix babylonica. Molecular Plant
Breeding 13 (1): 197-204. https://doi.org/10.13271/j.mpb.013.000197

Huang J, Liu L, Yang B, Yang CZ (2020) Distribution regularities of microsatellites in the
genome of great cormorant (Phalacrocorax carbo). Chinese Journal of Wildlife 41 (1):
108-114. https://doi.org/10.3969/j.issn.1000-0127.2020.01.015

Jansen HJ, Jansen HJ, Jong-Raadsen SA, Dufour S, Weltzien FA, Swinkels W,
Koelewijn A, Palstra AP, Palstra B, Spaink HP, van den Thillart GE, Dirks RP, Henkel CV
(2017) Rapid de novo assembly of the European eel genome from nanopore
sequencing reads. Scientific Reports 7: 1-13. https://doi.org/10.1038/s41598-01 7-07
650-6

Kajitani R, Toshimoto K, Noguchi H, Toyoda A, Ogura Y, Okuno M, Yabana M, Harada
M, Nagayasu E, Maruyama H, Kohara Y, Fujiyama A, Hayashi T, Itoh T (2014) Efficient
de novo assembly of highly heterozygous genomes from whole-genome shotgun short
reads. Genome Research 24 (8): 1384-1395. https://doi.org/10.1101/gr.170720.113
Kelkar YD, Tyekucheva S, Chiaromonte F, Makova KD (2008) The genome-wide
determinants of human and chimpanzee microsatellite evolution. Genome Research 18
(1): 30-38. https://doi.org/10.1101/gr.7113408

Liao ZY, Ma QY, Dai XG, Zhang DF, Li SX (2014) Microsatellite characters in Juglans
regia L. genome by high throughput sequencing technology. Journal of Northeast
Forestry University 42 (2): 65-68. https://doi.org/10.13759/j.cnki.dIxb.2014.02.016

14

Zhu Z et al

Liu SD, Xian WW (2009) Temporal and spatial patterns of the ichthyoplankton
community in the Yangtze Estuary and its adjacent waters. Biodiversity Science 17 (2):
151-159. https://doi.org/10.3724/SP.J.1003.2009.08194

Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold
Spring Harbor Laboratory Press, New York, 157-163 pp. [ISBN 0-87969-136-0]

Martin M (2011) Cutadapt removes adapter sequences from high-throughput
sequencing reads. EMBnet Journal 17 (1): 10-12. https://doi.org/10.14806/ej.17.1.200
Messier W, Li SH, Stewart CB (1996) The birth of microsatellites. Nature 381 (6582):
483. https://doi.org/10.1038/381483a0

Nelson JS, Grande TC, Wilson MVH (2016) Fishes of the World. 5th Edition. John Wiley
& Sons, Inc, Hoboken. [ISBN 978-1-11834-233-6] https://doi.org/
10.1002/9781119174844

Ni SS, Yang Y, Liu SF, Zhuang ZM (2018) Microsatellite analysis of Patinopecten
yessoensis using next-generation sequencing method. Progress in Fishery Sciences 39
(1): 107-113. https://doi.org/10.11758/yykxjz.20161209001

Pavey SA, Laporte M, Normandeau E, Gaudin J, Letourneau L, Boisvert S, Corbeil J,
Audet C, Bernatchez L (2017) Draft genome of the American eel (Anguilla rostrata).
Molecular Ecology Resources 17: 806-811. httos://doi.org/10.1111/1755-0998.12608
Ruan XH (2009) Development, characterization and application of microsatellite
markers in Turbot. The dissertation of a doctor of pharmaceutical chemistry. Medical
College, Ocean University of China, Qingdao.

Samadi S, Artiguebielle E, Estoup A, Pointier JP, Silvain JF, Heller J, Cariou ML, Jarne
P (1998) Density and variability of dinucleotide microsatellites in the parthenogenetic
polyploid snail Melanoides tuberculata. Molecular Ecology 7 (9): 1233-1236. https://

doi.org/10.1046/j.1365-294x.1998.00405.x
Schl6otterer C (2000) Evolutionary dynamics of microsatellite DNA. Chromosoma 109

(6): 365-371. https://doi.org/10.1007/s004120000089

Schorderet DF, Gartler SM (1992) Analysis of CoG suppression in methylated and
nonmethylated species. Proceedings of the National Academy of Sciences 89 (3):
957-961. https://doi.org/10.1073/pnas.89.3.957

Sinden RR, Potaman VN, Oussatcheva EA, Pearson CE, Lyubchenko YL,
Shlyakhtenko LS (2002) Triplet repeat DNA structures and human genetic disease:
dynamic mutations from dynamic DNA. Journal of Bioscience 27 (Suppl 1): 53-65.
https://doi.org/10.1007/BF02703683

Song Q, Guo XG, Chen DL (2019) Characterization of microsatellite DNA loci and
design of candidate primers to amplify these regions for Phrynocephalus forsythii by
using 454 GS FLX. Sichuan Journal of Zoology 38 (5): 512-520. https://doi.org/
10.11984 /j.issn.1000-7083.20190010

Sun B, Bao YX, Zhao QY, Zhang LL, Hu ZY (2009) Methods for obtaining microsatellite
loci: A review. Chinese Journal of Ecology 28 (10): 2130-2137. URL: http://Awww.cjae.
netv/EN/Y2004/V15/109/1580

Tang RY, Su MY, Yang WS, Xu JJ, Wang T, Yin SW (2022) Analysis of microsatellite
distribution characteristics in the channel catfish (/cta/urus punctatus) genome.
Progress in Fishery Sciences 43 (2): 89-97. https://doi.org/10.19663/j.issn2095-9869 .
20210126002

Genomic microsatellite characteristics analysis of Dysomma anguillare (Anguilliformes, ... 15

Tautz D, Renz M (1984) Simple sequences are ubiquitous repetitive components of
eukaryotic genomes. Nucleic Acids Research 12 (10): 4127-4138. https://doi.org/10.
1093/nar/12.10.4127

Temnykh S, DeClerck G, Lukashova A, McCouch §S, Lipovich L, Cartinhour S (2001)
Computational and experimental analysis of microsatellites in rice (Oryza sativa L.):
Frequency, length variation, transposon associations, and genetic marker potential.
Genome Research 11 (8): 1441-1452. https://doi.org/10.1101/gr.184001

Thiel T, Michalek W, Varshney R, Graner A (2003) Exploiting EST databases for the
development and characterization of gene-derived SSR-markers in barley (Hordeum
vulgare L.). Theoretical and Applied Genetics 106: 411-422. https://doi.org/10.1007/
$00122-002-1031-0

Wang YM, Zhang FY, Zhao M, Ma CY, Zhang LZ, Ma LB (2019) The complete
mitochondrial genome of Dysomma anguillare (Anguilliformes, Synaphobranchidae)
with phylogenetic consideration. Mitochondrial DNA Part B 4 (1): 1688-1689. https://
doi.org/10.1080/23802359.2019.1604103

Wang YR, Yang W, Ren XL, Jiang DN, Deng SP, Chen HP, Zhu CH, Li GL (2020)
Distribution patterns of microsatellites and development of polymorphic markers from
Scatophagus argus genome. Journal of Guangdong Ocean University 40 (4): 7-14.
https://doi.org/10.3969/j.issn.1673-9159.2020.04.002

Wierdl M, Dominska M, Petes TD (1997) Microsatellite instability in yeast: dependence
on the length of the microsatellite. Genetics 146 (3): 769-779. https://doi.org/10.1093/
genetics/146.3.769

Xu JJ, Zheng X, Li J, Yin SW, Wang T (2020) Distribution characteristics of whole
genome microsatellite of Pelteobagrus fulvidraco. Genomics and Applied Biology 39
(12): 5488-5498. https://doi.org/10.1341 7/j.gab.039.005488

Xu JJ, Zheng X, Zhang XY, Wang T, Yin SW (2021) Analysis of distribution
characteristics of microsatellites in four genomes of puffer fish. Genomics and Applied
Biology 40 (4): 1441-1451. https://doi.org/10.13417/j.gab.040.001441

Yang R, Tian SQ, Gao CX, Dai LB, Wang SC (2020) Effects of lipid removal on the
stable isotopes of Dysomma anguillaris in the offshore waters of southern Zhejiang.
Journal of Fishery Sciences of China 27 (9): 1085-1094. httos://doi.org/10.3724/SP.J.
1118.2020.20027

Yang TY, Huang XX, Ning ZJ, Gao TX (2021) Genome-wide survey reveals the
microsatellite characteristics and phylogenetic relationships of Harpadon nehereus.
Current Issues in Molecular Biology 43 (3): 1282-1292. https://doi.org/10.3390/cimb
43030091

Yang WS, Tang RY, Su MY, Xu JJ, Wang T, Yin SW (2021) Analysis of microsatellite
distribution characteristics in the whole genome of Bagarius yarrelli. Journal of Nanjing
Normal University, Engineering and Technology Edition 21 (3): 62-68. https://doi.org/
10.3969/j.issn.1672-1292.2021.03.009

Zhang B, Tang QS (2003) Feeding habits of six species of eels in East China Sea and
Yellow Sea. Journal of Fisheries of China 27 (4): 307-314. URL: https:/Avww.china-
fishery.com/scxuebao/article/abstract/20030404?st=article issue

Zhao RR, Lu ZC, Cai SS, Gao TX, Xu SY (2021) Whole genome survey and genetic
markers development of crocodile flathead Cociella crocodilus. Animal Genetics 52 (6):
891-895. https://doi.org/10.1111/age.13136

16

Zhu Z et al

Zhao SL, Xu HX, Zhong JS (2016) Zhejiang marine ichthyology. Zhejiang Science and
Technology Press, Hangzhou. [ISBN 978-7-53417-152-9]

Zhao X, Liu YX, Du XQ, Ma SY, Song N, Zhao LL (2022) Whole-genome survey
analyses provide a new perspective for the evolutionary biology of shimofuri goby,
Tridentiger bifasciatus. Animals 12 (15): 1914. https://doi.org/10.3390/ani12151914