# Development of a polymerase chain reaction (PCR)-based genetic sex identification method in the chub mackerel *Scomber japonicus* and blue mackerel *S. australasicus*

Reoto Tani<fnref n="1" /> · Wataru Kawamura<fnref n="1" /> · Tetsuro Morita<fnref n="2" /> · Christophe Klopp<fnref n="3" /> · Marine Milhes<fnref n="4" /> · Yann Guiguen<fnref n="5" /> · Goro Yoshizaki<fnref n="1" />,<fnref n="2" /> · Ryosuke Yazawa<fnref n="1" />

Received: 3 June 2021 / Accepted: 16 August 2021 / Published online: 30 August 2021

© The Author(s) 2021, corrected publication 2022

## Abstract

Chub mackerel *Scomber japonicus* and blue mackerel *S. australasicus* have become important aquaculture target species in Japan. For an efficient and stable aquaculture production of mackerels, it is desirable to work with a female-biased broodstock and thus to be able to manage precisely their sex ratio. As these two mackerel species do not have any external sexual dimorphism, direct biopsy of the gonads or measurement of sex hormone plasma levels is used in research to determine their phenotypic sex. However, these methods are associated with handling stress that results in gonadal atresia. Besides, it is difficult to identify sex using these methods in immature individuals. Therefore, genetic sexing would be desirable in these species to overcome these problems. In this study, we developed molecular sex identification tools for both chub and blue mackerels using sex-specific single-nucleotide variations (SNVs) identified by pool-sequencing (pool-seq) analyses. Genomic DNA was isolated from 30 males and females, in each species, and sequenced as pooled samples. Results of these pool-seq analyses identified a clear XX/XY male heterogametic sex-determination system in blue mackerel and a ZZ/ZW female heterogametic system in chub mackerel. Using this pool-seq information, we designed primer sets for genetic sex identification in both species. Results demonstrated that these primer sets are robust and accurate in assessing the genotypic sex of both blue and chub mackerels (accuracy > 99.5% in the chub, and > 94.5% in the blue mackerel) in various geographical samples of different ages. This method can now be applied for the efficient management of sex ratios in both mackerel aquaculture populations and wild-caught animals for fisheries management.

**Keywords** Chub mackerel *Scomber japonicus* · Blue mackerel *S. australasicus* · Molecular sexing · Broodstock management · Pool-seq

## Introduction

Chub mackerel *Scomber japonicus* and blue mackerel *S. australasicus* are distributed in Japan's coastal waters. Both species are major targets for commercial fisheries in Japan and have been selected as aquaculture targets recently (Mendiola et al. 2008; Hashimoto et al. 2019). Hatchery-reared breeding of mackerels has also been established recently, and commercialization of artificial seedlings has started in Japan (Matsuyama et al. 2005; Murata et al. 2005; Shiraishi et al. 2005). However, gamete collection in mackerels remains difficult due to the uncertainty of maturation in captivities (Amezawa et al. 2018). This difficulty is mainly due to the frequent occurrence of female mackerels

<figure><img src="image_1.png" /></figure>

<footnote>Ryosuke Yazawa
ryazawa@kaiyodai.ac.jp</footnote>

<footnote n="1">¹ Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan Minato-ku, Tokyo 108-8477, Japan</footnote>

<footnote n="2">² Institute for Reproductive Biotechnology for Aquatic Species, Tokyo University of Marine Science and Technology, 4-5-7 Konan Minato-ku, Tokyo 108-8477, Japan</footnote>

<footnote n="3">³ INRAE, Sigenae, Genotoul Bioinfo, Toulouse, France</footnote>

<footnote n="4">⁴ GeT-PlaGe, INRAE, Genotoul, Castanet-Tolosan, France</footnote>

<footnote n="5">⁵ INRAE, LPGP, Rennes, France</footnote>
whose maturation is retarded due to physiological stress in the rearing environment. To overcome this problem, two approaches can be explored. The first one is to develop a better method to induce female final maturation in mackerel, and several efficient maturation induction methods have been developed recently (Amezawa et al. 2018; Ohga et al. 2020). The second approach is to work with an increased number of females that could be used as broodstock candidates. However, identifying the sex of these two *Scomber* species with morphological characteristic is impossible as they do not possess any sexual dimorphism in their external appearance (Kobayashi et al. 2011; Amezawa et al. 2018). Techniques such as direct biopsy of the gonads by catheterization or measurement of sex hormone plasma levels have therefore been used to determine sex in these two mackerel species (Amezawa et al. 2018). However, mackerels have a low tolerance for such invasive samplings, and the handling stress induced by these manipulations occasionally results in ovarian atresia. Therefore, there is a need for minimally invasive approaches allowing the identification of females in aquaculture.

One of the less invasive techniques of sex determination is the genetic testing method. This method detects sex differences within sex-determining regions of sex chromosomes. Because these genetic tests can be performed using small fin-clip samples or skin mucus swiping, stress can be reduced to a minimum during sample collection. Previously, various molecular marker techniques, such as amplified fragment length polymorphisms, random amplified polymorphic DNAs (RAPDs), and simple sequence repeats (SSR), have been used to identify sex-linked molecular markers in fish. By employing these sex-linked DNA markers, sex identification methods have been developed in various species such as the African catfish *Clarias gariepinus* (Kovács et al., 2000), rainbow trout *Oncorhynchus mykiss* (Felip et al. 2005), half-smooth tongue sole *Cynoglossus semilaevis* (Chen et al. 2007), yellowtail *Seriola quinqueradiata* (Fuji et al. 2010), Nile tilapia *Oreochromis niloticus* (Lee et al. 2011), Pacific halibut *Hippoglossus stenolepis* (Galindo et al. 2011), barred knifejaw *Oplegnathus fasciatus* (Xu et al. 2013), and tiger puffer *Takifugu rubripes* (Matsunaga et al. 2014). With the advent of novel next-generation sequencing methods, many new approaches have been recently developed to identify sex-specific markers, and sex-specific markers have been isolated from various species, such as rockfishes *Sebastes carnatus* and *Sebastes chrysomelas* (Fowler and Buonaccorsi 2016), Pacific bluefin tuna *Thunnus orientalis* (Suda et al. 2019), and spotted knifejaw *Oplegnathus punc-tatus* (Li et al. 2020). However, for non-model species such as mackerels, the construction of high-resolution genetic linkage maps or reference genome sequences is still expensive and time-consuming; therefore, a more practical method is required. Alternatively, genome sequencing of pools of

individuals provides genome-wide polymorphism data at a considerably lower cost than sequencing many individual genomes (Schlötterer et al. 2014). This technique is considered an easy and cost-saving approach to identify sex-linked DNA markers, such as single-nucleotide variations (SNVs) in non-classical model species.

Therefore, we applied this approach to identify sex-specific SNVs in chub and blue mackerels and used this information to develop a polymerase chain reaction (PCR)-based method for genetic sex identification in these two species.

## Materials and methods

### Sample fish and DNA extraction

In November 2018, a set net was used to catch chub and blue mackerel in Tateyama Bay, Chiba, Japan. A total of 120 fishes were used, 30 for each of the four groups: female and male chub mackerels, and female and male blue mackerels. Gonads of these mackerels were inspected to identify the phenotypic sex of each individual. Fin-clips were taken from the pectoral fins of chub and blue mackerel and used for DNA extraction. Gentra Puregene (Qiagen GmbH, Dusseldorf, Germany) and NucleoSpin Kits for Tissue (Macherey-Nagel, Duren, Germany) were used according to the manufacturer's instructions to obtain high-molecular-weight DNA. When preparing DNA for sequencing, the DNA concentration was quantified using a NanoDrop ND2000 spectrophotometer (Thermo Scientific, Wilmington, DE). Species identification was conducted using a multiplex PCR kit "Saba checker-I" (SCOTS, Saga, Japan) according to the manufacturer's instructions to amplify species-specific genomic DNA regions of blue or chub mackerel in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS1) regions.

### Sequencing of male and female DNA pools

For each of the four groups described above, the same amount of DNA from each of the 30 fish was pooled and used as a sample for library construction. Library construction and sequencing were conducted at the GeT-PlaGe core facility, INRA Toulouse, France (http://get.genotoul.fr/en/). Libraries were constructed using the TruSeq nanokit (Illumina, FC-121-4001) according to the manufacturer's instructions. After the library preparation, sequencing was performed on a NovaSeq S4 lane (Illumina, San Diego, CA) using a paired-end 2×150 nt mode with Illumina NovaSeq Reagent Kit following the manufacturer's instructions. There were approximately 29 G base pairs of raw read for the male pool library and 51 G base pairs of raw read for the female pool library in chub mackerel, and 31 G base pairs of raw

<figure><img src="image_2.png" /></figure>
read for the male pool library and 39 G base pairs of raw
read for the female pool library in blue mackerel.

Identification of sex-specific SNVs in the de novo
assemblies

Illumina paired-end reads of two Scomber species were assembled using DiscovarDeNovo with default parameters. Reads filtered by Sickle (ver. 1.33: ≥ Q30) from the male and female pools were remapped to a genome sequence obtained from the above-mentioned assemblies using Bowtie2 (ver.2.3.5.1). SAMtools (ver. 0.1.20) was used to convert the resulting SAM sequence alignment files to BAM formats, followed by sorting, indexing, and merging. Subsequently, BCFTOOLS (ver. 0.1.20) was used to call the variants, after which sex-specific SNVs were selected using VCFTOOLS (ver. 0.1.17, minQ 10, minDP 4, max-missing 0.2). To further screen sex-specific SNVs, the following conditions were applied to the results obtained by the VCFTOOLS: no polymorphism on the homozygous side and approximately 50% identity on the heterozygous side, > 25 reads of male and female each mapped, nonrepetitive regions, and several sex-specific SNVs residing in a neighboring region (<500 bp) without strand bias. Integrative Genomics Viewer (ver. 2.8.2, Robinson et al. 2011) was used to confirm each condition in sex-specific SNV candidates.

Development of PCR-based sex identification
with sex-specific SNVs

Specific primers were designed based on the sex-specific
SNVs (Online Resource, Table S1). Then, first primer
screening was performed using three male and three female
samples of each species, respectively. PCR amplification was
conducted in a 25-µL reaction volume containing 1× PCR
Buffer II, 200 µM dNTPs, 1.5 mM MgCl₂, 1.25 U of Ampli-
Taq Gold DNA polymerase (Thermo Fisher Scientific, MA,
USA), 50 ng of template DNA, and 1 µM of each primer.
Similarly, thermal cycling conditions were one cycle of
95 °C for 8 min, then 35 cycles of 94 °C for 30 s, 55 °C for
15 s, 72 °C for 1 min, followed by a final elongation step at
72 °C for 7 min. Amplified PCR fragments products were
then electrophoresed on a 2.0% agarose gel. PCR amplifica-
tion with some primer sets was conducted using HiDi DNA
polymerase (myPOLSBiotec, Konstanz, Germany) that
efficiently amplifies primers that are completely matched
at the 3'-end and discriminate templated DNA containing
SNVs (Drum et al. 2014). By using HiDi DNA polymer-
ase, PCR amplification was conducted in a 25-µL reaction
volume tube containing 1×HiDi Buffer, 200 µM dNTPs,
2.5 U HiDi DNA polymerase, 50 ng template DNA, and
1 µM of each primer. The PCR mixtures were then cycled
under the following conditions: initial denaturation at 95 °C

for 2 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for
10 s, and 72 °C for 45 s. Verification of the PCR products
was observed afterward under ultraviolet light after electro-
phoresis on a 2.0% agarose gel. Species identification was
performed as well using a multiplex PCR kit, “Saba checker-
I” (SCOTS, Saga, Japan).

Confirmation of PCR-based sex identification
with various populations of mackerels

To confirm the reproducibility of this technique in various
regional populations and/or year-class, chub mackerel and
blue mackerel samples were collected from various areas in
Japan (Fig. 1, Online Resource, Table S1). DNA samples
were then extracted and identified species by Saba checker-
I as described above. All samples were identified as chub
mackerel or blue mackerel; there were no hybrids. DNA
samples were used for the PCR-based sex identification.
Identification of the phenotypic sex of each individual was
performed by morphological inspection of gonads.

<figure><img src="image_3.png" /><figcaption>**Fig. 1** Schematic figures for the sampling areas. Upper maps for chub mackerel samples, and lower maps for blue mackerel samples. The number in the boxes indicates the number of individuals collected from each area</figcaption></figure>
# Results

## De novo assemblies and identification of sex-specific SNVs

The blue and chub mackerel sequencing resulted in 233 million and 270 million paired-end reads corresponding to 70 Gb and 80 Gb of sequence data, respectively (DRA012454). The partial de novo assemblies for blue and chub mackerel were obtained as summarized in Table 1. In total, 929,890,313 bp were assembled in 2,307,454 contigs, and 825,218,221 bp were assembled in 2,043,150 contigs that were larger than 200 bp for each species. Reads from the male and female pools were then remapped to these de novo assemblies. Similarly, sex-specific SNVs were screened as summarized in Table 2. These selected SNVs showed a significant association with males in blue mackerel while showing a significant association with females in chub mackerel. The results of this analysis fit with a XX/XY male heterogametic sex-determination system in blue mackerel and a ZZ/ZW female heterogametic system in chub mackerel.

## Development of a PCR-based sex identification technique in chub and blue mackerels

Twenty-six primer sets (Online Resource, Table S2) designed based on the above-mentioned sex-specific SNVs were screened using three male and three female samples in each species. According to the results in Fig. 2, two primer sets Jpn1 and Jpn2 in chub mackerel and Aus1 and Aus2 in blue mackerel (Table 3) had 100% identification accuracy with phenotype sex. The polymorphisms between the female and male allelic sequences of sex-specific primer regions are

<figure><img src="image_4.png" /><figcaption>**Fig. 2** Confirmation and validation of the sex-specific PCR primer sets. Female-specific fragment amplified by primer sets “Jpn1” and “Jpn2.” Male-specific fragment amplified by primer sets “Aus1” and “Aus2.” “Saba checker I”: multiplex PCR that amplified both blue mackerel- and chub mackerel-specific regions in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS1) region for species discrimination</figcaption></figure>

shown in Fig. 3. Also, using the primer sets Jpn1 and Jpn2 for the chub mackerel and Aus1 and Aus2 for blue mackerel, a medium-scale confirmation was performed with 208 samples of chub mackerel, and 118 samples of blue mackerel, respectively, collected from various areas in Japan. Consistency between the phenotypic and genetic sexes identified using PCR for each primer set is summarized in Table 4. In the chub mackerel, the primer set Jpn1 showed 98.9% identification accuracy in males, and 100% in females, while the primer set Jpn2 showed 100% identification accuracy in males, and 97.2% in females (Table 4). On the other hand, in blue mackerel, the primer set Aus1 showed 92.6% identification accuracy in males and 96.9% in females, whereas the primer set Aus2 showed 94.4% accuracy in males and 96.9% in females (Table 5). With mackerel samples collected in 2018, 2019, and 2020, respectively (Tables 4 and 5), the primer set Jpn1 in chub mackerel showed 100%, 100%, and 98.7% identification accuracy, respectively, while the primer set Jpn2 provided 99.1%, 100%, and 98.7%, identification accuracy, respectively. Similarly, in the blue mackerel

<table><caption>**Table. 1** Summary of de novo assemblies</caption><thead><tr><th></th><th>Chub mackerel</th><th>Blue mackerel</th></tr></thead><tbody><tr><td>Number of scaffolds</td><td>20,43,150</td><td>23,07,454</td></tr><tr><td>Total size of scaffolds (bp)</td><td>82,52,18,221</td><td>92,98,90,313</td></tr><tr><td>Longest scaffold (bp)</td><td>1,06,937</td><td>1,28,293</td></tr><tr><td>Shortest scaffold (bp)</td><td>200</td><td>200</td></tr></tbody></table>

<table><caption>**Table. 2** Summary of the sex-specific SNVs identification</caption><thead><tr><th></th><th></th><th>No. of paired reads</th><th>No. of remapped reads</th><th>No. of sex-specific SNV candidates</th></tr></thead><tbody><tr><td rowspan="2">Blue mackerel</td><td>Female</td><td>12,94,44,376</td><td>34,784,821 (30.00%)</td><td rowspan="2">571</td></tr><tr><td>Male</td><td>10,38,40,913</td><td>29,451,144 (30.95%)</td></tr><tr><td rowspan="2">Chub mackerel</td><td>Female</td><td>17,14,31,237</td><td>39,898,431 (25.89%)</td><td rowspan="2">116</td></tr><tr><td>Male</td><td>9,88,32,713</td><td>21,952,779 (24.85%)</td></tr></tbody></table>
<table><caption>**Table. 3** Summary of PCR primer sets</caption><thead><tr><th rowspan="2">Target species</th><th rowspan="2">Primer set</th><th colspan="2">Primer SQs</th><th rowspan="2">Amp. size</th><th rowspan="2">Enzyme</th></tr><tr><th>Fwd</th><th>Rev</th></tr></thead><tbody><tr><td rowspan="2">Blue mackerel</td><td>Aus1</td><td>CTAGACTGAGGCTTGGGATG</td><td>ACTTTAGTCCCACAGCACGG</td><td>130 bp</td><td>HiDi</td></tr><tr><td>Aus2</td><td>TGTTAGTTACAGTATAGCTT</td><td>ACGGAGGCACTTGTCTTTAC</td><td>176 bp</td><td>Ex-taq</td></tr><tr><td rowspan="2">Chub mackerel</td><td>Jpn1</td><td>TAGATGTTGTTAACTTGTGT</td><td>TACGTCTGATCCATGGTCAG</td><td>140 bp</td><td>Ex-taq</td></tr><tr><td>Jpn2</td><td>ACACCATCTCACTAGGAAGA</td><td>GAGGAGGAGGTGGCGGTGGT</td><td>135 bp</td><td>HiDi</td></tr></tbody></table>

(A)

Jpn1:

P: TAGATGTTGTTAACTTGTGT

M: TAGACGTTGTTAACTTGGGGGAGTGTGGTACAGATAATGGAGCCTCGGCTGTTGAACAAGATGAACATCTGGTTGTAGCATCATTGCTGT

F: TAGAYGTTGTTAACTTGKGKGAGTGTGGTACAGATAATGGAGCYTCGGCTGTTGAACAAGATGAACATCTGGTTGTAGCATCATTGCTGT

*

* *

*

P: CTGACCATGGATCAGACGTA

M: CCCCGACCAAAAAATCTAAATCAGGGGGTTGTGACCATGGATCAGACGTA

F: CCCCGACCAAAAAATCTAAATCAGGGGGTTSTGACCATGGATCAGACGTA

*

(B)

Jpn2:

P: ACACCATCTCACTAGGAAGA

M: ACACCATCTCACTAGGACGGGGAGACCACCCCTCAGCTTTTGTAAAGCAGGCATCTACTAGCAGAGCTTGCATTACATCTCCAACTCCCA

F: ACACCATCTCACTAGGAMGRGGAGACCACCCCTCAGCTTTTGTAAAGCAGGCATCTACTAGCAGAGCTTGCATTACATCTCCAACTCCCA

* *

P: ACCACCGCCACCTCCTCCTC

M: ACCTACAATCTCCACCTCCTCCACCGCCTCCCCCACCTCCTCCTC

F: ACCTACAATCTCCACCTCCTCCACCRCCWCCSCCACCTCCTCCTC

* * *

(C)

Aus1:

P: CTAGACTGAGGCTTGGGATG

M: CTAGACTGAGGCTTGGGATGGAGAGTGGGGTTTAGGCTGGCTTCTGTGATCAAGGAGGGTGCACTGTGGCTTGGGCTGGGCCAGCGGAAG

F: CTAGACTGAGGCTTGGGATGGAGAGTGGGGTTTAGGCTGGCTTCTGTGATCAAGGAGGGTGCACTGTGGCTTGGGCTGGGCCAGCGGAAG

P: CCGTGCTGTGGGACTAAAGT

M: GCTGGGCTGCTGGGGTCTTTMCGTGCTGTGGGACTAAAGT

F: GCTGGGCTGCTGGGGTCTTTACGTGCTGTGGGACTAAAGT

*

(D)

Aus2:

P: TGTTAGTTACAGTATAGCTT

M: TGTTAGTTACAGTATAGCTTAAATGTGATTGATGAATTTATTCTGTGAAGGCAACCTAGCCCCACAGTGGCAGATACGGAAATAAGTGGA

F: TGTTAGTTA-----TAGCTTAAATGTGATTGATGAATTTATTCTGTGAAGGCAACCTAGCCCCACAGTGGCAGATACGGAAATAAGTGGA

*****

P: GTAAAGACAAGTGCCTCCGTG

M: GAGGAAAACACAAATGTAAATTAGTCTAAGCAGTAAGTGTAGCTCTAGATAAACCTGACTGGCTGTAAAGACAAGTGCCTCCGTG

F: GAGGAAAACACAAATGTAAATTAGTCTAAGCAGTAAGTGTAGCTCTAGATAAACCTGACTGGCTGTAAAGACAAGTGCCTCCGTG

**Fig. 3** Sequence alignment of the female-specific fragment and sex-specific primer designs “Jpn 1” (a) and “Jpn 2” (b) for the chub mackerel. Sequence alignment of the male-specific fragment and sex-specific primer designs “Aus1” (c) and “Aus2” (d) for the chub

mackerel. “P” indicates the sequences for the primers. “M” and “F” indicate the male allele and female allele sequences, respectively. The asterisks indicate the polymorphisms between female and male allelic sequences
<table><caption>**Table 4** Summary of sex identification using various population samples in chub mackerel</caption><thead><tr><th rowspan="3">Sampling<br>Place</th><th rowspan="3">Date</th><th colspan="2">No. of samples</th><th colspan="4">Identity accuracy (%)*</th></tr><tr><th rowspan="2">Male</th><th rowspan="2">Female</th><th colspan="2">Jpn1</th><th colspan="2">Jpn2</th></tr><tr><th>Male (%)</th><th>Female (%)</th><th>Male (%)</th><th>Female (%)</th></tr></thead><tbody><tr><td>Hokkaido</td><td>2020</td><td>2</td><td>4</td><td>100.0</td><td>100.0</td><td>100.0</td><td>75.0</td></tr><tr><td>Akita</td><td>2020</td><td>21</td><td>15</td><td>100.0</td><td>100.0</td><td>100.0</td><td>93.3</td></tr><tr><td>Niigata</td><td>2020</td><td>7</td><td>7</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td></tr><tr><td>Chiba</td><td>2018</td><td>41</td><td>37</td><td>97.6</td><td>100.0</td><td>100.0</td><td>97.3</td></tr><tr><td>Setouchi</td><td>2020</td><td>7</td><td>5</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td></tr><tr><td>Ishikawa</td><td>2020</td><td>0</td><td>4</td><td>–</td><td>100.0</td><td>–</td><td>100.0</td></tr><tr><td>Yamaguchi</td><td>2020</td><td>7</td><td>13</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td></tr><tr><td>Oita</td><td>2019</td><td>7</td><td>13</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td></tr><tr><td>Kagoshima</td><td>2020</td><td>7</td><td>11</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td></tr><tr><td>Total</td><td>–</td><td>99</td><td>109</td><td>98.9</td><td>100.0</td><td>100.0</td><td>97.2</td></tr></tbody></table>

<table><caption>**Table 5** Summary of sex identification using various population samples in blue mackerel</caption><thead><tr><th rowspan="3">Sampling<br>Place</th><th rowspan="3">Date</th><th colspan="2">No. of samples</th><th colspan="4">Identity accuracy*</th></tr><tr><th rowspan="2">Male</th><th rowspan="2">Female</th><th colspan="2">Aus1</th><th colspan="2">Aus2</th></tr><tr><th>Male (%)</th><th>Female (%)</th><th>Male (%)</th><th>Female (%)</th></tr></thead><tbody><tr><td>Akita</td><td>2020</td><td>3</td><td>8</td><td>100.0</td><td>87.5</td><td>100.0</td><td>100.0</td></tr><tr><td>Ishikawa</td><td>2020</td><td>9</td><td>11</td><td>100.0</td><td>100.0</td><td>100.0</td><td>100.0</td></tr><tr><td>Chiba</td><td>2018</td><td>34</td><td>38</td><td>91.1</td><td>97.4</td><td>94.1</td><td>97.4</td></tr><tr><td>Kagoshima</td><td>2020</td><td>8</td><td>7</td><td>87.5</td><td>100.0</td><td>87.5</td><td>85.7</td></tr><tr><td>Total</td><td>–</td><td>54</td><td>64</td><td>92.6</td><td>96.9</td><td>94.4</td><td>96.9</td></tr></tbody></table>

samples collected in 2018 and 2020, the primer set Aus1 showed 95.7% and 94.4% identification accuracy, respectively, while the primer set Aus2 provided 95.7% and 95.8% identification accuracy, respectively.

Discussion

In this study, we successfully identified two female-specific DNA makers in chub mackerel, and two male-specific DNA makers in blue mackerel, respectively. Using these sex-linked DNA makers, we developed a PCR method to identify the sex of these two mackerel species with high accuracy, i.e., over 95%. The accuracy is high enough for the efficient sex management of the parental fish population in mackerel aquaculture. First, the method can be implemented using fin clipping with minimum physical stress, compared with conventional sex determination techniques conducted by observing a portion of the gonads biopsied by catheter or by measuring the sex hormones in plasma. In the conventional methods, excessive stress caused by sample collection often prevents proper gonadal maturation,

especially in female broodstock, and can be an obstacle to artificial seedling production, as mackerel, in particular, are known to be vulnerable to physical stress and can die from handling (Amezawa et al. 2018). Therefore, in most cases of artificial seed production of mackerel, wild adult fish close to maturity are used since fish farmers tend to avoid maintaining parent fish for long periods. In these situations, the low-invasive sex identification methods established in this study can be effective and practical in mackerel aquaculture to manage the sex ratio of broodstock without retardation of gonadal maturation in parent fish.

Second, this method can identify the gender of mackerel at any stage of maturity with a high degree of accuracy. In contrast, it is difficult to identify the sex of immature fish using the above-mentioned conventional methods such as biopsy of the gonads or measuring plasma concentrations of sex hormones. Therefore, in mackerel, where there is no clear difference in external morphology between males and females, the method developed in this study is the only way to identify the sex of immature individuals to date. Also, since hatchery-reared breeding of mackerel has been used as broodstock recently instead of wild fish, it is also crucial to

<footnote>*Concordance rate between the phenotypic and genetic sex identified by PCR-based method developed in the present study</footnote>

<footnote>*Concordance rate between the phenotypic and genetic sex identified by PCR-based method developed in the present study</footnote>
know the sex ratio of a potential parent population as early
as possible in this case. While all mackerel males spontane-
ously mature and produce gametes under captive conditions,
not all females will produce eggs, even when hormones are
administered to induce maturation. Thus, the PCR-based sex
identification method developed in this study makes it pos-
sible to sort young and immature mackerels into sexes with
high accuracy. Collecting the required number of female-
biased parental candidates is also made possible, although
identification PCR accuracy developed in this study was
not 100%. Besides, using female-biased broodstock, it is
possible to produce artificial seed effectively and stably to
avoid wasting space, labor, and costs on excessive parental
maintenance.

While the chub mackerel population is divided into the
Pacific and Tsushima stocks, and blue mackerel is divided
into the Pacific and East China Sea stocks, it was reported
that spawning grounds for both stocks overlap in both spe-
cies (Yukami et al., 2009). Since the results showed that the
sex identification accuracy of PCR was consistently high in
both chub and blue mackerel samples with regional differ-
ences, the two above-mentioned stocks were tested. There-
fore, it is expected that the primer sets used in this study can
be applied for sex prediction in most chub and blue mackerel
populations distributed around Japan. This observation can
be a big advantage, since some sex-linked DNA markers
could be applied to only a limited pedigree (Agawa et al.
2015). Similarly, it was reported that the genetic structure of
chub mackerel's population along China's coast was signifi-
cantly different from that of the Japanese population (Cheng
et al. 2015; Yan et al. 2015). Moreover, blue mackerel is
widely distributed in tropical and subtropical waters of the
Pacific Ocean from Japan to northern Australia (Collette
and Nauen 1983). Therefore, further studies should confirm
whether this study's methods can be applied for these geneti-
cally distant populations.

Since we have succeeded in producing feminized and
masculinized mackerel by exogenous hormonal treatment
(Tani et al. 2018), it was expected that mackerels poten-
tially possess the ability for sex reversal from their genetic
sex. Studies have shown that various fish exhibit environ-
mental sex determination (ESD), where environmental fac-
tors can influence phenotypic sex during larval or juvenile
development or both (Miyoshi et al. 2020). However, in
this study, PCR-based molecular sex determination results
with various samples showed high consistency (> 95%)
between genetic and phenotypic sex in both chub mack-
erel and blue mackerel. These results, therefore, indicated
that chub and blue mackerel rarely reversed their genetic
sex in their natural environment, at least in the population
caught around Japan. It should thus be considered that
chub and blue mackerel possessed a rather strict genotypic
sex-determination system, with no or low ESD. Another

possibility is that they rarely experienced an extreme envi-
ronmental condition that reversed their sex in their natural
environment.

Based on the results of this study, we concluded that chub
mackerel has a female heterogametic ZZ/ZW sex-determin-
ing system. This was expected as triploid chub mackerel
showed a tendency for an all-female phenotype (Tani et al.
2018). Unexpectedly and interestingly, our analysis also
revealed that blue mackerel has a male heterogametic (XX/
XY) sex determination system. Turnovers of sex determi-
nation systems among closely related teleost species have
been already reported (Devlin and Nagahama 2002, Gam-
merdinger et al. 2018). It is, however, surprising that these
two *Scomber* species that are considered sister species with a
species divergence time estimated as a few million years ago
(Miya et al., 2013) possess opposite sex determination sys-
tems. In the Japanese medaka and its closely related species,
a similar turnover of sex chromosomes and sex determining
systems have been reported (Takehana et al. 2007, 2008).
The comparative analyses in *Oryzias* species have therefore
identified seven non-orthologous sex chromosomes, with six
species having a heterozygous XX/XY system, and two pos-
sessing heterozygous ZZ/ZW systems, respectively (Take-
hana et al. 2008; Kikuchi and Hamaguchi 2013). However,
whether the sex-linked SNVs of chub and blue mackerel are
located on the same or different chromosomes is currently
unknown as we do not have a good-quality genome reference
in these species. Further studies in mackerels, with more
genomic resources, should provide novel insights into the
mechanisms of sex chromosome turnover in teleost. From
previous studies in Scombridae, it has been reported that
the Pacific bluefin tuna possess a male heterozygous XX/
XY system (Suda et al. 2019), and it was speculated that
eastern little tuna *Euthynnus affinis* possess a female het-
erozygous ZZ/ZW system (Yazawa et al. 2019). This infor-
mation, therefore, suggests that there is great variety in the
sex determination systems of Scombridae species.

In this study, we identified sex-linked DNA makers in
chub and blue mackerel and successfully established a PCR-
based sexing method employing these markers. Since this
technique requires a small fin-clip and short handling time,
and thus minimum physical stress compared with conven-
tional methods such as gonad biopsy and plasma sex hor-
mone measurement, this novel method would help maintain
the health of stress-susceptible mackerels and contribute to
the proper maturation and spawning of the broodstock. Also,
since this method can determine sex with high accuracy
regardless of the maturity stage, it can be applied to produce
a broodstock population with a desired male-female ratio.
Taken together, the PCR-based sexing method established
in this study can be seen as a practical solution for a more
stable and well-planned artificial seed supply and would help
to significantly sophisticate the mackerel's aquaculture.
**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s12562-021-01548-z.

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