Molecular characterization of OSR1 in Pinctada fucata martensii and association of allelic variants with growth traits

Aquaculture 516 (2020) 734617

Contents lists available at ScienceDirect

Aquaculture journal homepage: www.elsevier.com/locate/aquaculture

Molecular characterization of OSR1 in Pinctada fucata martensii and association of allelic variants with growth traits

T

Chuangye Yanga,b, Jingmiao Yanga, Ruijuan Haoa, Xiaodong Dua,b, Yuewen Denga,b,∗ a b

Fisheries College, Guangdong Ocean University, Zhanjiang, 524088, China Pearl Breeding and Processing Engineering Technology Research Centre of Guangdong Province, Zhanjiang, 524088, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Growth traits Odd-skipped related 1 Pinctada fucata martensii Single nucleotide polymorphisms

Odd-skipped related 1 (OSR1) from Pinctada fucata martensii (PmOSR1) was cloned in this study. PmOSR1 contained an open reading frame measuring 951 bp that encoded a polypeptide of 316 residues. Domain analysis also revealed that PmOSR1 had a typical ZnF_C2H2 domain, indicating high identity with OSR1 protein sequence from Crassostrea gigas (55.25%) and Mizuhopecten yessoensis (65.48%). The expression pattern analysis of PmOSR1 revealed its wide expression in the tissues and developmental stages. PmOSR1 also presented a higher expression in the fast-growing group and hybrid families than in the slow-growing group and inbred families. The polymorphism analysis of PmOSR1 revealed 45 single nucleotide polymorphisms (SNPs) in the genomic sequence, and association analysis presented four SNPs significantly associated with growth traits (namely, shell length, width, height, and shell weight; P < 0.05). Three SNPs out of the four SNPs were shaped as a haploblock, and haplotype AGT showed significantly higher growth traits than that of haplotype AACAA. Therefore, PmOSR1 could be used as a growth candidate gene for the selective breeding of P. f. martensii.

1. Introduction Growth performance is a common concern to increase production of shellfish. Growth traits that are quantitative traits have been widely researched on basis of genetic linkage map (Li and He, 2014; Ren et al., 2016; Wang et al., 2016). A genetic map with sufficient density and resolution is an essential prerequisite for positioning candidate genes (Tian et al., 2015; Yu et al., 2015). Linkage maps have been constructed for many aquaculture species to date (Nie et al., 2017; Wang et al., 2015; Xia et al., 2010; Baranski et al., 2014). For bivalves, genetic maps have been constructed for the location of growth, biomineralization, color, disease resistance, and other important economic traits (Jiao et al., 2014; Wang et al., 2016; Shi et al., 2014). In our previous studies, one genetic map with 4463 markers that covered 14 linkage groups was conducted, and potential markers associated with growth traits were identified (Du et al., 2017) to explore growth-associated markers and genes for selective breeding. Utilizing the genetic map of P. f. martensii constructed with RAD-seq method, one odd-skipped related 1 (OSR1) showed its association with the shell length of pearl oyster (Du et al., 2017). OSR1 is a zinc finger transcription factor and initially reported in vertebrates because of its distinctive expression in the intermediate mesoderm, kidney formation, embryonic patterning, renal structure, ∗

and tissue morphogenesis (Wang et al., 2005; James et al., 2006; Mudumana et al., 2008; So and Danielian, 1999; Tena et al., 2007). In aquaculture, several OSR1 genes were detected through the genome, transcriptome, and other sequencing in aquatic animals, such as Oncorhynchus kisutch, Mytilus galloprovinciali, Crassostrea virginica, and C. gigas, but studies on their special functions are rarely reported (Kim et al., 2016; Pasquier et al., 2016; Zhang et al., 2012; Murgarella et al., 2016; Gomez-Chiarri et al., 2015; Wang et al., 2017). Zebrafish OSR genes can act as a relay within the genetic cascade of fin bud formation by controlling the expression of the signaling molecule Wnt2ba in the intermediate mesoderm (Neto et al., 2012). Zebrafish osr1 activity is required to limit the endoderm differentiation from the mesendoderm, and in the absence of osr1, excess endoderm alters mesoderm differentiation, shifting the balance from kidney toward vascular development (Mudumana et al., 2008). Pearl oyster P. f. martensii is naturally distributed in South China, Japan, and Australia (Lucas, 2008). It is widely cultured for roundnucleated pearl production. The cultured nucleated pearls are obtained by transplanting a mantle graft from a donor pearl oyster with a nucleus into a host pearl oyster (Zhao et al., 2012). The growth of the host pearl oyster is important for pearl quality (Wada and Komaru, 1996; Wang et al., 2013). Researchers are also interested in breeding for fast growth, color, and disease-resistant populations, which are suitable for pearl

Corresponding author. Fisheries College, Guangdong Ocean University, Zhanjiang, 524088, China. E-mail address: [email protected] (Y. Deng).

https://doi.org/10.1016/j.aquaculture.2019.734617 Received 7 July 2019; Received in revised form 15 October 2019; Accepted 18 October 2019 Available online 22 October 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.

Aquaculture 516 (2020) 734617

C. Yang, et al.

PCR) analysis was performed with DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific) by using the primers described in Supplemental Table 1. The expression levels of PmOSR1 in different tissues, development, families, and groups were calculated using the 2−ΔCt method with glyceraldehyde 3-phosphate dehydrogenase as the reference gene (Hao et al., 2018a). The data of P. f. martensii development transcriptome were from http://gigadb.org/dataset/view/id/100240/File_ page/11.

production (Deng et al., 2011, 2013). Single nucleotide polymorphisms (SNPs) with few genotyping errors and spontaneous mutations have also become important DNA markers for genetic diversity and breeding studies (Du et al., 2016). The SNP detection of the candidate genes to obtain potential markers for specific traits in breeding has been widely used in aquaculture (Lei et al., 2019; Guo et al., 2012; Fan et al., 2017). Therefore, the identified OSR1 gene in the pearl oyster from genetic map was cloned and analyzed in the present study to show its function and polymorphism, which are vital for pearl oyster breeding.

2.4. SNP genotype of PmOSR1 2. Materials and methods A total of 206 pearl oysters were sampled from the base stock. The adductor muscle of individuals was obtained and preserved in 75% ethanol for DNA extraction. We used the TIANGEN Marine Animal DNA Kit to extract the total genomic DNA from the adductor muscles. The 206 pearl oysters were genotyped through targeted resequencing. All identified SNPs were compared with the genome of the pearl oyster P. f. martensii (Du et al., 2017; http://gigadb.org/dataset/view/id/100240/ File_page/11). Expected heterozygosity (HE), observed heterozygosity (HO), and allele frequency were evaluated by PopGene 32 (version 3.2) according to the SNP information of PmOSR1. Polymorphism information content (PIC) was calculated with PIC_CALC (version 0.6). PIC can be divided into three types, namely, low (PIC < 0.25), medium (0.25 < PIC < 0.5), and high polymorphisms (PIC > 0.5). LD among SNPs (r2) within the PmOSR1 was calculated and visualized using the Haploview 4.2 program. The growth traits of different genotypes were compared by using SPSS version 19.0.

2.1. Experimental animals Pearl oysters P. f. martensii used in the present study were obtained from a commercial farm (20°250′ N, 109°570′ E) in Xuwen, Zhanjiang, Guangdong Province, China. During analysis of the expression pattern in tissues, eight pearl oysters were sacrificed with the mantle (M), adductor muscle (A), hepatopancreas (HE), gill (GI), gonad (Go), and foot (F). After fertilization, developmental samples, including the egg (E), fertilized egg (Fe), blastula (B), gastrula (G), trochophore (T), D-stage larvae (D), early umbolarvae (EU), and eyed larvae (EL) with three replicates were collected. For the sample of different families, a 292 complete diallel cross was made between the two fullsib families (M and N) to produce four families. The four families were named as follows: A, an inbred family produced by sister–brother mating from family M; B, a hybrid family with the female parent from family M and the male parent from family N; C, a reciprocal hybrid family with the female parent from family N and the male parent from family M; and D, an inbred family produced by sister–brother mating from family N. The inbred and hybrid families were developed in March 2014 and obtained by Yang et al. (2018). Twenty slow-growing oysters and twenty fastgrowing oysters from base stock were randomly selected according to the shell length of individuals for analysis of PmOSR1 in different groups. The adductor muscles of the four families and two groups were obtained and stored in liquid nitrogen to analyze the PmOSR1 expression levels.

2.5. PmOSR1 SNP verification An SNP (g.53337657) was selected to verify another population comprising 83 pearl oysters. Molecular detection was based on tetraprimer amplification refractory mutation system-polymerase chain reaction (PCR) for different individual adductor muscles. The PmOSR1 genotyping of P. f. martensii was performed. The primers used are presented in Supplemental Table 1. 2.6. Data analysis

2.2. Cloning and bioinformatic analysis of PmOSR1 gene The data analysis of the expression levels of tissues, development and families was performed with ANOVA in SPSS 19.0 (IBM, Chicago, IL, USA). The expression levels of PmOSR1 in the different groups were compared using t-test. The significance level for all analyses was P < 0.05.

The sequence of the OSR1 gene was gained from the data of the P. f. martensii genome (Du et al., 2017). Rapid amplification of cDNA ends reaction was performed to obtain the 5′ and 3′ ends of the target sequence with a SMART RACE cDNA amplification kit (TaKaRa, Dalian, China). The primers are shown in Supplemental Table 1. Then, the PmOSR1 full-length was attained with DNAMAN software. The bioinformatic analyses of the PmOSR1 sequence was performed for prediction using the open reading frame (ORF) Finder (https://www.ncbi. nlm.nih.gov/orffinder/) for ORF analysis. SMART (http://smart.emblheidelberg.de/smart/set_mode.cgi) was used for domain analysis of the detected peptides of PmOSR1. Clustal X program (http://www.ebi.ac. uk/Tools/msa/clustalo/) was used for multiple alignments with the selected OSR1 genes from other species. Phyre2 online http://www. sbg.bio.ic.ac.uk/phyre2/protocol (Kelley et al., 2015) was used for the advanced structure prediction of the OSR1 domain from P. f. martensii, C. gigas (EKC18512.1) (Zhang et al., 2012), and Mizuhopecten yessoensis (XP_021341163.1), and Chimera 1.8.1 to display the model.

The full length of PmOSR1 was 1732 bp with 107 bp 5′-untranslated region (UTR) and 674 bp 3′-UTR (Fig. 1A). The genomic structure of the DNA sequence of PmOSR1 revealed five exons (Fig. 1B). Moreover, complete PmOSR1 cDNA sequence presented an ORF of 951 bp and encoded a polypeptide of 316 residues, with an estimated molecular mass of 36.54 kDa. The deduced amino acid sequence of PmOSR1 featured three typical ZnF_C2H2 [Zinc Finger_Cys(2)His(2)] domains located at 230–252, 258–280, and 286–308 aa.

2.3. PmOSR1 expression pattern analysis

3.2. Homologous analysis and PmOSR1 structure

Total RNA was isolated from tissues, developmental samples, four families, and two groups by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instruction and verified using the method reported by Hao et al. (2018a). Then, cDNA was synthesized with an M-MLV First-strand cDNA Synthesis Kit (Invitrogen). Quantitative reverse transcription-polymerase chain reaction (qRT-

The PmOSR1-deduced protein sequence was homologous to OSR1. The homologous analysis of PmOSR1 was performed using Clustal X2 software. The results were compared with sequences of the OSR1 from C. gigas (EKC18512.1) and M. yessoensis (XP_021341163.1). PmOSR1 exhibited conserved function domain with other OSR1 (Fig. 2). The PmOSR1 shared 55.25% identity with C. gigas and 65.48% identity with

3. Results 3.1. Cloning and bioinformatic analyses of PmOSR1

2

Aquaculture 516 (2020) 734617

C. Yang, et al.

Fig. 1. Nucleotide sequence analysis of PmOSR1. (A) shows mucleotide sequence and aa of PmOSR1. The 5′-untranslated region (UTR) and 3′-UTR are indicated with small letters; Capital letters represent the ORF and the deduced amino acid (aa) sequences; nucleotides with borders correspond to the initiation codon (ATG) and stop codon (TGA); the sequence with yellow background represents the ZnF_C2H2 domain. (B) presents the genomic structure and mRNA of PmOSR1. The gene contains five exons. The 3′ and 5′ UTR (untranslated region, orange) and exons encoding the amino acid sequences (blue) are shown relative to their lengths in the cDNA sequences obtained. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3

Aquaculture 516 (2020) 734617

C. Yang, et al.

Fig. 2. Multiple sequence alignment of PmOSR1 aa sequence. “*” indicates the conserved amino acid. “:” signifies amino acid with strong similarity. The residues, which are highly conserved or invariant in other members of the Cyc-Cys/His-His class of finger proteins, are indicated by the yellow background and the consensus sequence. The numbers on the right presented the total amino acid of each protein. Accession number for sequences used in this alignment is as follows: C. gigas (EKC18512.1), M. yessoensis (XP_021341163.1). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.3. Expression analysis of PmOSR1

M. yessoensis. The predicted structural organization of OSR1 among different species (C. gigas OSR1 [EKC18512.1] and M. yessoensis OSR1 [XP_021341163.1]) also displayed the conservation of PmOSR1 protein sequence compared with other species (Supplemental Fig. 1).

The expression pattern of PmOSR1 in different tissues showed the wide existence of it in P. f. martensii. PmOSR1 mRNA was expressed in the mantle, adductor muscle, hepatopancreas, gill, foot, and gonad of P. f. martensii (Fig. 3A). The gill had a significantly higher expression level than other tissues. In the developmental stage, PmOSR1 was

Fig. 3. Expression pattern of PmOSR1 from P. f. martensii. (A) Expression pattern from different tissues. M: Mantle; F: Foot; A: Adductor muscle; GI: Gill; GO: Gonad; He: Hepatopancreas; Different letters indicate significant differences (P < 0.05) determined through one-way ANOVA, and the bar represents standard deviation. (B) Expression pattern of PmOSR1 in development stages from Pinctada fucata martensii. Note: E: Egg; Fe: fertilized egg; B: blastula; G: gastrula; T: trochophore; D: D-stage larvae; EU: early umbo larvae; EL: eyed larvae; Black shows the relative expression and RPKM represented with orange. (C) PmOSR1 expression in inbred families and hybrid families. A and D are inbred families. B and C are hybrid families. (D) PmOSR1 expressions in the Fast-growing and Slow-growing groups. Bars with different superscripts indicate significant differences (P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 4

Aquaculture 516 (2020) 734617

C. Yang, et al.

Table 1 Genetic diversity based on SNP polymorphisms in PmOSR1. Number

Locus

Ho

He

HWE

PIC

SNP1 SNP2 SNP3 SNP4 SNP5 SNP6 SNP7 SNP8 SNP9 SNP10 SNP11 SNP12 SNP13 SNP14 SNP15 SNP16 SNP17 SNP18 SNP19 SNP20 SNP21 SNP22 SNP23 SNP24 SNP25 SNP26 SNP27 SNP28 SNP29 SNP30 SNP31 SNP32 SNP33 SNP34 SNP35 SNP36 SNP37 SNP38 SNP40 SNP41 SNP42 SNP43 SNP44 SNP45 SNP46 Average

53330110 G > A(206) 53330124 A > T(206) 53330165 C > T(206) 53330180 T > G(206) 53330243 C > G(206) 53330298 T > C(206) 53330305 A > AT(206) 53332829T > TACTTATACAATAA(206) 53332861 G > A(206) 53332969 G > T(206) 53332972 T > C(206) 53332987 G > A(206) 53333057 A > T(206) 53333607 A > G(206) 53333612 A > T(206) 53333623 G > A(206) 53333624 C > A(206) 53333686 A > AT(206) 53333710 A > G(206) 53333761 A > T(206) 53333929 C > T(206) 53334195 T > C(206) 53334228 A > AT(206) 53336412 G > A(206) 53336496 C > T(206) 53336597 C > A(206) 53336611 ATT > A, AT(206) 53337349 T > G(206) 53337354 T > TA(206) 53337358 G > T(206) 53337465 A > G(206) 53337547 A > G(206) 53337563 A > C(206) 53337566 A > C(206) 53337652 A > AAC(206) 53337657 G > A(206) 53337662 A > T(206) 53338021 C > T(206) 53338090 T > G(206) 53338093 T > C(206) 53338203 GAAAA > G, GAAA(206) 53338575 G > GA(206) 53338590 A > G(206) 53338627 C > T(206) 53338643 T > C(206)

0.1311 0.5243 0.0340 0.0340 0.5728 0.5825 0.2087 0.0583 0.0097 0.0388 0.0680 0.6942 0.5291 0.0680 0.0583 0.0437 0.0583 0.0097 0.0437 0.0583 0.5534 0.5340 0.1262 0.0437 0.0194 0.6262 0.7184 0.8155 0.4417 0.4417 0.6602 0.5097 0.3592 0.3592 0.4806 0.4806 0.4612 0.0874 0.1359 0.1602 0.6068 0.0000 0.4175 0.0437 0.0388 0.2877

0.1395 0.4932 0.0335 0.0335 0.4955 0.4965 0.2319 0.0567 0.0097 0.0382 0.0748 0.4833 0.4814 0.0658 0.0567 0.1910 0.0567 0.0097 0.0428 0.0567 0.4693 0.4668 0.1185 0.0428 0.0193 0.4312 0.5294 0.4932 0.3557 0.3557 0.4642 0.4773 0.3311 0.3311 0.4118 0.4118 0.3855 0.0838 0.1270 0.1477 0.5409 0.0000 0.4528 0.0428 0.0382 0.2461

0.3783 0.3652 0.8186 0.8186 0.0248 0.0127 0.1482 0.6805 0.9606 0.7906 0.1738 0.0000 0.1533 0.6270 0.6805 0.0000 0.6805 0.9606 0.7628 0.6805 0.0099 0.0382 0.3437 0.7628 0.9031 0.0000 0.0000 0.0000 0.0005 0.0005 0.0000 0.3285 0.2212 0.2212 0.0162 0.0162 0.0047 0.5245 0.3046 0.2190 0.0001

0.1294 0.3710 0.0329 0.0329 0.3721 0.3726 0.2046 0.0549 0.0097 0.0373 0.0718 0.3659 0.3649 0.0635 0.0549 0.0417 0.0549 0.0097 0.0417 0.0549 0.3586 0.3572 0.1112 0.0417 0.0190 0.3376 0.4715 0.3710 0.2919 0.2919 0.3558 0.3628 0.2757 0.2757 0.3264 0.3264 0.3106 0.0801 0.1187 0.1365 0.4798 – 0.3497 0.0417 0.0373 0.2016

0.2610 0.7628 0.7906 0.3510

Mutations

S S

S

S S S

S S

Note: Ho: the observed heterozygosity; He: the expected heterozygosity; PIC: the polymorphism information content; HWE: Hardy-Weinberg equilibrium; S: synonymous. Table 2 Correlation of SNPs in PmOSR1 with growth traits (mean ± SD). Number

Locus

Genotype

Sample number

Shell length(cm)

SNP31

53337465 A > G(206)

SNP35

53337652 A > AAC (206)

SNP36

53337657 G > A(206)

SNP37

53337662 A > T (206)

AA GA GG AA AAAC AACAAC AA GA GG AA TA TT

63 136 7 10 99 97 97 99 10 105 95 6

5.07 4.81 4.87 5.30 4.88 4.88 4.88 4.88 5.30 4.90 4.86 5.50

± ± ± ± ± ± ± ± ± ± ± ±

0.08a 0.05b 0.27ab 0.22a 0.06b 0.07b 0.07a 0.06a 0.22b 0.06a 0.06a 0.24b

Note: Mean values with the same letter within a column are not significant different (P > 0.05).

5

Shell width(cm) 1.99 1.91 1.93 2.08 1.93 1.92 1.92 1.93 2.08 1.93 1.93 2.14

± ± ± ± ± ± ± ± ± ± ± ±

0.03a 0.02b 0.0ab 0.08a 0.02b 0.02b 0.02a 0.02a 0.08b 0.02a 0.02a 0.09b

Shell height(cm) 6.14 5.88 5.86 6.50 5.98 5.90 5.90 5.98 6.50 5.93 5.96 6.81

± ± ± ± ± ± ± ± ± ± ± ±

0.10a 0.07b 0.36ab 0.29a 0.09b 0.08b 0.08a 0.09a 0.29b 0.08a 0.09a 0.33b

Shell weight(g) 28.42 25.89 25.64 33.14 26.70 26.10 26.10 26.70 33.14 26.38 26.54 35.84

± ± ± ± ± ± ± ± ± ± ± ±

1.23a 0.87b 3.83ab 3.82a 1.00b 1.00b 1.00a 1.00a 3.82b 0.97a 1.03a 4.08b

Aquaculture 516 (2020) 734617

C. Yang, et al.

had larger growth traits than genotypes AA and GA. Genotypes AA and TA in g. 53337662 A > T had smaller traits than that of the TT genotype. Haplotype analysis was conducted according to the four identified SNPs which were significantly associated with growth traits. The haploblock structure of the four SNPs was formed (Fig. 4A), and one block was detected with SNP35 (g. 53337652 A > AAC), SNP36 (g. 53337657G > A), and SNP37 (g. 53337662A > T). Haplotype association analysis for growth traits was performed, and we found three haplotypes, namely, AACAA, AGT, and AGA. Haplotype AGT had a higher number of growth traits than haplotype AACAA (P < 0.05; Supplemental Table 2, Fig. 4B, 4C, 4D and 4E).

ubiquitously expressed throughout the developmental stage (Fig. 3B). In addition, a high expression level of PmOSR1 was detected in the blastula and gastrula, thereby corresponding with the result of developmental transcriptome analysis. The expression pattern analysis of PmOSR1 mRNA from four families showed a significantly high expression level in the hybrid families (Fig. 3C). PmOSR1 expression pattern in fast- and slow-growing groups revealed that PmOSR1 presented a significantly higher expression level in the former than in the latter (P < 0.05, Fig. 3D). 3.4. PmOSR1 polymorphisms In the polymorphism analysis of PmOSR1, 206 individuals in the base stock were sequenced through targeted resequencing. SNPs were identified on the basis of the multiple alignments of PmOSR1 sequences. A nucleotide site with an alternative base in five or more individuals was counted as a putative SNP locus. Forty-five SNPs were identified (Table 1), and the polymorphism analysis of the identified SNPs presented that HO ranged from 0 to 0.8155, whereas HE ranged from 0 to 0.5409. PIC classification indicated that 24 loci displayed low polymorphism level, and 21 loci presented medium polymorphism level. Hardy–Weinberg equilibrium (HWE) analysis showed that 17 loci deviated significantly from HWE (P < 0.05).

3.6. One selected SNP verification in another population SNP (g. 53337657 G > A) was selected for the genotype of 83 individuals, which were chosen for SNP verification. Tetra-primer ARMSPCR was used to detect the genotypes of individuals. The obtained genotype frequency was similar to the abovementioned results (Table 3). The association analysis of the growth traits of these pearl oysters with genotype revealed the higher growth traits in genotype GG than genotype AA and GA (Table 3). 4. Discussion

3.5. Association analysis between SNPs and growth traits As a zinc finger transcriptional factor, the protein sequence of OSR1 has three C2H2-type zinc fingers (Katoh, 2002). The C2H2 class comprised proteins containing, as the basic structural unit, pairs of cysteines and histidines separated by a loop of 12 amino acids (Evans and Hollenbergt, 1988). In the present study, the PmORS1 analysis showed that its primary sequences presented three ZnF_C2H2 domains and conserved motif (-F/Y-C–C—F———L–H—H—— motif) of OSR1 corresponding to the result of Katoh (2002) and Evans and Hollenbergt (1988). At the same time, the OSR1 comparison from different bivalves displayed their primary and advanced structure conservation, thereby

Association analysis between growth traits of the 206 individuals randomly selected from base stock and SNPs were performed, and four SNPs showed significant association with growth traits (P < 0.05). Table 2 illustrates the comparison results of the genotypes in the SNPs significantly associated with growth traits. For instance, the growth traits in the genotype AA of g.53337465 A > G were significantly larger than those in the genotype GA. The growth traits in the genotype AA of g. 53337652 A > AAC were significantly larger than those in the genotypes AAAC and AACAAC. Genotype GG in g.53337657 G > A

Fig. 4. Linkage disequilibrium analysis for the four SNPs in PmOSR1 significantly associated with growth traits of pearl oyster. A show the haploblock structure of the four SNPs. B, C, D and E show the growth traits (shell length, shell width, shell height, and shell weight) of different genotypes, respectively. Bars with different superscripts indicate significant difference (P < 0.05). 6

Aquaculture 516 (2020) 734617

C. Yang, et al.

Table 3 SNP (g. 53337657 G > A) verification in another population (mean ± SD). Number SNP36

Locus 53337657 G > A(83)

Genotype AA GA GG

Sample number 56 24 3

Shell length(cm) a

5.45 ± 0.64 5.41 ± 0.62a 6.49 ± 0.27b

Shell width(cm) a

1.84 ± 0.24 1.83 ± 0.27a 2.27 ± 0.05b

Shell height(cm) a

5.26 ± 0.70 5.18 ± 0.73a 6.18 ± 0.11b

Shell weight(g) 8.65 ± 2.94a 8.46 ± 2.71a 12.37 ± 1.36b

Mean values with the same letter within a column are not significant different (P > 0.05).

width, height, and weight. Thus, the alteration of the genetic sequence may lead to the variation in different correlated growth traits (Liu et al., 2002) and suggested that different growth traits may be controlled by the same genetic regulatory mechanism. Synonymous mutations do not change the kinds of amino acids, protein structure, and active characteristics but influence the biological function by regulating the gene expression or changing protein conformation (Cancela et al., 2010; Sauna et al., 2007). Synonymous mutations can affect the mRNA splicing, stability, structure, and translation efficiency of tRNA, thereby affecting the function of the protein synthesis (Wang, 2014). In the present study, the identified SNPs in PmOSR1 showed eight synonymous mutations. To determine whether the SNPs influenced the growth of pearl oysters, we conducted association analysis between the SNPs and growth traits, and one SNP (g. 53337465 A > G) as synonymous mutation was significantly associated with growth traits of pearl oyster, indicating its potential in breeding design. A haplotype is a physical arrangement of SNP alleles along a chromosome (Nazari et al., 2016; Sun et al., 2015). With the accessibility of SNP markers, haplotypes play important roles in genetic association studies (Cong et al., 2013; Liu et al., 2016; Andrade et al., 2017). The analysis of haplotypes is informative in association studies, because they provide a better representation of the actual gene diversity than SNPs (Duffy et al., 2007). In the present study, four SNPs significantly associated with the growth traits of pearl oyster were used to conduct haplotype studies, and a haplotype with three SNPs was obtained. The haplotype AGT revealed significantly high growth traits. These findings indicated the potential of AGT as marker for breeding with growth traits as a subject.

further indicating that the gene cloned in the present study was PmOSR1. These results showed the potential OSR1 function. In vertebrates, Osr1 and Osr2 exist and present a dynamic expression pattern in mouse embryogenesis, branchial arches, kidneys, and limbs (So and Danielian, 1999; Lan et al., 2001). Zebrafish OSR genes displayed important roles in development, especially that of the fin and kidney (Neto et al., 2012; Mudumana et al., 2008). In invertebrates, some OSR gene sequence information has been submitted online, but reports on OSR gene cloning and function are few. In the present study, PmOSR1 was expressed in the development of pearl oyster and in different tissues, thereby indicating its wide existence in the pearl oyster. Furthermore, PmOSR1 revealed higher expression level in the blastula and gastrula than in other developmental states which indicated PmOSR1 presented its function in development, especially early development. Transcriptome analysis of the two inbred families (A and D) and their reciprocal hybrid families (B and C) of P. f. martensii showed significantly varied growth traits of the four families (P < 0.05), and hybrid C had the highest values for different traits (Yang et al., 2018). OSR1 gene was obtained from the differentially expressed genes (DEGs) for comparisons between the hybrid and inbred families, and which was consistent with the result of the present study. In the previous studies of our group, we sequenced genes of the pearl oysters from experimental populations with different growths for their metabolome (PmEGFR) by searching SNP correlating with growth or some other six genes detected with DEGs to identify genes that contributed to the growth of pearl oysters and showed higher expression level in the fast-growing group than in the slow-growing group (Yang et al., 2018; Hao et al., 2018a and b, 2019; Wang et al., 2018, 2019). In the present study, PmOSR1 also showed significantly higher expression in the fast-growing group than that in the slow-growing group, indicating it may participate in the growth of pearl oyster, and the results corresponded with those of the quantitative trait locus analysis (Du et al., 2017). As a third-generation molecular marker, SNP can promote the development of analysis for genetic architecture of complex traits and improve the selection efficiency in animals (Mastrangelo et al., 2014). Sixteen SNP loci were developed for population genetics in P. fucata (Huang et al., 2014), and their genetic variation parameters (i.e., Ho, He, and PIC) had the same range with the values of most SNPs obtained in our study. Thirty-two SNP loci identified in Pf-MSTN cDNA revealed that 21 of these 32 loci were polymorphic, and the PIC value varied from 0.0739 to 0.3750. This result was also similar to the PIC of SNPs from PmOSR1. Therefore, the SNPs identified in PmOSR1 will be useful for future studies in investigating their utility in marker-assisted selection for P. f. martensii breeding. The association between the markers and specific traits in a population can determine the marker associated with a trait (Nazari et al., 2016). A SNP (c. 1815C.T) in the 39 UTRs of the TGF-b type I receptor gene from Zhikong scallops (Chlamys farreri) was identified, and scallops with genotype TT had higher growth trait values than those with genotype CC or CT in a full-sib family (Guo et al., 2012). Six SNPs were identified in the promoter region of an MSTN gene from scallop Chlamys nobilis, and association analysis showed significant effects of SNP g.-579A/C on body mass, soft-tissue mass, and adductor muscle mass (Fan et al., 2017). In the present study, four SNPs from PmOSR1 were significantly associated with growth traits, such as shell length,

5. Conclusion A gene named PmOSR1 possibly function in the growth of pearl oyster, as revealed from the analysis of genetic map and transcriptome. Then, we cloned the full length of PmOSR1 with conserved primary and advanced structures compared with other species. Expression analysis showed that PmOSR1 widely existed in all detected tissues during development. PmOSR1 also presented significantly high expression level in the fast-growing group. Forty-five SNPs were found in the genomic sequence of PmOSR1, and four SNPs showed significant association with growth traits. Haplotype AGT significantly revealed high growth traits. This study provided useful molecular information on the biological function of PmOSR1, thereby indicating a potential genetic mechanism in regulating pearl oyster growth and providing a candidate marker for the selective breeding design and genetic improvement of pearl oyster P. f. martensii. Declaration of competing interest No conflicts of interest, flnancial or otherwise, are declared by the authors. Acknowledgements This work was supported by “Innovation Team Project” (Grant number: 2017KCXTD016) from the Department of Education of 7

Aquaculture 516 (2020) 734617

C. Yang, et al.

Guangdong Province, the Science and Technology Department of Guangdong Province (Grant number: 2018A030310666, 2017A030307024 and 2017A030303076), the China Agriculture Research System (Grant number: CARS-049).

following whole-genome duplication. Mar. Genom. 25, 33–37. Lan, Y., Kingsley, P.D., Cho, E.S., Jiang, R., 2001. Osr2, a new mouse gene related to drosophila odd-skipped, exhibits dynamic expression patterns during craniofacial, limb, and kidney development. Mech. Dev. 107, 175–179. Lei, C., Li, J.H., Zheng, Z., Du, X.D., Deng, Y.W., 2019. Molecular cloning, expression pattern of β-carotene 15, 15-dioxygenase gene and association analysis with total carotenoid content in pearl oyster Pinctada fucata martensii. Comp. Biochem. Physiol. B 229, 34–41. Liu, S.X., Palti, Y., Gao, G.T., Rexroad III, C.E., 2016. Development and validation of a SNP panel for parentage assignment in rainbow trout. Aquaculture 452, 178–182. Liu, X.L., Chang, Y.Q., Xiang, J.H., Song, J., Ding, J., 2002. Analysis of effects of shell size characters on live weight in Chinese scallop Chlamys Farreri. Oceanol. Limnol. Sinica 33, 673–677 (In Chinese Abstract). Li, Y.G., He, M.X., 2014. Genetic mapping and QTL analysis of growth-related traits in Pinctada fucata using restriction-site associated DNA sequencing. PLoS One 9, e111707. Lucas, J.S., 2008. Environmental influences, pp.187–222. In: The Pearl Oyster, Southgate, P.C. and Lucas, J.S. Elsevier, Amsterdam, Netherland. Mastrangelo, S., Di Gerlando, R., Tolone, M., Tortorici, L., Sardina, M.T., Portolano, B., 2014. Genome wide linkage disequilibrium and genetic structure in Sicilian dairy sheep breeds. BMC Genet. 15 (1), 1–10. Mudumana, S.P., Hentschel, D., Liu, Y., Vasilyev, A., Drummond, I.A., 2008. Odd skipped related1 reveals a novel role for endoderm in regulating kidney versus vascular cell fate. Development 135, 3355–3367. Murgarella, M., Puiu, D., Novoa, B., Figueras, A., Posada, A., Canchaya, C., 2016. A first insight into the genome of the filter-feeder mussel Mytilus galloprovincialis. PLoS One 11 (3), e0151561. Nazari, S., Jafari, V., Pourkazami, M., Miandare, H.K., Abdolhay, H.A., 2016. Association between myostatin gene (MSTN-1) polymorphismand growth traits in domesticated rainbow trout (Oncorhynchus mykiss). Agri Gene 1, 109–115. Neto, A., Mercader, N., Gomez-Skarmeta, J.L., 2012. The osr1 and osr2 genes act in the pronephric anlage downstream of retinoic acid signaling and upstream of wnt2b to maintain pectoral fin development. Development 139 (2), 301–311. Nie, H.T., Yan, X.W., Huo, Z.M., Jiang, L.W., Chen, P., Liu, H., Ding, J.F., Yang, F., 2017. Construction of a high-density genetic map and quantitative trait locus mapping in the manila clam Ruditapes philippinarum. Sci. Rep. 8, 11953. Pasquier, J., Cabau, C., Nguyen, T., Jouanno, E., Severac, D., Braasch, I., Journot, L., Pontarotti, P., Klopp, C., Postlethwait, J.H., Guiguen, Y., Bobe, J., 2016. Gene evolution and gene expression after whole genome duplication in fish: the phylofish database. BMC Genomics 17 (1), 368. Ren, P., Peng, W.Z., You, W.W., Huang, Z.K., Guo, Q., Chen, N., He, P.R., Ke, J.W., Gwo, J., Ke, C.H., 2016. Genetic mapping and quantitative trait loci analysis of growthrelated traits in the small abalone Haliotis diversicolor using restriction-site-associated DNA sequencing. Aquaculture 454, 163–170. Sauna, Z.E., Kimchi-Sarfaty, C., Ambudkar, S.V., Gottesman, M.M., 2007. Silent polymorphisms speak: how they affect pharmacogenomics and the treatment of cancer. Cancer Res. 67, 9609–9612. Shi, Y.H., Wang, S., Gu, Z.F., Lv, J., Zhan, X., Yu, C.C., Bao, Z.M., Wang, A.M., 2014. Highdensity single nucleotide polymorphisms linkage and quantitative trait locus mapping of the pearl oyster, Pinctada fucata martensii dunker. Aquaculture 434, 376–384. So, P.L., Danielian, P.S., 1999. Cloning and expression analysis of a mouse gene related to Drosophila odd-skipped. Mech. Dev. 84 (1), 157–160. Sun, X.J., Shin, G., Hedgecock, D., 2015. Inheritance of high-resolution melting profiles in assays targeting single nucleotide polymorphisms in protein-coding sequences of the Pacific oyster Crassostrea gigas: implications for parentage assignment of experimental and commercial broodstocks. Aquaculture 437, 127–139. Tena, J.J., Neto, A., Calle-Mustienes, E.D.L., Bras-Pereira, C., Casares, F., GómezSkarmeta, J.L., 2007. Odd-skipped genes encode repressors that control kidney development. Dev. Biol. 301 (2), 518–531. Tian, M.L., Li, Y.P., Jing, J., Mu, C., Du, H.X., Dou, J.Z., Mao, J.X., Li, X., Jiao, W.Q., Wang, Y.F., Hu, X.L., Wang, S., Wang, R.J., Bao, Z.M., 2015. Construction of a highdensity genetic map and quantitative trait locus mapping in the sea cucumber Apostichopus japonicas. Sci. Rep. 5, 14852. Wada, K.T., Komaru, A., 1996. Color and weight of pearls produced by grafting the mantle tissue from a selected population for white shell color of the Japanese pearl oyster Pinctada jixata martensii (Dunker). Aquaculture 142, 25–32. Wang, H.F., 2014. SNPs Identification of Growth-Related Genes and Correlation Analysis on Growth Traits in Sinipercid Species. Sun Yat-Sen University, Guangzhou. Wang, J., Li, L., Zhang, G., 2016. A high-density SNP genetic linkage map and QTL analysis of growth-related traits in a hybrid family of oysters Crassostrea gigas × Crassostrea angulata using genotyping-by-sequencing. G3-Genes Genom Genet. 6, 1417–1426. Wang, L., Wan, Z.Y., Bai, B., Huang, S.Q., Chua, E., Lee, M., Pang, H.Y., Wen, Y.F., Liu, P., Liu, F., Sun, F., Lin, G., Ye, B.Q., Yue, G.H., 2015. Construction of a high-density linkage map and fine mapping of QTL for growth in Asian seabass. Sci. Rep. 5, 16358. Wang, Q.H., Lu, Y.Z., Deng, Y.W., Du, X.D., 2013. Correlation analysis on pearl thickness, weight and morphology of Pinctada martensii. J. Guangdong Ocean Univ. 33 (3), 18–21. Wang, Q., Lan, Y., Cho, E.S., Maltby, K.M., Jiang, R., 2005. Odd-skipped related 1 (Odd 1) is an essential regulator of heart and urogenital development. Dev. Biol. 288, 582–594. Wang, Q.H., Hao, R.J., Zhao, X.X., Huang, R.L., Zheng, Z., Deng, Y.W., Chen, W.Y., Du, X.D., 2018. Identification of EGFR in pearl oyster (Pinctada fucata martensii) and correlation analysis of its expression and growth traits. Biosci. Biotechnol. Biochem. 82 (2), 1–8. Wang, S., Zhang, J.B., Jiao, W.Q., Li, J., Xun, X.G., Sun, Y., Guo, X.M., Huan, P., Dong, B.,

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.aquaculture.2019.734617. References Andrade, E.S., Fracasso, N.C.A., Strazza, P.S., Simoes, A.L., Mendes, C.T., 2017. Associations of OCA2-HERC2 SNPs and haplotypes with human pigmentation characteristics in the Brazilian population. Leg. Med. 24, 78–83. Baranski, M., Gopikrishna, G., Robinson, N.A., Katneni, V.K., Shekhar, M.S., Shanmugakarthik, J., Jothivel, S., Gopal, C., Ravichandran, P., Kent, M., Arnyasi, M., Ponniah, A.G., 2014. The development of a high density linkage map for black tiger shrimp (Penaeus monodon) based on cSNPs. PLoS One 9, e85413. Cancela, M.L., Bargelloni, L., Boudry, P., Boulo, V., Dias, J., Huvet, A., Laizé, V., Lapègue, S., Leite, R., Mira, S., Nielsen, E.E., Planas, J.V., Roher, N., Sarropoulou, E., Volckaert, F.A.M., 2010. Genomic approaches in aquaculture and fisheries. In: Introduction to Marine Genomics. Springer, Totowa, New Jersey, USA, pp. 213–286. Cong, R.H., Li, Q., Kong, L.F., 2013. Polymorphism in the insulin-related peptide gene and its association with growth traits in the Pacific oyster Crassostrea gigas. Biochem. Syst. Ecol. 46, 36–43. Deng, Y.W., Fu, S., Du, X.D., Wang, Q.H., Huang, H.L., Liu, D., 2013. Fertilization, hatching, survival, and growth of third-generation colored pearl oyster (Pinctada martensii) Stocks. J. Appl. Aquac. 25, 113–120. Deng, Y.W., Gao, Y.Z., Chen, W.Y., Du, X.D., Lu, J., 2011. Comparison of growth performance and genetic diversity of pearl oyster (Pinctada martensii) families in a breeding program. Afr. J. Agric. Res. 6 (31), 6530–6536. Duffy, D.L., Montgomery, G.W., Chen, W., Zhao, Z.Z., Le, L., James, M.R., Hayward, N.K., Martin, N.G., Sturm, R.A., 2007. A three-single-nucleotide polymorphism haplotype in intron 1 of OCA2 explains most human eye-color variation. Am. J. Hum. Genet. 80, 241–252. Du, F.K., Xu, G.C., Li, Y., Nie, Z.J., Xu, P., 2016. Development of SNP markers and validation 31 SNPs in Coilia nasus. Conserv. Genet. Resour. 9 (1), 99–101. Du, X.D., Fan, G.Y., Jiao, Y., Zhang, H., Guo, X.M., Huang, R.L., Zheng, Z., Bian, C., Deng, Y.W., Wang, Q.H., Wang, Z.D., Liang, X.M., Liang, H.Y., Shi, C.C., Zhao, X.X., Sun, F.M., Hao, R.J., Bai, J., Liu, J.L., Chen, W.B., Liang, J.L., Liu, W.Q., Xu, Z., Shi, Q., Xu, X., Zhang, G.F., Liu, X., 2017. The pearl oyster Pinctada fucata martensii genome and multi-omic analyses provide insights into biomineralization. GigaScience 6 (8), 1–12. Evans, M.E., Hollenbergt, S.M., 1988. Zinc fingers: gilt by association. Cell 52 (1), 1–3. Fan, S.G., Xu, Y.H., Liu, B.S., He, W.Y., Zhang, B., Su, J.Q., Yu, D.H., 2017. Molecular characterization and expression analysis of the myostatin gene and its association with growth traits in Noble scallop (Chlamys nobilis). Comp. Biochem. Physiol. B 212, 24–31. Gomez-Chiarri, M., Warren, W.C., Guo, X.M., Proestou, D., 2015. Developing tools for the study of molluscan immunity: the sequencing of the genome of the eastern oyster, Crassostrea virginica. Fish Shellfish Immunol. 46 (1), 2–4. Guo, H.H., Bao, Z.M., Li, J.Q., Lian, S.S., Wang, S., He, Y., Fu, X.T., Zhang, L.L., Hu, X.L., 2012. Molecular characterization of TGF-b type I receptor gene (Tgfbr1) in Chlamys farreri, and the association of allelic variants with growth traits. PLoS One 7 (11), e51005. Hao, R.J., Du, X.D., Yang, C.Y., Deng, Y.W., Zheng, Z., Wang, Q.H., 2019. Integrated application of transcriptomics and metabolomics provides insights into unsynchronized growth in pearl oyster Pinctada fucata martensii. Sci. Total Environ. 666, 46–56. Hao, R.J., Wang, Z.M., Yang, C.Y., Deng, Y.W., Zheng, Z., Wang, Q.H., Du, X.D., 2018b. Metabolomic responses of juvenile pearl oyster Pinctada maxima to different growth performances. Aquaculture 491, 258–265. Hao, R.J., Zheng, Z., Wang, Q.H., Du, X.D., Deng, Y.W., Huang, R.L., 2018a. Molecular and functional analysis of pmchst1b in nacre formation of Pinctada fucata martensii. Comp. Biochem. Physiol. B 225, 13–20. Huang, X.D., Wu, S.Z., Guan, Y.Y., Li, Y.G., He, M.X., 2014. Identification of sixteen single-nucleotide polymorphism markers in the pearl oyster, Pinctada fucata, for population genetic structure analysis. J. Genet. 93, e1–e4. James, R.G., Kamei, C.N., Wang, Q., Jiang, R., Schultheiss, T.M., 2006. Odd-skipped related 1 is required for development of the metanephric kidney and regulates formation and differentiation of kidney precursor cells. Development 133, 2995–3004. Jiao, W.Q., Fu, X.T., Dou, J.Z., Li, H.D., Su, H.L., Mao, J.X., Yu, Q., Zhang, L.L., Hu, X.L., Huang, X.T., Wang, Y.F., Wang, S., Bao, Z.M., 2014. High-resolution linkage and quantitative trait locus mapping aided by genome survey sequencing: building up an integrative genomic framework for a bivalve mollusc. DNA Res. 21, 85–101. Katoh, M., 2002. Molecular cloning and characterization of OSR1 on human chromosome 2p24. Int. J. Mol. Med. 10, 221–225. Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N., Sternberg, M.J.E., 2015. The phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10 (6), 845–858. Kim, J.H., Leong, J.S., Koop, B.F., Devlin, R.H., 2016. Multi-tissue transcriptome profiles for coho salmon (Oncorhynchus kisutch), a species undergoing rediploidization

8

Aquaculture 516 (2020) 734617

C. Yang, et al.

Sci. Rep. 5, 15612. Zhao, X.X., Wang, Q.H., Jiao, Y., Huang, R.L., Deng, Y.W., Wang, H., Du, X.D., 2012. Identification of genes potentially related to biomineralization and immunity by transcriptome analysis of pearl sac in pearl oyster Pinctada martensii. Mar. Biotechnol. 14 (6), 730–739. Zhang, G.F., Fang, X.D., Guo, X.M., Li, L., Luo, R.B., Xu, F., Yang, P.C., Zhang, L.L., Wang, X.T., Qi, H.G., Xiong, Z.Q., Que, H.Y., Xie, Y.L., Holland, P.W., Paps, J., Zhu, Y.B., Wu, F.C., Chen, Y.X., Wang, J.F., Peng, C.F., Meng, J., Yang, L., Liu, J., Wen, B., Zhang, N., Huang, Z.Y., Zhu, Q.H., Feng, Y., Mount, A., Hedgecock, D., Xu, Z., Liu, Y.J., Domazet-Loso, T., Du, Y.S., Sun, X.Q., Zhang, S.D., Liu, B.H., Cheng, P.Z., Jiang, X.T., Li, J., Fan, D.D., Wang, W., Fu, W.J., Wang, T., Wang, B., Zhang, J.B., Peng, Z.Y., Li, Y.X., Li, N., Wang, J.P., Chen, M.S., He, Y., Tan, F.J., Song, X.R., Zheng, Q.M., Huang, R.L., Yang, H.L., Du, X.D., Chen, L., Yang, M., Gaffney, P.M., Wang, S., Luo, L.H., She, Z.C., Ming, Y., Huang, W., Zhang, S., Huang, B.Y., Zhang, Y., Qu, T., Ni, P.X., Miao, G.Y., Wang, J.Y., Wang, Q., Steinberg, C.E., Wang, H.Y., Li, N., Qian, L.M., Zhang, G.J., Li, Y.R., Yang, H.M., Liu, X., Wang, J., Yin, Y., Wang, J., 2012. The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490 (7418), 49–54.

Zhang, L.L., Hu, X.L., Sun, X.Q., Wang, J., Zhao, C.T., Wang, Y.F., Wang, D.W., Huang, X.T., Wang, R.J., Lv, J., Li, Y.L., Zhang, Z.F., Liu, B.Z., Lu, W., Hui, Y.Y., Liang, J., Zhou, Z.C., Hou, R., Li, X., Liu, Y.C., Li, H.D., Ning, X.H., Lin, Y., Zhao, L., Xing, Q., Dou, J.Z., Li, Y.P., Mao, J.X., Guo, H.B., Dou, H.Q., Li, T.Q., Mu, C., Jiang, W.K., Fu, Q., Fu, X.T., Miao, Y., Liu, J., Yu, Q., Li, R.J., Liao, H., Li, X., Kong, Y.F., Jiang, Z., Chourrout, D., Li, R.Q., Bao, Z.M., 2017. Scallop genome provides insights into evolution of bilaterian karyotype and development. Nat. Ecol. Evol. 1 (5), 0120. Wang, Z.M., Liang, F.L., Huang, R.L., Deng, Y.W., Li, J.H., 2019. Identification of the differentially expressed genes of Pinctada maxima individuals with different sizes through transcriptome analysis. Reg. Stud. Mar. Sci. 26, e100512. Xia, J.H., Liu, F., Zhu, Z.Y., Fu, J.J., Feng, J.B., Li, J.L., Yue, G.H., 2010. A consensus linkage map of the grass carp (Ctenopharyngodon idella) based on microsatellites and SNPs. BMC Genomics 11, 135. Yang, J.M., Luo, S.J., Li, J.H., Zheng, Z., Du, X.D., Deng, Y.W., 2018. Transcriptome analysis of growth heterosis in pearl oyster Pinctada fucata martensii. Febs. Open Bio. 8 (11), 1794–1803. Yu, Y., Zhang, X.J., Yuan, J.B., Li, F.H., Chen, X.H., Zhao, Y.Z., Huang, L., Zheng, H.K., Xiang, J.H., 2015. Genome survey and high-density genetic map construction provide genomic and genetic resources for the Pacific White Shrimp Litopenaeus vannamei.

9